Download PhD Thesis Non-thermal technologies for the disinfection of food

University of Patras
School of Medicine
Msc Program
“Biomedical Sciences”
PhD Thesis
Non-thermal technologies for the disinfection of food
and risk assessment for Public Health
BIRMPA ANGELIKI
Agronomist (Food Scientist), MSc
Patras, 2014
Πανεπιστήμιο Πατρών
Τμήμα Ιατρικής
Μεταπτυχιακό Πρόγραμμα
«Βασικές Ιατρικές Επιστήμες»
Διδακτορική Διατριβή
Εναλλακτικές Τεχνολογίες Απολύμανσης Τροφίμων
και εκτίμηση κινδύνου για την Δημόσια Υγεία.
ΜΠΙΡΜΠΑ ΑΓΓΕΛΙΚΗ
Γεωπόνος (Επιστήμης & Τεχνολογίας Τροφίμων), MSc
Πάτρα, 2014
True science teaches, above all, to doubt and be ignorant.
Miguel de Unamuno (1864-1936) Spanish writer and philosopher
3 Member Committee
Associate Professor Apostolos Vantarakis (Supervisor)
Professor Michalis Leotsinidis
Professor Chrissanthy Papadopoulou
7 Member Committee
Associate Professor Apostolos Vantarakis (Supervisor)
Professor Michalis Leotsinidis
Professor Chrissanthy Papadopoulou
Associate Professor Maria Kapsokefalou
Professor Iris Spiliopoulou
Professor John Zarkadis
Senior Lecturer James Lyng
Acknowledgements
I owe my deepest gratitude to my supervisor, Associate Professor Apostolos Vantarakis.
I am heartily thankful to him for his warm encouragement, motivated guidance, and
considerable support from the start to the completion in my research work. This thesis
would not have been possible without his excellent insight and creative ideas as well as
extreme patience and diligence during my dissertation.
I would also like to give my great appreciation to my committee members, Professor M.
Leotsinidis, with his valuable comments and suggestions during the experimental part of
the thesis as well as during writing of the PhD thesis, and Professor C. Papadopoulou,
for her helpful suggestions and valuable comments in my thesis research.
I am also grateful to the members of my 7-member committee Associate Professor
Maria Kapsokefalou, Professor Iris Spiliopoulou-Sdougkou and Professor John Zarkadis
for their valuable suggestions in my study and research work. I would like to give my
special thanks to the member of my 7-member committee Senior Lecturer Dr. James
G.Lyng, who gave me the opportunity to have a great experience under his supervision
in the laboratory in School of Agriculture Food Science and Veterinary Medicine of
College of Life Sciences in UCD Dublin, during my Erasmus placement June-September
2012, in Dublin.
I would also like to extend my sincere appreciation to many people who have given me a
huge help in my research progress. They are Postdoctoral Researchers Dr. Petros
Kokkinos with his suggestions throughout the phD thesis as well as with his warm
encouragement when it was needed. Thanks go also to Dr. Panos Ziros, MSc student
Maria Bellou, PhD student Spiros Paparrodopoulos, Panagiotis Pitsos, Postodoctoral
Researcher Dr. Eleni Sazakli and MSc student Efi Kougia. Furthermore, I am also
grateful to my lab mates and many of my colleagues for their kindly helps and great
support, MSc student Maria Tselepi and Gavriil Vasilopoulos. I would also like to give
my special thanks to Dr. Paul Whyte (Senior Lecturer of School of Veterinary Medicine
and Veterinary Science Centre, Dublin) who guided me and gave his special suggestions
during my Erasmus placement June-September 2012, in Dublin.
Moreover, I would like to express my gratitude to Professor Petros Groumpos and his
PhD student Antigoni Anninou for our collaboration to construct and implement the
mathematical model of this PhD thesis.
i
Thanks goes also to graduate students Wanda and Katell who gave me valuable help
during my lab work in UCD, Dublin. I would also like to thank Assistant Professor Dr.
Panagiotis Skandamis for his warm acceptance to the lab of Quality Assurance in
Agricultural University of Athens as well as Dr .Vassiliki Sfika in Department of
Quality Control, Regional Centre of Plant Protection and Quality Control Achaias,
Ministry of Rural Development and Food.
Finally, I am forever indebted to my parents, my sister and Vassilis for their
understanding, endless patience as well as their encouragement and support when it was
most required.
Abstract
Fruits and vegetables are considered as part of a healthy diet and lifestyle. However,
concerns have arisen regarding the microbiological safety of Ready To Eat (RTE)
produces due to a number of foodborne outbreaks associated with pathogens. Although
strict practices for controlling the safety of RTE produce have been implemented in the
fresh produce industry, the current commercial operations rely on a wash treatment with
water or with an antimicrobial agent as the only step for reducing microbial populations
on fresh produce. However, washing with common sanitizers has been demonstrated to
achieve no more than 1-2 log 10 reduction in pathogen populations. Recently, much
research effort has been put into development to provide multiple-hurdle techniques
which enhance produce safety. Thus, non-thermal technologies for the inactivation of
microorganisms are of increasing interest to the food industry for the control of spoilage
and safety, thus for assuring public health.
In this study, the effects of non-thermal disinfection processes, Near UV-Visible light
(NUV-Vis), Continuous Ultraviolet Light (UV 254 nm), High Intensity Light Pulses
(HILP), Ultrasound (US), as well as conventional sodium hypochlorite (SH) disinfection
solutions were used. The effect of the above technologies was tested against bacteria
(Escherichia coli, Staphylococcus aureus, Salmonella Enteritidis and Listeria innocua)
and viruses (Human Adenovirus). More precisely, the bacteria that were used were: E.
coli
K12,
E.
coli
NCTC
9001
(representative
microorganisms
for
the
Enterohaemorrhagic foodborne pathogen E. coli O157:H7), S. aureus NCTC 6571, L.
innocua NCTC 11288 (as a surrogate microorganism for the common foodborne
pathogen L. monocytogenes), S. Enteritidis NCTC 6676 and HAdV (indicator virus
selected as a surrogate of HAV and norovirus).
The main scope of this work was to study the efficacy of three light technologies on
liquid suspensions. Then, the effect of UV, US, SH and combined technologies were
evaluated on their efficiency to disinfect inoculated romaine lettuce, strawberries and
cherry tomatoes. Furthermore, the effect of the above technologies on quality (color) and
physicochemical
characteristics
of
the
RTE
produces
was
evaluated.
The
physicochemical characteristics tested were Total Antioxidant Capacity (TAC), Total
Phenolic Content (TPC) and Ascorbic Acid (AA) concentration.
This study demonstrates that the use of alternative non-thermal technologies is effective
for inactivation of microorganisms in fresh RTE foods and could be used as an
alternative to traditional chlorine immersions. However, the effect of UV and US on
iii
quality and nutritional quality retention of RTE foods should be considered before its
use as a disinfection technique.
As far as non-thermal light technologies are concerned, HIPL treatment inactivated both
E. coli and L. innocua more rapidly and effectively than either continuous UV-C or
NUV-vis treatments. With HILP at a distance of 2.5 cm from the lamp, E. coli and L.
innocua populations were reduced by 3.07 and 3.77 log 10 CFU/mL respectively after a 5
sec treatment time, and were shown to be below the limit of detection (<0.22 log10
CFU/mL) following 30 sec exposure to HILP (106.2 J/cm2).
Treatment of lettuce with UV reduced significantly the population of E. coli, S.aureus, S.
Enteritidis and L. innocua by 1.75, 1.21, 1.39 and 1.27 log 10 CFU/g, respectively.
Furthermore, more than a 2- log 10 CFU/g reduction of E. coli, S. Enteritidis and
L.innocua was achieved with US. In strawberries, UV treatment reduced bacteria only
by 1–1.4 log 10 CFU/g. The maximum reductions of microorganisms, observed in
strawberries after treatment with US, were 3.04, 2.52, 5.24 and 6.12 log 10 CFU/g for E.
coli, S. aureus, S. Enteritidis and L. innocua, respectively. Finally, cherry tomatoes
exhibited the best results when treated with non-thermal technologies. For instance,
3.16, 2.62, 3.29, 3.16 log 10 CFU/g for E. coli, S. aureus, S. Enteritidis and L. innocua,
respectively, were achieved when US treatment was used. UV treatment resulted in 2.39,
2.05, 2.62, 2.56 log 10 CFU/g reduction of the above microorganisms. The combined
technologies of alternative followed by conventional disinfection technologies resulted
in 2-3.50 log 10 CFU/g reduction for lettuce and strawberries. However, cherry tomatoes
exhibited greater reductions (3.28-4.78 log 10 CFU/g reduction). Finally, 1-2 log 10 CFU/g
log reduction was achieved for lettuce and strawberries when RTE foods were immersed
in NaOCl 200ppm solutions, and greater reductions (3-4 log 10 CFU/g) were achieved
for cherry tomatoes.
It was observed that HAdV was inactivated faster when chlorine treatment was used.
However, UV non thermal technology found to be more effective for disinfection of
HAdV compared to US, achieving a log 10 reduction of 2.13, 1.25 and 0.92 for lettuce,
strawberry and cherry tomatoes respectively when UV treatment for 30 minutes was
implemented, whereas, US treatment for the same treatment period achieved a log 10
reduction of 0.85, 0.53 and 0.36 log 10 respectively. The sequential use of US and UV
was found to be more effective and less time consuming, than when the treatments were
used alone, indicating the existence of an additive effect.
Treatment with UV and US, for time periods (up to 30 min) did not significantly (p >
0.05) change the color of RTE foods tested.
Moreover, it was indicated that no
significant differences (p>0.05) were observed as far as TAC is concerned when
conventional treatments at different treatment times were used. However, when
alternative disinfection treatments were used, an increase in TAC concentration was
obvious from the first minutes of treatment. TPC concentration remained constant or
was slightly decreased when RTE foods were immersed in NaOCl solutions. However,
TPC increased significantly (p<0.05) in all RTE foods when UV and US alternative
disinfection technologies were used. The vit.C content of RTE foods did not exhibit any
significant changes during different treatments. However, vit.C was slightly decreased
(p<0.05) when treatments of more than 30 minutes for US, UV and combinations of
UV+US occurred.
Furthermore, a computerized model was proposed based on critical points which are
important during the production of lettuce. More precisely the development of a
Decision Support System (DSS) using the theory of Fuzzy Cognitive Maps (FCMs), in
order to diagnose the importance of critical control points (concepts) for the food safety
and hygiene during the production of salad vegetables (lettuce), was implemented. The
methodology described, extracts the knowledge from experts with different scientific
background and exploits their experience on the process of lettuce production. The
results of this study show that the present software tool can be explored and problems
that can arise during the food production chain can be prevented.
Generally, it was noted that the effect of each disinfection method is dependent upon the
treatment time tested and the type of food. Treatment with UV and US reduced the
numbers of selected inoculated bacteria on lettuce, strawberries and cherry tomatoes,
which could be good alternatives to other traditional and commonly used technologies
such as chlorine and hydrogen peroxide solutions. These results suggest that UV and US
might be promising, non-thermal and environmental friendly disinfection technologies
for fresh RTE produce industry.
Taking everything into consideration, disinfection technologies play an important role in
commercial practice in order to prevent the survival of pathogens and lower the risk of
contamination thus assuring public health. However, nutritional and quality properties
are essential as they can provide a protective role against the development and
progression of many diseases and must be considered for the selection of disinfection
process parameters.
v
ΠΕΡΙΛΗΨΗ
Εκτεταμένη Περίληψη στα Ελληνικά
Εισαγωγή
Η κατανάλωση φρούτων και λαχανικών αποτελεί μέρος μίας υγιεινούς δίαιτας και
διατροφικού προφίλ γενικότερα. Η Μεσογειακή διατροφή αποτελεί ένα μοντέρνο τρόπο
διατροφής η οποία έχει τις ρίζες της στις χώρες της Μεσογείου, όπως η Ελλάδα, η
Ισπανία, η Πορτογαλία και η Νότια Ιταλία. Τα βασικά συστατικά που την απαρτίζουν
είναι το λάδι, τα λαχανικά, τα δημητριακά, τα φρούτα, τα ψάρια, τα γαλακτοκομικά, και
η μικρή κατανάλωση κρέατος (Noah and Truswell, 2001). Ως εκ τούτου, λαχανικά όπως
το μαρούλι, οι τομάτες είναι κύρια συστατικά μιας ισορροπημένης διατροφής. Επίσης,
φρούτα όπως οι φράουλες προτιμώνται από εκείνους που θέλουν να ακολουθούν την
Μεσογειακή Διατροφή αλλά και από αυτούς που θέλουν να προσέχουν την υγεία τους.
Το μαρούλι (Lactuca sativa L.) καταναλώνεται κυρίως ως σαλάτα και αποτελεί μία
πλούσια πηγή συστατικών ευεργετικών για την υγεία όπως τα φαινολικά, η βιταμίνη C,
τα καροτενοειδή και οι χλωροφύλλες (Nicolle et al., 2004). Περιλαμβάνει πολλά
μακροστοιχεία (π.χ K, Na, Ca και Mg) και ιχνοστοιχεία (π.χ Fe, Mn, Cu, Zn και Se), τα
οποία αποτελούν σημαντικά συστατικά μια σωστής διατροφής (Kawashima & Soares,
2003). Το μαρούλι αποτελεί επίσης μία καλή πηγή φωτοσυνθετικών χρωστικών και
άλλων φυτοχημικών τα οποία ωφελούν την διατροφή και διαδραματίζουν σπουδαίο
ρόλο στην παρεμπόδιση πολλών οξειδωτικών- σχετιζόμενων με το στρες- ασθενειών
(Llorach et al., 2008). Οι φράουλες, είναι πλούσιες σε μία σειρά φυτοχημικών, ιδιαίτερα
των φαινολικών συστατικών, κατέχοντας υψηλή αντιοξειδωτική ικανότητα (Häkkinen et
al., 1999, Koponen et al., 2007). Επίσης έχουν μεγάλη περιεκτικότητα σε βιταμίνη C
(60-100 mg/100 g τροφίμου) και σε ανθοκυανίνες, ειδικά πελαργονιδίνη-3-γλυκοζίτη
(pg-3-gluc) και κυανιδίνη-3-γλυκοζίτη (CyA-3-gluc). Ως εκ τούτου, η φράουλα
θεωρείται ως μια σημαντική διαιτητική πηγή ενώσεων που προάγουν την υγεία
(Koponen et al., 2007). Οι τομάτες αποτελούν μία καλή πηγή βιταμινών (βιταμίνη Α,
βιταμίνη C, και άλλων βιταμινών) καθώς και μεταλλικών στοιχείων (νάτριο, ασβέστιο,
φώσφορος, σίδηρος), και ινών, πρωτεϊνών και λιπών. Η τομάτα είναι μία καλή πηγή
αντιοξειδωτικών όπως το λυκοπένιο. Είναι γνωστό ότι το λυκοπένιο και οι ίνες είναι
ευεργετικές στην ανθρώπινη υγεία, όταν καταναλώνονται ως μέρος μια ισορροπημένης
διατροφής (Canene-Adams et al., 2005). Σύμφωνα με μελέτες το λυκοπένιο έχει
vii
χαρακτηριστεί για τις αντιφλεγμονώδεις, αντιμεταλλαξιγόνες και αντικαρκινικές
ιδιότητές του (Boon et al., 2010). Επιπλέον, το λυκοπένιο είναι γνωστό για τη μείωση
του κινδύνου αδενώματος, και την προώθηση λειτουργικότητας του ανοσοποιητικού
συστήματος (Kun et al., 2006). Συνιστάται, 6-15 mg πρόσληψη λυκοπενίου για την
βελτίωση της υγείας (Kun et al., 2006). Οι διαλυτές φυτικές ίνες ρυθμίζουν τη γλυκόζη
στο αίμα και τα επίπεδα χοληστερόλης (Weickert and Pfeifer, 2008). Ενώ οι αδιάλυτες
φυτικές ίνες προάγουν την κάθαρση και βοηθούν εναντίον πολλών καρκίνων όπως ο
καρκίνος του παχέος εντέρου (Alvarado et al., 2001). Στα προϊόντα τομάτας, η βιταμίνη
C και οι πολυφαινόλες έχουν αναφερθεί ότι είναι τα κύρια υδρόφιλα αντιοξειδωτικά
συστατικά, ενώ η βιταμίνη Ε και τα καροτενοειδή αποτελούν κυρίως το υδρόφοβο
κλάσμα (Hsu, 2008).
Πολλές επιδημιολογικές μελέτες έχουν συσχετίσει την κατανάλωση φρούτων και
λαχανικών με τον προστατευτικό ρόλο τους ενάντια σε πολλές ασθένειες (Hannum,
2004). Ρίζες οξυγόνου, μπορεί να αντιδράσουν με λίπη, πρωτεΐνες και DNA. Ο ρόλος
των αντιοξειδωτικών που υπάρχουν στα φρούτα και στα λαχανικά είναι να διατηρούν τα
χαμηλά επίπεδα των ελευθέρων ριζών είτε παρεμποδίζοντας την εμφάνισή τους, είτε
ευνοώντας την αποσύνθεσή τους (Hancock et al., 2007).
Παρόλα αυτά, το τελευταίο διάστημα, λόγω του αυξανόμενου αριθμού τροφιμογενών
ασθενειών σε όλο τον κόσμο, επικρατεί ανησυχία σχετικά με την μικροβιολογική
ασφάλεια των τροφίμων αυτών. Τρόφιμα «έτοιμα προς κατανάλωση (ready-to-eat)»,
θεωρούνται ότι ανήκουν στην κατηγορία «υψηλού κινδύνου». Τα συγκεκριμένα
τρόφιμα δεν επιδέχονται κάποια θερμική ή άλλη επεξεργασία θανάτωσης παθογόνων
μικροοργανισμών.
Η μικροβιολογική ασφάλεια των τροφίμων και των τροφιμογενών ασθενειών αποτελούν
περίπλοκα ζητήματα, καθώς περισσότερες από 200 γνωστές ασθένειες είναι γνωστό ότι
μεταδίδονται μέσω των τροφίμων. Οι κύριοι λόγοι μετάδοσης τροφιμογενών ασθενειών
είναι η επιμόλυνση με βακτήρια, ιούς, παράσιτα, μύκητες.
Στις Η.Π.Α, το μέσο ετήσιο κόστος που σχετίζεται με βακτηριακές και παρασιτικές
τροφιμογενείς λοιμώξεις εκτιμάται στα 6.5 δισεκατομμύρια δολάρια (Buzby & Roberts,
1996, Tauxe, 2002). Ο Tauxe (2002) αναφέρει ότι ανάμεσα στις καταγεγραμμένες
τροφιμογενείς λοιμώξεις, οι βακτηριακές λοιμώξεις ευθύνονται για ένα περίπου 30%
των περιπτώσεων, οι ιολογικές για το 67% και οι παρασιτικές για το 3%. Νοσηλεία στο
νοσοκομείο πραγματοποιήθηκε λόγω λοιμώξεων από βακτήρια (60%), από ιούς (35%)
και από παράσιτα (5%). Τέλος, θάνατος είναι δυνατό να προέλθει από βακτήρια (72%),
ιούς (7%) και παράσιτα (21%). Έχει αναφερθεί ότι πέντε τροφιμογενείς παθογόνοι
μκροοργανισμοί (E. coli O157:H7, Salmonella, Campylobacter, Listeria, and
Toxoplasma) είναι υπεύθυνοι για 3.5 εκατομμύρια κρούσματα, 33.000 νοσηλείες και
1.600 θανάτους ετησίως στις Η.Π.Α (Tauxe, 2002).
Μέχρι σήμερα στις βιομηχανίες τροφίμων εφαρμόζονται μία σειρά αυστηρών
πρακτικών απολύμανσης για τον έλεγχο της ασφάλειας των έτοιμων προς κατανάλωση
τροφίμων. Οι πρακτικές αυτές περιλαμβάνουν ξεπλύματα με τρεχούμενο νερό ή με
αντιμικροβιακά
διαλύματα.
Επιστημονικές
μελέτες
όμως
καταδεικνύουν
την
ανικανότητα επαρκούς απολύμανσης παθογόνων μικροοργανισμών που υπάρχουν στα
τρόφιμα, με τις συνήθεις πρακτικές που εφαρμόζονται σήμερα. Για το λόγο αυτό, νέες
τεχνικές πολλαπλών εμποδίων έχουν αρχίσει να εφαρμόζονται με σκοπό την
διασφάλιση της δημόσιας υγείας.
Τα τρόφιμα επεξεργάζονται με διάφορες τεχνολογίες με σκοπό την μείωση και την
απομάκρυνση πιθανών παθογόνων ή άλλων βιολογικών κινδύνων που μπορεί να
υπάρχουν στα τρόφιμα. Οι κλασικές τεχνολογίες απολύμανσης όπως παστερίωση ή
αποστείρωση, χρησιμοποιούνται με σκοπό την απενεργοποίηση ή τη θανάτωση των
μικροβίων.
Η απολύμανση με χλώριο αποτελεί μία ευρέως διαδεδομένη και οικονομική μέθοδο
απολύμανσης η οποία χρησιμοποιείται για την απολύμανση νερού και τροφίμων (EPA,
1999c). Διαφορετικές μορφές χλωρίου μπορούν να χρησιμοποιηθούν στην απολύμανση
των τροφίμων όπως: διοξείδιο του χλωρίου, υποχλωριώδες νάτριο, υποχλωριώδες
ασβέστιο, κ.α (Park et al., 2008). Παρόλα αυτά το χλώριο μπορεί να αντιδράσει με
οργανικές
ουσίες
σχηματίζοντας
οργανοχλωρoπαράγωγα,
δηλαδή
έτσι
ενώσεις
τοξικές
που
χημικές
ανήκουν
στην
ουσίες
όπως
κατηγορία
των
τριαλογονοµεθανίων (THM’s) (McDonnel and Russell, 1999).
Οι εναλλακτικές, μη-θερμικές τεχνολογίες απολύμανσης έχουν αποδειχθεί ότι είναι
ικανές να επιτύχουν απολύμανση μικροβίων χωρίς την έκθεση των τροφίμων σε
θερμότητα. Έχει επίσης βρεθεί ότι οι τεχνολογίες αυτές διατηρούν τα διατροφικά και
οργανοληπτικά χαρακτηριστικά των τροφίμων, επεκτείνοντας τον χρόνο ζωής τους και
διατηρώντας την εξωτερική τους εμφάνιση (Butz and Tauscher, 2002).
ix
Τέτοιες τεχνολογίες είναι τα παλλόμενα ηλεκτρικά πεδία (PEF), η υπεριώδης
ακτινοβολία (UV), το παλλόμενο φως υψηλής έντασης (HILP), υπέρηχοι (US), εγγύς
υπεριώδης φως (NUV light), ιονίζουσα ακτινοβολία, όζων, υψηλή υδροστατική πίεση
(HPP)κ.α (Mohd. Adzahan and Benchamaporn, 2007).
Η ακτινοβολία στο εγγύς υπεριώδες 395± 5 nm, δρα διεγείροντας ενδογενή μόρια
πορφυρίνης παράγοντας μονήρες οξυγόνο (1O 2 ), που καταστρέφει τα κύτταρα και έτσι
θανατώνονται οι μικροοργανισμοί (Elman and J. Lebzelter, 2004, Feuerstein et al. 2005,
Maclean et al. 2008b, Murdoch et al., 2012, Lipovsky et al. 2010).
Η
υπεριώδης
ακτινοβολία
όταν
διαπερνά
την
κυτταρική
μεμβράνη
των
μικροοργανισμών και απορροφάται από τα κυτταρικά συστατικά τους (DNA, RNA),
τους καθιστά ανίκανους να πολλαπλασιαστούν. Το κατάλληλο μήκος κύματος το οποίο
μπορεί να προκαλέσει ζημιά στο μικροβιακό DNA ή RNA είναι περίπου 254 nm. Όταν
το γενετικό υλικό των κυττάρων απορροφά την ενέργεια από την υπεριώδη ακτινοβολία
σχηματίζονται διμερή πυριμιδίνης μεταξύ γενετικών βάσεων πυριμιδίνης στην ίδια
αλυσίδα DNA. Χάρη σε αυτό το δεσμό διμερών στην αλυσίδα του DNA, οι
μικροοργανισμοί προσβάλλονται με τέτοιο τρόπο ώστε ο διαχωρισμός των κυττάρων
και επομένως ο πολλαπλασιασμός τους να είναι αδύνατος. Έτσι, ο μικροοργανισμός
γίνεται αβλαβής και θανατώνεται (Guerrero- Beltrán and Barbosa-Cánovas, 2004). Αν
και οι περισσότεροι μικροοργανισμοί προσβάλλονται από την υπεριώδη ακτινοβολία, η
ευαισθησία τους ποικίλλει, καθώς εξαρτάται από την αντίσταση στη διείσδυση της
υπεριώδους ακτινοβολίας. Η χημική σύνθεση του κυτταρικού τοιχώματος και το πάχος
του καθορίζουν την αντίσταση των μικροοργανισμών στην υπεριώδη ακτινοβολία. Η
αποτελεσματικότητα της απολύμανσης με υπεριώδη ακτινοβολία επηρεάζεται από την
ποσότητα – δόση της υπεριώδους ενέργειας που απορροφάται από το μικροοργανισμό.
Η δόση της ακτινοβολίας εξαρτάται από την ένταση της παρεχόμενης ακτινοβολίας
(ενέργεια, mW), τον χρόνο κατά τον οποίο ο μικροοργανισμός εκτίθεται σε αυτήν
(διάρκεια ακτινοβολίας, sec) και είναι αντιστρόφως ανάλογη με την επιφάνεια του
υγρού στο οποίο εφαρμόζεται (cm2).
Το παλλόμενο φως υψηλής έντασης (HILP) αποτελεί μία αναδυόμενη μη-θερμική
τεχνολογία απολύμανσης, η οποία χρησιμοποιεί μικρής διάρκειας (100–400 𝜇𝜇s) αλλά
υψηλής έντασης φως (200–1100 nm) (Marquenie et al., 2003, Woodling and Moraru,
2007, Gomez-Lopez et al., 2007). Ο τρόπος δράσης βασίζεται στην φωτοχημική δράση
της υπεριώδους ακτινοβολίας η οποία προκαλεί διμερισμό της θυμίνης οδηγώντας στον
θάνατο των κυττάρων (Muňoz et al., 2012, Gomez-Lopez et al., 2007, Rajkovic et al.,
2010).
Εξ’ ορισμού οι υπέρηχοι συνιστούν κύματα υψηλής συχνότητας που μεταφέρουν πίεση
κατά τη διέλευσή τους σε ένα μέσο. Αυτό έχει ως αποτέλεσμα τη δημιουργία περιοχών
χαμηλής και υψηλής πίεσης. Η διακύμανση αυτή της πίεσης αναφέρεται ως πλάτος
πίεσης (amplitude) και είναι ανάλογο της ποσότητας ενέργειας που εφαρμόζεται στο
σύστημα. Στην περίπτωση που οι διακυμάνσεις της πίεσης είναι αρκετά υψηλές (3.000
ΜΡa), τότε ένα υγρό μέσο μπορεί να αποδομηθεί και να έχουμε το σχηματισμό
μικροφυσαλίδων αερίου και ατμού. Το φαινόμενο αυτό είναι γνωστό ως σπηλαίωση
(cavitation),
ενώ
οι
φυσαλίδες
είναι
δυνατόν
να
διασπώνται
και
να
επαναδημιουργούνται συνεχώς επιφέροντας αλλαγές στη δομή του μέσου που υφίσταται
την
επίδραση
των
υπερηχητικών
κυμάτων,
απενεργοποιώντας
έτσι
τους
μικροοργανισμούς από την επιφάνεια των τροφίμων (Bilek and Turantas, 2013). Το
κύριο πλεονέκτημα των υπερήχων για τη βιομηχανία τροφίμων είναι ότι θεωρούνται
μια ευρέως αποδεκτή τεχνολογία από το ευρύ καταναλωτικό κοινό, λόγω της ασφάλειάς
τους, της μη τοξικότητάς τους και της φιλικότητάς τους προς το περιβάλλον.
Σκοπός της παρούσας εργασίας
Πολλές επιδημίες που προέρχονται από τροφιμογενείς λοιμώξεις έχουν καταγραφεί τον
τελευταίο καιρό, καθώς επίσης πολλές ανακλήσεις προϊόντων συμβαίνουν. Αποτελεί
λοιπόν αναγκαιότητα η εξάλειψη των παθογόνων από τρόφιμα λόγω του υψηλού
κινδύνου, του υψηλού ποσοστού θνησιμότητας καθώς και της οικονομικής επιβάρυνσης
που προκαλούν οι ασθένειες (π.χ λιστερίωση, σαλμονέλλωση).
Στην παρούσα μελέτη μη-θερμικές, εναλλακτικές τεχνολογίες απολύμανσης ελέγχθηκαν
όπως: Φως κοντά στην υπεριώδη ακτινοβολία σε μήκος κύματος 395±5 nm (NUV-Vis
light), συνεχής υπεριώδης ακτινοβολία σε μήκος κύματος 254nm (Continuous UV
light), υψηλής έντασης παλμοί φωτός (HILP), υπέρηχοι (ultrasound). Επίσης, η
συμβατική και κλασική μέθοδος της εμβάπτισης σε υποχλωριώδες νάτριο (sodium
hypochlorite solution) εφαρμόστηκε. Τέλος, συνδυασμοί εναλλακτικών, καθώς και
εναλλακτικών με κλασικές μεθόδους πραγματοποιήθηκαν. Ο σκοπός ήταν ο έλεγχος της
εφαρμογής των τεχνολογιών αυτών στα τρόφιμα με σκοπό τη διασφάλιση της δημόσιας
υγείας των καταναλωτών.
xi
Όλες οι παραπάνω μέθοδοι εφαρμόστηκαν σε τρόφιμα έτοιμα προς κατανάλωση όπως
μαρούλι, φράουλες και τοματίνια τύπου cherry, τα οποία αγοράστηκαν από τοπικό
σουπερμάρκετ και εμβολιάστηκαν με παθογόνους μικροοργανισμούς οι οποίοι
αποτέλεσαν το αρχικό μικροβιακό φορτίο. Οι μικροοργανισμοί οι οποίοι εμβολιάστηκαν
ήταν βακτήρια που έχουν συναντηθεί στα εν λόγω τρόφιμα όπως E. coli, S. aureus, S.
enteritidis και L. innocua καθώς και ο αδενοιός (HAdV35). Πιο συγκεκριμένα τα
στελέχη που χρησιμοποιήθηκαν ήταν: E. coli K12, E. coli NCTC 9001 (ως
μικροοργανισμοί δείκτες για το εντεροαιμοραγικό παθογόνο E. coli O157:H7), S. aureus
NCTC 6571, L. innocua NCTC 11288 (ως μικροοργανισμοί δείκτες για το παθογόνο
Listeria monocytogenes), S. Enteritidis NCTC 6676 και HAdV (ιός δείκτης για τους ιούς
HAV και norovirus).
Πιο συγκεκριμένα, στο πρώτο μέρος της διατριβής, τρεις τεχνολογίες απολύμανσης
(NUV-Vis, Continuous UV, HILP) χρησιμοποιήθηκαν όσον αφορά την απολύμανση
των μικροοργανισμών δεικτών (E. coli και L. innocua), τα οποία εμβολιάστηκαν σε
υγρά διαλύματα (MRD Buffer). Ο σκοπός ήταν να ελεγχθεί η απολυμαντική δράση των
τεχνολογιών αυτών, χρησιμοποιώντας διαφορετικές εντάσεις φωτός.
Στο δεύτερο μέρος της διατριβής, τα τρόφιμα εμβολιάστηκαν με διάφορα βακτήρια (E.
coli, S. aureus, S. Εnteritidis, L. innocua) και έναν αδενοϊό με σκοπό να ελεγχθεί η
απολύμανσή τους, έπειτα από τη χρήση του χλωρίου, του υπεριώδους φωτός και των
υπερήχων, καθώς επίσης και συνδυασμών τους. Επίσης, πραγματοποιήθηκαν πειράματα
με διαφορετικές αρχικές συγκεντρώσεις βακτηρίων, με σκοπό τον περεταίρω έλεγχο της
απολυμαντικής δράσης των παραπάνω τεχνολογιών. Τέλος, τα αποτελέσματα της
συγκέντρωσης των ιών που προήλθαν από τη χρήση αλυσιδωτής αντίδρασης
πολυμεράσης σε πραγματικό χρόνο (Real-Time PCR), επιβεβαιώθηκαν με τη χρήση
καλλιεργειών κυττάρων. Τέλος,
έπειτα από την επεξεργασία των τροφίμων με τις
μεθόδους απολύμανσης, αποθηκεύτηκαν τα τρόφιμα στο ψυγείο για διάστημα 15
ημερών, και ελέγχθηκε το μικροβιακό τους φορτίο έπειτα από 3, 7 και 15 ημέρες.
Στο τρίτο μέρος της διατριβής, η επίδραση των παραπάνω μεθόδων σε επιλεγμένες
διατροφικές παραμέτρους και παραμέτρους ποιότητας μελετήθηκε. Για το σκοπό αυτό,
ελέγχθηκαν πριν και έπειτα από την χρήση των τεχνολογιών: η ολική αντιοξειδωτική
τους ικανότητα, η περιεκτικότητά τους σε ολικά φαινολικά, η συγκέντρωση ασκορβικού
οξέος και η ένταση του χρώματός τους.
Στο τέταρτο μέρος της διατριβής, χρησιμοποιήθηκε ένα μοντέλο πρόβλεψης για την
ασφάλεια των τροφίμων. Το μοντέλο αποτελεί ένα μοντέλο λήψης απόφασης το οποίο
χρησιμοποιήθηκε στην παρούσα διατριβή με σκοπό την λήψη απόφασης σε ένα
πολύπλοκο σύστημα μιας καθετοποιημένης εταιρείας μαρουλιού. Στην συγκεκριμένη
περίπτωση, 9 κρίσιμα σημεία κατά τη διάρκεια παραγωγής-επεξεργασίας και διάθεσης
λαχανικών επιλέχθηκαν για το σύστημα. Τα σημεία αυτά ήταν: εργατικό δυναμικόπροσωπικό, συστήματα ποιότητας και ασφάλειας τροφίμων, τοποθεσία-περιβάλλων
χώρος της μονάδας παραγωγής τροφίμων, φυτώριο μαρουλιού, έδαφος παραγωγής
μαρουλιού, διαδικασία συγκομιδής, διαδικασία μετά τη συγκομιδή, μεταφορά, πώληση.
Στη συνέχεια, τρεις ειδικοί βαθμολόγησαν τα 9 κριτήρια αυτά μεταξύ τους, ως προς την
ύπαρξη ή απουσία σχέσης τους με σκοπό την πρόβλεψη ασφάλειας του τελικού
προϊόντος. Το συγκεκριμένο μοντέλο βασίζεται στη θεωρία των ασαφών γνωστικών
δικτύων.
Στο τελευταίο κομμάτι της διατριβής, τα αποτελέσματα από τις μεθόδους απολύμανσης
συγκεντρώθηκαν, και με βάση τη μολυσματική δόση κάθε μικροοργανισμού στο τελικό
προϊόν που έχει καταγραφεί στην βιβλιογραφία, εξάχθηκαν συμπεράσματα σχετικά με
την αποτελεσματικότητα των μεθόδων σε σχέση με την διασφάλιση της δημόσιας
υγείας.
Αποτελέσματα
Τα αποτελέσματα της διατριβής απέδειξαν ότι οι εναλλακτικές, μη-θερμικές τεχνολογίες
απολύμανσης είναι αποδοτικές για την απενεργοποίηση των μικροοργανισμών σε
φρέσκα έτοιμα προς κατανάλωση τρόφιμα και μπορούν να εφαρμοστούν ως
εναλλακτικές στην διαδεδομένη απολύμανση με τη χρήση χλωρίου. Ιδιαίτερη έμφαση
αξίζει να δοθεί στα διατροφικά χαρακτηριστικά, πριν την επιλογή της τεχνολογίας
απολύμανσης, έτσι ώστε να μην υποβαθμίζονται τα ευεργετικά συστατικά των
τροφίμων αυτών για την ανθρώπινη υγεία.
Από τις μη-θερμικές τεχνολογίες φωτός, οι παλμοί υψηλής έντασης (HILP)
απενεργοποίησαν τους μικροοργανισμούς E. coli και L. innocua σε συντομότερο
χρονικό διάστημα σε σύγκριση με τις άλλες δύο τεχνολογίες απολύμανσης (Continuous
UV και NUV-Vis). Όταν το δείγμα τοποθετήθηκε σε μικρή απόσταση από την πηγή
φωτός (2.5 cm), οι μικροοργανισμοί E. coli και L. innocua μειώθηκαν κατά 3.07 and
3.77 log 10 CFU/mL αντίστοιχα μετά από χρόνο επεξεργασίας 5 δευτερόλεπτα. Έπειτα
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από χρόνο επεξεργασίας 30 δευτερολέπτων με την ίδια τεχνολογία και έντασης 106.2
J/cm2, οι μικροοργανισμοί ήταν κάτω από το όριο ανίχνευσής τους (<0.22 log 10
CFU/mL).
Η επεξεργασία με τη μη-θερμική τεχνολογία του υπεριώδους φωτός στο μαρούλι,
μείωσε σημαντικά τους πληθυσμούς των παρακάτω μικροβίων E. coli, S.aureus, S.
Enteritidis and L. innocua κατά 1.75, 1.21, 1.39 και 1.27 log 10 CFU/g, αντίστοιχα. Όταν
η μη-θερμική τεχνολογία των υπερήχων εφαρμόστηκε, μία λογαριθμική μείωση της
τάξης των 2 log 10 CFU/g για τους πληθυσμούς E. coli, S. Enteritidis και L. innocua
καταγράφηκε. Η τεχνολογία υπεριώδους ακτινοβολίας μείωσε το μικροβιακό φορτίο
κατά 1–1.4 log 10 CFU/g. Οι μέγιστες λογαριθμικές μειώσεις που παρατηρήθηκαν στη
φράουλα μετά την επεξεργασία με υπερήχους, ήταν 3.04, 2.52, 5.24 και 6.12 log 10
CFU/g για τους μικροοργανισμούς E. coli, S. aureus, S. Enteritidis και L. innocua,
αντίστοιχα. Τέλος, στα τοματίνια cherry, η απολύμανση με τη χρήση μη-θερμικών
τεχνολογιών, είχε τα καλύτερα αποτελέσματα. Πιο συγκεκριμένα η απολύμανση με τη
χρήση υπερήχων μείωσε το μικροβιακό φορτίο κατά 3.16, 2.62, 3.29, 3.16 log 10 CFU/g
για τους μικροοργανισμούς E. coli, S. aureus, S. Enteritidis και L. innocua, αντίστοιχα.
Αξίζει να αναφερθεί ότι η τεχνολογία υπεριώδους ακτινοβολίας, είχε ως αποτέλεσμα
την μείωση κατά 2.39, 2.05, 2.62, 2.56 log 10 CFU/g των παραπάνω μικροοργανισμών
αντίστοιχα. Στη συνέχεια εφαρμόστηκαν και στα τρία έτοιμα προς κατανάλωση τρόφιμα
συνδυασμοί των παραπάνω μη-θερμικών εναλλακτικών τεχνολογιών. Η μείωση του
μικροβιακού φορτίου με τη χρήση υποχλωριώδους νατρίου 200ppm ήταν 1-2 log 10
CFU/g log για το μαρούλι και τις φράουλες, ενώ μεγαλύτερες μειώσεις (3-4 log 10
CFU/g) καταγράφηκαν όταν τα τοματίνια απολυμάνθηκαν με χλώριο. Τέλος, οι
συνδυασμοί εναλλακτικών-συμβατικών τεχνολογιών είχαν ως αποτέλεσμα μία μείωση
της τάξεως των 2-3.50 log 10 CFU/g, για το μαρούλι και τις φράουλες, ενώ μείωση 3.284.78 log 10 CFU/g πραγματοποιήθηκε για τα τοματίνια τύπου cherry.
Κατά την απολύμανση του αδενοϊού, η πιο αποτελεσματική μέθοδος ανάμεσα σε όλες οι
οποίες εφαρμόστηκαν, αποδείχθηκε η χρήση χλωρίου. Από τις εναλλακτικές μηθερμικές τεχνολογίες, η χρήση υπεριώδους φωτός ήταν πιο αποδοτική σε σχέση με τους
υπερήχους, μειώνοντας το ιϊκό φορτίο κατά 2.13, 1.25 και 0.92 log 10 για τα μαρούλια,
τις φράουλες και τα τοματίνια αντίστοιχα όταν τα τρόφιμα αυτά εκτέθηκαν σε υπεριώδη
ακτινοβολία για χρονικό διάστημα 30 λεπτών. Αντιθέτως η μείωση του ιϊκού φορτίου
όταν εφαρμόστηκαν υπέρηχοι για 30 λεπτά ήταν 0.85, 0.53 και 0.36 log 10 για τα τρία
τρόφιμα αντίστοιχα. Αξίζει να αναφερθεί ότι η συνδυαστική χρήση υπεριώδους
ακτινοβολίας και υπερήχων ήταν πιο αποδοτική και λιγότερο χρονικά δαπανηρή, σε
σχέση με τη μεμονωμένη χρήση των δύο παραπάνω τεχνολογιών, όσον αφορά την
απολύμανση των τροφίμων από βακτήρια και ιούς, αποδεικνύοντας την αθροιστική τους
δράση.
Όσον αφορά τις παραμέτρους ποιότητας των τροφίμων, η χρήση εναλλακτικών
τεχνολογιών απολύμανσης (UV, US) για χρονικό διάστημα μικρότερο των 30 λεπτών,
δεν άλλαξε σημαντικά (p>0.05) το χρώμα των τροφίμων. Επίσης, δεν παρατηρήθηκαν
στατιστικά σημαντικές διαφορές (p>0.05) στην ολική αντιοξειδωτική ικανότητα των
τροφίμων όταν χρησιμοποιήθηκε η συμβατική τεχνολογία απολύμανσης με χλώριο.
Όταν οι εναλλακτικές τεχνολογίες χρησιμοποιήθηκαν, μία αύξηση στην συγκέντρωση
των ολικών αντιοξειδωτικών ήταν εμφανής από τα πρώτα λεπτά της απολύμανσης. Η
περιεκτικότητα σε ολικά φαινολικά παρέμεινε σταθερή ή μειώθηκε ελαφρώς όταν τα
τρόφιμα απολυμάνθηκαν με την χρήση χλωρίου. Αντιθέτως, όταν τα τρόφιμα
απολυμάνθηκαν με τις εναλλακτικές τεχνολογίες απολύμανσης η περιεκτικότητά τους
σε φαινολικά συστατικά αυξήθηκε σημαντικά (p<0.05). Τέλος, η περιεκτικότητα σε
βιταμίνη C δεν μεταβλήθηκε κατά τη διάρκεια των διαφόρων τεχνολογιών. Όταν ο
χρόνος απολύμανσης με τις διάφορες τεχνολογίες ξεπέρασε τα 30 λεπτά ή όταν
συνδυάστηκαν οι εναλλακτικές τεχνολογίες μεταξύ τους για συνολικό διάστημα 30
λεπτών, σημαντική μείωση της περιεκτικότητας της βιταμίνης C παρατηρήθηκε
(p<0.05).
Το υπολογιστικό μοντέλο που χρησιμοποιήθηκε βασίστηκε σε κρίσιμα σημεία τα οποία
θεωρούνται σημαντικά σε μια καθετοποιημένη μονάδα παραγωγής μαρουλιών. Πιο
συγκεκριμένα, αναπτύχθηκε ένα σύστημα λήψης απόφασης με τη χρήση των ασαφών
γνωστικών δικτύων. Ο σκοπός ήταν η διάγνωση και ο έλεγχος των κρίσιμων σημείων
ελέγχου σε μία παραγωγική μονάδα, έτσι ώστε να διασφαλιστεί η υγιεινή και η
ασφάλεια των τροφίμων. Η μεθοδολογία που εφαρμόστηκε, χρησιμοποιεί την αληθινή
γνώση και εμπειρία ειδικευμένων στην παραγωγική διαδικασία της παραγωγής των
μαρουλιών. Αποδείχθηκε ότι η χρήση ενός τέτοιου μοντέλου μπορεί να προβλέψει και
να αποτρέψει προβλήματα που μπορεί να συμβούν κατά τη διάρκεια της παραγωγικής
διαδικασίας, έτσι ώστε να διασφαλιστεί η υγεία και η ασφάλεια των καταναλωτών. Το
μοντέλο λήψης απόφασης εφαρμόστηκε σε τρεις διαφορετικές περιπτώσεις της ίδιας
μονάδας παραγωγής μαρουλιών, όπου εξάχθηκαν αποτελέσματα σχετικά με την
ασφάλεια του τελικού προϊόντος.
xv
Στο τελευταίο μέρος των αποτελεσμάτων της διατριβής, βιβλιογραφικά δεδομένα
σχετικά με τη μολυσματικότητα των παραπάνω βακτηρίων και ιών συλλέχθηκαν από
διάφορους φορείς (FDA, PHAC, European pathogen fact sheet). Στη συνέχεια, με βάση
τα αποτελέσματα των μεθόδων απολύμανσης που εφαρμόστηκαν στα τρόφιμα,
συμπεράσματα εξήχθηκαν για την ικανότητα των μεθόδων αυτών να εφαρμοστούν στα
τρόφιμα και να διασφαλίσουν την δημόσια υγεία. Έτσι στο μαρούλι, παρατηρήθηκε ότι
η συνδυαστική τεχνολογία των υπερήχων ακολουθούμενη από χλώριο αποτέλεσε την
καλύτερη τεχνολογία απολύμανσης των βακτηρίων, ενώ η υπεριώδης ακτινοβολία και
το χλώριο είναι αποτελεσματικές τεχνολογίες για την απολύμανση των ιών. Για τις
φράουλες, οι υπέρηχοι, και η συνδυαστική τεχνολογία US+NaOCl, ήταν ικανές να
απολυμάνουν επαρκώς τα βακτήρια, ενώ η υπεριώδης ακτινοβολία αποδείχθηκε ως η
πιο αποτελεσματική τεχνολογία για την απολύμανση του αδενοιού στη φράουλα. Στα
τοματίνια τύπου cherry σχεδόν όλες οι τεχνολογίες ήταν ικανές να τα απολυμάνουν
ικανοποιητικά. Η συνδυαστική τεχνολογία US+NaOCl αποδείχθηκε αποδοτική για την
απολύμανση των βακτηρίων ενώ το χλώριο για τους ιούς.
Σε γενικές γραμμές παρατηρήθηκε ότι η επίδραση των τεχνολογιών απολύμανσης
εξαρτάται από την τεχνολογία, το χρόνο επεξεργασίας και το είδος του τροφίμου. Τα
αποτελέσματα της διατριβής αυτής απέδειξαν τις εναλλακτικές τεχνολογίες ως ικανές
και φιλικές προς το περιβάλλον τεχνολογίες που μπορούν να εφαρμοστούν στα τρόφιμα
με σκοπό την διασφάλιση της υγείας των καταναλωτών.
Συμπεράσματα
Τα αποτελέσματα της παρούσας μελέτης απέδειξαν ότι οι εναλλακτικές, μη-θερμικές
τεχνολογίες απολύμανσης, επέδρασαν σε μία αποτελεσματική μείωση του μικροβιακού
πληθυσμού τόσο των υγρών διαλυμάτων όσο και των τροφίμων.
Από τις εναλλακτικές τεχνολογίες φωτός, το παλλόμενο φως υψηλής έντασης αποτελεί
την πιο αποδοτική μέθοδο για την απολύμανση των βακτηρίων E.coli και L.innocua.
Επίσης αυτή η τεχνολογία είχε ως αποτέλεσμα την πιο γρήγορη και εντατική
απολύμανση σε σχέση με τις άλλες δύο τεχνολογίες φωτός που εφαρμόστηκαν. Η
εξαιρετική απόδοση της τεχνολογίας αυτής πιθανόν οφείλεται στην υψηλότερη
διεισδυτική ικανότητα της συγκεκριμένης πηγής φωτός καθώς και της μεγαλύτερης
ισχύος εκπομπής σε σχέση με τη συνεχή υπεριώδης ακτινοβολία και την εγγύς υπεριώδη
ακτινοβολία (NUV-vis). Η υψηλή ένταση του φωτός από την τεχνολογία παλλόμενου
φωτός, απέδιδε μία ένταση φωτός κατά 100 φορές μεγαλύτερη από τις άλλες
τεχνολογίες, στον ίδιο χρόνο λειτουργίας. Περισσότερη μελέτη χρειάζεται να
πραγματοποιηθεί σε τρόφιμα ώστε να διεξαχθούν συμπεράσματα εάν η τεχνολογία αυτή
δημιουργεί παραπροϊόντα στα τρόφιμα αυτά. Μπορεί να εξαχθεί το συμπέρασμα ότι η
εγγύς υπεριώδη ακτινοβολία είναι μία υποσχόμενη μέθοδος για τη χρήση της σε
βιομηχανίες τροφίμων, αυξάνοντας έτσι την παραγωγικότητα τους.
Οι εναλλακτικές τεχνολογίες (UV, US) που εφαρμόστηκαν στην απολύμανση των
τροφίμων αποτελούν εναλλακτικές στις ήδη υπάρχουσες διαδεδομένες τεχνολογίες
απολύμανσης. Μπορούν δηλαδή να εφαρμοστούν από τις βιομηχανίες τροφίμων, ως
τεχνολογίες χαμηλού κόστους και χαμηλής κατανάλωσης ενέργειας, καθώς δεν
απαιτούν ιδιαίτερο εξοπλισμό. Η απόδοση των τεχνολογιών αυτών εξαρτάται από την
δόση, τον χρόνο έκθεσης, την επιφάνεια του τροφίμου. Οι υπέρηχοι ήταν αποδοτικότερη
μέθοδος για την απολύμανση των βακτηρίων, ενώ η υπεριώδης ακτινοβολία πιο
αξιόπιστη για την απολύμανση των ιών. Κάποιες πιθανές μεταβολές του χρώματος των
τροφίμων μπορούν να ελεγχθούν, εφόσον χρησιμοποιηθούν οι κατάλληλες συνθήκες
των τεχνολογιών, διατηρώντας έτσι αναλλοίωτα τα χαρακτηριστικά των τροφίμων. Οι
συνδυαστικές τεχνολογίες είναι υποσχόμενες, εφόσον επιβεβαιωθεί η απουσία
παραπροϊόντων χλωρίου στα τελικά προϊόντα.
Επίσης, πρέπει να σημειωθεί ότι δεν μπορεί να γίνει μία απευθείας σύγκριση
τεχνολογιών απολύμανσης εάν δεν ληφθούν υπόψη και άλλες παράμετροι όπως το
χρώμα και διατροφικές παράμετροι. Στην παρούσα μελέτη αποδείχθηκε ότι υπάρχει
θετική ή μηδαμινή επίδραση στην ποιότητα των τροφίμων έπειτα από τις περισσότερες
τεχνολογίες που εφαρμόστηκαν.
Οι μικροβιολογικές και οι μοριακές αναλύσεις μπορούν να εφαρμοστούν στον ποιοτικό
έλεγχο μιας διατροφικής αλυσίδας. Ωστόσο, τα αποτελέσματα των αναλύσεων αυτών
είναι χρονοβόρα (μικροβιολογικές αναλύσεις) και πολλές φορές οικονομικά ασύμφορα
(μοριακές αναλύσεις). Επίσης, πολλές φορές τα αποτελέσματα των αναλύσεων
εξαρτώνται από την ακρίβεια καθώς και το καλιμπράρισμα του εξοπλισμού που
χρησιμοποιείται. Για τον σκοπό αυτό προτάθηκε και το θεωρητικό μοντέλο το οποίο
μπορεί να παρέχει ένα πρώτο έλεγχο-εκτίμηση της ποιότητας του τροφίμου που
παράγεται σε μία μονάδα παρασκευής-επεξεργασίας. Το μοντέλο αυτό εφαρμόστηκε
στα πλαίσια της παρούσας διατριβής πρώτη φορά σε μονάδα επεξεργασίας μαρουλιών.
xvii
Βασίζεται στη θεωρία των ασαφών γνωστικών δικτύων και αποτελεί μία απλή, φθηνή,
φιλική, πραγματικού χρόνου και εύκολη προσέγγιση για την πιθανή εκτίμηση της
ποιότητας-ασφάλειας του μαρουλιού. Επιπροσθέτως, το συγκεκριμένο μοντέλο θα
μπορούσε να αξιοποιηθεί από τις Αρχές Ελέγχου Τροφίμων, με σκοπό να αποκτούν μία
πρώτη εκτίμηση των προϊόντων που πρόκειται να επιθεωρήσουν.
Οι τροφιμογενείς λοιμώξεις παρουσιάζουν μία διαρκώς αυξανόμενη τάση, και
βασίζονται στην αυξανόμενη παραγωγή και ζήτηση τροφίμων και ιδιαίτερα έτοιμων
προς κατανάλωση τροφίμων. Οι καταναλωτές αντιλαμβάνονται την ασφάλεια των
τροφίμων ως «δεδομένο», και για το λόγο αυτό η βιομηχανία τροφίμων πρέπει να
εξασφαλίσει την «ποιότητα» των παραγόμενων προϊόντων. Η αυξημένη ζήτηση των
καταναλωτών για τα συγκεκριμένα τρόφιμα βασίζεται στην προμήθεια τροφίμων με
«μηδενικό» ή «ανύπαρκτο» κίνδυνο και το κόστος για την επίτευξη του γεγονότος
αυτού δεν θα πρέπει να λαμβάνεται υπόψη εφόσον ο στόχος παραμένει πάντα η
διασφάλιση της δημόσιας υγείας.
Λαμβάνοντας υπόψη όλα τα αποτελέσματα της παρούσης διατριβής, οι εναλλακτικές
τεχνολογίες απολύμανσης διαδραματίζουν ένα σπουδαίο ρόλο και αποτελούν βιώσιμες
τεχνολογίες οι οποίες μπορούν να ενταχθούν στην καθημερινή πρακτική με σκοπό να
παρεμποδίσουν την ανάπτυξη των μικροοργανισμών και να μειώσουν το κίνδυνο
μόλυνσης, διασφαλίζοντας έτσι την δημόσια υγεία. Ιδιαίτερη προσοχή πρέπει να δοθεί
στην επιλογή των κατάλληλων συνθηκών των τεχνολογιών ώστε να διατηρηθούν τα
απαραίτητα διατροφικά και ποιοτικά χαρακτηριστικά των τροφίμων, τα οποία έχουν
ευεργετικές επιδράσεις στην υγεία των καταναλωτών.
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Table of Contents
Abbreviations ................................................................................................................ 7
List of Figures ............................................................................................................... 9
List of Graphs ............................................................................................................. 14
List of Tables ............................................................................................................... 15
INTRODUCTION....................................................................................................... 17
Chapter 1: Literature Review ..................................................................................... 19
1.1 Fresh produce and Mediterranean diet ............................................................... 19
1.1.1 Lettuce................................................................................................................. 20
1.1.2 Strawberries ........................................................................................................ 22
1.1.3 Tomatoes ............................................................................................................. 23
1.2. Nutritional, Quality and Health Aspects of fresh ready-to- eat (RTE) fruits and
vegetables .................................................................................................................... 25
1.2.1
Antioxidant Compounds................................................................................ 27
1.2.1.1 Determination of Antioxidants ..................................................................... 28
1.2.2
Phenolic Compounds..................................................................................... 31
1.2.2.1 Determination of TPC (total phenolic content) ............................................ 31
1.2.3
Ascorbic Acid ................................................................................................ 33
1.2.3.1 Determination of ascorbic acid..................................................................... 34
1.2.4
Quality Aspects of Foods .............................................................................. 36
1.2.4.1 External Quality-Color ................................................................................. 36
1.3 Foodborne Pathogens and Foodborne diseases................................................. 37
1.3.1
Global challenge and increased frequency of foodborne diseases. ............... 37
1.3.2
Hazards during the food production chain .................................................... 39
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1.3.3
Microbial colonization on fresh produce surfaces .........................................40
1.3.4 Foodborne illness and foodborne disease outbreaks ...........................................42
1.3.5 Foodborne Bacteria ..............................................................................................44
1.3.5.1 Escherichia coli.............................................................................................44
1.3.5.2 Staphylococcus aureus ..................................................................................47
1.3.5.3 Salmonella spp. .............................................................................................49
1.3.5.4 Listeria spp. ...................................................................................................51
1.3.6 Foodborne Viruses...............................................................................................53
1.3.6.1 Noroviruses ...................................................................................................55
1.3.6.2 Hepatitis ........................................................................................................56
1.3.6.3 Adenoviruses .................................................................................................57
1.3.7
Pathogens and Infectious Dose ......................................................................59
1.3.8
Methods for detection of foodborne pathogens .............................................61
1.4 Infection and Disinfection .................................................................................. 64
1.4.1
The Bacterial Cell and Antimicrobial Interaction ..........................................65
1.4.2 The virus genome and infectivity ........................................................................66
1.4.3
Conventional Food Processing/Preservation Technologies ...........................67
1.4.3.1 Chemical Methods ........................................................................................67
1.4.3.1.2 Organic Acids ............................................................................................69
1.4.3.1.3 Peroxyacetic Acid ......................................................................................70
1.4.3.1.4 Hydrogen Peroxide ....................................................................................71
1.4.3.2 Physical Methods ..........................................................................................71
1.4.3.2.1 Heat Processing ..........................................................................................71
1.4.3.2.2 Radio Frequency (RF) and Microwave Heating (MH) ..............................72
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Birmpa Angeliki
1.4.3.2.3 Ohmic Heating .......................................................................................... 72
1.4.4 Non-thermal Technologies (Alternative Technologies) ..................................... 73
1.4.4.1 Ultraviolet Light (UV).................................................................................. 73
1.4.4.2 High Intensity Light Pulses (HILP).............................................................. 77
1.4.4.3 Near UV-Vis Light (NUV-Vis) .................................................................... 78
1.4.4.4 Ultrasound .................................................................................................... 79
1.4.5 Other Methods .................................................................................................... 82
1.4.5.1 Ozone............................................................................................................ 82
1.4.5.2 Pulsed Electric Fields ................................................................................... 83
1.4.5.3 High Pressure Processing ............................................................................. 84
1.4.5.4 Electrochemical (Cold Plasma) Method....................................................... 85
1.4.6
Biological control .......................................................................................... 86
1.5 Control of foodborne diseases .............................................................................. 86
1.5.1
Public Health Surveillance ............................................................................ 87
1.5.2 Food Legislation ................................................................................................. 89
1.5.3 Guidelines for the microbiological quality of RTE foods in Greece .................. 90
1.5.4 Predictive Models-Risk Assessment Support Systems and Public Health ......... 91
AIM OF THE STUDY ................................................................................................ 95
Chapter 2. MATERIALS AND METHODS .............................................................. 97
2.1 In Vitro Experiments with 3 Light Technologies .................................................. 98
2.1.1 Equipment ....................................................................................................... 98
2.1.2 Disposables- Plasticwares ............................................................................... 98
2.1.3 Culture Media .................................................................................................. 99
2.1.4 Solutions for microbiological analysis ............................................................ 99
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2.1.5 Disinfection Light Treatments .......................................................................100
2.1.6 Microbiological analysis ................................................................................103
2.2 Food Disinfection .................................................................................................104
2.2.1 Equipment ......................................................................................................104
2.2.2 Disposables- Plasticwares ..............................................................................104
2.2.3 Culture Media ................................................................................................105
2.2.4 Solutions for microbiological analysis ...........................................................106
2.2.5 Solutions for virus concentration ...................................................................107
2.2.6 Bacterial Strains .............................................................................................107
2.2.7 Cell lines and virus Adeno-35 stock ..............................................................108
2.2.8 Bacterial Preparation ......................................................................................108
2.2.9 Sample Selection ............................................................................................108
2.2.10 Sample preparation ......................................................................................109
2.2.11 Bacterial Cocktail .........................................................................................109
2.2.12 Sample Inoculation ......................................................................................109
2.2.13 Virus inoculation ..........................................................................................110
2.2.14 Disinfection Treatments ...............................................................................110
2.2.15 Storage conditions ........................................................................................112
2.2.16 Microbiological Analysis .............................................................................113
2.2.17 Bacteria Enumeration ...................................................................................113
2.2.18 Analysis for Detection of Viruses ................................................................114
2.2.19 Evaluation of disinfection with different initial bacteria cocktail................118
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Birmpa Angeliki
2.2.20 Culture Assay for HAdV35 ......................................................................... 119
2.3 Food Quality parameters ...................................................................................... 120
2.3.1 Color Measurement ....................................................................................... 120
2.3.2 Physicochemical Parameters ......................................................................... 121
2.4 A user-friendly theoretical mathematical model for the prediction of food safety in
a food production chain ............................................................................................. 125
2.4.1 Selection of critical points ............................................................................. 125
2.4.2 Decision Making Support System in lettuce’s safety using fuzzy cognitive
maps........................................................................................................................ 129
STATISTICS ............................................................................................................. 130
Chapter 3. RESULTS ............................................................................................... 133
3.1 In Vitro Experiments with 3 Light Technologies ................................................ 135
3.2 Food Disinfection................................................................................................. 141
3.2.1 Bacteria Disinfection ..................................................................................... 141
3.2.2 Adenovirus Disinfection................................................................................ 156
3.2.3 High and Low Initial Load Disinfection Treatments .................................... 161
3.2.4 Storage Conditions ........................................................................................ 164
3.3 Food Quality parameters ...................................................................................... 169
3.3.1 Color .............................................................................................................. 169
3.3.2 Physicochemical Parameters ......................................................................... 185
3.4 A user-friendly theoretical mathematical model for the prediction of food safety in
a food production chain ............................................................................................. 195
3.5 Assessment of disinfection technologies based on infectivity doses ................... 202
Chapter 4. DISCUSSION ......................................................................................... 204
4.1 In Vitro Experiments with 3 Light Technologies ................................................ 206
4.2 Food Disinfection................................................................................................. 209
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4.2.1 Bacteria Disinfection......................................................................................210
4.2.2 Adenovirus Disinfection ................................................................................221
4.2.3 High and Low Initial Load Disinfection Treatments .....................................225
4.2.4 Storage Conditions .........................................................................................226
4.3 Food Quality parameters.......................................................................................229
4.3.1 Color...............................................................................................................229
4.3.2 Physicochemical Parameters ..........................................................................231
4.4 A user-friendly theoretical mathematical model for the prediction of food safety in
a food production chain ..............................................................................................238
4.5 Assessment of disinfection technologies based on infectivity doses....................242
Chapter 5. CONCLUSIONS AND FUTURE RECOMMENDATIONS................ 246
5.1 Conclusions...........................................................................................................246
5.2 Future Recommendations .....................................................................................248
References ................................................................................................................. 252
APPENDIX ............................................................................................................... 302
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Birmpa Angeliki
Abbreviations
AA: Antioxidant Activity
ABTS: 2,2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid
ANOVA: Analysis of Variance
AW: Water Activity
CDC: Center for Disease Control and Prevention
CFU: Colony Forming Units
DPPH: 2,2-diphenyl-1-picryl-hydrazyl assay
EFSA: European Food Safety Authority
ELISA: Enzyme-linked Immunosorbent assay
EPR: Electronic Paramagnetic Resonance
FAO: Food and Agriculture Organization
FDA: Food and Drug Administration
Fe3+-TPTZ: ferric tripyridyltriazine
FRAP: Ferric Reducing Antioxidant Power
GA: Gallic Acid
GAP: Good Agriculture Practice
GHP: Good Hygienic Practice
GMP: Good Manufacturing Practice
HACCP: Hazard Analysis Critical Control Point
HAV: Hepatitis A Virus
HEV: Hepatitis E Virus
ID: Infective/Infectious Dose
ORAC: Oxygen-Radical Absorbance Capacity
RTE: Ready-To-Eat
SEs: Staphylococcal Enterotoxins
SSC: Soluble Solid Contents
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TEAC: Trolox Equivalent Antioxidant Capacity
TPC: Total Phenolic Content
TP: Total Phenolics
TRAP: Total Radical-Trapping Antioxidant Parameter
TSST: toxic-shock syndrome toxin
USDA: United States Department of Agriculture
US: Ultrasound
UV: Ultraviolet
WHO: World Health Organization
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Birmpa Angeliki
List of Figures
Chapter 1
Figure 1.1: Reported Foodborne Outbreaks in vegetables (on the left), and in fruits (on
the right)………………………………...………………………..……………………..20
Figure 1.2.3.1 Oxidation of L-ascorbic to dehydro-L-ascorbic acid followed by evolution
into
products
lacking
biological
activity…………………………………………………………...……………………...33
Figure 1.3.2.1:Ways of contamination of raw fruits and vegetables with pathogenic
microorganisms………………………………..……………..………...……………….39
Figure 1.3.4.1 Number of multistate foodborne disease outbreaks, by year and pathogen
— Foodborne Disease Outbreak Surveillance System, United States, 1998–
2008………………………………..…………..…………..……………………………43
Figure 1.3.5.1: E. coli on fresh ready to eat lettuces........................................................46
Figure 1.3.5.3.1: Taxonomic scheme of Salmonella serovars………………………….49
Figure 1.3.6.3.1: Adenovirus…………………………………………………………....58
Figure 1.4.1.1: Schematic illustration of cell wall structures of microbial pathogens. (a)
Gram-negative bacteria, (b) Gram-positive bacteria………………………..…….…….66
Figure 1.4.2.1: Effects of capsid of virus when different situations occur…….………..67
Figure 1.4.4.1.1: Electromagnetic Spectrum……….…………..……………………….74
Figure 1.4.4.1.2:Structure of DNA before and after absorbing a photon of UV
light………………………...……………………………………………………………76
Figure 1.4.4.1.3: UV chamber……………………………………………..…………....76
Figure 1.4.4.2.1: Equipment of HILP...............................................................................78
Figure 1.4.4.2.2: Internal Part of pulsed Light with a Data Logger……………...……..78
Figure 1.4.4.3.1: High intensity near ultraviolet/visible (NUV–vis) 395±5 nm light unit
…………………………………………………………………………………………..79
Figure 1.4.4.4.1: Ultrasonic Cleaning Bath………………………………………..…....80
Figure 1.4.4.4.2: Ultrasonic Probe or horn…………………………………….………..80
Figure 1.4.4.4.3: Ultrasound cup-horn………………………………………………….80
Figure 1.4.4.6.1: Schematic Representation of PEF equipment……….………………..84
Figure 1.4.4.7.1: High pressure processing Unit……………………….……………….85
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Figure 1.5.1.1: Cycle of the public health prevention (Tauxe, 2002)………...…………88
Figure 1.5.1.2: Surveillance pyramid………………………………………….….….....89
Chapter 2
Figure 2.1.5.1.1: NUV Vis Equipment……………………………………….………100
Figure 2.1.5.2.1: Layout of UV treatment unit I, housing for W lights; 2, safety interlock;
3, treatment chamber with dimensions (length, width, and height) of 790 by 390 by 345
mm; 4, UV lights (95 W) 500 mm in length………………………………..…..……101
Figure 2.1.5.2.2: Custom made UV equipment……………………………...……….101
Figure 2.1.5.3.1: HILP Unit……………………………………………..…………...102
Figure 2.2.14.3.1: Ultrasound equipment……………………………...………………112
Figure 2.2.18.4.1: Schematic Presentation of the procedure of Nucleic Acid
Extraction……………………………………………………………….……………...117
Figure 2.4.1.1 Flow Chart of Lettuce/ Leafy Greens Production.................................128
Chapter 3
Figure 3.1.1: Survival curves of E. coli suspended in maximum recovery diluent (MRD)
placed at: 3cm (∆), 12cm (☐), 23 cm (○) and L. innocua placed at: 3cm (▲), 12cm (■)
and 23cm (●) from the high intensity near ultraviolet/ visible (NUV–vis) 395±5 nm light
source
(Results
expressed
as
mean
log10
CFU/mL)……………………………………………………………………………....135
Figure 3.1.2: Survival curves of E. coli suspended in maximum recovery diluent (MRD)
placed at: 6.5 cm (∆), 17cm (☐), 28.5 cm (○) and L. innocua placed at: 6.5cm (▲),
17cm (■) and 28.5cm (●) from continuous UV light source (Results expressed as mean
log10 CFU/mL)……………………………………………………………..…………136
Figure 3.1.3: Survival curves of E. coli suspended in maximum recovery diluent (MRD)
placed at: 2.5 cm (∆), 8cm (☐), 11.5 cm (○), 14 cm (◊) and L. innocua placed at: 2.5cm
(▲), 8cm (■), 11.5cm (●) and 14cm (♦) from high Intensity pulsed light source (Results
expressed as mean log10 CFU/mL)…………………………………...………….……136
Figure 3.1.4: Mean Log cfu/mL E. coli on MRD after treatment at the same dosages at
shortest distance with 3 different light equipments: NUV-vis (■), Continuous UV (■)
and High Intensity Light Pulses (■). ………………………………………...……….137
Figure 3.1.5: Mean log cfu/mL L. innocua on MRD after treatment at the same dosages
at shortest distance with 3 different light equipments: NUV-vis (■), Continuous UV (■)
and High Intensity Light Pulses (■)……………………………………...……………138
Figure 3.1.6: Mean Temperature increase (ΔΤ ᵒC) for NUV-Vis light technology at
distances : 3cm (▲), 12cm (■) and 23cm (●)………………………………...……….139
Figure 3.1.7: Mean Temperature increase (ΔΤ ᵒC) for UV light technology at distances:
6.5cm (▲), 17cm (■) and 28.5cm (●)……………………………………….…….......139
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Birmpa Angeliki
Figure 3.1.8: Mean Temperature increase (ΔΤ ᵒC) for HILP light technology at
distances: 2.5cm (▲), 8cm (■), 11.5cm (●) and 14cm (♦)…………………………….139
Figure 3.2.1.1.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E.
coli, S. aureus, S. enteritidis, L. innocua inoculated on fresh romaine lettuce……...…141
Figure 3.2.1.1.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S.
enteritidis, L. innocua inoculated on fresh romaine lettuce………..…………………..142
Figure 3.2.1.1.3: Disinfection Efficiency of combined alternative and conventional
disinfection technologies (US+NaOCl 50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm,
UV+NaOCl 200ppm) on E.coli, S.aureus, S. Enteritidis, L. innocua inoculated on fresh
romaine lettuce…………………………………………………………….……..……143
Figure 3.2.1.1.4: Disinfection Efficiency of combined alternative disinfection
technologies on E.coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh romaine
lettuce……………………………………………………………………….………….144
Figure 3.2.1.2.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E.
coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries………..….146
Figure 3.2.1.2.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S.
Enteritidis, L. innocua inoculated on fresh strawberries……………………...…….147
Figure 3.2.1.2.3: Disinfection Efficiency of combined alternative and conventional
technologies (US+NaOCl 50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm,
UV+NaOCl 200ppm) on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on
fresh strawberries…………………………………………………………………...….148
Figure 3.2.1.2.4: Disinfection Efficiency of combined alternative technologies on E. coli,
S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries…………..…..149
Figure 3.2.1.3.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E.
coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh cherry tomatoes…….…150
Figure 3.2.1.3.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S.
Enteritidis, L. innocua inoculated on fresh cherry tomatoes………………….……..151
Figure 3.2.1.3.3: Disinfection Efficiency of combined alternative and conventional
technologies (US+NaOCl 50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm,
UV+NaOCl 200ppm) on E. coli, S. aureus, S. Enteritidis, L.innocua inoculated on fresh
cherry tomatoes……………………………………………………...…………..……..153
Figure 3.2.1.3.4: Disinfection Efficiency of combined alternative technologies on E. coli,
S. aureus, S. Enteritidis, L.innocua inoculated on fresh cherry tomatoes…………...154
Figure 3.2.2.1: Standard Curve based on the entire hexon region of Ad35 cloned into
pBR322…………………………………………………………………………...……156
Figure 3.2.2.2: Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and
Cherry tomatoes (light grey bars) and single step conventional Disinfection
Treatments…………………………………………………………………..…………158
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Figure 3.2.2.3: Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and
Cherry tomatoes (light grey bars) and single step Alternative Disinfection
Treatments…………………………………………………………….……………….159
Figure 3.2.2.4: Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and
Cherry tomatoes (light grey bars) and combined Disinfection Treatments....................160
Figure 3.2.2.5: The results of Real Time PCR were also evaluated with cell cultures
observed under an Epifluorescence microscope…………………………….…………163
Figure 3.2.4.1: E. coli, S. aureus, S. Enteritidis and L. innocua populations inoculated on
romaine lettuce before and after selected disinfection treatments during storage for 15
days at 6°C……………………......................................................................................164
Figure 3.2.4.2: E. coli, S. aureus, S. Enteritidis and L. innocua populations inoculated on
strawberries before and after selected disinfection treatments during storage for 15 days
at 6°C…………………………………………………………………………………..166
Figure 3.2.4.3: Populations of E. coli, S. aureus, S. Enteritidis and L.innocua inoculated
on cherry tomatoes before and after selected disinfection treatments during storage for
15 days at 6°C…………………………………………………………..……………168
Figure 3.3.2.1: TAC of Romaine Lettuce before and after conventional, alternative and
combined disinfection technologies…………………………………………….……..185
Figure 3.3.2.2: TPC of Romaine Lettuce before and after conventional, alternative and
combined disinfection technologies……………………………………………….…..186
Figure 3.3.2.3: AA of Romaine Lettuce before and after conventional, alternative and
combined disinfection technologies……………………………………….……..……187
Figure 3.3.2.4: TAC of Strawberries before and after conventional, alternative and
combined disinfection technologies……………………………………………...……188
Figure 3.3.2.5: TPC of Strawberries before and after conventional, alternative and
combined disinfection technologies………………………………………………...…189
Figure 3.3.2.6: AA of Strawberries before and after conventional, alternative and
combined disinfection technologies……………………………………………...……190
Figure 3.3.2.7: TAC of Cherry Tomatoes before and after conventional, alternative and
combined disinfection technologies…………………………………………….……..191
Figure 3.3.2.8: TPC of Cherry Tomatoes before and after conventional, alternative and
combined disinfection technologies…………………………………...………………192
Figure 3.3.2.9: AA of Cherry Tomatoes before and after conventional, alternative and
combined disinfection technologies……………………………….…………………..193
Figure 3.4.1. The FCM Model…………………………………………........…………197
Figure 3.4.2: Fuzzy Cognitive Map………………………………………..…………..197
Figure 3.4.3: Subsequent values of concepts till convergence of 1st case……………199
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Figure 3.4.4: Subsequent values of concepts till convergence of 2nd expert…….…....200
Figure 3.4.5: Subsequent values of concepts till convergence of 3rd expert….......…..201
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Table of Contents
List of Graphs
Graph 1.3.5.3.1. Time trend of salmonellosis notification rate, Mandatory Notification
System, Greece, 2004-2012.Hellenic Center for Disease Control and Prevention ……50
Graph 1.3.5.3.2. Annual notification rate (cases/100,000 population) of salmonellosis by
age
group,
Mandatory
Notification
System,
Greece,
20042012…………………………………………………………………………………….50
Graph 1.3.5.4.1. Notification rate of listeriosis by age group and gender in Greece,
Mandatory Notification System, 2004-2012…………………………………………...53
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Birmpa Angeliki
List of Tables
Chapter 1
Table 1.1 Most Commonly Recognized Foodborne Pathogens……………….…..……17
Table 1.3.4.1: Number of reported foodborne diseases-outbreaks, cases and deaths in
United States, 1998-2002………………………………………………………..……...43
Table 1.3.4.2: Number of multistate foodborne disease outbreaks, by year and pathogenFoodborne
Disease
Outbreak
Surveillance
System,
Greece,
20042012……………………………………………………………………………….…….44
Table 1.3.5.1 : Five categories of the diarrheagenic E. coli…………………….………45
Table 1.3.5.3.1 Number of notified cases of salmonellosis per year, Mandatory
Notification System, Greece, 2004-2012………………………………………...……..50
Table 1.3.5.4.1. Annual number of notified cases and notification rate of listeriosis in
Greece, Mandatory………………………..……………………...……………….…….53
Table 1.3.6.1: Enteric viruses and clinical syndromes………………...………………..54
Table 1.4.4.1.1: Ranges, Wavelengths and Characteristics of different types of
UV)………………………………………………………………………………….…..74
Table
1.4.4.4.1:
Advantages-Disadvantages
of
electrochemical
ultrasonic
apparatuses………………………………………………………………………....…....80
Chapter 2
Table 2.1.5.3.1: Calculated exposure time (sec) of non-thermal light technologies at
selected distances from the light source.(NUV-Vis: NUV–vis light; UV: Ultraviolet
Light; HILP: High Intensity Light Pulses, *Samples that are not analyzed due to high
temperature, NT: Not tested samples)……………………..……………….…………102
Table 2.2.14.1.1: Sodium Hypochlorite Treatments…………………………………..110
Table 2.2.14.2.1: UV treatments……………………………………….………….…..111
Table 2.2.14.3.1: Various ultrasound treatments…………………………….……..….111
Table 2.2.14.4.1: Combined Treatments……………………………………....…...….112
Table 2.2.16.1: ISO Methods…………………………………………………….…….113
Table 2.2.18.5.1: Working solutions of primers and probe……………..……………..117
Table 2.2.18.5.2: Volumes of reagents for PCR mix…………...……………………..118
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Chapter 3
Table 3.2.3.1: High and Low Inocula on lettuce and disinfection with selected
treatments………………………………………………………..……………………..161
Table 3.2.3.2: High and Low Inocula on strawberries and disinfection with selected
treatments……………………………………………………………………………....161
Table 3.2.3.3: High and Low Inocula on cherry tomatoes and disinfection with selected
treatments ………………………………………………………………………..…....162
Table 3.3.1: Values are average ± standard deviation of at least three experiments and
represent the color parameters of romaine lettuce after each processing time with each
disinfection method: NaOCl 50ppm, NaOCl 200ppm, UV: Ultraviolet irradiation (254
nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30 W/L), US+NaOCl
50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm, UV+NaOCl 200ppm,
UV+US………………………………………………………….……………………..173
Table 3.3.2: Values are average ± standard deviation of at least three experiments and
represent the color parameters of strawberries after each processing time with each
disinfection method : NaOCl 50ppm, NaOCl 200ppm, UV: Ultraviolet irradiation (254
nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30 W/L), US+NaOCl
50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm, UV+NaOCl 200ppm,
UV+US…………………………………………………………………………….…..178
Table 3.3.3: Values are average ± standard deviation of at least three experiments and
represent the color parameters of cherry tomatoes after each processing time with each
disinfection method: NaOCl 50ppm, NaOCl 200ppm, UV: Ultraviolet irradiation (254
nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30 W/L), US+NaOCl
50ppm, US+NaOCl 200ppm, UV+NaOCl 50ppm, UV+NaOCl 200ppm,
UV+US…………………………………………………………..…………………….183
Table 3.4.1: Evaluation of three experts, where W: Weak, M: Medium, S: Strong, VS:
Very Strong………………………………………………………………….………....196
Table 3.5.1: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different
disinfection methods at the longest exposure times and infectious doses for each
microorganism inoculated in lettuce. Stars show the severity of infection: low infectivity
(*), medium infectivity (**), high infectivity (***)…………………………..……….202
Table 3.5.2: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different
disinfection methods at the longest exposure times and infectious doses for each
microorganism inoculated in strawberries. Stars show the severity of infection: low
infectivity (*), medium infectivity (**), high infectivity (***)………………………..203
Table 3.5.3: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different
disinfection methods at the longest exposure times and infectious doses for each
microorganism inoculated in cherry tomatoes. Stars show the severity of infection: low
infectivity (*), medium infectivity (**), high infectivity (***)…………………......…203
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INTRODUCTION
The market of fresh produce is increasing constantly and this can be attributed to the
consumers’ tendency for healthy and convenient foods, due to the fact that positive
effects to human health have been attributed to consumption of fresh produce (Gilbert,
2000, Ragaert et al., 2004). Organizations such as the World Health Organization
(WHO), Food and Agriculture Organization (FAO), United States Department of
Agriculture (USDA), and European Food Safety Authority (EFSA) recommended an
increase of consumption of fresh ready-to-eat (RTE) fruit and vegetables as they are
correlated with a decrease in the risk of cardiovascular diseases and cancer (Allende et
al., 2006). Moreover, the Mediterranean diet is followed by many people nowadays, due
to its positive effects on health. Many epidemiological studies suggest that food involved
in Mediterranean diet may be linked to a reduction in coronary heart disease risk. There
is also evidence that the antioxidants present in selected foods, improve cholesterol
regulation and LDL cholesterol reduction, and moreover anti-inflammatory and antihypertensive effects are correlated with Mediterranean diet (Covas, 2007).
RTE produce is defined as washed, bite-size, and packaged fresh fruit and vegetables,
which allow consumers to eat healthy on the run and to save time on food preparation. In
fact, the availability of fresh-cut fruits in automated vending machines existing in public
places constitutes an excellent strategy to improve the nutritional quality of snacks and
convenience of foods in a time when obesity and nutrition-related illnesses affect large
percentages of the population (Olivas and Barbosa-Cánovas, 2005).
Microbiological safety of foods and foodborne illness are complex issues since there are
more than 200 known diseases that are transmitted through foods. Primary causative
agents of foodborne illness are bacteria, viruses, parasites and molds (table 1.1).
Bacteria
Viruses
Parasites
Molds
Listeria Monocytogenes
Norovirus
Giardia lamblia
Aspergillus spp.
Salmonella spp.
Rotavirus
Cryptosporidium parvum
Penicillium spp.
Campylobacter spp
Astrovirus
Toxoplasma gondii
Fusarium spp.
Escherichia coli O157:H7
Hepatitis A virus
Cyclospora cayetanensis
Staphylococcus aureus
Trichinella spiralis
Clostridium perfrigens
Table 1.1 Most Commonly Recognized Foodborne Pathogens (Parikh, 2007)
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Introduction
In United States, the annual patient-related costs of the principal bacterial and parasitic
foodborne infections have been estimated at $6.5 billion or more (Buzby and Roberts,
1996, Tauxe, 2002). Tauxe (2002) reported that among the established foodborne
infections, bacterial infections accounted for an estimated 30% of cases, viral infections
count for 67% and parasitic infections for 3%. Hospitalizations are attributed to bacteria
(60%), viruses (35%) and parasites (5%). Finally, bacteria accounted for 72%, viruses
for 7% and parasite for 21% of deaths respectively. It is reported that five foodborne
pathogens – E. coli O157:H7, Salmonella, Campylobacter, Listeria, and Toxoplasma –
together cause an estimated 3.5 million cases, 33,000 hospitalizations and 1600 deaths
each year in United States (Tauxe, 2002).
The symptoms of foodborne illnesses range from mild gastroenteritis to life threatening
neurologic, hepatic, and renal syndromes. Center for Disease Control and Prevention
(CDC) has estimated approximately 76 million illnesses, 325,000 hospitalizations, and
5,000 deaths in the U.S. each year, out of which approximately 14 million illnesses,
60,000 hospitalizations, and 1,800 deaths were due to known foodborne pathogens.
Moreover, CDC recognized Salmonella, Listeria monocytogenes, and Toxoplasma
gondii as leading causes of death since they were responsible for 1,500 deaths each year.
For instance, more than 75% of deaths were caused by known pathogens however they
accounted for only approximately 11% of total cases of foodborne illness (Mead et al.,
1999).
In the environment, bacteria and viruses can be found in contaminated animal or water
sources. Animal droppings may be used to fertilize crops while a contaminated water
source may be used to irrigate or wash plants (Solomon et al., 2003). Contamination can
also be initiated by infected servers (Berrang et al., 2008).
Microorganisms have been shown to enter fruits and vegetables through various
pathways such as through the stomata, stem, stem scar, or calyx (Zhuang et al., 1995,
Seo and Frank, 1999). They can enter physically damaged fruits and vegetables through
punctures, wounds, cuts, and splits during maturation, harvesting, or processing.
Bacterial soft rot of fruits and vegetables can also increase the likelihood of
contamination
Page 18
with
pathogens
(Zhuang
et
al.,
1995).
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Birmpa Angeliki
Chapter 1: Literature Review
1.1 Fresh produce and Mediterranean diet
The Mediterranean diet is a modern nutritional pattern inspired by the traditional dietary
patterns of Greece, Spain, Portugal and Southern Italy. The Mediterranean diet pyramid
is based on food patterns typical of Crete, much of the rest of Greece, and southern Italy
in the early 1960s, where adult life expectancy was among the highest in the world and
rates of coronary heart disease, certain cancers, and other diet-related chronic diseases
were among the lowest (Willett et al., 1995). The principal ingredients of this diet are
based on high consumption of olive oil, legumes, cereals, fruits, and vegetables,
moderate to high consumption of fish, dairy products (mostly cheese and yogurt),
moderate wine consumption, and low consumption of meat and meat products (Noah
and Truswell, 2001). Thus, lettuce and tomatoes are important ingredients of a balanced
every day diet, as well as strawberries, which are selected among other fruits and
vegetables, by those who adhere to Mediterranean diet and are interested in following a
healthy lifestyle.
Flavonoids from vegetable and fruits intake appear to be inversely related to coronary
heart disease (CHD) mortality (Hertog et al., 1995, Rimm et al., 1996). Furthermore,
catechin, a naturally occurring flavonoid, has been linked to the prevention of human
plasma oxidation and to inhibition of oxidation of low-density lipoprotein (Lotito and
Fraga, 1998). Thus, flavonoids as well as other antioxidants are responsible for the
protective effects of the Mediterranean diet, rich in vegetable, fruit and wine against
CHD (Evans et al., 1995, Mangiapane et al., 1992). Moreover, Mediterranean diet has
also been linked with reduced obesity. The potential role of fiber-rich diets, and of
components other than fiber but included in the same foods (fruits and vegetables) such
as antioxidants are known to exert the important effect on weight regulation (Park et al.,
2005). Thus, Mediterranean diet is recommended for people who face problems with
obesity (Bes-Rastrollo et al., 2006).
However, contamination of fresh fruits and vegetables is of special concern, because
they are consumed raw, without any type of microbiologically lethal processing, thus are
prone to a number of pathogens. Ramos et al. (2013) has extensively studied the
outbreaks that have been reported for fresh produce (figure 1.1).
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Literature Review
Figure 1.1: Reported Foodborne Outbreaks in vegetables (on the left), and in fruits (on the right)
(Ramos et al., 2013)
The majority of pathogens implicated in produce-related outbreaks are transmitted via
the fecal-oral route (Johnston et al., 2006). Microbial contamination of fruits and
vegetables can occur during plant growth, harvesting, transport, processing, distribution
and marketing, or during processing at home (Beuchat, 1998). Produce contaminated
with pathogens cannot be completely disinfected by washing or rinsing the product in an
aqueous solution (Rodgers and Ryser, 2004). The prevention of produce contamination
with human pathogens is the only practical and effective means of ensuring safe produce
for human consumption. Therefore a growing need for effective sanitizer treatments to
reduce the number of microbial pathogens on produce to safer levels is raised.
1.1.1 Lettuce
Lettuce is one of the most consumed vegetable worldwide with a global production of
about 24 million tons in 2011 (FAOSTAT, 2011). Mean daily consumption of lettuce in
Europe is 22.5 g, which represents about 6.5% of the total dietary intake of vegetables
(WHO, 2003). In Greece 84000 tons of lettuce was produced in 2008, in a cultivation
area of 5500 hectares (EL.STAAT, 2014). In Peloponnesus half of the above production
occurs (around 37000 tons) (EL.STAAT, 2014).
Lettuce (Lactuca sativa L.) is one of the most popular vegetable for human consumption,
especially in salad, and is considered as a good source of health-promoting compounds
such as phenolics, vitamin C, folates, carotenoids and chlorophylls (Nicolle et al., 2004).
It contains several macrominerals (e.g. K, Na, Ca and Mg) and trace elements (e.g. Fe,
Mn, Cu, Zn and Se) which are essential for human nutrition (Kawashima and Soares,
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Non-thermal technologies for the disinfection of food and risk assessment for Public Health
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2003). It is also known as a good source of photosynthetic pigments (chlorophylls and
carotenoids) and other phytochemicals that benefit nutrition and have a significant role
in the prevention of several oxidative stress-related diseases (Llorach et al., 2008).
Romaine lettuce is consumers’ favorite leafy vegetable for its crispness, good aroma,
tender appearance and high concentration in many health-promoting compounds
(Llorach, et al., 2008). Lettuce leaves consists of vascular and photosynthetic tissues
(Toole et al., 2000), where a thick white mid-rib (vascular tissue) constitutes the
majority of the leaf (photosynthetic tissue). The leaves are tightly wrapped and
interlocked providing a crispy texture to lettuce (Toole et al., 2000). Toole et al. (2000),
exhibited that photosynthetic and vascular tissues possess different phenolic metabolism
as well as textural characteristics. Vascular tissue has lower polyphenol oxidase,
peroxidase and phenylamonium lyase activity than photosynthetic tissue (Fukumoto et
al., 2002). However the potential for the development of “browning” is higher in
vascular tissues, especially in the outer than the inner leaves probably due to the fact that
vascular tissues have a higher total volume and cut area (Fukumoto et al., 2002).
Lettuce can be found as whole-heads and as fresh-cut lettuce. Indeed, in the fresh-cut
industry, fresh-cut lettuce is one of the most important products. However, the physical
damage or wounding caused during preparation, increases the rate of biochemical
reactions responsible for changes in visual quality (color, texture and browning) and
phytochemicals (vitamin C content and phenolic content) (Saltveit, 2003).
In the specie Lactuca sativa there are seven main groups of cultivars: butterhead lettuce,
iceberg or crisphead lettuce, romaine/cos lettuce, cutting lettuce, stalk lettuce, latin
lettuce and oilseed lettuce. Among them, romaine lettuce is the most popular minimally
processed leafy vegetable. Its consumption has increased dramatically in recent years
due to the fact that consumers need more convenient and less time consuming products,
which also seem fresh and possess healthy characteristics (Ragaert et al., 2004, Rico et
al., 2007).
Lettuce head has been classified as a commodity with moderate respiration rate.
However, cutting lettuce for minimally processing enhances its respiration rate (Kader
and Saltveit, 2003). Respiration rate has been associated with the perishability of the
product. Therefore it is assumed that higher respiration rate reduces the shelf-life of
lettuce. It has been demonstrated that the increase in respiration rate is higher in
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Literature Review
transverse than longitudinal cut sections. In addition, other methods of preparation such
as shredding lettuce increases the respiration rate in comparison to cutting lettuce with a
sharp knife or tearing by hand due to less damage that is caused to the tissue (Kader and
Saltveit, 2003, Lopez et al., 2014).
1.1.2 Strawberries
Greece has increased its exports in strawberries, as an increasing demand all over the
worlds exists. The largest percentage of strawberries is exported to Germany, Italy,
Hungary, Romania, Bulgaria, Russia, Serbia (EL.STAAT, 2014). In 2013, 34.118.653
kg of strawberries produced in Greece, were exported (EL.STAAT, 2014). Moreover,
the strawberry is an attractive fruit, with benefits to human health, due to its high content
of vitamin C, anthocyanins and flavonols, and high antioxidant activity (OdriozolaSerrano et al., 2010, Tiwari et al., 2009, Alexandre et al., 2012).
The garden strawberry (Fragaria × ananassa) comes from the genus Fragaria
(collectively known as the strawberries) belonging to the family of Rosaceae. It is
cultivated worldwide for its fruit. The fruit has a characteristic aroma, bright red color,
juicy texture, and sweetness. It is consumed in large quantities, either fresh or prepared
in foods as fruit juice, pies, ice creams, milkshakes, and chocolates. Strawberry is rich in
soluble sugars such as glucose, fructose and sucrose. Organic acids and soluble pectins
also contribute to soluble solid content of strawberry (Pelayo-Zaldívar et al., 2005). The
Soluble Solid Contents (SSC) continually increase in the fruit during its development but
the main changes occur between 21 and 28 days after fruit set, during fruit ripening
period (Montero et al., 1996). SSC is used as an indicator for fruit taste. However, the
relative proportions of sugar components such as glucose, fructose and sucrose may
influence the perception of sweetness.
Compared to other fruits, berries possess high antioxidant capacity and are rich in a
variety of phytochemicals, such as phenolic compounds (Häkkinen et al., 1999,
Koponen et al., 2007). Furthermore, strawberries are extremely rich in vitamin C (60100 mg/100 g FW) and in anthocyanins, especially pelargonidin-3-glucoside (pg-3-gluc)
and cyanindin-3-glucoside (cya-3-gluc). However, variability in concentration of the
fruit components is often observed. Therefore, strawberry is considered as an important
dietary source of health promoting compounds (Koponen et al., 2007).
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Birmpa Angeliki
Numerous epidemiological studies have shown that consumption of fruits and vegetables
has a protective effect against degenerative diseases (Hannum, 2004). Oxygen radicals
which are produced with ageing of the fruit can react with lipids, protein and DNA. The
antioxidants which exist in fruits and vegetables are able to maintain low cellular levels
of oxygen radicals by preventing their formation, scavenging them or promoting their
decomposition (Hancock et al., 2007).
However, strawberries are highly susceptible to mechanical injury, physiological
disorders, fungal activity and water loss (Romanazzi et al., 2013). The storage period
and the shelf life of strawberries are very short due to perishability and susceptibility to
rot-causing pathogens (Aday, 2014). The most severe postharvest diseases of
strawberries are gray mould (Botrytis cinerea Pers.ex.Fr.) and Rhizopus rot (Rhizopus
stolonifer Ehrenb.Fr.Vuill.) which cause severe losses during storage and transport from
cold store to the market (Vardar and Ilhan, 2012).
1.1.3 Tomatoes
Tomato (Solanum Lycopersicum) is an herbaceous fruiting plant and is consumed
worldwide with an annual production mass of more than 140 million tons (Faostat,
2009). Greece is selected among the 14 processing countries which represent 92% of
global production. Greece total exports in tomatoes were around 20.000.000 kg in year
2013 (EL.STAAT, 2014). In Greece tomatoes are grown from Autumn until Spring and
Summer in colder climates. Tomato has become one of the most widely grown
vegetables with the ability to survive in diverse environmental conditions. Moreover,
tomato grows better in fertile, well-drained soils, with pH 6 and ambient temperatures of
25°C (Rice et al., 1987).
Tomato fruit is a good source of vitamins (vitamin A, vitamin C, folate and other trace
vitamins) and minerals (potassium, calcium, phosphorus, iron), also containing a fibre
and a very small amount of protein and fat. Tomato is also a good source of antioxidants,
such as lycopene. Lycopene and fibres are beneficial to health when consumed in a diet
(Canene-Adams et al., 2005). According to researches, lycopene has anti-inflammatory,
antimutagenic and anticarcinogenic properties (Boon et al., 2010). Moreover, lycopene
is also known for reducing the risk of adenoma, and promoting immune system
functionality (Kun et al., 2006). The aforementioned health benefits are related to the
singlet oxygen and free radical scavenging properties of lycopene (Canene-Adams et al.,
Page 23
Literature Review
2005). It is recommended, 6-15 mg lycopene intake for improved health (Kun et al.,
2006). Soluble fibre modulates blood glucose and cholesterol levels (Weickert and
Pfeifer, 2008). Insoluble fibre promotes laxation and helps against many cancers such as
the cancer of colon (Alvarado et al., 2001). In tomato products, vitamin C and
polyphenols are reported to be the major antioxidant hydrophilic components, whereas
vitamin E and carotenoids mainly constitute the hydrophobic fraction (Hsu, 2008).
Cherry tomato is a very small variety of tomato. Cherry tomatoes range in size from a
thumbtip up to the size of a golf ball, and can range from being spherical to slightly
oblong in shape. The oblong cherry tomatoes often share characteristics with plum
tomatoes, and are known as grape tomatoes. The berry tomato is regarded as a botanical
variety of the cultivated berry Solanum lycopersicum var. cerasiforme (Raffo et al.,
2006).
In Mediterranean countries, cherry tomatoes are largely used for fresh consumption, thus
their commercial importance is continuously increasing (Leonardi et al., 2000a). Cherry
tomatoes are grown in unheated greenhouses, which have no climate control systems
and are covered with plastic film. As a consequence, the development and the ripening
of cherry tomatoes happen under varying climatic conditions. It has been reported that
cherry tomato plants grown in greenhouse under high light, exhibited an approximately
two-fold greater soluble phenols content (rutin and chlorogenic acid) than low-light
plants (Raffo et al., 2006). The temperature and the light intensity are factors that
influence the quality attributes of tomato, such as appearance, firmness, texture, dry
matter and sensory properties. Furthermore, environmental factors can also affect the
antioxidant content of tomatoes (Dumas et al., 2003, Lee and Kader, 2000).
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1.2. Nutritional, Quality and Health Aspects of fresh
ready-to- eat (RTE) fruits and vegetables
The nutritional quality and health-beneficial properties of plant products have been
studied by many research groups because of the growing consumer’s interest in recent
years in including healthy foods in their everyday diet. Fruits and vegetables are rich in
ingredients such as phytosterols, vitamins, minerals, fibers, water, isoflavones, lycopene,
etc. (Hasler et al., 2004). Eating fruits and vegetables also reduces blood pressure, boosts
the immune system, detoxifies contaminants and pollutants, and reduces inflammation in
the human body (Wang and Lin, 2000). Thus, researchers support the recommendation
of consuming five or more servings of fruits and vegetables daily (Crecente-Campo et
al., 2012, Hung et al., 2004). In fact, the nutritional profile of many fruits and vegetables
is influenced by many factors, such as cultural practices, preharvest conditions (climate,
temperature), maturity, post-harvest handling and food processing (Rekika et al., 2005,
Wang et al., 2002).
Many epidemiological studies have shown the inverse relationship between the
consumption of fresh RTE fruits and vegetables and the incidence of chronic diseases
such as cardiovascular diseases and certain types of cancer (Maynard et al., 2003,
Temple and Gladwin, 2003, Trichopoulou et al., 2003). The above effect is attributed to
bioactive or phytochemical compounds that are present in the foods and are involved in
certain biological actions, resulting in health beneficial effects (Prior and Cao, 2000).
Therefore, the consumption of fruits and vegetables includes not only nutrients essential
to life (carbohydrates, proteins, fats, vitamins, etc.), but also phytochemical or bioactive
compounds (carotenoids, phenolics, vitamins A, C, and E, fiber, glucosinolates,
organosulfur compounds, sesquiterpenic lactones, etc.) (Duthie et al., 2000, Eastwood
and Morris, 1992, Knekt et al., 2000, Lampe and Peterson, 2002, Ling and Jones, 1995,
Piironen et al., 2000, Plaza et al., 2006a, 2006b, Sanchez-Moreno et al., 2006a, 2006b,
Simon et al., 2001, Skimola and Smith, 2000).
Other researchers also emphasize that the health benefits of lettuce have been attributed
to the presence of phenolic compounds, fiber and vitamin C content (Nicolle et al.,
2004). A regular intake of antioxidant compounds from lettuce is useful to improve the
lipid status and to prevent lipid peroxidation in tissues. Generally, differences in
chlorophyll and anthocyanin concentrations, as well as in phenolics and antioxidant
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properties in the lettuce leaves have been observed in different lettuce cultivars that
range in color (green and yellow to deep red) (Ozgen and Sekerci, 2011). Generally red
lettuce has been found to have a higher content in total phenolics (TP) and in total
antioxidant capacity (TAC) than green lettuce (Llorach et al., 2008). Studies have shown
that, the cultivar, the planting date and the growing conditions may alter the phenolic
content and antioxidant capacity of lettuce (Liu et al., 2007). Moreover, it is known that
phytochemicals and antioxidant capacity in lettuce may differ from outer to inner leaves.
Thus, consumers tend to eat from middle part of lettuce head towards to inner since
these parts look fresher, crispier and tender without knowing the real phytonutrient
contents of these parts (Ozgen and Sekerci, 2011).
Strawberries are a rich source of antioxidants and a common and important fruit
consumed in the Mediterranean diet because of their high content in essential nutrients
and beneficial phytochemicals, and also due to healthy substances that are observed in
human health (Giampieri et al., 2012). These phytochemicals are essentially flavonoids
(mainly anthocyanins, with flavonols providing a minor contribution), hydrolysable
tannins (ellagitannins and gallotannins) phenolic acids (hydroxybenzoic acids and
hydroxycinnamic acids), and also condensed tannins (proanthocyanidins) (Cerezo et al.,
2010). The color expression of strawberry fruits is associated with concentration and
composition of anthocyanins. Anthocyanins in strawberries are the major known
polyphenolic compounds. Pelargonidin-3-glucoside is the main anthocyanin in
strawberries, whereas cyanidin-3-glucoside exists in smaller proportions. However, the
composition of strawberries varies according to genotype (Tulipani et al., 2008).
Research that has been conducted with strawberry extracts, exhibited a high level of
antioxidant capacity against free radical species (Li et al., 2014). Thus, strawberries may
have an effective role in decreasing the risk of cancer and in preventing various human
diseases caused by oxidative stress. On the other hand, strawberries are highly
susceptible to infection by pathogens and spoil rapidly after harvesting. Thus, low
temperature storage is effective in order to reduce decay and maintain the overall
acceptable quality of strawberries. However, there is strong evidence that the levels of
anthocyanin and aroma compounds in strawberries remain low at lower temperatures
than at higher temperature storage conditions (Jin et al., 2011).
Tomatoes (Solanum lycopersicum L.) commonly used in the Greek diet, are a major
source of antioxidants and contribute to the daily intake of a significant amount of these
molecules. In fact, tomato fruit is rich in diverse antioxidant molecules, such as
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carotenoids (especially lycopene), phenolics, flavonoids, vitamin C, vitamin E and
tocopherols (Mitchell et al., 2007). Lycopene (the red pigment of tomato), phenolics and
flavonoids have received great interest during the last few years because of their
antioxidant properties in relation to free radicals, suggesting protective roles in reducing
risk of chronic diseases, such as cancer and cardiovascular disease (Rice-Evans et al.,
1996). It is known that the skin of some tomato fruits contains significantly higher levels
of phenolics, flavonoids, lycopene, ascorbic acid and antioxidant activity than pulp and
seed fractions (Toor and Savage, 2005). These results led researchers to propose the peel
enrichment of tomato-based products as a means of increasing the nutritional value of
tomato pastes and enhancing carotenoids intake (Reboul et al., 2005). The tomato skin is
not only a valuable source of nutrients, but also acts as a protective organ by helping to
preserve the nutritional value of the remaining parts of the fruit. It maintains the physical
integrity of tomato and prevents flesh deterioration, by avoiding a direct contact with air
and, thus, preventing both dehydration and oxidation of sensitive chemical compounds.
Capanoglu et al. (2010) have showed that the direct contact of the tomato pulp with
oxygen can have detrimental effects to ascorbic acid, lycopene and phenolic content and
this is why nitrogen-conditioned packaging for tomato derivatives is used. Moreover, the
skin also prevents the direct incidence of light on the pulp, which has been linked to the
deterioration of bioactive compounds (Lee and Chen, 2002). Finally, tomato seeds are
edible and rich in bioactive compounds and minerals (Toor and Savage, 2005),
nevertheless they are usually discarded specially in the preparation of tomato
derivatives. Recently, studies have shown that consumption of the natural gel found in
tomato seeds can help to maintain a healthy blood circulation by preventing blood from
clotting (Vinja et al., 2014).
1.2.1 Antioxidant Compounds
An antioxidant compound delays or inhibits oxidation of an oxidizable substrate (lipids,
proteins, and DNA). Among the most important antioxidants present in foods are
vitamins C and E, carotenoids, and phenolic compounds (Martin-Belloso and SolivaFortuny, 2011).
Augmented antioxidant activity (measured using the ABTS•+ or DPPH radical
scavenging methods) has been reported in tissues of various vegetables in response to
stress caused by mechanical damage (peeling and cutting). This has been associated with
an increase or decrease in concentrations of phenolic compounds more than vitamin C
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concentration (Reyes et al., 2007). Phenolic compounds confer antioxidant capacity
(Proteggente et al., 2002), and as a consequence this capacity is one of the major reasons
why increased consumption of fruits and vegetables has been recommended as
beneficial to health (Prior and Cao, 2000).
Many methods (FRAP, DPPH, TEAC, TRAP, ORAC, EPR) are available for analyzing
antioxidant activity, with different concepts, mechanisms of action, ways of expressing
results, and applications (Huang et al., 2005, Frankel and Meyer, 2000).
1.2.1.1 Determination of Antioxidants
1.2.1.1.1 FRAP Method (Ferric Reducing Antioxidant Power)
This method is based on the reducing ability of an iron complex Fe+3-TPTZ (2,4,6-Tri(2Pirydil)-s-triazine) which is colorless, to a blue colored ferrous iron when antioxidantsand phenolic compounds are present. This conversion is owned to the transfer of an
electron from the antioxidant complex. Frap values are obtained by comparing the
absorbance change at wavelength of 593 nm in test reaction mixtures with those
containing ferrous ions of known concentration. The bigger the difference of absorbance
is, the greater the capacity of the antioxidant to reduce TPTZ-Fe+3 to TPTZ-Fe+2 is. The
results are expressed in μmol Fe+2/l and the absorbance is measured with a UV-Vis
spectrophotometer (Benzie and Strain, 1996).
The scheme that follows represents the above reaction that takes place:
Antioxidant compound
TPTZ-Fe+3 (colorless)
Fe+2-TPTZ
T=37°C, pH=3.6
The measurement of the difference of absorbance indicates the antioxidant capacity of
the sample.
1.2.1.1.2 DPPH Method (2,2-diphenyl-1-picryl-hydrazyl assay)
This method is based on the measurement of capacity of antioxidant compounds, such as
phenolic compounds, to bind to the stable radical DPPH. This radical is converted to
hydrazine which reacts with compounds such as phenolic compounds that can give one
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hydrogen atom from its molecule. The solutions’ reaction takes place in the presence of
DPPH solution for a period of time. The absorbance is then measured at 515-528 nm.
The reduction of absorbance of the DPPH solution is correlated with color alteration,
which indicates the enhanced capacity of DPPH, thus the enhanced capacity of being
bound from the antioxidant compound. This “effectivity” can be calculated:
%DPPH radical scavening = ((Αbs initial – Αbs sample)/Αbs initial) * 100,
where ABS initial is the DPPH absorbance, ABS sample is the absorbance of both
DPPH and of the sample after a certain period of time.
The higher the reduction of the absorbance gets, the bigger the antioxidant capacity of a
sample is, binding a larger amount of DPPH radicals. The DPPH method is useful, offers
repeatability, and is mainly used for the TAC determination in food and drink extracts
(Sánhez-Moreno, 2002).
1.2.1.1.3 ΤΕΑC Method (Trolox Equivalent Antioxidant Capacity)
TEAC or ABTS method is based on the antioxidant’s inhibition in the absorbance of
2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS+) which shows characteristic
absorption at 734 nm. ABTS+ is a green-blue chromophor, which is reduced to ABTS,
when antioxidants are present. The stable solution ABTS+ is based on the reaction of
aqua solution ABTS with potassium persulfate solution. Then the mix must remain in
dark conditions for 12-16 h. The initial method is based on the activation of
metmyoglobin with Η 2 Ο 2 through the creation of ferrymyoglobin, which after oxidize
ABTS to form ABTS+.
In this method the forming ABTS+ are mixed with the sample (which contains
polyphenols) and the inhibition’s percent of absorbance is measured at 734 nm, which
determines the quantity of polyphenols. Trolox is used as a standard, and this name has
been attributed to the method TEAC. The results are stated as “equivalents Trolox”,
which are defined as the solution concentration Trolox (mmol/l), with a dynamic
antioxidant equivalent in 1mmol/l of sample’s solution. This method has been used not
only in vitro but also in vivo (Sánhez-Moreno, 2002).
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1.2.1.1.4 TRAP Method (Total Radical-Trapping Antioxidant Parameter)
This method was first introduced by Wayner et al. (1985) in order to determine the
antioxidant level of human’s plasma. R-phycoerythrin (R-PE) is a phycobiliprotein of
red color. A correlation exists between the antioxidant activity and the damage of
fluorescence of R-PE. This method is based on the protection that antioxidants offer to
the damage of fluorescence of R-PE (lag-phase) during controlled peroxidase reaction.
The kinetic reaction takes place in 38οC and is recorded during 1h with a
spectrophotometer. TRAP values (values in μΜ), are calculated from the length of lag
phase and are expressed as molecules Trolox, which have the same antioxidant capacity
in 1l of plasma (Sánhez-Moreno, 2002).
1.2.1.1.5 ORAC Method (Oxygen-Radical Absorbance Capacity)
This method is commonly used for the determination of antioxidant capacity in plasma
and tissues. It is based on the initial experiment that was carried out by Delange and
Glazer (1989) and it is based on unique properties of phycoerythrin, which is used as a
target of free radicals. According to this method, TAC is determined through the
calculation of area under curve, until the moment of flection of fluorescence of
phycoerythrin curve, in the presence of antioxidants. For the creation of hydroxyl
radicals, radical AAPH is used. ORAC combines the inhibition time and the inhibition
rate of free radicals from antioxidants by using area under curve technique. In other
words, the calculation of the area, is used in order to have a quantitative analysis. The
results are expressed as ORAC units or Trolox equivalents. (Sánhez-Moreno, 2002).
This method shows a sensitivity against hydrogen transfer, whereas FRAP against
electron transfer and TEAC against both (Aguilar-Garcia et al., 2007).
1.2.1.1.6 ΕPR Method (Electronic paramagnetic resonance)
This is a spectroscopic method which is used for the tracing of free radicals and the
determination of antioxidant capacity in vitro, in fruits, vegetables, olive oil, juices and
other plant foods. This method is based on microwave irradiation and tracing of free
radicals with niter-oxides directly, or indirectly when resonance happens. Free radicals
are traced after determination of height and breadth of spectrum vertices, and from this
area, quantification of these radicals occurs. Antioxidant capacity of these foods is
calculated through the use of standard radical TEMPOL and the method is standardized
using Trolox (Papadimitriou et al., 2005).
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1.2.2 Phenolic Compounds
Phenolic compounds are secondary metabolites produced by vegetables, which possess a
benzene ring in their chemical structure with hydroxyl groups that are responsible for
their activity. The polyphenols present a wide variety of structures including simple
molecules
(monomers
and
dimers),
and
polymers
(tannins).
Abundant
are
hydrocinnamic acids (C6-C3), benzoic acids (C6-C1), flavonoids (C6-C3-C6),
proanthocyanidins (C6-C3-C6)n, stilbenes (C6-C2-C6), lignanes (C6-C3-C3-C6), and
lignines (C6-C3)n (Scalbert et al., 2005). Phenolics have been widely studied for their
relationship with the quality characteristics of plant foods such as color, because many
of them are pigments (such as anthocyanins) responsible for the color of grapes,
cherries, plums, strawberries, raspberries, etc. Moreover, phenolic compounds when
oxidized cause enzymatic browning, which is responsible for a large percentage of loss
of quality in plant foods during processing and storage. They are also widely known for
their contribution to the flavor and aroma of plant foods. Flavones, flavonols, flavanols,
flavanones, anthocyanins, and isoflavones are common in fruits and vegetables. The
basic characteristic of most existing flavonoids in fruits and vegetables is the antioxidant
and radical scavenger activity (Rice-Evans et al., 1996).
It is well known that the intake of foods rich in phenolics, and most particularly in
flavonoids, are correlated with a low incidence of cardiovascular diseases and some
types of cancer (Duthie et al., 2000, Skibola and Smith, 2000). The more widely studied
foods include tea, spices, and grape derivatives, and also some fruits such as apples and
berries (strawberries, raspberries, blackberries, etc.). Furthermore, flavonoids are known
for
their
anti-inflammatory,
antiallergic,
antiviral,
hypocholesterolemic,
and
anticarcinogenic activities (Knekt et al., 2000, Skibola and Smith, 2000). Maas and
Galleta, (1991) found that red grapes, blackberries, raspberries, and strawberries are rich
in hydroxybenzoic acids, particularly in ellagic acid that has been shown to have a
protective effect against cancer.
1.2.2.1 Determination of TPC (total phenolic content)
Several analytical methods have been proposed for the determination of flavonoids. Due
to the importance of phenolics in prevention of a multitude of diseases, a series of in
vitro methods have been developed in order to be able to detect phenolics accurately.
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1.2.2.1.1 Spectrophotometric Method (Folin-Ciocalteau)
With this method the quantitative determination of total phenolics takes place. An
Ultraviolet-visible (UV-vis) spectrophotometer is used and is based on a colorimetric
oxidation/reduction reaction. The oxidizing agent is Folin-Ciocalteau reagent. The
Folin–Ciocalteu phenol reagent is used to obtain a crude estimate of the amount of
phenolic compounds present in an extract. Phenolic compounds undergo a complex
redox reaction with phosphotungstic and phosphomolybdic acids present in the reagent
to phosphotungstic/phosphomolybdic phenolic complex, which has blue color and exists
in alkaline conditions.
Phenolics + alkaline + FC reagent  blue colored product, Abs 765 nm
With this quantitative determination, total phenolics can be calculated through a gallic
acid standard curve, as it is not always feasible to find every single phenolic compound
separately. The results are expressed as mg gallic acid/g of food weight (Skerget et al.,
2004, Sokmen et al., 2005).
1.2.2.1.2 Standard curve of catechin
This method is based on the creation of standard solutions of catechin of different
concentrations. Then, a standard curve of catechin is prepared, which shows the
concentration of catechin in correlation with absorbance. After that, the absorbance of
the sample is determined and from the standard curve of catechin, the concentration of
total phenolics in mg catechin/g is finally calculated. For the determination of total
phenolics in different plant extracts, vegetables and fruits, the standard solutions of
catechin are prepared in distilled water or in the same extract of plant (Kivits et al.,
1997).
1.2.2.1.3 Terbium sensitized fluorescence Method
This method is based on the fluorescence sensitization of terbium (Tb3+) by
complexation with flavonols (quercetin as a reference standard) (at pH 7.0), which
fluoresces intensely with an emission maximum at 545 nm. Quercetin and terbium
cations (at pH 7.0) form a stable complex and the resulted emission at 545 nm can be
used for the determination of the total phenols concentration expressed in terms of
“quercetin equivalent”. This fluorimetric method is very simple, precise, rapid and more
sensitive than the other methods that have been used for the determination of total
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phenols (Benzie and Strain, 1996). The developed method was easily applied to the
determination of total phenols in tea infusions, tomato and apple juice with excellent
reproducibility (Shaghaghi et al., 2008).
1.2.3 Ascorbic Acid
Vitamin C belongs to the group of water-soluble vitamins and is one of the most
important micronutrients, 90% of which derive from the intake of fruit and vegetables.
Structurally, vitamin C is composed of chenodiol conjugated with the carbonyl group in
the lactone ring. In the presence of oxygen, ascorbic acid is oxidized to dehydroascorbic
acid. The latter has the same vitamin activity but is more unstable, so that the activity
can readily be lost through lactone hydrolysis and formation of 2,3-diketogulonic acid
(figure 1.2.3.1). Numerous epidemiological studies have shown a strong correlation
between the health effects of consuming fruit and vegetables and their content in vitamin
C. Vitamin C (ascorbic acid, ascorbate) is an effective antioxidant against free radicals.
Vitamin C (ascorbic acid-AA + dehydroascorbic acid-DAA) content of fruit and
vegetables depends on the species, cultivar, climatic conditions, agricultural practices,
ripeness, and of course postharvest handling (Lee and Kader, 2000).
The vitamin C concentration increases in vegetable tissues through the action of light
during growth of the plant. On the other hand, the principal cause of vitamin C
degradation in vegetables is storage at high or inappropriate postharvest temperatures.
Vitamin C content starts to decline as soon as the product is harvested. Levels depend
significantly on the type of vegetable and the processing and storage conditions. The
amount of vitamin C that is degraded increases with storage temperature and time
(Davey et al., 2000).
Figure 1.2.3.1: Oxidation of L-ascorbic to dehydro-L-ascorbic acid followed by evolution into
products lacking biological activity (Martin-Belloso and Soliva-Fortuny, 2011).
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1.2.3.1 Determination of ascorbic acid
Various methods have been employed for the analysis of ascorbic acid in food, including
electrochemical (Calokerinos and Hadjiioannou, 1983), spectrophotometric (Liu et al.,
1982), spectrofluorimetric (Sánchez-Mata et al., 2000) and chromatographic methods.
1.2.3.1.1 HPLC method
High-performance liquid chromatography (HPLC) is a very efficient method in ascorbic
acid analysis of fruits, vegetables or beverages (Quirós et al., 2009) and it also has some
advantages, such as the increase of selectivity, sensitivity and the elimination of
interferences, specificity, easy operation, high accuracy, good repeatability and
reproducibility, a relative short analysis time, unambiguous identification of AA and
isoAA (Valente et al., 2014). Reversed-phase, bonded-phase NH 2 , ion-exchange or ionpair reversed columns have been the most commonly employed columns for ascorbic
acid analysis. Regarding the way of detection, AA can be easily detected by UV at
wavelengths between 245 nm and 254 nm. Although, UV detectors are usually included
in HPLC systems and are simpler and faster than others, UV-HPLC methods have been
validated to be used for vitamin C determination in foods. Most of these methods have
been validated in beer, wine and fruit beverages. Reliability has been satisfactory for all
the evaluated UV-HPLC methods, reducing agents and fruits. In every case, suitable
linearity, sensitivity, precision and accuracy through recovery for AA and vitamin C
analysis in strawberries, tomatoes and apples were obtained (Odriozola-Serrano et al.,
2007).
1.2.3.1.2 2,6 Dichlorophenol-indophenol titration method
Of the many methods that have been proposed for the direct chemical determination of
ascorbic acid in plants and animal tissues, those based upon the reduction of 2,6
dichlorophenol indophenol have been adopted. The indophenol method depends upon
the fact, that ascorbic acid is the major or only natural tissue component which reduces
the dye rapidly in an acid solution (e.g. pH 2 to 4). It is essential that four major
precautions should be observed if accurate results are to be obtained in the titration
against 2,6-Dichlorophenolindophenol method. These are: (1) representative sampling,
(2) complete extraction, (3) prevention of oxidation, (4) rapid performance of the
titration itself (Kallner, 1986).
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1.2.3.1.3 Analytical Voltammetry
Analytical voltammetry is an electrochemical method in which the changes of
electrolysis current are measured when a gradually increasing voltage is applied to the
cell. Conditions are adjusted so that the analyte is oxidized or reduced selectively at one
of the electrodes in the cell. Voltammetric analysis using different electrodes
(conventional electrodes, microdisc electrodes, microband and multiple microband
electrodes and carbon paste electrodes). A wax-impregnated graphite electrode (WIGE)
is used as the indicator electrode. During the electrolysis, ascorbic acid donates electrons
to the indicator electrode and the voltammetric experiment exhibits an oxidation
(anodic) current step. The quantity of electricity involved in the oxidation peak is
directly proportional to the concentration of ascorbic acid. Therefore, an unknown
concentration of ascorbic acid can be determined (Arya et al., 2000).
1.2.3.1.4 Bromatometric method
The determination of ascorbic acid is also performed by a direct bromatometric method.
During titration, KBrO 3 standard solution is added drop wise to the acidic solution of
vitamin C and KBr and the liberated bromine reacts quantitatively. Methyl orange is
used to indicate the end point. Because it has a higher standard redox potential than that
of L-ascorbic acid the liberated bromine first oxidizes the L-ascorbic acid and when the
end point of the titration is reached, the first drop of excess amount of bromate forms
elementary bromine which oxidizes the indicator (decolorization), too (Arya et al.,
2000).
1.2.3.1.5 Oxidation-reduction reaction
Furthermore, ascorbic acid can be determined in food by using an oxidation-reduction
reaction. The redox reaction is preferable to an acid-base titration because a number of
other species in juice can act as acids, but relatively few interfere with the oxidation of
ascorbic acid by iodine. The solubility of iodine is increased by complexation with
iodide to form triiodide. Triiodide then oxidizes ascorbic acid to dehydroascorbic acid.
The endpoint is indicated by the reaction of iodine with starch suspension, which
produces a blue-black product. As long as ascorbic acid is present, the triiodide is
quickly converted to iodide ion, and no blue-black iodine-starch product is observed.
However, when all the ascorbic acid has been oxidized, the excess triiodide (in
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equilibrium with iodine) reacts with starch to form the expected blue-black color (Arya
et al., 2000).
1.2.4 Quality Aspects of Foods
1.2.4.1 External Quality-Color
External quality is the most important and direct sensory quality attribute of food
products. In general terms, the external quality of fruits and vegetables is evaluated by
considering their color, texture, size, shape, and the visual defects (Costa et al., 2011).
The outer appearance of fruits and vegetables affects their point-of-sale value and
consumers’ buying behavior, and sometimes the defective, infected and contaminated
fruits and vegetables can spread the infection or contamination to the sound products
even the whole batch, thus causing great economic losses, and safety problems (GomezSanchis et al., 2008). Visual observation can give information about the color of
different foodstuffs. The limitations of visual observation may be overcome by the
instrumental evaluation of color and color differences according to the system of color
measurement established by the Commission International d' Eclairage (CIE)
(Clydesdale, 1978). This system is based on the reflectance spectrophotometric
instrumentation (Clydesdale, 1978, Hunter and Harold, 1987). In this CIE system
surface color of fruits and vegetables can be easily measured and may be regarded in a
three-dimensional space in which each color has a unique location. As a consequence,
colors are measured in terms of their fundamental tristimulus values (X, Y and Z). These
values, can be further used to calculate L*, a* and b* (CIE-Lab) color space values
(Hunter and Harold, 1987). L* indicates lightness, a* indicates hue on a green (-) to red
(+) axis, and b* indicates hue on a blue (-) to yellow (+) axis (Clydesdale, 1978).
Moreover, CIE-Lab space measures two important functions:
(a) the hue angle, H°= (tan b/a)-l, which is recommended by the CIE as the
psychometric correlation of the visually perceived attribute of hue.
(b) the total color difference, which can be calculated as ΔE =(ΔL2+ Δa2+ Δb2)
1/2
,
which expresses a color difference between two colors by a single function number
(Hunter and Harold, 1987).
The above measurements of total color difference have been found to be quantitative,
repeatable and reproducible. The above instrumental system has been employed to
assess colors in fruits and in the food industry (Yang et al., 1990). A hand-held
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tristimulus reflectance colorimeter (Minolta CR-200) can be used, in order to measure
the color on different foodstuffs, after its calibration with a white standard tile (L
=97.92, a =-0.45, b=2.12).
1.3 Foodborne Pathogens and Foodborne diseases
1.3.1 Global challenge and increased frequency of foodborne
diseases.
The occurrence and epidemiology of foodborne diseases in a population is the result of
complex interactions among environmental, cultural and socioeconomic factors
(Johnston et al., 2006).
Growing international trade in food industries, migration and travel are factors that
accelerate the spread of dangerous pathogens and contaminants in food products thus
increasing our universal vulnerability (Tauxe et al., 2010).
It must be mentioned that through globalization of food distribution, the health of people
in different countries can be affected, by consuming contaminated food. Once the food is
contaminated, a recall of literally tons of food products is inevitable. This can lead to
considerable economic losses in production, as well as damage to tourist industry
development at a country level. It must be emphasized that many global foodborne
diseases have resulted from foods that are produced in developed countries. For
example, the global spread of Salmonella serotype Enteritidis (now the most important
Salmonella serotype in many countries) and potentially also the spread of multiply
antibiotic-resistant strains of Salmonella serotype Typhimurium, has initially started
from developed countries (Hendriksen et al., 2011).
Furthermore, changing food consumption trends (e.g. eating more meals away from
home, including greater use of salad bars), increased global trade in raw fruits and
vegetables, as well as increased international travel in general, could also increase the
risk of produce-associated foodborne diseases (Stine et al., 2005, Beuchat, 1998,
Beuchat et al., 2001). Finally, the susceptibility of the public to foodborne diseases, is
changing due to increased numbers of people who are elderly, immunocompromised or
suffer from chronic diseases. This change in social demographics is likely to lead to
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increased risk of illness associated with the consumption of raw produce that otherwise
may contain levels of pathogens innocuous to healthy individuals (Beuchat, 1998).
The current need for a healthier way of life has led to an increasing demand for fresh
RTE foods, without additives but with high nutritional value, included antioxidants and
free radicals. The last decades an increasing number of industries produce packaged
foods, which can be consumed easily not only at house but also at work. Taking also into
account their accessible-low price, minimally processed fresh RTE fruits and vegetables
offer big advantages to consumers (Artés et al., 2009).
While the prevention of contamination of fruits and vegetables during production,
transport, processing and handling, still remains an important issue, much improvement
is needed in some parts of the world, in order to ensure hygienic production of fruits and
vegetables. Furthermore, many microbial contaminants are present in the nature and are
part of the environment, thus fruits and vegetables may be inadvertently contaminated
(Beuchat, 2001).
The high quality of minimally processed foods is mainly related to 5 main
characteristics: color, texture, flavour, nutritional value and finally food safety.
However, it must be emphasized that food safety constitutes the most critical point
(Artés et al., 2009).
The steps required to prepare fresh cut produce can lead to a rapid increase of
microorganisms, some of which may be potentially harmful to human health. Although
RTE foods are processed minimally, the destroyed plant structure and thus the increase
of the aging rate of plant tissues can lead to an increase of plant resistance and to
microbial spoilage (Artés-Hernández et al., 2007).
Moreover, the shelf-life of freshly cut products is influenced by several factors. These
are: pre-processing factors (crop varieties, cultivation, harvesting and ripening stage),
processing factors (chilling, cutting, cleaning, conditioning, cutting, peeling, decoring,
washing, disinfection, drainage, irrigation, drying, packaging) and distribution
conditions (temperature, relative humidity) (Artes, 2004).
In addition, during the production of safe fresh produces, the materials that enter the
processing chain (processing and packaging treatments) result in higher microbial load.
The suppression of microbial growth during processing, as well as the prevention of
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infection after processing, must be taken seriously into account (Artés and Allende,
2005).
In developing countries, the use of untreated wastewater and manure as fertilizers for the
production of fruits and vegetables are important factors, which contribute to food
contamination that causes numerous foodborne disease outbreaks (Hedberg et al., 1994).
The increased application of improperly composted manures to soils in which fruits and
vegetables are grown, changes in packaging technology such as the use of modified or
controlled atmosphere and vacuum packaging, extended time between harvesting and
consumption, also play an important role (Beuchat et al., 2002). The use of properly
composted manure and properly treated irrigation and spray waters, as well as safe and
pathogen-free water for washing, can minimize the risk of contamination of fruits and
vegetables with microbial pathogens. Moreover, good hygienic practice during
production and transport, everyday sanitizing of harvesting equipment and transport
vehicles, as well as the application of good hygienic practice during processing and
preparation are important factors that must be taken into account (Beuchat, 2001).
1.3.2 Hazards during the food production chain
Prevention of contamination of fruits and vegetables with pathogenic microorganisms
should be the goal of everyone involved in both the pre-harvest and post-harvest phases
of delivering produce to the consumer. However, this seems a very difficult task, since
some pathogens normally exist in the soil and may therefore be present on the surface of
fruits and vegetables when they are harvested (figure 1.3.2.1) (Beuchat, 1996).
Extremely important is the elimination of animals and insects from processing, storage,
marketing and food-service facilities. The highest level of hygiene must be maintained
and practiced by all handlers (including consumers) of fresh RTE foods, from the field
to the table.
Figure 1.3.2.1: Ways of contamination of raw fruits and vegetables with pathogenic
microorganisms (Beuchat, 1996).
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Agricultural systems are rich in microorganisms (soil inhabitants) that are observed on
whole fruit or vegetable surfaces and are responsible for maintaining a dynamic
ecological balance. The dissemination of these microbes is facilitated by vectors, like
soil particles, airborne spores, and irrigation water (figure 1.3.2.1). In most cases,
bacteria and fungi that arrive on the developing crop plant are responsible for crop
damage (Andrews and Harris, 2000). The microorganisms normally present on the
surface of raw fruits and vegetables may consist of chance contaminants from the soil or
dust of the environment, or bacteria or fungi that have grown and colonized by utilizing
nutrients that exist in the surface of plant tissues. Among the groups of bacteria that are
commonly found on fruits and vegetables are coliforms or faecal coliforms e.g.
Klebsiella and Enterobacter, Salmonella, Listeria monocytogenes, S. aureus, C.
botulinum, Yersinia enterocolitica, Campylobacter, Bacillus cereus (Beuchat, 1996).
1.3.3 Microbial colonization on fresh produce surfaces
Generally, microorganisms contaminate the surfaces of fresh produce mostly via water
or soil. Each type of microbe has its own method of attachment and can vary even within
different strains of the same bacteria (Katsikogianni and Missirlis, 2004, Rivas et al.,
2005, Takeuchi and Frank, 2000). The factors that are responsible for an effective
attachment of bacterial cells to a surface are: bacterial hydrophobicity, surface charge,
cell surface structures (flagella, pili, curli), outer membrane proteins, and bacterial
growth conditions (Van Houdt and Michiels, 2005). The produce surface exhibits
hydrophobicity, due to epicuticular wax of produces (Koch et al., 2008). Thus the water
has a reduced access to the produce, due to the hydrophobicity (Wang et al., 2009).
Surface composition, surface charge, and surface free energy of plant also play a role in
bacteria adhesion. Enteric pathogens prefer to grow or survive, in special distinct and
localized spots on plant surfaces which are rich in nutrients like sucrose, amino acids
and nitrates (Jaeger et al., 1999). The viability of microorganisms also is dependent on
changes in temperature and osmotic conditions within the same day, and on poor
nutrition (Heaton and Jones, 2008). There seems to be an optimal adhesion temperature
for each microorganism (Gorski et al., 2003). Finally, different bacteria have exhibited
different attachment to different fresh produce surfaces (cut edge and stomata versus
intact tissue) or plants (Barak et al., 2008, Seo and Frank, 1999).
A large number of fruits and vegetables present nearly ideal conditions for the survival
and growth of many types of microorganisms. Their internal tissues are rich in nutrients
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and vegetables and have a neutral pH. Their structure is comprised mainly of
polysaccharides, such as cellulose, hemicellulose, and pectin. Microorganisms have the
ability to exploit the host using extracellular lytic enzymes that degrade these polymers
to release water and the plant’s other intracellular constituents for use as nutrients for
their growth. Moreover, microbes are capable of colonizing and creating lesions on
healthy, undamaged plant tissues, they also can enter plant tissues during fruit
development, either through the calyx (flower end) or along the stem, or through various
specialized water and gas exchange structures of leafy matter (Tournas, 2005).
Moreover, in order to achieve a successful establishment to fruits and vegetables,
microbes need to overcome multiple natural protective barriers. Fruits and vegetables
possess an outer protective epidermis, which is covered by a natural waxy cuticle layer
containing the polymercutin. The microorganisms firstly identify and recognize the plant
surface, secondly employ strategies to achieve irreversible attachment to the plant
surface, and finally internalize and colonize to the tissue. It must be mentioned that
external damage such as bruising, cracks, and punctures facilitates the establishment and
growth conditions of the microbes to fresh produce. Thus, microorganisms will arrive
within open wound sites at the packing facility, and through shedding from the
asymptomatic wound, cross-contamination during different treatments (handling,
culling, washing, sorting, and packing before storage) occurs (Barth et al., 2009).
Food surface topology plays a major role in how and where bacteria attach to fresh
produce. Studies show bacteria to prefer to attach to cuts in the leaves, lenticels,
trichomes, locations around veins on leaves, and stomata in plants (Burnett et al., 2000,
Kroupitski et al., 2009, Seo and Frank, 1999).
In general terms, rough, highly-textured surfaces with deep crevices can easier harbour
soil, thus an increased number of microorganisms can be hosted. On the other hand,
smooth surfaces of fruits and vegetables can host a smaller amount of microorganisms.
The presence of a pathogen is minimized when the rind, skin or peel is removed before
consumption. Furthermore, microorganisms may have become trapped on the inner
complicated surfaces of leaves of certain vegetables, thus they can not be removed by
routine cleaning practice (Barth et al., 2009).
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1.3.4 Foodborne illness and foodborne disease outbreaks
Foodborne disease can be defined as any infectious disease or toxic nature disease
caused by consumption of food. The number of foodborne disease outbreaks, by year
and pathogen is recorded by Foodborne Disease Outbreak Surveillance System (table
1.3.4.1, table 1.3.4.2, figure 1.3.4.1) Foodborne disease outbreak can be defined as: a)
the disease that is observed, when the number of cases of a particular disease exceeds the
expected number and as b) the occurrence of two or more cases of a similar foodborne
disease, which results from the ingestion of a common food. Sporadic case can be
defined as the case that cannot be linked epidemiologically to other cases of the same
illness (WHO, 2008). Foodborne diseases outbreaks can be further divided to commonsource outbreaks, propagated outbreaks or mixed outbreaks (HCDCP, 2012).
Foods are implicated for transmitting infections through foodborne infections and
foodborne poisoning. Foodborne infections such as cholera, typhoid fever, dysentery are
transferred through food. Food plays the key role of the first step in the chain of
transmission of infection. In foodborne poisoning, the pathogen is usually multiplied
inside the food, and this step is necessary in order to reach the adequate infective dose
and/or to produce toxin. Surveys of minimally-processed fruit and vegetables have
demonstrated that fresh-cut produce could harbor high counts of bacteria and also
foodborne pathogens such as Salmonella, L. monocytogenes, Aeromonas hydrophila and
E. coli O157:H7 (table 1.3.4.1) (Abadias et al., 2008, Beuchat, 1996).
S. aureus is related to staphylococcal food poisoning by ingestion of preformed toxins
generated in food (Fueyo et al., 2001). The incubation time of staphylococcal infection
may be only 30-min while salmonellosis needs an incubation time of 15-48 hours
depending on the infecting dose. Microorganisms that cause food poisoning are different
from those that cause food spoilage. Foods that contain pathogens have pleasant
organoleptic properties, compared to foods that contain spoilage microorganisms which
have unpleasant taste, smell and appearance without necessarily being hazardous to
health.
To minimize the risk of infection or intoxication associated with the consumption of raw
fruits and vegetables, potential sources of contamination from the environment to the
table should be identified and specific measures and interventions to prevent and/or
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minimize the risk of contamination should be considered and correctly implemented
(Beuchat, 1998).
Table 1.3.4.1: Number of reported foodborne diseases-outbreaks, cases and deaths in United
States, 1998-2002 (CDC, 2013).
Figure 1.3.4.1: Number of multistate foodborne disease outbreaks, by year and pathogen Foodborne Disease Outbreak Surveillance System, United States, 1998–2008 (CDC, 2013).
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DISEASE
2004
2005
2006
2007
2008
2009
2010
2011
2012
1327
1062
886
709
810
406
299
471
405
Hepatitis A
52
160
120
282
119
89
58
41
74
Sighellosis
62
26
28
48
19
38
33
47
91
Typhic-Paratyphic
20
20
16
18
11
4
10
7
6
Listeriosis
3
8
7
10
1
4
10
9
11
Infection by EHEC
2
0
1
1
0
0
1
1
0
0
0
0
0
0
1
0
0
0
Salmonellosis
(non-
typhic-paratyphic)
Fever
(STEC/VTEC)*
Allantiasis
Table 1.3.4.2: Number of multistate foodborne disease outbreaks, by year and pathogen Foodborne Disease Outbreak Surveillance System, Greece, 2004–2012 (HDCP, 2012).
1.3.5 Foodborne Bacteria
1.3.5.1 Escherichia coli
Escherichia coli are Gram-negative, non-spore-forming, facultatively anaerobic rods
bacteria of family Enterobacteriaceae. They are typically mesophilic and grow from 710°C up to 50°C (optimum 37°C). The minimum aw for growth is 0.95 and pH 4.4-8.5
(WHO, 2008). Several different pathotypes of pathogenic E. coli have been categorized
based on their virulence properties and disease-causing mechanisms (table 1.3.5.1)
(Kaper et al., 2004).
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Table 1.3.5.1: Five categories of the diarrheagenic E. coli. (Zhou, 2010).
Escherichia coli are classified as a genetically diverse species with the majority of its
members being nonpathogenic and part of the natural gut microflora of humans and
animals. Fruits and vegetables can be contaminated with E. coli in the field or during
post-harvest handling. E. coli O157:H7 is a strain of enterohemorrhagic E. coli. The
growth of this strain in the human intestine produces a large quantity of toxins that can
cause severe damage to the lining of the intestine and other organs of the body. Many
outbreaks have been linked to lettuce (Ackers et al., 1996), apple cider (CDC 1996a),
radish sprouts, (Nathan, 1997) and alfalfa sprouts (CDC, 1997). Enterohemorrhagic E.
coli can grow on fruits and vegetables like cantaloupe and watermelon cubes, shredded
lettuce, sliced cucumbers and apple cider (Abdul-Raouf et al., 1993), causing health
problems. An outbreak was linked to contaminated spinach in which 200 people were
affected, more than half of which were hospitalized, and three died (Anonymous, 2006).
The world’s largest reported E. coli O157:H7 outbreak occurred in Japan in 1996 and
was linked to the consumption of white radish sprouts, where approximately 6,000
school children were infected and 17 died (Michino et al., 1999). In 1997, white radish
sprouts were once again implicated in another E. coli O157:H7 outbreak in Japan,
affecting 126 people and resulting in one death (Taormina et al., 1999).
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Figure 1.3.5.1: E. coli on fresh ready to eat lettuces (Bermúdez-Aguirre and Barbosa-Cánovas,
2013).
Enterohemorrhagic E. coli (EHEC) was first recognized as a human pathogen in 1982
when it was identified as the cause of two outbreaks of hemorrhagic colitis. Those
outbreaks were associated with undercooked hamburgers served at fast food restaurants
(Smith, 2005). E. coli O157:H7 produces toxins that cause a mild non-bloody diarrhea or
an acute grossly bloody diarrhea which is known as hemorrhagic colitis (Doyle, 1991).
In patients such as children and elderly people, E. coli O157:H7 infection can progress
to hemolytic uremic syndrome (HUS), which is a severe postdiarrheal systemic
complication. The toxins of E. coli have been referred to as shiga-like toxins (verotoxin,
verocytotoxin). E. coli O157:H7 has been isolated from undercooked ground beef and
from other products including fresh RTE fruits and vegetables (Griffin and Tauxe, 1991,
Smith et al., 2003). However, it must be stated that outbreaks related with fresh produce
are obvious in restaurants which are accounted for 40% of the E. coli O157:H7
outbreaks associated with fresh produce, and approximately 47% was responsible for
cross-contamination during food preparation (Rangel et al., 2005). For instance, Stine et
al., (2005) found that the survival of E. coli O157:H7 was enhanced under high humidity
conditions. E. coli O157:H7 was found to survive for 12 days at 4°C on lettuce (figure
1.3.5.1), bean sprouts and dry coleslaw (Francis and O'Beirne, 2002). E. coli O157:H7
inoculated on lettuce increased significantly when it was kept at 12°C for 14 days.
Whereas, modified atmosphere packaging had little or no effect on the survival or
growth of E. coli O157:H7 (Abdul-Raouf et al., 1993).
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Enterohaemorrhagic E. coli infection is the rarest stated foodborne illness notifiable in
Greece. The declared average annual incidence of infection with EHEC for the period
2004-2011 was 0.06 / 1,000,000 people per year (total declared 6 cases), whereas the
average declared suspicious cases in countries of both the European Union and EEA /
EFTA (European Economic Area / European Free Trade Association), was consistent
with the latest published data, 6.60 cases per 1,000,000 inhabitants. Serotype O157: H7
has been implicated in the largest percentage epidemics worldwide, however different
serotypes have emerged, such as the recent large outbreak in Germany in May 2011 with
the responsible serotype O104:H4 (HCDCP, 2012).
1.3.5.2 Staphylococcus aureus
S. aureus is a principal cause of gastroenteritis resulting from the consumption of
contaminated food. Staphylococcal food poisoning is due to the absorption of
staphylococcal enterotoxins prior existed in the food (Trudeau, 2012). Lettuce, parsley,
radish, salad vegetables and seed sprouts are among the RTE foods that have been
reported for Staphylococcus outbreaks (Abadias et al., 2012, FAO/WHO, 2008, Olaimat
and Holley, 2012, Ramos et al., 2013).
S. aureus is a facultative anaerobic Gram-positive coccal bacterium, also known as
"golden staph" and Oro staphira. Optimal temperature for Staphylococcus growth is
within the range of 30 - 37°C. Moreover, growth can be observed at 7 - 48°C and at pH
values between 4.0 and 9.8 (Vilhelmsson, 2000). S. aureus produces a wide variety of
extracellular proteins that may play an important role in its virulence as a human
pathogen. These include hemolysins, nucleases, proteases, lipases, hyaluronidase,
collagenase, leukocidins, exfoliative toxins, and pyrogenic toxin superantigens which
include toxic shock syndrome toxin-1 (TSST-1), epidermolysins and the staphylococcal
enterotoxins (Vilhelmsson, 2000).
S. aureus is a major human pathogen and is potentially able to infect any tissue of the
human body, causing from skin infections to life-threatening diseases. Methicillin
resistant S. aureus (MRSA) has been a topic of concern for several years, being a large
burden for most healthcare institutions around the world, with higher mortality,
morbidity and financial costs compared to methicillin-susceptible S. aureus (MSSA)
(Gould, 2006). The MRSA rates have been increased rapidly worldwide during the last
decades (Stefani et al., 2012).
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Staphylococcal food poisoning is reported as the third most prevalent cause of foodborne
illness worldwide (Normanno et al., 2005, Zhang et al., 1998). There are two major
aggravations to its presence: the toxins production and the antimicrobial resistance. S.
aureus produces more than 30 different extracellular byproducts and staphylococcal
toxins like: pyrogenic toxin superantigens (PTSAgs), exfoliative toxins, leukocidins and
other toxins. The family of PTSAgs includes staphylococcal enterotoxins (SEs), SE-like
(SEl) toxins, exfoliative toxins and toxic shock syndrome toxin-1 (TSST-1) (Lina et al.,
2004). Generally, five classical antigenic SE types (SEA, SEB, SEC, SED, and SEE) are
known. Recently, new types of SEs toxins (SEG, SEH, SEI, SElJ, SElK, SElL, SElM,
SElN, SElO, SElP, SElQ, SElR, SElU, SElU2, and SElV) have been also reported
(Thomas et al., 2006, Peles, 2007). SEs are the main cause of food poisoning that occur
after ingestion of foods contaminated with S. aureus by improper handling and
subsequent storage at elevated temperatures. Human handling of food products as well
as infection/colonization of livestock or farm workers have been described as
mechanisms for the contamination of food with S. aureus (Greig et al., 2007). S. aureus
can contaminate food by direct contact through body, through skin fragments, or through
respiratory droplets produced when people cough and sneeze. Most S. aureus foodborne
illness results from food contamination by food handlers, meat grinders, knives, storage
containers, and cutting blocks. While low levels of the S. aureus bacterium exist in many
foods, proper food handling techniques can prevent further contamination or growth in
the food, thus preventing toxin production. Illness is caused by enterotoxin-producing S.
aureus. About 100 - 200 ng of the enterotoxin are adequate to produce illness, and
because the toxins are heat-resistant, cooking is not capable of maintaining the food safe
(Anderson et al., 1996).
Symptoms are of rapid onset and include nausea and violent vomiting, with or without
diarrhea, and abdominal pain within 1-6 h post-consumption of contaminated foods. The
illness is usually self-limiting and only occasionally is severe enough to lead to
hospitalization (Argudín et al., 2010).
Foodborne outbreaks attributed to S. aureus include a variety of foods such as meat,
milk, and cheese (Asao et al., 2003, Guven et al., 2010, Rall et al., 2008), dairy products
(Normanno et al., 2007) and RTE foods (Oh et al., 2007) .
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1.3.5.3 Salmonella spp.
The genus Salmonella belongs to the family of Enterobacteriaceae. Salmonella are Gram
negative, facultative anaerobe bacteria that diverged from E. coli approx. 100 – 160
million years ago and acquired the ability to invade host cells (Müller, 2012). Based on
DNA relatedness, Salmonella is today divided into the two species S. enterica and S.
bongori (figure 1.3.5.3.1). Major clinical symptoms associated with humans are enteric
(typhoid) fever caused by S. enterica serovar Typhi and Paratyphi, and gastroenteritis
caused by non typhoidal Salmonellae (NTS). Salmonella may be divided in three groups
based on their association with human and animal hosts (Armon et al., 1997, Chavant et
al., 2007). The first group is characterized by specificity for the human host. The second
group consists of organisms that are usually adapted to specific animal hosts. The third
group consists of un-adapted Salmonellae that cause disease in humans and a variety of
animals. Most Salmonellae are included into the third group with Salmonella enterica
serovar Typhimurium (S. Typhimurium) being the most common. There are over 2,463
recognized serovars of Salmonella (Brenner et al., 2000) with Typhimurium and
Enteritidis being the most prevalent nontyphoidal Salmonella serovars isolated from
human salmonellosis cases in the U.S.A (Andrews-Polymenis et al., 2009).
Figure 1.3.5.3.1: Taxonomic scheme of Salmonella serovars (Müller, 2012).
Outbreaks of salmonellosis have been linked to a wide variety of fresh fruits and
vegetables including apple, cantaloupe, alfalfa sprout, mango, lettuce, unpasteurized
orange juice, tomato, melons, celery and parsley, bean sprouts (Krause et al., 2001,
Mahon et al., 1997). Two outbreaks caused by S. Typhimurium in 2006 and 2008 in
USA were linked to contaminated tomatoes and peanut butter, respectively, led to
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enormous economic loss and 714 confirmed cases of salmonellosis according to CDC
(CDC, 2013).
The average annual incidence of disease of Salmonelosis declared in Greece, based on
the mandatory declaration system diseases (HCDCP), for the years 2004-2012, was 6.6
cases per 100,000 population (table 1.3.5.3.1, graph 1.3.5.3.1). A higher incidence is
observed in children, especially in the age group 0-4 years (graph 1.3.5.3.2) (HCDCP,
2012).
Table 1.3.5.3.1: Number of notified cases of
salmonellosis
per
year,
Mandatory
Notification System, Greece, 2004-2012.
(http://www.keelpno.gr - 7/5/2013 )
Graph 1.3.5.3.1: Time trend of salmonellosis
notification rate, Mandatory Notification
System, Greece, 2004-2012.Hellenic Center
for Disease Control and Prevention
(HCDCP, 2012)
Graph 1.3.5.3.2: Annual notification rate (cases/100,000 population) of salmonellosis by age
group, Mandatory Notification System, Greece, 2004-2012 (HCDCP, 2012).
The routes of transmission of Salmonella to humans are the environment and the contact
with animals or the person-to-person contact but in industrialized countries, Salmonella
are mostly transmitted through contaminated food (Müller, 2012). Common food
products that enhance the Salmonella transmission are fresh meat and eggs (Wegener et
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al., 2003). Salmonella in the food can directly originate from the farm reservoirs.
However, Salmonella can enter at any stage of the food chain through contamination
from the environment, other foods, animals or humans. The endpoint of the food chain is
the kitchen of the consumers, restaurants or canteens. The main risk factors for
Salmonella outbreaks have been associated to undercooking, improper storage and
cross-contamination. Cross-contamination is one of the most important factors and needs
special attention, in order to eliminate the spread of the bacteria thus protecting the
contamination of RTE foods (Müller, 2012).
1.3.5.4 Listeria spp.
Listeria monocytogenes is a short, Gram-positive, non-sporeforming rod, with tumbling
end-over-end motility at room temperature. It is a catalase positive, oxidase negative,
facultative anaerobe with slight β–hemolysis on blood agar. L. monocytogenes has been
known to survive refrigeration, freezing, heating, and drying, which creates obstacles for
the food industry (CFSAN, 2001). It has optimum growth at 32-35°C, but can survive
and multiply at refrigeration temperatures. It has been found in raw foods, such as fruits,
vegetables, and uncooked meats, and has been associated with outbreaks in raw milk, ice
cream, raw meats, and RTE meat and cheese products.
The genus Listeria, together with the genus Brochotrix, belongs to the Listeriaceae
family, the order Bacillales, the class Bacilli and the phylum Firmicute (Wheeler et al.,
2000). Today the genus comprises the following seven species: L. monocytogenes, L.
innocua, L. ivanovii subsp. ivanovii and, L. ivanovii subsp. londoniensis, L. seeligeri, L.
welshimeri,and L. grayi. Recently described specie is L. marthii (Graves et al., 2009).
L. monocytogenes has been linked to infection due to consumption of contaminated
prepackaged salads, cabbage, lettuce, celery, and tomatoes (Berrang et al., 1989). L.
monocytogenes is a major public health concern due to its high mortality rate (∼20%)
and its ability to grow at refrigeration temperatures (Gandhi and Chikindas, 2007, Mead
et al., 1999). Food safety is one of the top eleven priorities of the World Health
Organization, which has called for systematic and more aggressive steps to reduce the
risk of foodborne diseases due to microbial contamination (WHO, 2000).
L. monocytogenes is responsible for approximately 2,500 illnesses and 500 deaths in the
United States each year (CDC, 2000). Most healthy adults have few or no symptoms, as
the disease generally affects those with compromised immune systems. In at-risk
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populations, flu-like symptoms including fever, headache, nausea, vomiting, and
diarrhea appear about 12 hours or more after ingestion. After several days, the more
serious symptoms appear, including meningitis, encephalitis, septicemia, and
intrauterine/cervical infections that may result in spontaneous abortion in pregnant
women (CFSAN, 2001). Generally, mortality rates for listeriosis may be as high as 80%
for neonatal infections, and 50-70% for meningitis and septicemia patients (CFSAN,
2001). The infective dose of L. monocytogenes is currently unknown, although it appears
to be above 100 viable cells, depending on pathogen strain and susceptibility of the host
(Roberts, 1994). While pasteurization and cooking methods used by processors can kill
L. monocytogenes, post-processing contamination may occur because the organism is so
resilient in the environment.
Listeria innocua is a Gram-positive bacterial strain closely related to L. monocytogenes.
It is non-sporeforming short rod, catalase positive, oxidase negative, and facultatively
anaerobic. However, L. innocua does not produce β-hemolysis on blood agar (Poysky et
al., 1993). This characteristic has been one of the few tests available for the
differentiation of Listeria species. The apparent difference between L.innocua and L.
monocytogenes is the lack of pathogenicity of L. innocua. There have been over 1000
worldwide cases of human foodborne illness associated with L. monocytogenes in the
last 35 years (CFSAN, 2001). On the other hand, no cases of human illness associated
with L. innocua have been reported until recently. However, the presence of L. innocua
indicates the potential for L. monocytogenes contamination. Thus, L. innocua strain is
used to model the behavior of L. monocytogenes. The ubiquitous nature of L.
monocytogenes makes it nearly impossible to completely eradicate from an environment,
thus many researchers and processors are understandably hesitant to introduce a
pathogen into their working environment for fear of future contamination problems.
(Benech et al., 2002, Dykes et al., 2003, Olasupo et al., 2004, Scannel et al., 2001,
Sommers et al., 2002).
Listeriosis is the foodborne disease caused by L. monocytogenes. The case fatality rate
of listeriosis is high compared to other foodborne diseases. It mainly affects pregnant
women, newborns, the elderly and immunocompromised adults (HCDCP, 2012). In
total, 64 cases of listeriosis were reported in Greece from 2004 to 2012 (table 1.3.5.4.1).
The highest mean annual notification rate of the disease regarded the age group of ≥ 65
years old (2.41/1,000,000 population) and the age group of 0-4 years old (0.62/1,000,000
population) (graph 1.3.5.4.1). Generally, notification rate of listeriosis is low in Greece.
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The mean notification rate in the EU and EEA/EFTA countries was 3.5 cases per
1,000,000 population for the year 2009 (HCDCP, 2012). Outbreaks from L.
monocytogenes are not so common compared with those caused by pathogens like
Salmonella. However, they receive considerable attention when they do occur because
they usually have some seriously affected cases and even deaths (Todd and Notermans,
2011). Moreover, foods related to listeria outbreaks are bean sprouts, cabbage, chicory,
cantaloupe, eggplant, lettuce, potatoes, radish and lettuce (Ramos et al., 2013).
Table 1.3.5.4.1: Annual number of
notified cases and notification rate of
listeriosis in Greece, Mandatory
Notification
System,
2004-2012
(http://www.keelpno.gr - 7/5/2013)
Graph 1.3.5.4.1: Notification rate of
listeriosis by age group and gender in
Greece,
Mandatory
Notification
System, 2004-2012 (HCDCP, 2012).
1.3.6 Foodborne Viruses
Fruits and vegetables are vehicles for viral infection. In Europe, viruses were responsible
for 10.2% of the foodborne outbreaks during 2006 and were pointed out as the second
most common causative agent, after Salmonella (EFSA, 2007). Lettuce (Rosenblum et
al., 1990), diced tomatoes and raspberries (Reid and Robinson, 1987) as well as
strawberries (CDC, 1997) have been contaminated with Hepatitis A. Moreover,
Hernandez et al. (1997) have declared that lettuce contaminated with sewage could be a
vehicle for hepatitis A virus and rotavirus. An outbreak caused by hepatitis A, associated
with green salad onions was reported in the United States in 2003 (Chancellor et al.,
2006). It must be stated that cases of foodborne disease caused by Norwalk-like viruses
(i.e. Small Round Structured Viruses, or SRSV) have been also reported (Bean and
Griffin, 1990). NoV outbreaks have been reported linked to leafy greens (Ethelberg et
al., 2010, Gallimore et al., 2005). Soft red fruits have also been implicated in NoV
outbreaks (Maunula et al., 2009). Rapid alerts related to the detection of NoV in lettuce
and soft red fruits have been noted (RASSF, 2010). Hepatitis A and Norwalk-like
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viruses are the most commonly documented viral food contaminants (FDA, 2001).
Numerous viruses can be found in the human intestinal tract (table 1.3.6.1).
Table 1.3.6.1: Enteric viruses and clinical syndromes (Koopmans et al., 2002)
Studies have shown that viruses may present for weeks or even months on vegetable
crops and in soils that have been irrigated or fertilized with sewage wastes. For instance,
viruses introduced onto green onions remained stable for over 14 days (Kurdziel et al.,
2001). In general, Rotaviruses, astroviruses, enteroviruses (polioviruses, echoviruses and
coxsackie viruses), parvoviruses, adenoviruses and coronaviruses have been reported to
be transmitted by foods on occasion (Cliver, 1994).
The food- and waterborne viruses can be divided into three disease categories:
1. viruses that cause gastroenteritis (e.g. astrovirus, rotavirus, the enteric
adenoviruses, and the two genera of enteric caliciviruses, i.e. the small round
structured viruses or ‘Norwalk-like viruses’ (NLV), and typical caliciviruses or
‘Sapporo-like viruses’ (SLV),
2. fecal orally transmitted hepatitis viruses: hepatitis A virus (HAV), hepatitis E
virus (HEV) .
3. viruses which cause other illnesses, e.g. enteroviruses (Koopmans et al., 2002).
More precisely, human noroviruses and hepatitis A virus (HAV) are the most important
human foodborne viral pathogens with regard to the number of outbreaks and people
affected (Bozkurt et al., 2014). Scallan et al. (2011) reported that an estimated 80-90%
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of all non-bacterial outbreaks of gastroenteritis reported each year are due to human
noroviruses and HAV. These viruses are generally environmentally stable, survive
adverse conditions and are resistant to extreme pH conditions and enzymes of the
gastrointestinal tract (D’Souza et al., 2006). They are known to have low infectious
doses, of as few as 10 infectious particles, which can cause illness (CDC, 2012).
1.3.6.1 Noroviruses
Norovirus is a genus of the Caliciviridae family. The NoV genome is approximately 7.5
kb in length and contains three open reading frames. NoV infection causes acute
vomiting, diarrhea, fever and abdominal cramps (Koopmans, 2008). Cases typically
become symptomatic 24–48 h after infection, and the illness typically resolves after 4872 h (Teunis et al., 2008).
Foods can become contaminated with pathogens at any point during production,
processing, and preparation (Greig et al., 2007). NoV outbreaks, are associated with
food handlers and poor personal hygiene practices (Baert et al., 2009b, Dominguez et al.,
2010).
The stability and persistence of NoV is also a contributing factor to food and waterborne
outbreaks. Food products provide varying degrees of protection or antiviral activity,
depending on their properties. FCV has been shown to survive for 7 days on ham, 3–5
days on lettuce, 1–5 days on cantaloupe, 3–4 days on bell peppers, and 1 day on
strawberries (Mattison et al., 2007, Stine et al., 2005), although it is rapidly inactivated
in the acidic environment of mussels (Hewitt and Greening, 2004). Temperature control
is a key parameter for control of bacterial pathogens in food but not for eliminating NoV
(Baert et al., 2009).
Fresh fruits and vegetables may also be contaminated with NoV during production,
processing or distribution. Contaminated irrigation water or wash water is the vehicle for
transferring NoV to fresh products (Cheong et al., 2009, Mara and Sleigh, 2010), and
surrogate viruses have been shown to attach and persist on fruit and vegetable surfaces
(Mattison et al., 2007, Urbanucci et al., 2009, Wei et al., 2010). NoV has been
implicated as the cause of outbreaks of gastroenteritis from salads, cantaloupe and
frozen raspberries (Allwood et al., 2004, Bowen et al., 2006, Ethelberg et al., 2010,
Gallimore et al., 2005b, Maunula et al., 2009). NoV genomes have also been detected in
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up to 6% of prepackaged salads (Mattison et al., 2010). Cooking could be an effective
control measure for NoV contamination but is not applicable to the fresh RTE fruit and
produce category. Washing in clean water can reduce levels of NoV contamination from
1 to 3 log 10 (Baert et al., 2008b). The most effective intervention is the prevention
measures (Mattison et al., 2010). Moreover, appropriate treatment of irrigation and wash
water can inactivate NoV (Baert et al., 2009). Surveillance networks may detect point
source foodborne outbreaks, and this information can be used to prevent or limit the
further spread of disease (Koopmans et al., 2003).
1.3.6.2 Hepatitis
The viruses which cause hepatitis can be divided into enterically transmitted viruses
(HAV, HEV), and parenterally transmitted hepatitis viruses (hepatitis B, C, D, G)
(Koopmans et al., 2002).
HAV are small, non-enveloped spherical viruses, measuring between 27 and 32 nm in
diameter. They contain a single (positive) stranded RNA genome of approximately 7.5
kb in length (Koopmans et al., 2002). The majority of infections occur in early
childhood and virtually all adults are immune. However, young children generally
remain asymptomatic. In developed countries, however, HAV infections become less
common as a result of increased standards of living. Infection with HAV can produce
asymptomatic or symptomatic infection after an incubation period of 30 days. The
illness caused by HAV infection is characterized by symptoms that can include fever,
headache, fatigue, nausea and abdominal discomfort (Koopmans et al., 2002, Koopmans
et al., 2004).
Outbreaks associated with food, particularly raw produce, contaminated before reaching
the food service establishments have been recognized increasingly in recent years (CDC,
1997). Fresh RTE produce appears to be contaminated during harvest, which could
occur from handling by virus-infected humans (Koopmans et al., 2002). Approximately
10% of the virus particles can easily be transferred from faecally contaminated fingers to
foods and surfaces (Bidawid et al., 2000b). Furthermore, a low relative humidity favours
the survival of HAV and human rotavirus (HRV) (Mbithi et al., 1991). In another study
it was stated that HAV remained infectious in dried faeces for 30 days when stored at 25
°C and 42% relative humidity. HAV vaccination is recommended for foodhandlers,
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although the use of stringent personal hygiene is better preferred, in order to prevent
infections (Koopmans et al., 2004).
Many produce items have been implicated in HAV linked outbreaks including
blueberries, strawberries, lettuce, green onions, raspberries, and semi-dried tomatoes
(Calder et al., 2003, Donnan et al., 2012, Rosenblum et al., 1990). In 2003, the largest
documented HAV outbreak in the United States was associated with contaminated green
onions consumed at a restaurant in Pennsylvania causing 601 cases of illness including 3
deaths and 124 hospitalizations (Wheeler et al., 2005). More recently, a multijurisdictional outbreak in Australia was linked to semi-dried tomatoes with over 562
reported cases (Donnan et al., 2012).
1.3.6.3 Adenoviruses
Adenoviruses are large, non enveloped particles that constitute a double-stranded
deoxyribonucleic acid (DNA) genome totally packaged in an icosahedral capsid, or
protein coat (figure 1.3.6.3.1). They have been isolated from mammalian species. There
are 51 HadV serotypes classified originally on the basis of their ability to be neutralized
by specific animal antisera (Crawford-Miksza and Schnurr, 1996). These can be further
subdivided into six species -or subgroups- (A to F) based on their G-C content of their
DNA and their capacity to agglutinate erythrocytes of human, rat and monkey as well as
on their oncogenicity in rodents (Crawford-Miksza and Schnurr, 1996, Wadell, 1984).
Species B is further subdivided into B1 and B2 (Segerman et al., 2003a). Species B1, C
and E mainly cause respiratory disease, whereas species B, D and E can induce ocular
disease. Species F is responsible for gastroenteritis and B2 viruses infect the kidneys and
urinary tract (Russell, 2005). Subgenus F includes Ad40 and Ad41, which are
considered to be the most important adenoviral species with respect to infantile
dysentery and are shed in high concentrations by infected children (Sattar et al., 2002,
Wadell, 1984).
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Figure 1.3.6.3.1: Adenovirus structure (http://signagen.com/).
Adenovirus type 35 (Ad35) strain, belonging to adenovirus subgroup B, was isolated in
1973 from the lungs and kidney of a 61-year old woman (Flomenberg et al., 1994)
Subgroup C includes adenovirus type 2 and adenovirus type 5. These are considered to
be endemic and account for over half of adenoviral infections. They mainly infect
children and result in both gastrointestinal and respiratory disease. Adenovirus types
from Species B (Ad3) and Species E (Ad 4) have been identified as causative agents of
recreational waterborne outbreaks of conjunctivitis and pharyngoconjunctival fever
(McMillan et al., 1992). Adenoviruses exhibit an increased stability in the environment
compared to other viruses. They are generally considered to be emerging human
pathogens (Yates et al., 2006).
Adenoviruses are nonenveloped, icosahedral particles consisting of a protein coat, or
capsid, surrounding a DNA-protein core. They range in size from 70-100 nm (Strauss
and Strauss, 2002). The protein coat contains several different types of proteins, mainly
hexons which are located at each vertex of the virus’s icosahedral coat, creating a penton
complex from which a fiber protein protrudes (Rux and Burnett, 1999) (figure 1.3.6.3.1).
Each adenovirus particle has 12 molecules of fiber protein which are responsible for the
attachment of viruses to their host cells. Adenovirus attaches to the coxsackie and
adenovirus receptor (CAR) on the surface of host cells (Bergelson et al., 1997).
According to Seth (1999a) adenovirus enters cells via receptor-mediated endocytosis,
during which the portion of the cell membrane containing the CAR and bound virus
become a membrane-bound vesicle. After binding to the cell surface, viral particles enter
endosomes within the host cell. When the endosomes lyse, viral particles are released to
the cytosol and travel along microtubules to the host cell’s nucleus, where adenoviral
DNA is replicated by the host cell’s DNA replication machinery. Then, new viral
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particles are formed and released from the cell. The virus travels from its attachment site
on the surface of the host cell to the nucleus in about 30-min (Greber et al., 1993).
Attachment of adenovirus to the cell surface involves mainly the CAR receptor and the
fiber protein (Strauss and Strauss, 2002).
1.3.7 Pathogens and Infectious Dose
Infectious dose or infective dose (ID) is the amount of pathogens (measured in number
of microorganisms) that are required to cause an infection in the host. ID varies
dramatically across pathogen species, as well as according to consumer's age and overall
health (Leggett et al., 2012).
Foodborne illness resulting from the consumption of any food is dependent upon a
number of factors. The produce must first be contaminated with a pathogen and the
pathogen must survive until the time of consumption at levels sufficient to cause illness.
The infective dose (minimum numbers of organisms necessary to cause foodborne
illness) is very low in many cases, which means that the microorganism needs only to
contaminate the food and survive without reproducing. For example, pathogenic
parasites and viruses are unable to multiply outside of a human or animal host and only
need to survive in sufficient numbers to cause illness (FDA, 2011).
The infectious dose of a foodborne pathogen depends on many variables including the
immune status of the host, the virulence and infectivity of the pathogen, the type and
amount of contaminated food consumed, the concentration of the pathogen in the food
and the number of repetitive challenges (EU, 1999). In general, infectious pathogens
may enter the body and invade or colonize host tissues. This requires some time (e.g.,
usually greater than 8 h for onset of illness). Toxigenic pathogens create food
“poisoning” situations by producing an enterotoxin in the food (Behling et al., 2010).
Risk assessment and impact of foodborne pathogens on the health of different
populations remains one of the goals of every country. For certain pathogens, such as
Listeria monocytogenes and Escherichia coli O157:H7, there are no feeding studies due
to ethical reasons, and the results from outbreaks are normally used to estimate the
infectious dose (Kothary and Babu, 2007).
The infectious dose for different serovars of Salmonella and strains of E. coli was quite
large (>105 organisms), while the infectious dose for some Shigella spp. seemed to be as
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low as less than 10 organisms (Kothary and Babu, 2007). The infectious dose of
salmonellae can vary, depending on the bacterial strain ingested as well as on the
immuno-competence of individuals. For serotypes not presenting particular adaptations
to an animal host, experimental studies showed that between 105 to 107 bacteria were
required to establish an infection. However, data from outbreaks of foodborne diseases
indicate that infections can be caused by ingestion of as few as 10-45 cells (Lehmacher
et al., 1995). It has repeatedly been reported that the infectious dose is lower when
salmonellae are present in food with a high content of fat or protein, substances which
protect bacterial cells against the low pH of gastric juices (Blaser and Newman, 1982).
An oral dose of at least 105 Salmonella Typhi cells are needed to cause typhoid in 50%
of human volunteers, whereas at least 109 S. Typhimurium cells (oral dose) are needed to
cause symptoms of a toxic infection. Infants, immunosuppressed patients, and those
affected with blood disease are more susceptible to Salmonella infection than healthy
adults (Todar, 2012). The infectious dose of S. Enteritidis can be relatively small, 100 to
1,000 organisms are enough to cause the infection in some people. Food prepared from
infected animals, insufficiently cooked and food contaminated prior to consumption are
the principal causes of infection (CDC, 2005).
The infective dose of ETEC for adults has been estimated to be at least 108 cells.
However, young and elderly people may be susceptible to lower levels. Because of its
high infectious dose, analysis for ETEC is usually not performed unless high levels of E.
coli have been found in a food. At least 106 EIEC organisms are required to cause illness
in healthy adults. The infectious dose for O157:H7 is estimated to be 10-100 cells, but
no information is available for other EHEC serotypes (FDA, 2011).
The infective dose for Listeria monocytogenes is uncertain, although it is generally
considered to be high (105-107) (Farber et al., 1996) for healthy individuals, with food
contamination rates of more than 1,000 cells/g being required. Due to the length of the
incubation period, it can be difficult to determine the numbers of bacteria in foods at the
time of consumption. An outbreak associated with frankfurters in the USA in 1998 is
thought to have been caused by product containing less than 0.3 cells/g, although it is
suspected that the strain involved may have been unusually virulent (Farber et al., 1996).
The probability of exposure to a higher dose (> 1.000 CFU) was large enough to account
for the observed rate of listeriosis.
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The infectious dose of S. aureus has been found to be at least 100,000 organisms in
humans (PHAC, 2011). Therefore, if enterotoxinogenic staphylococci are able to grow
in food to high numbers (more than 105 to 106 CFU/g or /ml) before they are killed there
is still a risk for intoxication with consumption.
As virus infectivity dose is concerned, it has been estimated that NLVs and HAV have
an infective dose of between 10 and 100 virus particles. Adenoviridae infectious dose is
>150 plaque forming units when given intranasally (PHAC, 2011).
1.3.8 Methods for detection of foodborne pathogens
Generally, there are standard conventional methods for determination of the foodborne
pathogens in food products including culture, microscopic, chemical and biological
methods (immunological, molecular genetic methods, gel diffusion). Moreover, there
exist more rapid methods including physical methods (biosensors, impedance,
microcalorimetry, flow cytometry, biosys instrument) and bioassays (Yeni et al., 2014).
Real-time PCR (rtPCR), microarrays, and biosensors have emerged as recent
developments in the field of pathogen testing (Mattingly et al., 1988).
Among the culture-based conventional methods, standard plate count is the longest
available detection and enumeration method. Culturing methods utilized to monitor for
the presence of indicator organisms are time consuming and labor intensive (Shannon et
al., 2007). As an alternative to standard plate count, incorporation of chromogenic and
fluorogenic substrates into culture media exists for the biochemical identification of
pathogenic microorganisms (Mattingly et al., 1988).
Emerging pathogens, that are not detectable by conventional microbiological methods,
can be detected with nucleic acid (DNA and RNA)-based assays for the differentiation
and identification of foodborne pathogens. These methods include polymerase chain
reaction (PCR), pulsed-field gel electrophoresis, ribotyping, plasmid typing, randomly
amplified polymorphic DNA (RAPD), restriction fragment length polymorphism
(RFLP) (Yeni et al., 2014). Most of them are available in commercial kits. Molecularbased detection methods, such as the polymerase chain reaction (PCR), amplifies
specific target DNA of the desired organism being investigated, and have exhibited huge
potential as routine and rapid analysis tools for the detection of specific micro-organisms
(Shannon et al., 2007). However, the PCR technique is unable to distinguish between
viable or dead cells, which could lead to false positive results (Moreno et al., 2011).
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Immunology-based methods and several types of biosensors are the new developing
techniques for detection of foodborne pathogens (Velusamy et al., 2010). For
immunodetection, which is based on antigen-antibody binding selection, several types of
antibodies can be used (conventional, heavy chain antibodies, polyclonal, monoclonal or
recombinant antibodies). For instance, detection of L. monocytogenes can be performed
via polyclonal antibodies (Jung, et al., 2003) and via monoclonal antibodies (Mattingly
et al., 1988), but for Salmonella detection, monoclonal antibodies (Yeni et al., 2014)
have been identified.
ELISA (enzyme-linked immunosorbent assay) is the most prominent method among
other types of immunological pathogen detection methods. It integrates the specificity of
antibodies and the sensitivity of simple enzyme assays by using antibodies or antigens
connected to an enzyme (Lazcka et al., 2007, Yeni et al., 2014).
Another promising rapid detection method is the Matrix-assisted laser desorption/
ionisation time of flight mass spectrometry (MALDI-TOF MS) (Biswas and Rolain,
2013). The initial capital cost for purchasing the equipment is high, however, this
technique seems cheap when utilised for the routine biotyping of bacteria (Biswas and
Rolain, 2013, Clark et al., 2013).
The 3M™Molecular Detection system is a new technology being extensively used in
food analysis for the detection of Listeria spp., Salmonella spp. and E. coli 0157:H7.
The detection system makes use of a loop-mediated isothermal amplification (LAMP)
method for the detection of foodborne pathogens. It then combines isothermal DNA
amplification with bioluminescence detection, which allows for specificity and
sensitivity in the testing for pathogens (Loff et al., 2014).
The culturing techniques that are commonly used for the detection and enumeration of
pathogens in foods include selective chromogenic media for detecting each bacteria.
A selective, chromogenic medium that can be used for the detection and enumeration of
E. coli in foods is TBX Agar (Oxoid, CM 0595). TBX Medium is a tryptone Bile Agar
which is based on a chromogenic agent -X-glucuronide- detecting glucuronidase
activity. The released chromophore in TBX Medium is insoluble and accumulates within
the cell. This ensures that colored target colonies are easy to be identified. Most E.
coli strains can be differentiated from other coliforms by the presence of the enzyme
glucuronidase. The chromogen in TBX Medium is 5-bromo-4-chloro-3-indolyl-beta-DPage 62
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glucuronide (X-glucuronide), and is targeted by this enzyme. Escherichia coli cells are
able to absorb this complex intact and intracellular glucuronidase splits the bond
between the chromophore and the glucuronide. The released chromophore is colored and
is accumulated within the cells, causing E. coli colonies to obtain a color of blue/green.
The tryptone provides the essential growth nutrients (nitrogen, vitamins, amino acids) to
the organisms whereas bile salts mixture inhibits Gram-positive organisms. Moreover, in
order to detect E. coli O157:H7 in foods, an enrichment stage, is ofter required, since the
number of cells is usually low, and injured cells are likely to be present. For this reason,
enrichment in modified trypticase soy broth (mTSB), supplemented with either
novobiocin or acriflavin, can be used (Taormina, 1998).
Baird Parker medium has been developed in order to detect S. aureus in foods. BairdParker (1962) developed this medium from the tellurite-glycine formulation. As a
nitrogen source for the organism, casein peptone and beef extract are added to the
medium. Yeast extract provides nitrogen as well as other important nutrients like Bcomplex vitamins. The selective agents glycine, lithium and potassium tellurite have
been carefully balanced to suppress the growth of most bacteria present in foods, without
inhibiting S. aureus. Egg yolk emulsion (Oxoid SR0047) makes the medium yellow and
opaque. S. aureus reduces potassium tellurite to form grey-black shiny colonies and then
produces clear zones around the colonies by proteolytic action. This clear zone with
typical grey-black colony is characteristic for S. aureus detection. Most strains of S.
aureus form opaque haloes around the colonies. This is probably due to the action of a
lipase. However, not all strains of S. aureus produce both reactions.
A selective medium that can be used for the isolation and identification of Salmonella is
Xylose-Lysine-Desoxycholate Agar (XLD Agar), which was originally formulated by
Taylor (1965). It relies on xylose fermentation, lysine decarboxylation and production of
hydrogen sulphide for the primary differentiation of shigellae and salmonellae from
non-pathogenic bacteria. Salmonella spp. are differentiated from non-pathogenic xylose
fermenters by the incorporation of lysine in the medium. Salmonellae exhaust the xylose
and decarboxylate the lysine, thus altering the pH to alkaline and mimicking
the Shigella reaction. However, the presence of Salmonella is differentiated from that
of shigellae by a hydrogen sulphide indicator. Sodium Thiosulfate and Ferric
Ammonium Citrate act as selective agents, allowing visualization of hydrogen sulfide
production under alkaline conditions.The high acid level produced by fermentation of
lactose and sucrose, prevents lysine-positive coliforms from reverting the pH to an
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alkaline value, and non-pathogenic hydrogen sulphide producers do not decarboxylate
lysine. The acid level also prevents blackening by these micro-organisms until after the
18-24 hour examination for pathogens. Sodium desoxycholate is incorporated as an
inhibitor in the medium. The sensitivity and selectivity of XLD. Agar exceeds that of the
traditional plating media e.g. Eosin Methylene Blue, Salmonella-Shigella and Bismuth
Sulphite agars, which tend to suppress the growth of shigellae (Dunn and Martin, 1971,
Taylor and Schelhart, 1967).
Listeria Selective Medium (Oxford Formulation) is recommended for the detection of
Listeria in foods. It is based on the formulation described by Curtis et al. (1989). The
medium utilizes: (i) the selective inhibitory components lithium chloride, acriflavine,
colistin sulphate, cefotetan, cycloheximide or amphotericin B and fosfomycin, (ii) the
indicator system aesculin and ferrous iron for the isolation or differentiation of Listeria
monocytogenes. Listeria monocytogenes hydrolyses aesculin, producing black zones
around the colonies due to the formation of black iron phenolic compounds derived from
the aglucon. Ferric Ammonium Citrate aids in the differentiation of Listeria spp. Since
all Listeria spp. hydrolyze esculin, the addition of ferric ions to the medium will detect
the reaction. A blackening of the colony and surrounding medium in cultures containing
esculin-hydrolyzing bacteria results from the formation of 6,7-dihydroxycoumarin which
reacts with the ferric ionsGram-negative bacteria are completely inhibited (Fraser and
Sperber, 1988). Most unwanted Gram-positive species are suppressed, but some strains
of enterococci grow poorly and exhibit a weak aesculin reaction, usually after 40 hours
incubation. Typical Listeria colonies are almost always visible after 24 hours, but
incubation should be continued for a further 24 hours to detect slow-growing strains.
1.4 Infection and Disinfection
Traditional thermal treatments are a basic pillar in the food industry providing required
safety profiles and extensions of shelf-life of the product. However, attention must be
given to losses of desired organoleptic properties and damage to temperature labile
nutrients and vitamins. Consequently, the food industry is interested in alternative or
combined approaches in order to achieve the objectives of disinfection. Thus, nonthermal technologies have been designed to meet the required food product safety
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standards or shelf-life demands while minimizing the effects on its nutritional and
quality attributes.
1.4.1 The Bacterial Cell and Antimicrobial Interaction
The bacterial cell interacts with biocides through: the cell wall, the cytoplasmic
membrane and the cytoplasm. However, a biocide can interact with one, two or three
regions of the bacterial cell in order to have a successful antimicrobial effect. The cell
wall is comprised of an open network of peptidoglycan (with a lipopolysaccharide
overlayer for Gram-negative bacteria) that serves as an excellent target for
antimicrobials. Thus, the presence of the outer envelope adds an additive resistance of
Gram negative bacteria to antimicrobials when compared to Gram-positive bacteria
(Denyer and Maillard, 2002) (figure 1.4.1.1). The peptidoglycan layer and its associated
anionic polymers provide access to the membrane of microorganisms to molecules with
molecular weights ranging from 30- 57 KDa. Antimicrobials such as hydrogen peroxide,
phenols, alcohols, aldehydes, QACs and biguanides are small enough, thus their cross to
the cell wall is easy (Lambert, 2002). Porins, which are large protein structures
embedded within the outer membrane, not only allow the diffusion of cellular nutrients
but can also serve as channels to hydrophilic biocides of molecular weight less than 600
Da (Denyer and Maillard, 2002). The cytoplasmic membrane is available to biocide
attack as it acts as a rich matrix of balanced interactions between phospholipid and
enzymatic/structural proteins where intracellular homeostasis and transport/metabolisms
are maintained (Denyer and Stewart, 1998). The cytoplasm is considered to be the most
common target for biocides due to the presence of catabolic and anabolic processes.
Damage to the membrane can take place due to physical disruption, dissipation of the
proton motive force and inhibition of membrane associated enzyme activity (Maillard,
2002). Hypochlorous acid reacts with a wide variety of biological molecules including
proteins, DNA, cholesterol, lipids, free thiols and sulfides (Hawkins et al., 2003,
Noguchi, et al., 2002). The bactericidal activity of hypochlorite has been attributed to the
formation of secondary products (chloramines), which react with subcellular compounds
such as ammonia ions and organic amines (Miche and Balandreau, 2001). However,
chloramines are toxic compounds capable of diffusing through cell membranes and
reaching intracellular components such as DNA. Consequently, hydrogen bonding is
disrupted, resulting in the dissociation of DNA (Hawkins et al., 2003). Studies have
demonstrated that radicals that result from the decomposition of chloramines on
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hypochlorous acid treated proteins can cause damage to other substrates as well. The
oxidation of proteins by hypochlorous acid can lead to side chain modification. Many of
these reactions occur primarily with thiols, sulfides, amines, amides and aromatic rings
(Hawkins et al., 2003). The effectiveness of disinfectants like H 2 O 2 , and NaOCl, is
dependent on concentration, pH, temperature, microbial strain, and the presence of
organic materials.
Figure 1.4.1.1: Schematic illustration of cell wall structures of microbial pathogens. (A) Grampositive bacteria, (B) Gram-negative bacteria (Yin et al., 2013).
1.4.2 The virus genome and infectivity
The virus must be able to attach to the host cell in order to inject its material into the
host cell thus being able to replicate. When pasteurization is used as a disinfection
method, the heat that is produced inhibits the bonding with the host cell. Thus, the virus
in incapable of recognizing the host and is unable of attaching to it. In general terms
chlorine and ozone affect the viral protein material, whereas UVC affects the genomic
material more than the protein material (Nuanualsuwan and Cliver, 2003, ThurstonEnriquez et al., 2005, Toropova et al., 2008). When chlorine dioxide is used, the
reactions take place in the genome and proteins, and byproducts that further react with
aminoacids and as a concequence nucleotides are formed (Pecson et al., 2009). When
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radiation is used, chemical reactions are created that can destroy the genome, inhibiting
virus replication. Radiation leads to direct photolysis of photolabile virus components,
thus the virus protein capsid is broken. When the capsid is broken, there is no way for
the virus to inject its material into the host cell. Finally, when chlorination is used for
virus disinfection, the attack of chlorine to genome, prevents it from replication and
destroys its injection into the host cell (figure 1.4.2.1).
Figure 1.4.2.1: Effects of capsid of virus when different situations occur (Wigginton et al.,
2012).
1.4.3 Conventional Food Processing/Preservation Technologies
1.4.3.1 Chemical Methods
Chlorine compounds can be categorized to three different types: chlorine gas, chlorine
hypochlorites (e.g., sodium, calcium, lithium and potassium) and chlorine releasing
agents
(trichloroisocyanuric
acid,
sodium
dichloroisocyanurate,
dichlorodimethylhydantoin, chloramines T) (McDonnel and Russell, 1999). Chlorine
compounds are bactericidal and sporicidal (Russel, 1983). Their activity is mainly
related to their solubility, amount of available chlorine present and pH of the solution.
However, their activity is impaired by the presence of organic matter.
Hypochlorites are powerful oxidants and can induce lysis in Gram-negative bacteria by
affecting the cell wall (Maillard, 2002). The manufacture of sodium hypochlorite is a
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simple procedure that involves the reaction of chlorine with caustic soda in a batch or
continuous process (Gordon et. al., 1995). The reaction that takes place follows:
2 NaOH + Cl 2 → NaOCl + NaCl + H 2 O + Heat
Other commonly used terms include free available chlorine and available chlorine. Free
available chlorine (FAC) refers to the concentration of molecular chlorine (Cl 2 ),
hypochlorous acid (HOCl), and hypochlorite ion (OCl-) in water expressed as available
chlorine. Available chlorine is used to express the amount of chlorine in chlorine gas and
hypochlorite salts. Commercial grade sodium hypochlorite can be produced by
manufacturers at concentrations as high as 16% chlorine, with typical concentrations
ranging between 5 and 15% chlorine. The stability of a sodium hypochlorite solution is
affected by factors such as concentration, light, pH, temperature and heavy metals
(Casson and Bess, 2003). Liquid hypochlorite typically has a pH between 11 and 13. In
basic solution, hypochlorite ion (OCl-) decomposes to form chlorate ion (ClO3-) (a toxic
by-product). This is a second order process and involves the reaction of OCl- with
chlorite ion ClO2- (an intermediate ion). The reactions are the following (Bolyard and
Fair, 1992):
2OCl- → ClO2- + Cl- (slow reaction)
OCl- + ClO2- → ClO3- + Cl- (fast reaction)
At pH higher than 9, the HOCl is almost completely dissociated to OCl- (HOCl ↔ H+ +
OCL-), which results to a very poor disinfectant. Thus, lower pH is important for an
adequate disinfection. The reaction that takes place when sodium hypochlorite is added
to water is the following:
NaOCl+H 2 O → HOCl + Na+ + OHChlorine is an economical disinfectant and has been used in water and food disinfection
(EPA, 1999c). Chemicals that have been evaluated for use as disinfectant agents in food
produces, are chlorinated water and chlorine dioxide (Park et al., 2008). However, there
are also several disadvantages related to chlorine disinfection. Chlorine reacts with
organic and inorganic compounds and produces undesirable trihalomethanes (THMs)
and other carcinogenic disinfection by-products (DBPs), such as chloroform and
bromodichloromethane. Thus the use of chlorine has been associated with the formation
of carcinogenic compounds (EPA 1998a). Moreover, some foodborne pathogens have
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been shown to be more resistant to the lethal action of chlorination (Furata et al., 2004).
Furthermore, higher concentrations of chlorine can cause poor taste and odor in treated
produces. To improve food safety and maintain the freshness, the products are subjected
to appropriate washing systems containing chemical sanitizers. One of the most
commonly used disinfectants in food industry is chlorine (50–200 ppm). However, it is
reported that chlorine is not effective to inactivate internalized pathogens due to its
limited penetration into the internal complex areas of foods (Sapers, 2001).
The USFDA recommends 50–200 mg/l total chlorine at pH 6.0–7.5 and contact times of
1–2 min for this purpose (Beuchat, 1998). The International Fresh-Cut Produce
Association (IFPA) Model HACCP Plan for shredded lettuce suggests a chlorination of
100–150 mg /l total chlorine at pH 6.0–7.0 (Delaquis et al., 2004). Chlorinated water has
been used to wash and disinfect vegetables and fruits shortly after harvesting and at
various stages of handling and processing (Beuchat, 1998). The effectiveness of
treatment with water containing up to 200 mg/ml chlorine in reducing numbers of
naturally occurring microorganisms and pathogenic bacteria is minimal, often not
exceeding 2 log 10 on lettuce (Zhang and Farber, 1996) and tomatoes (Beuchat et al.,
1998, Wei et al., 1995, Zhuang et al., 1995). However, Sapers and Simmons (1998)
reported that the quality of some products may be degraded by browning induction (in
mushrooms and lettuce) or bleaching of anthocyanins (in strawberries and raspberries).
In order to improve efficacy, chemical treatments are combined with other disinfection
methods such as ultrasound (Seymour et al., 2002, Zhou et al., 2009), vacuum
infiltration (Sapers, 2001), and adding detergents or enzymes to dissolve extracellular
polymeric substances (Johansen et al., 1997).
1.4.3.1.2 Organic Acids
Organic acids, mainly citric, lactic and acetic acid, which are involved in GRAS
(Generally Recognized As Safe) status, have been investigated because of their ability to
inactivate foodborne pathogens (Akbas and Ölmez, 2007). Organic acids act rapidly and
kill a broad spectrum of bacteria. Moreover, they are effective within a wide temperature
range and are not affected by water hardness (Sagong et al., 2011). The antimicrobial
action of organic acids, in general, is attributed to the pH reduction in the environment,
and it changes widely among the organic acids (Ölmez and Kretzschmar, 2009).
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Antimicrobial properties of acetic acid have been shown to inactivate food-borne
pathogenic bacteria (Bell et al., 1997). Many studies have been conducted related to the
use of organic acids as disinfectants. It was reported that the fecal coliforms and the
coliforms were reduced by 1.0 and 2.0 log 10 CFU/g, respectively, on mixed salad
vegetables treated with 1.0% lactic acid (Torriani et al, 1997). Francis and O’Beirne
(2002) found that 1.0% citric acid solution reduced mesophilic population densities on
lettuce by about 1.5 log 10 CFU/g in 5 min. Dipping of fresh-cut iceberg lettuce in 0.5%
citric acid or 0.5% lactic acid solutions for 2 min were found to have the same effect like
100 ppm chlorine, in reducing the natural microbial population (Akbas and Ölmez,
2007).
Chang and Fang (2007) found that 5% acetic acid could reduce 3 log 10
population of E. coli O157:H7 in iceberg lettuce, however, may alter the sensory quality
by causing an unacceptable sour flavor. However, it is obvious that the antimicrobial
activity of organic acids varies in a wide range depending on the type of organic acid. In
general, the exposure times needed for a significant reduction in microbial load is
between 5 and 15 min. Moreover, organic acids also have disadvantages such as high
cost, odor, and corrosiveness. It must also be taken into consideration that the use of
organic acids for disinfection purposes in fresh RTE industry would have an impact on
the wastewater quality, characterized by high COD and BOD values in the wastewater
(Ölmez et al., 2009).
1.4.3.1.3 Peroxyacetic Acid
Peroxyacetic acid (PA), which is also referred to as peracetic acid, is an aqueous
quaternary equilibrium mixture of acetic acid and hydrogen peroxide (Dell’Erbaa et al.,
2007). Like ozone and chlorine dioxide, peroxyacetic acid is an effective disinfectant
able to kill pathogenic microorganisms in suspension at lower concentrations. Moreover,
it is important to note that, the efficacy of PA is not affected by the organic compounds
present in the process water, whereas the efficacy of chlorine is affected (Ruiz-Cruz et
al., 2007). PA has become and interesting disinfectant among chlorine-alternative
chemical disinfectants due to the fact that only harmless disinfection by-products have
been formed from its spontaneous decomposition (i.e acetic acid, water, oxygen)
(Dell’Erbaa et al., 2007). The US Code of Federal Regulations states that the use of
peroxyacetic acid in fruits and vegetables is allowed up to 80 ppm in wash water.
However, studies revealed that 80 ppm peroxyacetic acid in wash water is not sufficient
to obtain a substantial reduction in the microbial load of the fresh RTE fruits and
vegetables (Ruiz-Cruz et al., 2007, Vandekinderen et al., 2007).
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1.4.3.1.4 Hydrogen Peroxide
Hydrogen peroxide (H 2 O 2 ), also referred to as hydrogen dioxide, has both a
bacteriostatic and a bactericidal activity due to its strong oxidizing power and its
generation of cytotoxic species (Juven and Pierson, 1996). Although it is involved in the
GRAS status, its use in the food industry is limited only to some products (milk, dried
egg, starch, tea and wine) as an antimicrobial or bleaching agent in the range of 0.04–
1.25%. The effectiveness of hydrogen peroxide as a disinfectant and antimicrobial agent
has been established (Ölmez and Kretzschmar, 2009).
One of the main advantages of using H 2 O 2 as a disinfecting agent is that it produces no
residue as it is decomposed into water and oxygen by the enzyme catalase which is
naturally found in plants. The main drawback is its phytotoxicity, which has been
observed in some products like lettuce and berries. Studies revealed that the H 2 O 2
treatment induces extensive browning on some products (lettuce, mushrooms) (Ölmez
and Kretzschmar, 2009).
1.4.3.2 Physical Methods
1.4.3.2.1 Heat Processing
Foods are processed by various processing technologies to reduce or remove any
potential pathogen or biological hazard that might be introduced while handling or food
processing. Traditional food processing technologies, such as pasteurization and heat
sterilization, use heat in order to kill or inactivate microbiological contaminants.
Pasteurization is a mild heat treatment in which foodstuffs are heated to a temperature
lower than 100°C (Fellows, 2000). This process is used to minimize potential health
hazard through destruction of non-spore forming pathogenic microorganisms.
Pasteurization can kill 99-99.9% of spoilage micro-organisms and inactivate enzymes,
thus enhancing the shelf life of the product (Parikh, 2007).
The HTST pasteurization has been successfully implemented and has achieved a fivelog10 reduction, killing 99.999% of the number of viable micro-organisms. This is
considered adequate for destroying almost all yeasts, molds, and common spoilage
bacteria and also to ensure adequate destruction of common pathogenic, heat-resistant
organisms (Doyle et al., 2001).
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On the other hand, heat sterilization is a severe heat treatment in which foods are heated
at sufficiently high temperatures for prolonged time-periods to destroy microbial and
enzyme activity. As a result, sterilized foods are shelf-stable with more than six months
of shelf life (Fellows, 2000). A sterilized product may contain viable spores that cannot
grow due to environmental conditions, such as low pH, low water activity, etc. However,
it can be concluded that the product treated with heat sterilization is considered as
commercial sterile product (Parikh, 2007).
However, limitations exist when thermal disinfection technologies are used. Heat alters
or destroys nutrient components of foods that are responsible for the special flavor,
color, taste, or texture, and as a result they are perceived nowadays to have lower quality
and value (Fellows, 2000).
1.4.3.2.2 Radio Frequency (RF) and Microwave Heating (MH)
RF and MW systems operate by the same principle,forcing polar molecules such as
water, and ionic species to realign themselves by reversing and electric field around the
food products. The promise of rapid and volumetric heating has called the attention of
food industry for the potential use of dielectric systems. Industrial and experimental
applications of MW for food preservation, among others include sterilization and
pasteurization of ready to eat meals. RF systems could be particularly suitable for heat
processing of whole meat products (Pereira and Vicente, 2010).
1.4.3.2.3 Ohmic Heating
An ohmic heater also known as a joule heater is an electrical heating device that uses a
liquid’s own electrical resistance to generate the heat. Heat is produced directly within
the fluid itself by Joule heating as alternating electric current (I) is passing through a
conductive material of resistance (R), with the result energy generation causing
temperature rise (Sakr and Liu, 2014). OH technology is distinguished from other
electrical heating methods by the presence of electrodes contacting the foods (in
microwave and inductive heating electrodes are absent), the frequency applied
(unrestricted, except for the specially assigned radio or microwave frequency range), and
the waveform (also unrestricted, although typically sinusoidal) (Pereira and Vicente,
2010).
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1.4.4 Non-thermal Technologies (Alternative Technologies)
Consumers increasingly perceive fresh foods or minimally processed foods as healthier,
compared to heat-treated foods. Thus, the industry is now developing alternative
processing technologies. Novel non-thermal processing technologies have the ability to
inactivate microorganisms at ambient or near ambient temperatures, thereby avoiding
the process-induced changes that heat may have on the flavor, color and sensory, quality
and nutritional characteristics of foods. Therefore, alternative processing technologies
are now being used to effectively destroy any microbial threat, and to maintain at the
same time the quality and storage-stability of foods (Ahvenainen, 1996).
Non-thermal technologies are processing technologies able to achieve microbial
inactivation without exposing foods to adverse effects of heat. At the same time, an
extension of product shelf life and retention of their fresh-like physical, nutritional, and
sensory qualities is also achieved (Butz and Tauscher, 2002). These technologies include
pulsed or radio frequency electric fields (PEF/RFEF), ultraviolet light (UV), pulsed light
(PL), Near Ultraviolet Light (NUV Light), ultrasound (US), high pressure processing
(HPP), ionizing radiation, dense phase carbon dioxide (DPCO 2 ) and ozone (Mohd.
Adzahan and Benchamaporn, 2007).
Like any other process, non-thermal technologies can be combined with thermal or other
non-thermal processes as a hurdle technology or as a compliment to other processes. The
hurdle approach is widely used to produce minimally processed and microbiologically
stable food. The microbial stability is achieved by combining different hurdles to
increase the destruction of the microbial cytoplasmic membrane as well as preventing
cell repair of survivors from treatments (Leistner, 2000).
It is true that combining non-thermal processes with conventional preservation methods,
their antimicrobial effect is enhanced, and as a consequence lower process intensities as
well as shorter treatment times can be used, which is an advantage to the food industries
(Mohd. Adzahan and Benchamaporn, 2007, Ross et al., 2003).
1.4.4.1 Ultraviolet Light (UV)
Radiation from the UV region of the electromagnetic spectrum can be used for the
disinfection of food produce. The wavelength for UV processing ranges from 100 to 400
nm (figure 1.4.4.1.1) (Sastry et al., 2000).
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Figure 1.4.4.1.1: Electromagnetic Spectrum (Guerrero- Beltrán and Barbosa-Cánovas, 2004).
The UV light is easy to use and has been proved lethal to microorganisms (Bintsis et al.,
2000). The wavelength between 200 and 280 nm (UV-C) is considered germicidal
against microorganisms such as bacteria, viruses, protozoa, moulds and yeasts, and algae
(Bintsis et al., 2000), compared to other types of UV (table 1.4.4.1.2).
Table 1.4.4.1.1: Ranges, Wavelengths and Characteristics of different types of UV (GuerreroBeltrán and Barbosa-Cánovas, 2004).
The highest germicidal effect is observed between 250 and 270 nm. More precisely, the
wavelength of 254 nm (UV-C, generated by LPM lamps) is used for disinfection of
surfaces, water, and food products (Bintsis et al., 2000).
Mode of Action of Continuous UV to Microorganisms
In general terms, because the UV-C bactericidal effect is mainly observed at the nucleic
acid level, radiation absorbed by DNA can stop cell growth and lead to cell death
(Liltved and Landfald, 2000, Wright et al., 2000). In detail, the UV-C light absorbed by
DNA molecule, causes a physical shifting of electrons to render splitting of the DNA
bonds, delay of reproduction or cell death. Then, cross-linking effects between
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neighbouring thymine and cytosine (pyrimidine nucleoside bases) in the same DNA
strand occur (figure 1.4.4.1.3). The produced DNA photoproducts are the cyclobutyl
pyrimidine dimers. The cross-linking effects in the DNA are proportional to the amount
of UV-C light exposure (dosage, time). The consequence is the cell death due to the
blocked DNA transcription and replication as well as the blockage of many cellular
functions (Guerrero- Beltrán and Barbosa-Cánovas, 2004).
UV-C irradiation is also responsible for the production of DNA mutations in the injured
organism (Sastry et al., 2000). Photoreactivation can occur only when the UV-C injured
cells are exposed to wavelengths higher than 330 nm (Liltved and Landfald, 2000).
Protein factors (DNA repair genes) can repair the DNA damage (Yajima et al., 1995).
Moreover, the activation of the enzyme photolyase that monomerises the dimer species
(splitting of thymine and other pyridines) can photoreactivate the split nucleic acid
(Stevens et al., 1998). However, a dark environment can avoid photoreactivation of
irradiated products (Stevens et al., 1998) or restore cells exposed to UV-C light.
It must be mentioned, that the effect of UV radiation on microorganisms inoculated on
food surfaces may vary from species to species. In the same species it depends on many
factors such as strain, growth media, stage of culture (Chang et al., 1985, Wright et al.,
2000), density of microorganisms and other characteristics, such as type and
composition of the food. Generally, fungi and yeasts (large microorganisms) are more
resistant during UV disinfection (Bachmann, 1975).
UV-C light is also applied to fresh fruits, vegetables and roots before being stored in
order to reduce the initial count of microorganisms on the surface of the product and to
induce host resistance to the microorganisms. The beneficial effect of UV-C light on
fresh food products is called ‘hormesis’ and the agent (UV light) is called ‘hormetin’ or
‘hormetic effect’ (Stevens et al., 1997, Stevens et al., 1999). The hormetic effect of UVC light may cause the production of phenylalanine ammonia-lyase (PAL) that induces
the formation of phytoalexins (phenolic compounds). Phytoalexins’ role is the
improvement of the resistance of fruits and vegetables to microorganisms (D’hallewin et
al., 2000, Stevens et al., 1997, Stevens et al., 1998, Stevens et al., 1999).
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Figure 1.4.4.1.2: Structure of DNA before and after absorbing a photon of UV light (Koutchma,
2009).
UV intensity flux or irradiance is usually expressed in W/cm2, and the dose or radiant
exposure is expressed as J/m2 (Bintsis et al., 2000). The UV-C dose (D) is defined as:
D=I 254 nm *t
“Equation 1”
Where: D is the dose (J/cm2), I 254 is the intensity or dosage rate (W/cm2) and t is the
retention time in seconds (Chang et al., 1985, Morgan, 1989, Stevens et al., 1999).
In a flow system, the retention time is obtained as: t= Volume of chamber / Flow Rate
Figure 1.4.4.1.3: UV chamber (inside photo)
Among the advantages, it can be concluded that the photoinactivating process by UV-C
(figure 1.4.4.1.3) is a physical method which does not produce undesirable by-products
(Chang et al., 1985) that could change the sensory characteristics (odor, taste and color)
of the final product. Moreover, it does not generate chemical residues nor residual
radioactivity (compared to gamma radiation) to the final product (Morgan, 1989). It can
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be concluded that it is a non-thermal process that can be simply and effectively used at
low cost compared with other sterilization methods. It can be obtained by simply
applying UV-C to the desired food surface at low intensity for long periods or high
intensity for short periods of time (Morgan, 1989).
1.4.4.2 High Intensity Light Pulses (HILP)
High-intensity light pulses (HILP) (figure 1.4.4.2.1) is an emerging non-thermal
technology which uses short (100–400 𝜇𝜇s) high-power, intense pulses of broad-spectrum
light (200–1100 nm) and has been used to inactivate bacteria (vegetative cells and
spores), yeasts, moulds, and even viruses (Marquenie et al., 2003, Gomez-Lopez et al.,
2007, Woodling and Moraru, 2007). The mode of action of HILP on microorganisms is
based on the photochemical action of the UV-C part of the light spectrum that causes
thymine dimerization in the DNA chain preventing transcription and replication and
ultimately leading to cell death (Muňoz et al., 2012, Gomez-Lopez et al., 2007, Rajkovic
et al., 2010). Microbial inactivation using HILP has gained attention in recent years due
to lower energy consumption compared to conventional thermal processes (BarbosaCánovas et al., 1998). Depending on the energy delivered through each flash, the
distance between the lamps and the contaminated matrix, the targeted microorganism,
and even the nature of the contaminated matrix itself, HILP has been reported to result in
a 0.5 to 8 log 10 CFU/mL bacterial reduction (Hsu and Moraru, 2011). In addition, it has
also been shown that both the visible and infrared regions of HILP in combination with
its high peak power also contribute to the killing effect on microorganisms (Elmnasser et
al., 2007). High-intensity light pulse (HILP) is an emerging technology that has been
shown to be highly effective against a wide range of pathogenic microorganisms,
including S. aureus, E. coli O157:H7, S. enterica, and Cryptosporidium parvum (Bialka
and Demirci, 2007, Krishnamurthy et al., 2007, Lee et al., 2008). The germicidal action
of HILP has been also attributed to the localized elevated temperature due to the
simultaneous use of UVs and IR radiations leading to bacterial disruption (Dunn, 1995,
Nicorescu et al., 2013, Takeshita et al., 2003, Uesugi and Moraru, 2009).
The use of HILP to food products such as apple juice, milk, minimally processed
vegetables, berries, alfalfa seeds, hot dogs and salmon fillets have been studied with the
intention to extending shelf-life and/or inactivating pathogens (Bialka and Demirci,
2007, Gomez et al., 2012, Huang and Chen, 2014, Oms-Oliu et al., 2010, RamosVillarroel et al., 2011). Moreover, HILP treatment does not result in the development of
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volatile organic compounds or suspended airborne particulates. It is cost effective and
generally does not change the “special” characteristics of food matrices (Luksiene et al.,
2007).
However, there are some disadvantages that limit the HILP application to the fresh
produce industry. One issue is that HILP treatment causes substantial heating of the
samples, which might damage the quality of fresh produce. Another issue is that
microorganisms on an opaque food surface must directly face the HILP-strobe in order
to be inactivated due to the shallow penetration depth of HILP. In addition, samples
positioned in different parts of the HILP chamber (figures 1.4.4.2.1, 1.4.4.2.2) might be
exposed to different doses of HILP (Huang and Chen, 2014).
Figure 1.4.4.2.1: Equipment of HILP (www.xenoncorp.com)
Figure 1.4.4.2.2: Internal Part of pulsed Light with a Data Logger (www.xenoncorp.com).
1.4.4.3 Near UV-Vis Light (NUV-Vis)
Novel technologies utilizing visible wavelengths of light, in the violet/blue region of the
electromagnetic spectrum induces a phenomenon called “photodynamic inactivation
(PDI)”. Traditionally PDI has involved the use of dyes and other exogenous
photosensitiser molecules coupled with light exposure to induce inactivation of
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microorganisms, but recently natural photosensitiser molecules, particularly porphyrins
endogenous within microbial cells have been targeted (Murdoch et al., 2013).
NUV-vis light 395±5 nm (figure 1.4.4.3.1) is a safe, non-UV based disinfection
technology which is thought to act by stimulating endogenous microbial porphyrin
molecules to produce oxidizing reactive oxygen species (ROS), predominantly singlet
oxygen (1O 2 ) that damages cells leading to microbial death (Elman and Lebzelter, 2004,
Feuerstein et al. 2005, Lipovsky et al. 2010, Maclean et al. 2008b, Murdoch et al., 2012).
Exposure of microorganisms to visible light particularly at wavelengths of 405 nm, has
been shown to be effective in inactivating a range of bacteria, including Gram- positive
and Gram-negative bacterial species and antibiotic-resistant microorganisms such as
Methicillin-resistant Staphylococcus aureus, and its use has been suggested for a range
of decontamination applications (Dai et al. 2012, Dai et al., 2013, Enwemeka et al. 2008,
Guffey and Wilborn 2006, Maclean et al. 2008a, Maclean et al., 2009, Maclean et al.,
2010, Murdoch et al. 2012).
Figure 1.4.4.3.1: High intensity near ultraviolet/visible (NUV–vis) 395±5 nm light unit
(Haughton et al., 2012).
1.4.4.4 Ultrasound
Power ultrasound consists of pressure waves with a frequency from 20 kHz to 10 MHz
(Brondum et al., 1998, Butz and Tauscher, 2002). These cyclic sound pressure waves
have a frequency beyond the upper limit of human hearing. Ultrasound can be classified
to: a) low intensity ultrasound with a frequency range of 5-10 MHz with sound
intensities in the range of 0.1 to 1 W/cm2 (diagnostic ultrasound) and b) high intensity
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ultrasound with a frequency range of 20-100 kHz and a sound intensity ranging from 10
to 1,000 W/cm2 (McClements, 1995). Higher power ultrasound at lower frequencies is
referred as power ultrasound (Piyasena et al., 2003) which has been recognized as a
promising non-thermal processing technology, which can replace or complement
conventional thermal treatment in the food industry.
Ultrasonic apparatuses are divided to three categories. All of them have their advantages
and disadvantages which are summarized in table 1.4.4.4.1 and are shown in figures
1.4.4.4.1, 1.4.4.4.2, 1.4.4.4.3.
Table 1.4.4.4.1: Advantages-Disadvantages of electrochemical ultrasonic apparatuses (Zhou,
2010)
Figure
1.4.4.4.1:
Ultrasonic Cleaning Bath
(Elma-ultrasonic.com)
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Figure
1.4.4.4.2:
Ultrasonic Probe or horn
(www.hielscher.com)
Figure
1.4.4.4.3:
Ultrasound
cup-horn
(www.hielscher.com)
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
The mode of action of ultrasound is based on cavitation phenomenon. The general mode
of action is based on bubbles which pass through the liquid solution and create a series
of compression/rarefaction (expansion/collapse) cycles creating a negative pressure
affecting the molecules of the liquid. When the distance between the molecules exceeds
the minimum molecular distance, the liquid breaks down and a void is formed. In
successive cycles, voids or cavities continuously grow with a small amount of vapor
from the liquid (Bilek and Turantas, 2013). There are two types of cavitation: transient
and stable cavitation.
Transient cavitation is based on bubbles which are nonstable and collapse quickly in a
very short time period and then disintegrate into a mass of smaller bubbles. Moreover,
they are produced when sound intensity exceeds 10 W/cm2. The radius of bubbles
expands to at least twice of their initial size, and then the bubbles collapse violently on
compression into smaller bubbles. No mass transfer through the bubble by diffusion of
gas is produced due to the short lifetime of transient bubbles, whereas evaporation and
condensation of liquid might take place. The generation of extremely high temperature
(5,000ᵒC) and pressure (2,000 atm) within these bubbles is believed to play an important
role, thus causing sonoluminescence. The subsequent release of pressure from bubble
implosion creates shock waves which may be responsible for surface cleaning and
disinfection. A powerfull inrush of liquid to fill the void occurs due to the sudden
collapse of the bubble. This in turn produces shear forces in the surrounding bulk liquid
(Bilek and Turantas, 2013, Lauternborn and Ohl, 1997, Lee et al., 2005, Mason et al.,
2002).
Stable cavitation is based on bubbles which are non-linear, have some equilibrium size
during pressure cycles, and form large bubble clouds. These bubbles happen at low
sound intensities (1-3 W/cm2), containing mainly gas and some vapour, which oscillate,
often nonlinearly, during many acoustic cycles. Mass and heat transfer of gas through
the bubble by diffusion of gas occurs due to the longer lifetime. The stable bubbles like
transient bubbles are also accompanied by evaporation and condensation of liquid. The
stable bubbles can be transformed into transient bubbles, but the violence of their
implosion will be less than that of the transient bubbles due to the cushion effect of gas.
Stable bubbles can also continue to grow, float to the liquid-gas interface and be
expelled to air, which is the process of ultrasonic degassing (Mason et al., 2002).
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The mechanism of microbial killing is mainly due to increase of permeability of
membranes and lost selectivity, thinning of cell membranes, localized heating (Suslick,
1998), and production of free radicals (Bilek and Turantas, 2013, Butz and Tauscher,
2002, Fellows, 2000, Piyasena, 2003). Moreover, the chemical effect of a 20 kHz
ultrasound unit is correlated to the increase of inactivation of microorganisms due to the
antimicrobial mechanisms of hydroxyl radicals (Bilek and Turantas, 2013, Butz and
Tauscher, 2002). Many studies have shown that the temperature increase which is
localized inside a collapsing bubble, generates hydroxyl radicals (Bilek and Turantas,
2013, Suslick, 1989). According to researchers, the hydroxyl radical (OH−) is able to
react with the sugar-phosphate backbone of the DNA chain and cause the secession of
the phosphate-ester bonds and breaks in the double strand microbial DNA (Bilek and
Turantas, 2013). The effectiveness of a power ultrasound treatment is influenced by
many factors, including the frequency and intensity of ultrasound, the solvent, gas type
and content in working medium, treatment temperature, geometry of the reactor,
uniformity of the acoustic field in the treatment chamber, and externally applied pressure
(Zhou, 2010). In general, increasing ultrasonic frequency results in a decrease in the
intensity of cavitation in liquids. Recently, surface decontamination of fresh produce
with ultrasound has gained attention of many researchers (Alexandre et al., 2011,
Sagong et al., 2011, Seymoor et al., 2001). The advantages of ultrasound over heat
pasteurisation include: reduction of flavour loss, greater homogeneity, and possible
energy savings.
1.4.5 Other Methods
1.4.5.1 Ozone
Ozone (O 3 ) is a triatomic form of oxygen and is characterized by a high oxidation
potential that conveys bactericidal and viricidal properties (Kim et al., 1999). Moreover,
ozone has also been effective against fungi and protozoa (Khadre et al., 2001). Ozone
results from the rearrangement of atoms when oxygen (O 2 ) molecules are subjected to
high-voltage electric discharge. The product is a bluish gas with a characteristic pungent
odour and strong oxidizing properties. It is an environmentally friendly antimicrobial
agent which can be used in both gas and liquid phases in the food industry as it does not
produce toxic byproducts (Kim et al., 1999). The inactivation of bacteria by ozone is
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performed through an oxidation reaction. Inactivation of bacteria by ozone is a complex
process because ozone attacks numerous cellular constituents including proteins,
unsaturated lipids and respiratory enzymes in cell membranes, peptidoglycans in cell
envelopes, enzymes and nucleic acids in the cytoplasm, and proteins and peptidoglycan
in spore coats and virus capsids (Cho et al., 2010). Ozone also reacts with amino acids
and modifies purine and pyrimidine bases in nucleic acid (Scott and Lesher, 1963).
Ozone produces single strand breaks in DNA which cause extensive breakdown of
DNA, resulting in loss of cell viability. Ito et al. (2005) proposed that ozone caused
DNA backbone cleavage, due to production of hydroxyradicals. Ozone has been
approved as safe (GRAS) for treatment of bottled water and as a sanitizer for process
trains in bottled water plants (FDA, 1995). In 2001, ozone was approved as an
antimicrobial agent in foods in the USA (USFDA, 2001). Scott and Lesher (1963) found
that treatment with ozone causes alteration in E. coli cell membrane permeability leading
to leakage of cell contents. Thanomsub et al. (2002) also supported that bacteria were
inactivated by ozone as their cell membrane was destructed.
1.4.5.2 Pulsed Electric Fields (PEF)
The basic theory of microbial inactivation caused by PEF is based on electroporation of
cell membranes, causing reversible or irreversible pore formation depending on the
electric field intensity. The application of high voltage electric field (5–80 kV/cm) in
short electric pulses (1–100 μs) is known to disrupt the cell membrane by generating a
potential difference across cell membranes high enough to cause the membranes to
“break down” (Jeyamkondan et al., 1999). This phenomenon is based on the formation
of pores (electroporation). This results in loss of the semipermeability properties of the
cell membranes, altering homeostasis and causing cell death (Teopfl et al., 2006). PEF
treatment is carried out in liquid media, usually in continuous mode. However,
permeabilization takes place only if a certain level of the electrical energy is exceeded.
In order to profit from the advantages of PEF as a non-thermal technology, the
temperature at every location in the chamber should remain low enough not to damage
the valuable nutritive and sensorial qualities (figure 1.4.4.6.1).
Hence, a detailed
knowledge of temperature as well as field strength distributions in the chamber is
necessary for an efficient application of PEF (Cullen, 2012).
PEF has also been successfully combined with other non-thermal technologies such as
UV irradiation to achieve bacterial inactivation in food beverages (Noci et al., 2009,
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Walkling-Ribeiro et al., 2008), or with US (Halpin et al., 2014). Many researchers have
studied the effect of PEF on food (Jeyamkondan et al., 1999, Teopfl et al.,2006).
Figure 1.4.4.6.1: Schematic Representation of PEF equipment (www.foodengineeringmag.com,
www.intechopen.com)
1.4.5.3 High Pressure Processing
High pressure processing is another non-thermal method where the food is subjected to
elevated pressures (in the range of 100–1000 MPa) (figure 1.4.4.7.1) to achieve
inactivation of microorganisms and enzymes, without the severe degradation effects
associated with flavor and nutrients (Ramos et al., 2013). The factors that are responsible
for the successful implementation of this method are pressure, treatment time, and types
of enzymes and/ or microorganisms (Guerrero-Beltrán et al., 2005).
Moreover, high quality products with a fresher taste are produced, as there is little heat
damage to nutrients or natural flavors and colors. This effect can be attributed to the use
of ambient or even chill temperatures (Ramos et al., 2013). The type of composition of
the food can play an important role on the response of microorganisms during pressure
treatment. Carbohydrates, proteins, lipids and other food constituents can confer a
protective effect (Garcia-Graells et al., 1999). This is probably due to the fact that, in
contrast to heat, HPP does not denature covalent bonds, which in turn leaves primary
protein structure largely unaffected (Murchie et al., 2005).
Many researchers have already involved HPP in fruit and vegetable products processing.
This treatment provides high quality food with higher safety and extended shelf-life,
while maintaining similar characteristics to fresh products (Guerrero-Beltrán et al.,
2005). Currently, the food industry has used HPP for products such as guacamole,
cooked RTE meats, tomato-based salsa, fruit juices, whole-shell oysters, and other
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shellfish (Patterson, 2005). HPP food products are available in the United States,
Europe, and Japan. Furthermore, combining HPP with other microbial agents such as
lacticin 3147, lactoperoxidase and nisin has shown a synergistic effect regarding bacteria
inactivation. The combination of HPP with alternative non-thermal treatments for use as
a combined hurdle technology has also possibilities of enhancing the synergistic effect
(Considine et al., 2008).
Figure 1.4.4.7.1: High pressure processing Unit (www.ibebvi.be)
1.4.5.4 Electrochemical (Cold Plasma) Method
An emerging antimicrobial non-thermal technology for decontamination is the use of
non-thermal ionized gases (cold gas plasma). Briefly, plasma is composed of gas
molecules, which have been dissociated by an energy input. It is constituted by photons,
electrons, positive and negative ions, atoms, free radicals and excited or non-excited
molecules that, when combined together, have the ability to inactivate microorganisms
(Fernández et al., 2012).
The action is based on the use of electricity and a carrier gas, such as air, oxygen,
nitrogen, or helium. The primary modes of action are due to UV light and reactive
chemical products of the cold plasma ionization process. More precisely, an electrical
current is applied between an anode and cathode in an electrolytic solution containing
water and a solution of highly electronegative anions. A mixture of oxygen and ozone is
produced at the anode. The advantages associated with this method are the use of lowvoltage DC current, no feed gas preparation, reduced equipment size, possible
generation of ozone at high concentration and generation in water. The degree of
inactivation can be affected by the type of microorganisms, the inactivation medium,
number of cells, operating gas mixture, gas flow, and physiological state of cells, among
others (Bermúdez-Aguirre and Barbosa-Cánovas, 2013). Studies on produce had shown
that cold plasma is highly effective on the removal of surface human pathogens, such as
E. coli O157:H7 and Salmonella spp. (Fernández et al., 2013, Misra et al., 2011, Wang
et al., 2012).
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1.4.6 Biological control
The use of bacteria, viruses, and bioengineered compounds (e.g. enzymes, bacteriocins)
can prevent infections in fresh produce (Castaneda-Ramirez et al., 2011). Nonpathogenic bacteria and natural microflora on fresh produce encourage and discourage
the survival and proliferation of pathogenic bacteria (Cooley et al., 2006, Gragg and
Brashears, 2010, Heaton and Jones, 2008). Natural microflora and plant pathology play
an important role on encouragement or discouragement of pathogens. Moreover,
symbiosis has a key role in pathogen increase (Allen et al., 2009). Finally, the use of
bacteriophages is another biological control method that is of importance.
Bacteriophages are viruses that attack bacteria, degrade extracellular polymeric
substances (Warning and Datta, 2013), and undergo either a lytic or lysogenic cycle. E.
coli phages have been shown to significantly reduce the bacterial loads on cantaloupes,
lettuce (Sharma et al., 2009), tomatoes, spinach, broccoli, and ground beef (Abuladze et
al., 2008).
1.5 Control of foodborne diseases
Although preventing bacterial attachment and growth on the surface of fresh produces is
nearly impossible, contamination can be controlled and reversed. Removal of bacteria
from produce without lowering product quality can be facilitated through: mechanical
removal, chemical death, biological control, or alternative disinfection methods.
Mechanically removing bacteria is facilitated through the scrub of the surface. However,
this method is not very effective against internalized bacteria. Chemical death is a very
common method of removal, however, its use is continuously reduced due to the
production of by-products. Finally, alternative disinfection technologies are used
nowadays due to their enhanced effect against microorganisms (Warning and Datta,
2013).
Foodborne diseases once they emerge, they spread very fast. Thus, ways of reducing and
preventing foodborne diseases must be addressed. Improving on-farm sanitation and
biosecurity, antimicrobial use, and other good agricultural practices (GAP) can be of
paramount importance (Tauxe, 2002).
Spoilage microorganisms can be introduced to the crop on the seed itself, during crop
growth in the field, during harvest and postharvest handling, or during storage and
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distribution. The soil-borne spoilage microbes that occur on produce are the same
microorganisms that are present on harvesting equipment, on handling and packaging
equipment, in the storage facilities, and on food contact surfaces throughout the food
distribution chain. Therefore, through the use of good agricultural practices (GAP), early
intervention measures can be taken during crop development and harvesting. Examples
of GAPs include foliar fungicide application in the field, cross-contamination prevention
measures in the packaging equipment and storage facilities, and use of postharvest
fungicides. In 1998, FDA published the Guide to Minimize Microbial Food Safety
Hazards for Fresh Fruits and Vegetables, recommending GAPs that growers, packers,
and shippers must implement in order to address the common microbiological hazards
that may be associated with their operations (FDA, 1998). GAPs can be implemented in:
water, manure and municipal biosolids, worker health and hygiene, sanitary facilities,
field sanitation, packing facilities sanitation, transportation and traceability (Tauxe,
2002).
Furthermore, hazard analysis-critical control point (HACCP) strategies, and controlling
contamination during transport and storage are important. Training and certification of
foodhandlers, as well as regular handwashing could prevent many infections (Tauxe,
2002).
1.5.1 Public Health Surveillance
Public health surveillance is based on the implementation of disease prevention
programs. Surveillance is defined as a systematic collection of reports of specific health
events as they occur in a population (Tauxe, 2002). Surveillance defines the current
magnitude and burden of a disease for which prevention measures are in place. It
identifies unusual clusters or outbreaks of the disease, in order to take actions to control
them. Surveillance also measures the impact of control and prevention efforts, and it
serves to reassure the public that this critical part of public safety is in place (figure
1.5.1.1). Surveillance may ensure that immediate control measures are implemented, and
it may also identify areas that need more applied research so that better control measures
can be developed (Tauxe, 2002).
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Figure 1.5.1.1: Cycle of the public health prevention (Tauxe, 2002).
It is true that published data of foodborne diseases is reported much too late after the
events have occurred. This is the reason for limited data existence (Soon et al., 2011).
However, it is known that complete data from large countries with a number of levels of
government are difficult to obtain, due to local/regional/county or state burocracy.
Furthermore, resources to conduct full traceback investigation are often limited (Todd,
1990). There may be substantial under-reporting in mild and common illnesses as most
individuals for example regard diarrhea as an inconvenience rather than a symptom of
disease, and hence may not consult the doctor. In addition, the general practitioner must
order a stool culture, the laboratory must identify the etiologic agent and report the
positive results to the local or state public health institution (Soon et al., 2011). Taking
these limitations into consideration, the actual number of cases that occur is likely to be
greater than the number of cases that are reported, and a surveillance pyramid is created
(figure 1.5.1.2).
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Figure 1.5.1.2: Surveillance pyramid. The number of illnesses reported to public health
department is limited compared to the total number of illnesses (Soon et al., 2011).
It is well understood from the surveillance pyramid that for every case that is reported, it
has been estimated that 38 cases of salmonellosis occur. Once the food is implicated in
an outbreak, then a detailed review of its production process can reveal the points of
possible contamination sources. As a consequence, a multi-disciplinary approach is
needed by risk assessors, in order to identify as soon as possible the hazard, thus
developing strategies for eliminating it (Soon et al., 2011).
1.5.2 Food Legislation
Fresh cut RTE fruits and vegetables must adhere to the food laws of the country where
they are grown, harvested, processed, transported, and sold to consumers by caterers and
retailers. The applicable food law is important when they are subjected to be traded
internationally. The Codex Alimentarius Commission (CAC) is the international
intergovernmental organization for food standards, guidelines, and recommended
practices. Special attention must be given to good practices, especially to Good
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Agricultural Practices (GAPs) and Good Manufacturing Practices (GMPs), for the
successive stages in the food production chain. Good Hygienic Practices (GHPs) follow
the produce from the beginning to the end of the production chain. Systems based on the
Hazard Analysis and Critical Control Point (HACCP) principles are also applicable
throughout the production chain. Local Governments publish also guidance documents
to explain their legislation (Martín-Belloso and Soliva-Fortuny, 2011).
Generally, RTE foodstuffs should not contain microorganisms, toxins or metabolites in
quantities that present an unacceptable risk for human health. In order to contribute to
the protection of public health, Commission European Regulation (EC) No 2073/2005
establishes harmonized microbiological criteria for microorganisms, their toxins or
metabolites in certain foodstuffs and includes a number of implementing rules. Food
business operators at all stages of the food chain, i.e. primary production, processing,
manufacturing, distribution, retail and catering must comply with the relevant criteria.
Generally, compliance with Regulation on the Hygiene of Foodstuffs EC No.852/2004
as well as with the Directive 2000/13/EC, is of paramount importance.
1.5.3 Guidelines for the microbiological quality of RTE foods
in Greece
Due to the fact that RTE food is consumed in the same state as that in which it is
produced, sold and distributed, it must be given special attention to the microbiological
criteria. Enterobacteriaceae are useful indicators of hygiene and of post-processing
contamination of foods. Their presence in high numbers in RTE foods indicates that an
unacceptable level of contamination has occurred or there has been under processing
(e.g. inadequate cooking). The presence of E. coli in RTE foods is undesirable because it
indicates poor hygienic conditions which have led to contamination or inadequate heat
treatment. E. coli and Enterobacteriaceae are used as indicators of faecal contamination
of RTE foods (EC. No 2073/2005). Ideally E. coli should not be detected in RTE foods,
however, levels of E. coli exceeding 1000 CFU/g, are unacceptable and indicates a level
of severe contamination. Furthermore, contamination of RTE foods with coagulasepositive staphylococci is a result of human contact. Contamination should be minimized
through good food handling practices and growth of the organism prevented through
adequate temperature controls. The presence of coagulase-positive staphylococci is
considered as potentially hazardous, as this level of contamination may result in food
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borne illness. RTE foods should be free of Salmonella and Listeria (absent in fruits and
vegetables in a sample of 25g), as consumption of food containing these pathogens may
result in food borne illness (EC. No 2073/2005).
1.5.4 Predictive Models-Risk Assessment Support Systems and
Public Health
In a processing operation, the basic principles of GMPs, HACCP, sanitation and
documented operating procedures are commonly employed to ensure the production of
safe products (FDA, 2006). The complexity of food systems and the large number of
“critical points” in food production chain impose the necessity of the development of
mathematical models for the prediction of food safety as well as the prevention of
contamination. New practices, predictive models, methods and valuable tools have been
emerging as complements to decisions taken in Food Science problems (FDA, 2006).
Several types of models are used ranging from qualitative (e.g., tree structure) to
quantitative (e.g., microbial growth models) (Wijtzes et al., 1998). Prototype dynamic
models which describe the growth and inactivation of a microbial population as a
function of time and temperature have already been presented by Baranyi et al. (1996)
and Van Impe et al. (1992).
Quantitative microbiological risk assessment (QMRA), predictive modeling (PM) and
Hazard Analysis Critical Control Points (HACCP) have gained increased attention in
food microbiology recently. Structures and tools have been created in order to ensure
food safety by evaluating the safety of foodstuffs and predicting the effects of
intervention measures in food production processes. HACCP is typically linked to
industrial processes, whereas QMRA is more often used for public health purposes, in
order to elucidate ‘farm to table’ models. HACCP system and QMRA studies study the
potential bacterial growth and incorporate predictive food microbiology models.
Moreover, PM can quantify the increase or decrease of bacterial population sizes (Nauta,
2002).
QMRA is the quantitative estimation of the risks posed to public health when food and
pathogen are combined (Oscar, 2011). QMRA can be a useful tool in the development of
scientific-based strategies to manage risks and safeguard public health. QMRA is based
on: 1) hazard identification, 2) exposure assessment, 3) hazard characterization and 4)
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risk characterization (Codex, 1999). The development of QMRA models focusing on
fresh produce is important because RTE vegetables are mostly eaten raw, without a
definitive cooking step before consumption.
Modeling bacterial survival curves have become an important issue nowadays, due to
the increasing use of mild heat treatments for food products which have to guarantee the
safety of the products and due to the increasing use of risk analyses aiming to offer a
better control of the foodborne diseases (Geeraerd et al., 2000). Most microbial survival
curves have a non-log linear behavior. Availability of effective survival models is
needed if unbiased estimates of the probability of cross contamination are to be
estimated (Pérez-Rodríguez et al., 2013).
Predictive models are excellent tools for assessing and controlling food safety,
particularly when dynamic conditions are used (Ding et al., 2010). In the study of
Koseki and Isobe (2005), the Baranyi model (Baranyi and Roberts, 1994) and the
Ratkowsky model (Ratkowsky et al., 1982) were used in order to estimate the E. coli
O157:H7 growth on non-packaged iceberg lettuce, and to predict growth parameters
such as maximum growth rate, latent phase and maximum density of population, as a
function of temperature (5-25°C) (Posada-Izquierdo et al., 2013). Posada-Izquierdo et al.
(2013) have proposed a model which permits predictions over a wide range of
temperatures and also incorporates variability, thereby making it suitable for
Quantitative Risk Assessment (QRA) studies.McKellar and Delaquis (2011) have
developed a secondary death growth model based on data from different studies dealing
with E. coli O157:H7 growth in leafy vegetables. Predictive models that consider the
influence of stresses such as washing in chlorinated water of MAP would provide more
accurate and realistic estimates of risk (Posada-Izquierdo et al., 2013).
Meanwhile, Decision Support Systems (DSS) in the field of Food Science require
flexibility, autonomy, intelligence, reliability but above all should be trusted by people
related to Food Science. To fulfil all these diverse and difficult requirements, food
scientists investigate new models and techniques that will integrate and combine known
advanced theories and new techniques that will be the core of these sophisticated
systems (Halder et al., 2011). A Decision Support System (DSS) is defined as any
interactive computer - based support system for making decisions in any complex
system, when individuals or a team of people are trying to solve unstructured problems
on an uncertain environment. DSS are especially valuable in situations in which the
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amount of “scientific data’’ is prohibitive for the “human decision maker’’ to precede in
solving difficult problems (Groumpos, 2010). Advanced DSS can aid human cognitive
deficiencies by integrating various methodologies and tools utilizing a number of
different information sources in order to reach “acceptable decisions”. The benefits in
using DSS are that they increase efficiency, productivity, competitiveness, and offer cost
effectiveness and high reliability. This could give to a food science business a
comparative advantage over other competitors (Groumpos and Stylios, 2000).
Fuzzy Cognitive Maps are a combination of methods of fuzzy logic (FL) and neural
networks. Fuzzy logic develops multi-valued, non-numeric linguistic variables for
modelling human reasoning in an imprecise environment. FL has been applied in solving
problems in crop management, soil and water, food quality and safety, animal health and
behaviour, agricultural vehicle control, precision agriculture, greenhouse control,
agricultural machinery, food processing, air quality and pollution, agricultural facilities,
agricultural robotics, chemical application, and others such as natural resources
management and agricultural product design. Artificial Neural Networks (ANNs)
provide a way to emulate biological neurons to solve complex problems in the same
manner as the human brain. ANNs have the largest body of applications in agricultural
and biological engineering when compared with other soft computing techniques. ANNs
have been applied in solving problems in food quality and safety, crop, soil and water,
precision agriculture, animal management, post-harvest, food processing, greenhouse
control, agricultural vehicle control, agricultural machinery (Huang et al., 2010).
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AIM OF THE STUDY
The Health promotion and foodborne disease prevention remain important issues in the
21st century. Many outbreaks are reported and contaminated product recalls continue to
occur. It is, therefore, very important to eliminate pathogens from foods because of the
high risk, fatality rate, and economic burden of diseases (e.g listeriosis, salmonellosis
etc) caused by principal foodborne pathogens like E. coli, S. aureus, S. Enteritidis, L.
monocytogenes and HAV virus. Consumers nowadays prefer RTE foods, due to many
advantages that they offer. Moreover, Mediterranean diet based on consumption of fruits
and vegetables on an everyday basis remains popular. However, fruits and vegetables are
associated with a number of illness outbreaks of human pathogens. Outbreaks of E. coli,
S. aureus, S. Enteritidis, L. monocytogenes infections have been found to be associated
with consumption of RTE lettuce, strawberries and tomatoes.
The aim of the present study was to study the effect of different emerging, sustainable
disinfection technologies on the decontamination of artificially inoculated RTE foods.
The scope was to evaluate their effectiveness as promising technologies to be used by
food industries with the final insight to assure public health.
In the first part of the dissertation, an in vitro initial experiment took place. Liquids
inoculated with indicator microorganisms (E. coli and L. innocua), -one Gram positive
and one Gram megative-, representing potentially foodborne pathogens, were treated
with alternative, non-thermal technologies for different treatment times. Three light
technologies (NUV-Vis, Continuous UV, HILP) were used. In addition the disinfection
efficiencies of Gram negative and Gram positive microorganisms when different light
methods, as well as different dosages and treatment times of each method were used.
In the second part, the disinfection efficiency of minimally processed RTE fruits and
vegetables (lettuce, strawberry, cherry tomatoes) inoculated with the indicator pathogens
(E. coli, S. aureus, S. Enteritidis, L. innocua and HAdV) were investigated. The main
objective of this study was to study the effectiveness of different chemical sanitizers on
their capacity to adequately disinfect RTE foods. For this reason, conventional and
alternative disinfection technologies were used. Immersions in NaOCL solutions of a
low and a high concentrations with additional physical hurdles (UV, US), as well as
combinations of the above treatments were used. Moreover, different initial
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concentrations of the above microorganisms were inoculated, in order to evaluate the
disinfection efficiency of the above technologies. For adenovirus, culture assays were
performed in order to confirm the results obtained with PCR assay. Finally, after the use
of selected disinfection technologies, storage at 6°C of the above products followed for
15 days, and analysis of RTE produces were conducted at day 3rd, 7th and 15th.
In the third part of the phD thesis, the effect of the treatments on selected quality (color)
and physicochemical characteristics (total antioxidant capacity, total phenolic content,
ascorbic acid concentration) of the above RTE foods were investigated. The above
characteristics were measured before and after the treatments in order to evaluate their
possible change.
In the fourth part, a predictive model based on Decision Support Systems (DSS) in the
field of Food Science was constructed, in order to take decisions in the complex system
of a vertical lettuce company, where individuals or a team of people are trying to solve
unstructured problems on an uncertain environment. The DSS is based on Fuzzy
Cognitive Maps are a combination of methods of fuzzy logic (FL) and neural networks.
The beneficial tool will be of importance as it is a way to enhance the efficiency,
productivity, competitiveness of any production company as well as offer cost
effectiveness and high reliability. This risk assessment model could be a valuable tool to
a food science business as a comparative advantage over other competitors exists.
Finally, after gathering all the above results, and taking into account that preventing
bacterial attachment and growth on the surface of fresh produces is nearly impossible,
contamination can be controlled and reversed. Thus conclusions of assessment and
evaluation of the proposed disinfection technologies, based on infective doses for each
pathogen were taken into consideration, in order to assure public health.
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Chapter 2. MATERIALS AND METHODS
During the first experimental approach, in vitro experiments with liquids (MRD
solutions) inoculated with E. coli and L.innocua were conducted. Then, disinfection with
three light technologies (NUV, UV, HILP) took place and microbiological analysis
followed for enumeration of bacteria in the treated samples.
At the second experimental approach, RTE foods were used for the experiments.
Romaine lettuce, strawberries and cherry tomatoes were artificially inoculated with a
cocktail of bacteria (E. coli, S. aureus, S. Enteritidis, L. innocua) and virus (HAdV-35)
and then conventional, alternative and combined disinfection technologies followed for
different treatment times. Furthermore, the disinfection capacity was tested when
different concentrations of microorganisms were used. For adenovirus, culture assay was
used in order to confirm the results obtained with PCR. Moreover, the effect of storage
of the above treated RTE foods on their microbial load was evaluated for a period of 15
days.
During the third experimental approach, quality and physicochemical characteristics of
the treated RTE foods were evaluated. Color, TAC, TPC and AA content were tested
before and after the disinfection treatments.
The computerized model that was then proposed, as a fourth approach, was based on a
Decision Support System (DSS) that used the decisions of three Experts for the effect of
critical control points on a final safe product. The DSS is based on Fuzzy Cognitive
Maps, which are a combination of methods of fuzzy logic (FL) and neural networks.
All the results obtained throughout the study were evaluated for their significance with
different tests using SPSS 21.0 program.
During the experimental approaches, lab coat and gloves were mandatory, as they
assured that aseptic conditions were kept throughout the experiments. Moreover, the
RTE foods were kept in refrigeration in protective bags before, during and after the
experiments.
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2.1 In Vitro Experiments with 3 Light Technologies
2.1.1 Equipment
Equipment
Origin (Model, Company)
UV Chamber
Baro Applied Technology, Ireland
NUV-Vis Unit
AP Technologies, Bath, UK
HILP Unit
Xenon, USA
K-Type Thermocouple
Grant Instruments, Cambridge, UK
Grant Data Logger
Grant Instruments, Cambridge, UK
Incubator
Grant Instruments, Cambridge, UK
Colony Counter
IUL Instruments, Spain
Fridge
Tricity Bendix, UK
Freezer
Tricity Bendix, UK
Centrifuge
Beckman J2-HS, USA
Electronic Balance
Denver S-2002, Germany
Autoclave
Rodwell SS14 3SD, Biosciences, UK
Vortex
Genie 2, Scientific Industries, INC, K
2.1.2 Disposables- Plasticwares
Disposables-Glass-Plasticwares
Origin (Company)
Bottle top Dispenser
Pipettes (10 μL, 100 μL, 200 μL, 1 mL)
Micropipette plastic tips (1000 μL, 200 μL and 10 μL)
Gloves
Centrifuge and microcentrifuge tubes/bottles (1.5 mL, 15 mL, 50 mL)
Eppendorf
Sarstedt
VWR
Sarstedt
Gosselin, Villeurbanne,
France
Sarstedt
-
Stomacher bags
Glass tubes
Petri dishes 90mm diameter
Sterile glass spreaders
Beakers (600 mL, 1 mL)
Volumetric flasks (50 mL, 100 mL)
Conical flasks (500 mL, 1 L, 2 L)
Sterile Food Pincers
Pyrex Bottles (500 mL, 1 L)
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2.1.3 Culture Media
2.1.3.1 Selective Medium for E. coli (Tryptone Bile X-Glucuronide Medium (TBX,
Oxoid)
36.6 g of TBX Medium were suspended in 1 L of distilled water and boiled to dissolve
the medium. Then the medium was sterilized by autoclaving at 121°C for 15-min. The
medium was then cooled to 50°C until it was finally poured into sterile Petri dishes. The
plates were stored at 4°C.
2.1.3.2 Selective Medium for L. innocua (Listeria Selective Agar (Oxford
Formulation, Oxoid)
27.75 g of the Listeria Selective Agar Base (Oxford Formulation) were suspended in 500
ml of distilled water. The solution was dissolved gently to the boil point. It was then
sterilized by autoclaving it at 121°C for 15-min. It was then cooled to 50°C and
aseptically the content of one vial of Listeria Selective Supplement (Oxford
Formulation) was added, after diluting it in 50% ethanol:water solution. It was mixed
well and poured into sterile Petri dishes. The plates were stored at 4°C.
2.1.4 Solutions for microbiological analysis
2.1.4.1 Tryptone Soya Broth (TSB, Merck)
30 g were added to 1 L of water (purified), mixed well and distributed into final
containers. The medium was then sterilized by autoclaving at 121°C for 15 min. The
broth was stored at 4°C.
2.1.4.2 MRD (Maximum Recovery Diluent, Oxoid)
9.5 g of MRD were dissolved in 1 L of distilled water. The solution was dispensed into
the final containers and sterilized by autoclaving at 121°C for 15 min.
2.1.4.3 Microorganisms and Culture Preparation
Experiments were conducted using E. coli K12 (DSM 1607) and L. innocua (NCTC
11288). For inoculation of the model solutions, cultures of E. coli or L. innocua grown
overnight at 37°C in Tryptone Soya Broth, TSB (Oxoid) were used. The 24 h cultures
were then centrifuged for 10 min at 10,000 x g and the resulting pellets were washed and
Page 99
Materials and Methods
centrifuged twice in Maximum Recovery Diluent (MRD, Oxoid) before being mixed
together by resuspending in a final volume of 10 mL MRD. This resulted in mixed
culture cell suspensions of ~108 colony forming units per milliliter (CFU/mL). The
suspensions containing both E. coli and L.innocua inoculates were assessed for
susceptibility to three light technologies in a liquid matrix (MRD). Samples (10 ml) were
then placed into Petri dishes (50 mm diameter). After removal of covers, Petri dishes
containing the MRD solutions were subjected to different light equipments.
2.1.5 Disinfection Light Treatments
2.1.5.1 High intensity NUV–vis light unit
The NUV–vis light was produced by a light-emitting diode (LED) array (OD-2049)
(Opto Diode Corp) with a central wavelength of 395±5 nm, a bandwidth of 12 nm fullwidth at half maximum (FWHM) and a half intensity beam angle of 30°. The irradiance
(J * cm-2) of light emitted from the LED unit was measured using a UV–VIS Radiometer
(model no. RM12, Dr. Gröbel UV Electronik, GmbH, Ettlington, Germany) fitted with a
RM UV-A sensor (part no. 811030, Dr. Gröbel UV Electronik) (figure 2.1.5.1.1).
Distances of 3, 12 and 23 cm from the light source were chosen for treatments. The
corresponding energy intensities and time needed to achieve them are presented in table
2.1.5.3.1. Sample temperatures were measured during the treatment using a K-type
thermocouple attached to a Grant Data Logger to ensure that the maximum temperature
reached was non-lethal to the bacteria under the treatment times investigated (<50°C).
Figure 2.1.5.1.1: Schematic representation of NUV Vis Equipment
2.1.5.2 Continuous UV Equipment
The UV unit was a custom-made unit with intimal dimensions (length × width × height)
of 790 × 390 × 345 mm and consisting of four 95-W bulbs (Baro) 500 mm in length
(figures 2.1.5.2.1, 2.1.5.2.2). The UV dosages (J/cm2) were varied by altering the
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Birmpa Angeliki
distance of the sample (6.5, 17, and 28.5 cm) from the light source and by changing the
treatment time (table 2.1.5.3.1). Sample temperatures were measured during the
treatment using a K-type thermocouple attached to a Grant Data Logger to ensure that
the maximum temperature reached was nonlethal to the bacteria under the treatment
times investigated (<0˚C).
Figure 2.1.5.2.1: Layout of UV treatment unit; 2, safety interlock; 3, treatment chamber with
dimensions (length, width, and height) of 790 by 390 by 345 mm; 4, UV lights (95 W) 500 mm
in length.
Figure 2.1.5.2.2: Custom made UV treatment unit (outside and inside the chamber)
2.1.5.3 HILP (High Intensity Light Pulses) Unit
The HILP unit was a benchtop SteriPulse-XL system (Xenon, USA) (figure 2.1.5.3.1).
The system comprised a high-energy pulsed ultraviolet-visible flash lamp (Type C, 190
nm spectral cut-off point) delivering a maximum of 1.27 J/cm2. The pulse width
produced was 360 μs at a fixed pulse rate of 3 Hz. The pulse energy delivered to the
sample varied depending on its distance from the quartz window within the HILP
chamber. Distances of 2.5, 8, 11.5 and 14 cm were selected for treatments, in order to
achieve a wide spectrum of dosages varying between 0.18-106.2 J/cm2. The
corresponding dosages and time needed to achieve them are presented in table 2.1.5.3.1.
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Materials and Methods
During HILP treatment, samples were placed in an iced bath to minimize heating.
Sample temperatures were measured during the treatment using a K-type thermocouple
attached to a Grant Data Logger to ensure that the maximum temperature reached was
nonlethal to the bacteria under the treatment times investigated (<50˚C).
Figure 2.1.5.3.1: Representative Scheme of HILP Unit
NUV-VIS
23
UV
6.5
17
28.5
HILP
2.5
14
11.5
8
Distance from light source (cm)
12
3
Dose per treatment (J/cm2)
0.18
0.36
0.7
1.44
2.832
6
17.7
27
36
54
106.2
6
11
22
45
88
186
548
836
111
*
*
28
55
110
221
435
*
*
*
*
*
*
149
298
595
119
2341
*
*
*
*
*
*
30
60
120
240
472
*
*
*
*
*
*
36
72
144
288
566
*
*
*
*
*
*
45
90
180
360
708
*
*
*
*
*
*
NT
NT
NT
NT
0.8
NT
5
NT
NT
NT 30
0.1
0.2
0.4
0.8
NT
NT
NT
NT
NT
30
NT
0.3
0.6
NT
NT
5
NT
NT
30
NT NT
0.2
0.4
0.8
NT
NT
NT
NT
30
NT
NT NT
NT
Table 2.1.5.3.1: Calculated exposure time (sec) of non-thermal light technologies at selected
distances from the light source.(NUV-Vis: NUV–vis light; UV: Ultraviolet Light; HILP: High
Intensity Light Pulses, *Samples that were not analyzed due to high temperature, NT: Not tested
samples, due to inconsistency of correspondence of time and dose).
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2.1.6 Microbiological analysis
After treatment of liquid samples, the contents of each Petri dish were transferred to
sterile containers. Ten-fold dilution series were prepared in MRD and 0.1 mL of each
dilution was pour plated in duplicate using TBX (Oxoid) for E. coli and Listeria
Selective Agar (Oxford formulation, Oxoid) for L. innocua. The plates were incubated at
44˚C and 37˚C for 24 and 48 h respectively. Mean counts for each treatment were
calculated and converted to log 10 CFU/mL values with results for surviving numbers of
microorganisms in MRD expressed per mL (CFU/mL). The plates were then used to
enumerate viable cells in untreated controls and in samples following processing. The
survival of bacterial cells following illumination was monitored by counting their viable
number after exposure of the suspended bacteria to light. Bacterial cultures grown under
the same conditions but without light exposure served as controls. The results were
expressed as the logarithmic reduction (log N/N 0 ), where N 0 is the initial microbial load
and N the number remaining after treatment. All experiments were repeated at least three
times.
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Materials and Methods
2.2 Food Disinfection
2.2.1 Equipment
Equipment
Origin (Model, Company)
Ultrasound Bath
Ultraviolet Light
Ultraviolet Light Cabinet
Autoclave
Elmasonic P60, Elma
4 95-W lamps (Baero Hellas)
4 8-W lamps (Osram Germicidal G5)
Yamato Autoclave SM52
Stomacher
BagMixer, Interscience, St Nom la Bretêche,
France
Bag Rack
pH Meter
Refrigerator
Electronic Balance
Incubators
Real-time PCR platform
Waterbath
DNA/RNA UV-Cleaner
Colonies Counter
Vortex
Rocking platform
Incubator with CO 2
Dynal MPC-S Magnetic Particle Concentrator
Freezer
Block Heater
Refrigerated centrifuge(s) and rotor(s)
Biosafety cabinet class II
Centrifuge
Epifluorescence microscope
Interscience
Consort (C830)
Frigorex
GF 3000-EC A&D Instruments LTD
WTC Binder, Memmert
Stratagene MX 3005
GFL D3006
UVC/T-M-AR
WTC BZG 30
Scientific GE industries Bohemia n.y.11716
U.S.A Model K-550-GE
Environmental
Shaker-Incubator
ES-29
Biosan
Heal Force HF 90 Smart Cell
A13346, Invitrogen
Telstar Igloo
Stuart Scientific Bibby
Technolab Sigma 3K 30
Cytair 155, FluFrance
HermLe 2383 K
ZEISS
2.2.2 Disposables- Plasticwares
Disposables-Glass-Plasticwares
Origin (Company)
Automatic Pipet
Pipettes 10 μL, 100 μL, 200 μL, 1 mL
Micropipette plastic tips (1000 μL, 200 μL and 10 μL)
Gloves
Centrifuge and microcentrifuge tubes/bottles (1.5 mL, 15 mL,
50 mL)
Tubes (1.5 mL) with screw caps
Stomacher bags
Glass tubes
Petri dishes 90 mm diameter
Sterile glass spreaders
Beakers (600 mL, 1 L)
Pipetboy Plus, Technomara
Eppendorf
Sarstedt
VWR
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Sarstedt
Sarstedt
Gosselin, Villeurbanne, France
Sarstedt
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Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Disposables-Glass-Plasticwares
Origin (Company)
Volumetric flasks (50 mL, 100 mL)
Conical flasks (500 mL, 1 L, 2 L)
Sterile Food Pincers
96-well polypropylene plates
Optical adhesive covers
TC-12-well Plates with Lid
Pyrex Bottles (500 mL, 1 L)
Flasks
Agilent Technologies
Agilent Technologies
Cellstar, Greiner Bio-one
CellstarGreiner Bio-one
2.2.3 Culture Media
2.2.3.1 Selective Medium for E. coli (Tryptone Bile X-Glucuronide Medium (TBX),
Merck)
36.6 g of TBX Medium were suspended in 1 L of distilled water and boiled to dissolve
the medium. Then the medium was sterilized by autoclaving at 121°C for 15-min. The
medium was then cooled to 50°C until it was finally poured into sterile Petri dishes. The
plates were stored at 4°C.
2.2.3.2 Selective Medium for S. aureus (Baird-Parker Agar, Oxoid)
63 g were suspended in 1 L of distilled water and boiled to dissolve the medium. Then,
the medium was sterilized by autoclaving at 121°C for 15-min. Then the medium was
cooled to 50°C and were aseptically added 50 mL of Egg Yolk Tellurite Emulsion
(SR0054, Oxoid). The medium was finally mixed well before pouring into sterile Petri
Dishes. The plates were stored at 4°C.
2.2.3.3 Selective Medium for S. Enteritidis (Xylose-Lysine-Desoxycholate Agar
(XLD) Agar, Oxoid)
53 g were suspended in 1 L of distilled water. The medium was heated with frequent
agitation until boil. Then, it was transferred immediately to a water bath at 50°C.
Finally, it was poured into sterile Petri dishes as soon as the medium was cooled. The
plates were stored at 4°C.
2.2.3.4 Selective Medium for L. innocua (Listeria Selective Agar (Oxford
Formulation, Oxoid)
27.75 g of the Listeria Selective Agar Base (Oxford Formulation) were suspended in 500
mL of distilled water. The solution was dissolved gently to the boil point. It was then
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Materials and Methods
sterilized by autoclaving it at 121°C for 15-min. It was then cooled to 50°C and
aseptically the content of one vial of Listeria Selective Supplement (Oxford
Formulation) was added, after diluting it in 50% ethanol:water solution. It was mixed
well and poured into sterile Petri dishes. The plates were stored at 4°C.
2.2.4 Solutions for microbiological analysis
2.2.4.1 Tryptone Soya Broth (TSB, Merck)
30 g TSB were added to 1 L of water (purified), mixed well and distributed into final
containers. The medium was then sterilized by autoclaving at 121°C for 15-min. The
broth was stored at 4°C.
2.2.4.2 0.1% Peptone Saline Solution (Bacteriological Peptone (Oxoid)
8.5 g sodium chloride and 1 g bacteriological peptone were suspended in 1 L distilled
water. Then the pH was adjusted to 7.0±0.2. It was heated to dissolve the medium
completely. It was finally sterilized by autoclaving 121°C for 15-min. The solution was
stored at 4°C.
2.2.4.3 Buffered Peptone Water (Merck)
20.0 g of Buffered Peptone Water (ISO) were added to 1 L of distilled water. The
solution was mixed well and was distributed into final containers. It was sterilized by
autoclaving at 121°C for 15-min.
2.2.4.4 Sodium Hypochlorite Solutions
A low (50 ppm) and a high (200 ppm) concentration of sodium hypochlorite solution
was prepared, by using a stock NaOCl solution of 14%. To prepare a 50 ppm solution,
0.357 mL was added to 1 L of sterile water and the pH was adjusted to 6.5. For a 200
ppm solution 1.43 mL of stock solution was added to 1 L of sterile water and the pH was
adjusted to 6.5.
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2.2.5 Solutions for virus concentration
2.2.5.1 5X PEG/NaCl solution (50% (w/v) PEG 8000, 1.5M NaCl)
500 g PEG 8000 (Biochemica, AppliChem), 87 g NaCl (Sigma-Aldrich) and 450 mL
molecular grade water were added to a bottle. The solution was mixed with gentle
shaking/stirring until the solids were dissolved. Finally the final volume was adjusted to
1 L.
2.2.5.2 Chloroform:Butanol solution
Equal volumes of chloroform and butanol were added in a pyrex bottle. It was then
shaked to mix.
2.2.5.3 Tris Glycine 1% Beef Extract (TGBE) Buffer
12.1 g Tris base (Tris-hydroxymethylaminomethan, Merck), 3.8 g glycine (Glycine
Molecular biology Grade, AppliChem), 10 g beef extract powder (BBL, BD) and 1 L
molecular grade water were added to a bottle. They were mixed with stirring until the
solids are dissolved. The pH was adjusted to 9.5. It was then sterilized by autoclaving at
121°C for 15-min.
2.2.5.4 Phosphate Buffered Saline (PBS)
2 PBS tablets (Gibco) were added in 1 L deionised water. Using a magnetic stirrer, the
PBS tablets were dissolved in the deionised water. Finally, PBS solution was sterilized
in an autoclave at 121°C for 15-min.
2.2.6 Bacterial Strains
Bacterial strains used were Escherichia coli NCTC 9001, Staphylococcus aureus NCTC
6571, Salmonella Enteritidis NCTC 6676 and Listeria innocua NCTC 11288 (HPA,
Colingdale, U.K). Lenticules with the microorganisms were rehydrated in 9 mL of
peptone saline (0.1%) (Oxoid) and after 20-min, working cultures were streaked into
Tryptic Soy Agar (Oxoid), incubated at 37°C for 24 h, and stored at 4°C. Bacterial
strains were maintained as frozen stocks at -70ºC in the form of microorganism
protective beads (Mast DIagnostixa GmbH).
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Materials and Methods
2.2.7 Cell lines and virus Adeno-35 stock
Human adenovirus serotype 35stocks were cultivated in human lung carcinoma cell line
A549 cells. A549 cells were then cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM, Gibco, Grand Island, NY, US), containing 4,5 g/L D-Glucose, L-glutamine
and pyruvate supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco,
Grand Island, NY, US). A549 cells were cultured confluent (80-90%) in 175 cm3 flasks
at 37°C and 5% CO 2 , and infected with Adenoviruses serotype 35.Viruses were released
from cells by freezing and thawing the culturing flasks for 3 times. A centrifugation step
at 3000 × g for 20-min was applied to eliminate cell debris. The obtained supernatant
was ultracentrifuged for 1h at 34,500 × g and finally resuspended in PBS, quantified and
stored in 10 mL aliquots at −80 °C until used. The initial concentration of HAdV stock
suspensions were quantified by Real-Time PCR and were calculated as 108-109 genome
copies/mL.
2.2.8 Bacterial Preparation
Each bacterial type was cultured in 40 mL Tryptone Soya Broth (TSB) at 37°C for 18-20
h, harvested then by centrifugation at 4000 × g for 20-min at 4°C and washed three times
with buffered peptone water (BPW). The final pellets were resuspended in BPW,
corresponding to concentrations of approximately 107-108 CFU/mL, depending on
different microbe.
2.2.9 Sample Selection
A leafy green vegetable such as romaine lettuce (Lactuca sativa L. var. longifolia),
cherry tomatoes (Solanum lycopersicum var. cerasiforme) and strawberries (Fragaria x
ananassa) were selected as fresh ready to eat produces in order to study the level of
decontamination. These fresh produces were purchased from a local supermarket
(Patras, Greece) the day of the experiment and stored under refrigerated conditions (4ºC)
until the time of the experiment. As cherry tomatoes are concerned, a careful selection
was made in order to secure a uniform maturity stage and size of samples with a color of
light-red. Fruits with bruises, sign of infection or those different from the group were
discarded from the samples. Uniform, unblemished tomatoes having similar size and
color were finally selected.
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2.2.10 Sample preparation
All samples were rinsed with sterile water to remove some of the natural flora or any
other matter before treatment. For lettuce, two to three outer leaves were discarded and
the intact internal leaves were removed and weighted to give samples of 10 g. Likewise
pieces of whole strawberries without calyx were weighted to give final weight of 10 g.
Finally pieces of whole cherry tomatoes were weighted to give final weight of
approximately 10-11 g.
2.2.11 Bacterial Cocktail
Bacterial cocktails (E. coli, S. aureus, S. Enteritidis, L. innocua) were prepared by
mixing equal volumes of each bacterial type of high concentration in a 50 mL tube.
2.2.12 Sample Inoculation
A spot-inoculation method was used to inoculate the pathogenic bacteria on lettuce
leaves and strawberry pieces (Mahmoud, 2010). Briefly, 100 μL (10 drops) of bacterial
cocktail corresponding to 107-108 of each bacteria type was spotted with a micropipette
on the surface of each produce. The bacteria cocktail was evenly applied throughout the
skin surface of the strawberry, approximately midway between the calyx and cap (Bialka
et al., 2008). In lettuce, the cocktail was placed to the centre (abaxial) outer surface of
the lettuce, in order to simulate real conditions that can occur when contaminated
compost and irrigation water can be transferred to lettuce leaves (Oliveira et al., 2011).
100 μL (10 drops) of deionized sterile water was spotted on the surface of control
samples. To allow bacterial attachment, the samples were air dried on sterile aluminum
foil in a class II biosafety cabinet for 2 hours in 25°C prior to treatments. The fact that
the inoculums were attached to the vegetable surfaces was verified by comparing the
results with a control sample containing food only in BPW for 60-min. Whereas, a
dipping inoculation method was used to inoculate the pathogenic bacteria on cherry
tomatoes (Hadjok et al. 2008). Briefly, batches (10-11 g) of whole cherry tomatoes were
submerged in a beaker containing 300 mL of bacterial suspensions diluted in PBW
corresponding to 106–107 of each bacteria type and were agitated in a shaker for 1h. The
samples were then air-dried for 1h to allow bacterial attachment.
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Materials and Methods
2.2.13 Virus inoculation
The spot-inoculation method was used to inoculate the adenoviruses on the ready to eat
produces. Briefly, 100 μL (10 drops) of Adeno-35 corresponding to concentration of
108–109 infectious units/mL was spotted with a micropipette on 10 different areas of the
surface of each produce. After spiking, to allow viral attachment, the samples with
inocula were dried in a class II biosafety cabinet for 20-min at 22±2°C prior to
treatments.
2.2.14 Disinfection Treatments
2.2.14.1 Chlorine Treatment
For the chlorine treatment, the inoculated samples were immerged in a beaker containing
100 mL of chlorinated water (NaOCl solution) of 2 concentrations (50 ppm and 200
ppm) and were handed agitated for 3-min at 22±2°C. After that, the samples were
transferred to a beaker with sterile water and were left for another 3 minute period.
Finally, they were left on a sterile paper for drying. The sodium hypochlorite treatments
used are included in table 2.2.14.1.1.
Treatments
Sodium Hypochlorite (ppm)
Time (minutes)
1
50
1
2
50
3
3
50
5
4
200
1
5
200
3
6
200
5
Table 2.2.14.1.1: Sodium Hypochlorite Treatments selected throught the experiments.
2.2.14.2 Continuous UV Treatment
A UV cabinet with four UV-C (Osram Germicidal G5) lamps was used. The peak
emission of the lamps was 254 nm. The inoculated samples were placed in sterile petri
dishes and were placed at 8 cm distance from the lamps and treated for 1, 3, 5, 10, 20,
30, 45 and 60-min. The treatment was conducted at an intensity of 2 mW/cm2 at dosages
0.12, 0.36, 0.6, 1.2, 2.4, 3.6, 5.4 and 7.2 J/cm2. Throughout the experiments, the UV-C
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light intensity was kept constant, and the applied doses varied by altering the exposure
distance and time.
The UV dose (D) was calculated by using the equation 1 (1.4.4). The Intensity measured
in the UV chamber was 2 mW/cm2 in a 8 cm distance from the lamp. The Energy
(mJ/cm2) delivered was calculated according to the time (table 2.2.14.2.1).
Treatments
1
2
3
4
5
6
7
8
Intensity (mW/cm2)
2
2
2
2
2
2
2
2
Time (minutes)
1
3
5
10
20
30
45
60
Energy (J/cm2)
0,12
0.36
0.6
1.2
2.4
3.6
5.4
7.2
Table 2.2.14.2.1: Corresponding Energy-Intensity- Time for different UV treatments selected for
the experiments.
2.2.14.3 Ultrasound Treatment
For the Ultrasound treatment, a 5.75 L ultrasound tank (Elmasonic, Germany) was filled
with 3 L of distilled water and used at an operating frequency of 37 kHz and a power up
to 30 W/L. A glass beaker (600 mL) was placed in the US tank and filled with 9-fold
dilution of distilled water (figure 2.2.14.3.1). The ratio fruit (strawberry) or vegetable
(lettuce or cherry tomatoes) to distilled water for the ultrasound treatments was 1 part of
food (10 g) to 9-fold dilution of liquid. Inoculated lettuce leaves, strawberries and cherry
tomatoes were immersed in the glass beaker and processed with US at a constant
frequency of 37 kHz for 1, 3, 5, 10, 20, 30, 45 and 60-min. At least three replicates of
each treatment were performed (table 2.2.14.3.1).
Treatments
Ultrasound Frequency (kHz)
Time (minutes)
1
2
3
4
5
6
37
37
37
37
37
37
1
3
5
10
20
30
7
37
45
8
37
60
Table 2.2.14.3.1: Various ultrasound treatments for various treatment times.
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Materials and Methods
Figure 2.2.14.3.1: Ultrasound equipment outside and inside (Elmasonic P, Elma, Germany).
2.2.14.4 Combined Treatments
The combined treatments consisted of combinations of alternative technologies as well
as of combinations of alternative and conventional disinfection technologies (table
2.2.14.4.1).
Combined Disinfection Treatments
Treatments
Time(minutes)
Alternative+Conventional
Alternative+Conventional
Alternative+Conventional
Alternative+Conventional
Alternative+Conventional
Alternative+Conventional
Alternative+Conventional
Alternative+Conventional
Alternative+Conventional
Alternative+Conventional
Alternative+Conventional
Alternative+Conventional
Alternative+Alternative
Alternative+Alternative
Alternative+Alternative
UV+NaOCl
UV+NaOCl
UV+NaOCl
UV+NaOCl
UV+NaOCl
UV+NaOCl
US+NaOCl
US+NaOCl
US+NaOCl
US+NaOCl
US+NaOCl
US+NaOCl
UV+US
UV+US
UV+US
1+3
3+3
5+3
10+3
20+3
30+3
1+3
3+3
5+3
10+3
20+3
30+3
5+5
10+10
10+20
Alternative+Alternative
UV+US
20+10
Table 2.2.14.4.1: Combined Disinfection Treatments for various treatment times.
2.2.15 Storage conditions
After each treatment, samples were stored under refrigerated conditions (4±1 °C) in
refrigerator. The temperature of the fridge was monitored using a calibrated
thermometer. Samples were collected throughout different storage time intervals (3rd
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th
day, 7th day, 15 day) and analyzed in terms of bacterial populations for a total of 15
days storage.
Selected treatments (7 in total) were examined after 3 days of storage (3rd, 7th, 15th)
which were: UV (30-min), US (30-min), NaOCl 200 ppm (3-min), UV 30-min followed
by NaOCl 200 ppm 3 -min, US 30-min followed by NaOCl 200 ppm 3-min, UV 10-min
followed by US 20-min and UV 20-min followed by US 10-min.
2.2.16 Microbiological Analysis
For enumeration of bacteria, 10 g of treated lettuce, strawberry or cherry tomato sample
was transferred into a sterile stomacher bag containing 90 mL of Peptone Buffer water
(PBW) and homogenized in a stomacher for 2-min. One mL of the homogenized sample
was then 10-fold serially diluted in 9 mL of sterile PBW, and appropriate dilutions were
pour or spread-plated into appropriate selective media. All samples were analyzed
according to ISO standard methods (table 2.2.16.1).
Microorganisms
E. coli
S. aureus
S. Enteritidis
L. innocua
Selective
Culture
Media
TBX
Baird Parker
XLD
Oxford Listeria Agar
IncubationTemperature
ISO Method
44 ± 1 °C
37 ± 1 °C
37 ± 1 °C
37 ± 1 °C
16649-1:2001
6888-1:1999
6579:2002
11290:1996
Table 2.2.16.1: ISO Methods implemented throughout the experiments for E. coli, S. aureus, S.
Enteritidis and L. innocua enumeration.
2.2.17 Bacteria Enumeration
Reductions of bacteria were calculated on a per Gram of fruit and vegetable basis. Mean
counts for each treatment were calculated and converted to log 10 CFU/g values. The
results were then expressed as the logarithmic reduction (log N/N 0 ), where N 0 is the
initial microbial load and N the number remaining after treatment. All experiments were
repeated at least three times. Negative and Positive Controls were included.
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Materials and Methods
2.2.18 Analysis for Detection of Viruses
2.2.18.1 Virus concentration from fresh produce surfaces
The sample was processed by the method of Dubois et al. (2006) as described by
Kokkinos et al. (2012) with slight modifications.
•
Approximately 25 g of vegetable and fruit pieces were weighed and transferred
to a sterile beaker.
•
40 mL of TGBE buffer were added to each sample.
•
The samples were agitated at room temperature for 20-min by rocking at 60 rpm.
•
The eluate was decanted from the beaker through a strainer into one 50 mL or
two smaller centrifuge tubes.
•
The sample was centrifuged at 10,000 × g for 30-min at 4ºC. Then the
supernatant was decanted into a single clean tube.
•
The pH of the sample was adjusted to 7.2 with Hydrochloric acid (1 N and 0.1
N).
•
0.25 volumes of 5 × PEG/ NaCl solution were added and mixed by inversion,
then incubated with gentle rocking at 4ºC for 60-min.
•
The tube was then centrifuged at 10,000 × g for 30-min at 4ºC.
•
The supernatant was discarded.
•
It was centrifuged at 10,000 × g for 5-min at 4ºC to compact pellet.
•
Finally the pellet was resuspended in 500 μL PBS and 500 μL
chloroform:butanol solution (1:1) and mixed by vortexing.
•
It was allowed to stand for 5-min.
•
Finally it was centrifuged at 10,000 × g for 15-min at 4ºC and the aqueous phase
was transferred to a clean tube and stored at -20ºC.
2.2.18.2 Virus concentration from fruits
The procedure was the same with the vegetables. The only difference was indicated at
the initial phases. After 10-min of agitation at room temperature by rocking at 60 rpm
the pH of the eluate was checked. If the pH fall below 9.0 it was adjusted to 9.4 with
sodium hydroxide (4% w/v). The period of agitation was extended by 10-min for every
time the pH was adjusted. Then, the following procedure was the same and finally the
aqueous phase was transferred to a clean tube and stored at -20ºC.
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2.2.18.3 DNAseTreatment
An enzymatic digestion treatment was applied to reduce false positive results by
detection of free DNA when using qPCR analysis (Nuanualsuwan and Cliver, 2002).
Each analyzed sample was treated with DNase I (free – RNase) (DNase I, Molecular
Grade, Invitrogen), before DNA extraction to degrade DNA released from damaged
viral capsids, according to manufacturer's instructions, before the nucleic acid extraction.
Briefly, 400 μL of concentrated sample was added to 2.5 μL of DNase I and 97.5 μL
Reaction Buffer and incubated for 2 h at 37°C.
2.2.18.4 Nucleic Acid Extraction
The principle of the protocol used for Nucleic Acid Extraction is based on the Nuclisens
miniMAG (Biomerieux, Paris) using Boom technology and magnetic silica (figure
2.2.18.4.1). The steps followed for nucleic extraction are:
•
500 μL of the extract from soft fruits or vegetables were added into a clean
centrifuge tube.
•
4.5 mL of Nuclisens lysis buffer were added to the tube, and were mixed by
vortexing briefly.
•
The sample was incubated for 10-min at room temperature.
•
Centrifugation for 2-min at 1,500 × g was followed to ensure that entire sample
was brought down into the tube.
•
50 μL of well-mixed magnetic silica solution was added to the tube and mixed by
vortexing briefly.
•
Incubation for 10-min at room temperature followed.
•
Centrifugation for 2-min at 1,500 × g and then the supernatant was carefully
discarded.
•
400 μL wash buffer 1 was added and resuspension of the pellet by
pipetting/vortexing.
•
Transfer of suspension to a 1.5 mL screw-cap tube. (It was very important to
avoid creating foam at this stage. Very gentle pipetting should be done when
using wash-buffer 1, because of the GuSCN incorporated into it. This avoided
any loss of nucleic acids).
•
It was washed for 30 sec using the automated wash steps of the miniMAG
extraction systems.
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•
After washing silica was allowed to settle using magnet of the magnetic rack.
•
The supernatant was discarded.
•
Separation of the tubes from magnet followed.
•
400 μL of wash buffer 1 was added.
•
Pellet was resuspended and washed for 30 sec.
•
After second washing, silica was allowed to settle using magnet.
•
The supernatant was discarded.
•
Separation of the tubes from magnet.
•
500 μL wash buffer 2 were added.
•
Pellet was resuspended and washed for 30 sec.
•
Silica allowed tosettle using magnet.
•
The supernatant was discarded.
•
After this washing with Buffer 1, the total sample was transferred to a clean 1.5
mL tube to eliminate GTC residues.
•
Washing with Buffer 2 was repeated.
•
The tubes were separated from magnet.
•
500 μL wash Buffer 3 were added.
•
Washing for 15 sec was followed to allow silica to settle using magnet.
•
Supernatant was discarded.
•
50 μL elution buffer were added, and the tubes were transferred to thermoshaker
•
Incubation for 5-min at 60ºC was followed.
•
Tubes were placed in magnetic rack and allow silica to settle.
•
Transfer of the eluate to a clean tube.
•
Repetition of the step with elution Buffer (total volume of the eluate 100 μL).
•
The samples were retained at -80ºC for up to one week.
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Figure 2.2.18.4.1: Schematic Presentation of the procedure of Nucleic Acid Extraction
(Kokkinos et al., 2012).
2.2.18.5 PCR Quantification
The mix was prepared in DNA/RNA UV-Cleaner room according to the tables (tables
2.2.18.5.1 and 2.2.18.5.2).
Firstly, the stock volumes of primers and probes were
prepared (table 2.2.18.5.1).
Stock volume
H2O
Final
volume
Molarity
Primers (AdF, AdR)
225 μL
275 μL
500 μL
45 μΜ
Probe AdP1
56.25 μL
443.75 μL
500 μL
11.25 μΜ
Table 2.2.18.5.1: Working solutions of primers and probe.
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Then, the PCR mix was prepared according to the table 2.2.18.5.2.
Reagent
Working
Concentration
Final
Concentration
Volume
(μL)
Mix
2x
1x
12,5
Primer AdF
45
900 nM
0,5
Primer AdR
45
900 nM
0,5
Probe AdP1
11,25
225 nM
0,5
H2O
1
Total volume of PCR mix
15
Table 2.2.18.5.2: Volumes of reagents for PCR mix.
Once the mix has been prepared aliquots of 15 μL were added into each well. The total
volume for one reaction after addition of target was 25 μL (15 μL mix + 10 μL sample).
The samples were then added in duplicate in a separate area. DNA standard was added
as positive control (PAC) in duplicate. Finally, 10 μL of nuclease-free dd-water were
added in the Non-template control (NTC) wells. The assay included a NTC to prove mix
does not produce fluorescence. Whereas the PAC must be added to verify that the
reaction has worked and has not failed. The wells were then closed with adhesive cover.
The QPCR was performed in a real-time PCR platform, selecting the appropriate
parameters (considering the use of adhesive cover and the total volume in each well,
etc). Following activation of the UNG (2-min, 50°C) and activation of the AmpliTaq
Gold for 10-min at 95°C, 45 cycles (15 s at 95°C and 1 minute at 60°C) were performed.
Once the reaction is completed, the results are stored. The amount of DNA was
calculated as GC/mL and was converted to log 10 .
2.2.19 Evaluation of disinfection with different initial bacteria cocktail
The RTE foods were inoculated with different initial concentrations of bacteria and
virus. For bacteria cocktail experiments, serial dilutions of high cocktail inocula were
done, and then the RTE foods were inoculated with different initial concentrations.
Then, selected disinfection treatments followed and RTE foods were subsequently
microbiologically analyzed.
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2.2.20 Culture Assay for HAdV35
Briefly, A549 monolayers were incubated overnight in 12-well plates at 37 °C in 5%
CO 2 until they reached 90–100% of confluence. Thirty microliters (30 μL) of direct and
diluted samples were inoculated into each well and incubated for 90-min at 37 °C on a
shaking incubator. After that, the media with the inoculum was discarded and 1% FBSsupplemented with DPH (DMEM supplemented with hepes and pest). The flasks were
incubated for 3-4 days at 37 °C in 5% CO 2 . Finally, cells were observed under an
epifluorescence microscope for cytopathic effect. The theoretical detection limit of this
technique is 10 viral particles/mL and even viral particles that have lost their ability to
develop cytopathic effect may be still detected. The final result of each sample analyzed
was expressed as the geometric mean of the most probable number of cytopathic units
(MPNCU) per milliliter calculated for two independent replicates. All assays were
performed in triplicate and negative and positive controls were included.
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2.3 Food Quality parameters
2.3.1 Color Measurement
A colorimeter (CIE colorimeter) was used for color measurements. All the treated
samples were surface dried; they were positioned then in a plastic bag and held on ice
until all experiments have been completed. A lettuce, a strawberry piece and cherry
tomatoes (10 g) were then placed directly on the colorimeter sensor and measured in at
least three different points of the product. Three measurements were performed per
treatment and results were averaged. The L* parameter shows lightness to darkness and
ranges from 0 (black) to 100 (white). The a* parameter measures the degree of redness
(+a*) or greenness (-a*). The b* parameter indicates the degree of yellowness (+b*) or
blueness (-b*). The net color difference (ΔE*) and chroma or saturation index (C*) were
determined using L*, a* and b* values and were compared with the values of unprocessed
samples (Bermúdez-Aguirre and Barbosa-Cánovas, 2013). Finally, the Tomato Color
Index (TCI) and the Whiteness Index (WI) were calculated for cherry tomatoes and
lettuce respectively as described by Clément et al. (2008) and Obande et al. (2011). The
above calculations were calculated according to the following equations:
C*= (a*2 + b *2) ½
WI= 100 – [(100 - L*) 2 + a*2 + b*2]0.5
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2.3.2 Physicochemical Parameters
2.3.2.1 Equipment
Equipment
Origin (Model, Company)
Ultrasound Bath
Elmasonic P60, Elma
Absorption Spectrophotometer
Hitachi U-1900
Colorimeter
CIE Colorimeter
Electronic Balance
AND HR-300
pH meter
Consort (C830)
Waterbath
Edelstahl, RostFrei
Stirrer
Heidolph Ruhren
2.3.2.2 Disposables
Disposables, Glass-Plasticwares
Origin (Model, Company)
Burette
-
Spectrophotometer cuvettes
-
Plastic Tubes (15 mL, 50 mL)
Sarstedt
Conical Flasks
-
Filter papers
-
Pipettes 10 μL, 100 μL, 200 μL, 1 mL
Eppendorf
Micropipette plastic tips (1000 μL, 200 μL and 10 μL)
Sarstedt
2.3.2.3 Chemicals
Chemicals, Reagents
Origin (Company)
Acetone
Sodium Acetate
Acetic Acid Glacial 100%
TPTZ (2,4,6 Tris 2-Pyridyl-s-triazine)
FeCl3• 6H2O (Iron (III) chloride Hexahydrate)
Hydrochloric Acid Fuming 37%
Folin-Ciocalteau Reagent
Sodium Hydrogen Carbonate
Gallic Acid monohydrate
2,6 Dichlorophenolindophenol
Sodium salt hydrate
Meta-Phosphoric Acid
L-Ascorbic Acid
Sigma
Sigma
Merck
Sigma-Aldrich
AnalaR BDH
Merck
Merck
AnalaR
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
AnalaR BDH
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2.3.2.4 Methods
2.3.2.4.1 Samples extraction
The samples were extracted using 60% acetone (v/v) and distilled water. Each sample of
2 g was weighed into conical flask and 50 mL 60% acetone solution was added. The
samples were then placed in an ultrasound bath at temperature 30˚C for 2 hours. The
“extracts” were then filtered to obtain a clean solution. The extracts were finally left in a
dark place to cool.
2.3.2.4.2 Total antioxidant capacity
The total antioxidant capacity of samples was determined according to FRAP (Ferric
Reducing Antioxidant Power) of Benzie and Strain (1996), as introduced in section
1.2.1.1.1. This method is based on the reduction of Fe+3-TPTZ yellow complex to the
ferrous blue form (Fe+2) by the antioxidants of sample.
2.3.2.4.2.1 FRAP reagents
- Acetate buffer solution 0.3 Μ, pH=3.6.
In order to prepare this solution, 3.1 g of sodium acetate were mixed with 16 mL of
acetic acid and were diluted in 1 L of buffer solution with an adjusted pH. This solution
was prepared once and used throughout the experiments.
- Solution 40 mM HCl
1.6 mL of HCl Fuming 37% were added to 500 mL of water.
- Solution TPTZ 10 mM.
This solution was a solution of 10 mM TPTZ in 40 mM of HCL. This solution was
prepared every 2 days and was maintained in the fridge.
- Solution FeCl 3 •6H 2 0 20 mM
This solution was prepared by dilution of 1.3525g FeCl 3 •6H 2 0 in 250 mL of distilled
water. This solution was prepared once and was used throughout the experiments.
- FRAP reagent
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It was prepared by mixing 25 mL of acetate buffer solution, 2.5 mL of TPTZ solution
and 2.5 mL FeCl 3 solution. This solution was prepared on a daily basis, before the
experiments were carrying out. It was maintained in 37°C in a water bath. After the end
of experiments the solution FRAP was thrown away.
2.3.2.4.2.2 Total antioxidant capacity (TAC) determination
Briefly, 1 mL of sample was added to 5 mL FRAP solution and was left in a dark place
for 30-min. The absorbance was recorded after 30-min incubation at 595 nm. Control
samples were also measured. The results were based on a standard curve of
FeSO 4 •7H 2 0. The results were finally expressed in μmol Fe2+ equivalents per g of fresh
weight of food sample.
2.3.2.4.3 Total Phenolic Content (TPC) Determination
The determination of total phenolic content was measured according to the method of
Spanos and Wrolstad (1990) using Folin-Ciocalteau reagent. Briefly, 3 mL of each
filtered sample, plus 1 mL Folin-Ciocalteau reagent, plus 1mL sodium carbonate (7.5%)
were added. After incubation at room temperature in a dark place for 60-min, the
absorbance of the reaction mixture was measured at 765 nm against aquatic methanol
blank on a spectrophotometer. Standards were prepared from gallic acid in water. From
the standard curve, the total phenolic contents of samples were expressed as mg gallic
acid/g of fresh weight of food sample.
2.3.2.4.4 Determination of Vitamin C
The
determination
of
ascorbic
acid
was
made
by
titration
against
2.6-
dichlorophenolindophenol. The key point was to carry out the titration in an acid
environment and rapidly.
2.3.2.4.4.1 Reagents needed for the determination of Vitamin C:
- Ascorbic acid solution
Preparation of ascorbic acid solution 1 mg/mL in metaphosphoric acid solution.
-Metaphosphoric acid solution
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15 g HPO 3 were dissolved in 40 mL CH 3 COOH and 200 mL deionised water. Dilution
was made in 500 mL deionised water. HPO 3 was converted gradually to phosphoric
acid. This solution was unstable and was kept in fridge. It was thrown away after 1 week
in order to avoid the conversion into orthophosphoric acid.
- Standard solution 2.6 dichlorophenolindophenol
50 mg of meta sodium salt of 2.6 dichlorophenolindophenol were dissolved in 50 mL of
deionised water which had 42 mg NaHCO 3 content. The solution was diluted to 200 mL
water, then it was filtered from folded filter and it was kept in a dark bottle.
2.3.2.4.4.2 Ascorbic Acid determination
Food extracts (2 mL) were inserted in a conical flask. The titration was made rapidly
with standard solution of 2.6 dichlorophenolindophenol until a slight pink color
appeared. This pink color must be conserved for at least 5 seconds. The determination
was carried out in triplicates.
In the same time, the standard solution of ascorbic acid was titrated in order to find out
its content in ascorbic acid, more precisely in order to correspond the amount (mg) of
ascorbic acid to amount (1 mL) of pigment solution.
The Ascorbic Acid determination was made according to the following equation:
mg ascorbic acid/mL = (X – B) × (F/E) ×(V/Y)
X - average volume for test solution titration (mL)
B - average volume for test blank titration (mL)
F - mg ascorbic acid equivalent to 1.0 mL indophenol standard solution,
E - number of g assayed
V - volume of initial test solution
Y - volume of test solution titrated
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2.4 A user-friendly theoretical mathematical model for the
prediction of food safety in a food production chain
The purpose of the mathematical model was to focus on the construction and the use of
FCM in modelling a fit-for-purpose Decision Support System which diagnoses the
possibility of the cross-contamination of lettuce from production to the point of sale in a
vertical production company of vegetables.
2.4.1 Selection of critical points
Nine critical points were selected from three (3) experts, with different scientific
background, as they play a crucial role throughout the lettuce production procedure
(figure 2.4.1.1). The selection of critical points was based on background information
questionnaires, based on HACCP audit principles, which were completed for the
premise during the European FP7 project VITAL (Integrated Monitoring and Control of
Foodborne Viruses in European Food Supply Chains) (http://www.eurovital.org) aimed
to gather data on virus contamination with the aim of providing a basis for subsequent
quantitative viral risk assessment and recommendation of control measures. These
critical points were selected among others as the most important ones to be taken
seriously into consideration, in order to be able to estimate the infection risk for humans
through consumption of the leafy vegetables.
1. Labour, Manpower
Vegetable production is very labour intensive work which requires both dedication and
skill to effectively undertake it. Basic training in agronomic principles or experience in
the same field is very crucial. Moreover, hygiene training is of great importance.
Lettuce/leafy greens may be harvested mechanically or by hand and are almost always
consumed uncooked or raw. Because lettuce/leafy greens may be hand-harvested and
hand-sorted for quality, there are numerous “touch points” early in the supply chain and
a similar number of “touch points” later in the supply chain as the products are used in
foodservice or retail operations. Each of these “touch points” represents a potential
opportunity for cross-contamination (FDA, 2006). Emphasis in hand washing where
there is risk of contamination (e.g. before starting work, after using the bathroom, etc.)
must be given. Workers with any notifiable infectious disease must be excluded from
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work. Harvesting equipment (knifes) should be cleaned and/or sanitised daily. Suitable
protective clothing has to be worn by food workers, except for disposable gloves.
2. Quality and Safety of Food Systems
The existence of Good Agricultural Practices (Global, GAP) is a necessity in a vegetable
company. Internal and external auditing must be in place. Quality systems, i.e. ISO
22000, Sanitation Standard Operating Procedures (SSOPs) must be present throughout
the food supply chain. HACCP guidelines are also important so as to ensure the safe
production and handling of lettuce/leafy greens products from field to fork (FDA, 2006).
The Recommended International Code of Practice General Principles of Food Hygiene
(CAC, 2003) indicates that “Prior to application of HACCP to any sector of the food
chain, that sector should have in place prerequisite proGrams such as good hygienic
practices according to the Codex General Principles of Food Hygiene, the appropriate
Codes of Practice, and appropriate food safety requirements” (Garayoa, 2011).
3. Location-Surroundings of the growing field
Caution must be given with domestic animals which in primary production must not
have access or presence on the premises. Moreover, emphasis must be given to the place
of storage of raw manure and to the existence or not of any industrial, and/or farming
activity adjacent to the field. Fields that contain animal manure are more likely to be
contaminated with enteric pathogens because of their ability to survive in soils for
months or years (Doyle and Erickson, 2008). Faeces may naturally contain between 102
and 105 CFU/g E. coli and between 102 and 107 CFU/g Salmonella spp.
(Himathongkham et al., 1999, Olaimat and Holley, 2012).
4. Lettuce Nursery
The quality of lettuce nursery is of great importance. Melotto et al. (2006) reported that
phytopathogen infections, which occur frequently during field cultivation, could affect
the interaction between human pathogens and plants (Ge et al., 2013). Microbial and
chemical testing of products can be carried out in accredited laboratories on a scheduled
base and the results must be satisfactory. A labelling and traceability system as well as a
recall system must be in place.
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5. Produce Land
Lettuce grows best in fields that are level and well drained. Lettuce is highly sensitive to
salinity. High salinity causes one of the most widespread types of abiotic stress
worldwide, severely limiting crop productivity. Thus, the choice of soil with an
appropriate electrical conductivity is highly desirable. Land used for lettuce is often preirrigated before land preparation is completed to facilitate salt leaching. The variety and
seed selection as well as the seed handling are very important. The irrigation and the
cultivation procedure are key elements in the production of lettuce (Zhu, 2001).
6. Harvesting the crop
The handling technique that will be followed is important during the harvesting. Because
most lettuce undergoes little processing, great emphasis is placed on producing a high
quality product. It is essential that the product be free of pest damage and contamination
at harvest. Lettuce is open to contamination from a wide variety of sources that includes
manure amended soil, irrigation water, insects and wild animals (Warriner et al., 2009).
A wash step is applied in fresh-cut processing in an attempt to remove field acquired
contamination or at least prevent cross-contamination between batches (Barrera et al.,
2012, Gil et al., 2009, Nou et al., 2011). Lettuce trimming and coring-in-field (CIF) are
relatively recent industry developments designed to increase processing plant
production. This process significantly reduces shipping and waste disposal costs while
maintaining the market quality of lettuce (Brown and Rizzo, 2001). However, core
removal requires additional human handling per head in the field and exposes the
internal leaf tissues, increasing the risk of direct contamination, which is already high in
field environments (FAO/WHO, 2008). Cut leaf tissues, such as those resulting from
coring, provide a moist, nutrient rich environment especially conducive to direct and
rapid infiltration, and pathogen attachment, growth and survival (Takeuchi et al., 2000,
Yang et al., 2012).
7. Postharvest Processing
Field packaged lettuce can be packed "naked" in the carton, film-wrapped in perforated
or non-perforated cellophane; or bagged in perforated plastic bags. After harvest, the
lettuce is transported to a cooling shed and distribution centre where it is stored at low
temperatures and it must be shipped with 48 hours. Cross contamination combined with
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Materials and Methods
the growth of pathogens during storage are fundamental risk factors for listeriosis (Ding
et al., 2013, Hoelzer et al., 2012).
8. Transportation
The transport of fresh fruit and vegetables is a complicated topic. The equipment should
be maintained in good condition, and the cleaning frequency must be documented and
verified. The storage/carriage conditions afforded the produce should be such that
excessive water loss does not occur. Optimum transit temperature is around 0°C,
container temperature 1-2°C, and relative humidity 90-95%. Fast transportation with
minimum damage during shipment is vey important in successful marketing of
perishable. Although optimal storage temperature (0-2°C) is very useful for prolonging
the shelf-life of vegetables, the recommended temperature is not always maintained
during postharvest storage and normal temperature is usually found during the
transportation of lettuce from farm to retails since some of the transportation equipment
are open air vehicles (Ding et al., 2013, Yang et al., 2012).
9. Point of Sale
The lettuce packaging should be designed to preserve the content as fresh and safe as
possible. Its second function is to make the product look attractive to customers, using
colourful prints. In addition, cross contamination might be occurred during the period of
transport or storage in the market, restaurant and home (Ding et al., 2013). Moreover,
the final packing containers must be properly handled in order to prevent crosscontamination and be kept covered. The premises must be regularly cleaned according to
a documented cleaning plan.
C1. Labor, Manpower
C2. Quality and Safety of Food
Systems
C9. Point of Sale
C3. Location-Surroundings of
the growing field
C8. Transportation
C4. Lettuce Nursery
C7. Postharvest Processing
C5. Produce Land
C6. Harvesting the crop
Figure 2.4.1.1: Flow Chart of Lettuce/ Leafy Greens Production including 9 concepts (critical
points)
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2.4.2 Decision Making Support System in lettuce’s safety using fuzzy
cognitive maps
The concepts that were selected to be tested during the lettuce production procedure
were extracted from questionnaires that were filled from experts. The methodology
described extracts the knowledge from experts and exploits their experience of the
process. Each expert based on his/her experience knows the main factors that contribute
to the decision. Experts describe the existing relationship firstly as “negative” or
“positive” and secondly, as a degree of influence using a linguistic variable, such as
“low”, “medium”, “high” etc.
More specifically, the causal interrelationships among concepts are declared using the
variable influence which is interpreted as a linguistic variable taking values in the
universe of discourse U = [-1, 1]. Its term set T (influence) is suggested to be comprised
of nine variables. Using nine linguistic variables, an expert can describe the influence of
one concept on another in detail and can discern it between different degrees. The nine
variables used here are: T (influence) = {negatively very strong, negatively strong,
negatively medium, negatively weak, zero, positively weak, positively medium,
positively strong, and positively very strong}. With this method the purpose is to
diagnose and predict the effect of different factors during the lettuce production chain in
their contribution to a final safe fresh lettuce.
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STATISTICS
All experiments were carried out in triplicate. During each experiment two samples were
taken at any time to conduct microbial counts. The microbiological data were analyzed
in terms of log 10 (𝑁𝑁/𝑁𝑁 0 ), where 𝑁𝑁 is the microorganism load at a given time, and 𝑁𝑁 0
corresponds to the initial microbial load of untreated samples.
N is calculated from two successive dilutions using the following equation:
N=Σα / V (n 1 +0,1n 2 ) d
Σα: is the sum of the CFU counted on all the dishes retained from two successive
dilutions.
n 1 : is the number of dishes retained at the first dilution.
n 2 : is the number of dishes retained at the second dilution.
V: is the volume of inoculum, in millilitres, applied to each dish.
D: is the dilution factor corresponding to the first dilution retained
All the data were analyzed for statistical significance using SPSS 21.0 (SPSS Inc.,
Chicago, USA).
An assessment of the Normality of the data was done with Shapiro-Wilk test. Results
were then compared by an analysis of variance (ANOVA) followed by Tukey’s pairwise
comparison of the means with significance defined at the 𝑃𝑃 < 0.05 level.
Tukey's HSD test is a post-hoc test, meaning that it is performed after an analysis of
variance (ANOVA) test. The purpose of Tukey's HSD test is to determine which groups
in the sample differ. Thus, in the present study, Tukey Test determined the differences
between different groups of microorganisms, different groups of RTE foods and
different groups of disinfection technologies, as well as differences between
physicochemical and color values.
Moreover, Pearson coefficient was used for measuring correlation between values.
Correlation between different sets of data is a measure of how well they are related.
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Pearson Correlation in the present study was used in order to correlate different
microorganisms as far as their disinfection efficiency is concerned. The Pearson productmoment correlation coefficient is a measure of the strength of the linear relationship
between two variables. It is referred to as Pearson's correlation or simply as the
correlation coefficient. If the relationship between the variables is not linear, then the
correlation coefficient does not adequately represent the strength of the relationship
between the variables. Pearson's r can range from -1 to 1. An r of -1 indicates a perfect
negative linear relationship between variables, an r of 0 indicates no linear relationship
between variables, and an r of 1 indicates a perfect positive linear relationship between
variables. Figure 1 shows a scatter plot for which r = 1.
Finally, pairwise t-tests concern the comparison of the same group of individuals, or
matched pairs, being measured twice, before and after an “intervention”. In the present
study, the “intervention” was all the selected disinfection treatments for treatment times
ranging
from
1
to
60-min.
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Chapter 3. RESULTS
In this study four experimental approaches were conducted in order to evaluate food
safety and public health. The final aim at the fifth part was to assess the efficiency of
different disinfection technologies on their ability to reduce or totally disinfect
pathogens which may be present in ready to eat fresh produces, thus ensuring public
health.
The aim of the first experimental approach was to evaluate the effectiveness of three
non-thermal light technologies (NUV-Vis, continuous UV and HILP) on their ability to
inactivate two pathogens on a liquid matrix. The liquid matrix used for the disinfection
experiments was a liquid matrix (MRD solution). The indicator microorganisms that
were selected were Escherichia coli K12 and Listeria innocua. E. coli K12 was selected
as a representative microorganism for the enterohaemorrhagic foodborne pathogen E.
coli O157:H7 and L. innocua as a surrogate microorganism for the common foodborne
pathogen Listeria monocytogenes, respectively.
The second experimental approach involved the use of non-thermal technologies (UV
and US) as well as conventional sodium hypochlorite (NaOCl) solutions, in order to
evaluate the disinfection efficiency of three ready-to-eat produces (romaine lettuce,
strawberry and cherry tomatoes). The series of these disinfection technologies included
also combinations of the above technologies. More precisely, UV+US, UV+NaOCl and
US+NaOCl combined technologies were used. The disinfection efficiency was tested
against Gram positive and Gram negative microorganisms (E. coli, S. aureus, S.
Enteritidis, L. innocua) as well as adenovirus-35 which have been artificially inoculated
on the above fresh produces. Moreover, infectivity assays were conducted based on
different initial concentration inocula of RTE produces. For this reason, the RTE
produces were inoculated with different concentrations of the above bacteria and virus
and their disinfection efficiency with selected disinfection technologies was tested.
Furthermore, with the aim of investigating how the above pathogens survive during
refrigerated storage, the three fresh produces inoculated with the cocktail of the above
four microorganisms were treated with selected disinfection technologies and were kept
in refrigerated conditions for 15 days. The microbial load of romaine lettuce, strawberry
and cherry tomatoes was recorded after 3, 7 and 15 days of storage at 6˚C.
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In the third experimental approach, the quality and the physicochemical characteristics
of the above fresh ready-to-eat produces were tested before and after the use of
disinfection technologies. More precisely, color was recorded as a quality indicator and
TAC, TPC and AA were studied as valuable physicochemical characteristics of the fresh
produces.
The fourth approach was a computerized model, which was proposed, in order to
evaluate and explore problems that can arise during the food production chain and
predict the possibility of cross-contamination of fresh produce from production to the
point of sale in a vertical production company of vegetables. The final aim was to obtain
a risk assessment software tool to ensure food safety and public health.
Finally, conclusions based on infectivity doses for each pathogen and the results
obtained from the present study, were exported.
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3.1 In Vitro Experiments with 3 Light Technologies
The first light equipment that was used was NUV-vis 395 ± 5 nm. The inactivation rate
of E. coli (figure on the left) and L. innocua (figure on the right) was dose and time
dependent. Generally, it was observed that as the distance from the lamp was increased,
the time needed for inactivation for both microorganisms was longer (figure 3.1.1).
Figure 3.1.1: Survival curves of E. coli suspended in maximum recovery diluent (MRD) placed
at: 3 cm (∆), 12 cm (☐), 23 cm (○) and L. innocua placed at: 3 cm (▲), 12 cm (■) and 23 cm
(●) from the high intensity near ultraviolet/ visible (NUV–vis) 395±5 nm light source (Results
expressed as mean log 10 CFU/mL).
Moreover, when higher dosages were achieved (36 J/cm2), the inactivation rates of L.
innocua remained significantly higher with a maximum average log 10 CFU/mL
reduction of 2.74 achieved after 1115 sec of treatment, compared to that of E. coli where
the maximum average log 10 reduction after the same time was 1.37 log 10 CFU/mL (p <
0.05, n=12). The higher susceptibility of L. innocua was even observed at 23 cm
distance from the light source, giving a log reduction at the highest dose (2.832 J/cm2) of
1.10 log 10 CFU/mL. It was significantly greater (p<0.05) in comparison with the
corresponding reduction that was observed for E. coli which was 0.52 log 10 CFU/mL
(p>0.05).
Subsequently, experiments with continuous UV light source followed. The inactivation
rate of both E. coli and L. innocua were also dependent on treatment time and dose.
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Figure 3.1.2: Survival curves of E. coli suspended in maximum recovery diluent (MRD) placed
at: 6.5 cm (∆), 17 cm (☐), 28.5 cm (○) and L. innocua placed at: 6.5 cm (▲), 17 cm (■) and
28.5 cm (●) from continuous UV light source (Results expressed as mean log 10 CFU/mL).
A positive correlation was observed between log 10 reduction and duration of UV light
exposure. The highest reductions were achieved at the shortest distance from the lamp
(6.5 cm) and at an exposure time of 472 sec. More precisely, reductions of 2.66 log 10
CFU/mL and 3.04 log 10 CFU/mL were achieved for E. coli and L. innocua, respectively.
It must be stated that the susceptibility of the two microorganisms when this light
technology was used, was not significantly different (p = 0.749).
The third light equipment used in this first approach of disinfection experiments was the
High Intensity Light Pulsed (HILP) source. The results observed with this disinfection
equipment are quite promising and are illustrated in figure 3.1.3 for E. coli (on the left)
and L. innocua (on the right) respectively.
Figure 3.1.3: Survival curves of E. coli suspended in maximum recovery diluent (MRD) placed
at: 2.5 cm (∆), 8 cm (☐), 11.5 cm (○), 14 cm (◊) and L. innocua placed at: 2.5 cm (▲), 8 cm
(■), 11.5 cm (●) and 14 cm (♦) from high Intensity pulsed light source (Results expressed as
mean log 10 CFU/mL).
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In general, increased treatment times resulted in greater reductions for both E. coli and
L. innocua. The least susceptible microorganism was E. coli. A dosage of 17.7 J/cm2
resulted in log reductions of E. coli and L. innocua populations (3.07 and 3.77 log 10
CFU/mL, respectively). When a dosage of 54 J/cm2 (30 sec) was implemented,
reductions of 4.81 and 5.56 log 10 CFU/mL were achieved or E. coli and L. innocua,
respectively. At a dosage of 36 J/cm2, a degree of variation was observed between the
two tested microorganisms. For example, E. coli was reduced by 3.85 log 10 CFU/mL,
whereas L. innocua was reduced by 5.30 log 10 CFU/mL (p < 0.05, n=12). At 2.5 cm
distance, at longest exposure time (30 sec), both microorganisms were below the limit of
detection (<0.22 log 10 CFU/mL). The susceptibility of two microorganisms regarding
this light technology was also significantly different (p < 0.05, n=192).
In figure 3.1.4 comparisons between three light technologies for E. coli are illustrated.
The dosages that were used had a range between 0.18-106.2 J/cm2. Where no
measurement took place (e.g due to temperature increase) no bar exists.
Figure 3.1.4: Mean Log CFU/mL E. coli on MRD after treatment at the same dosages at shortest
distance with 3 different light equipments: NUV-vis (■), Continuous UV (■) and High Intensity
Light Pulses (■).
It can be observed that High Intensity Light Pulses was the most powerful technology as
far as the disinfection capacity on E. coli is concerned. Moreover, dosages greater than
2.832 J/cm2 were not appropriate for Continuous UV technology, due to the increase in
temperature (>30°C) that was observed.
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When low dosages were implemented (0.18, 0.36, 0.72, and 1.44 J/cm2), the observed
inactivation rates were similar for both E. coli and L. innocua (p > 0.05, n=396) (figures
3.1.4, 3.15). However, when a higher dose of 2.832 J/cm2 was delivered, L. innocua
exhibited a higher log reduction (1.25 log 10 CFU/mL) compared to E. coli (0.68 log 10
CFU/mL) after 88 sec of treatment (p < 0.05, n=84), which is around 2 times the
inactivation log of the more resistant bacterium of E. coli.
In figure 3.1.5 comparisons between three light technologies for L. innocua inactivation
are illustrated. The dosages that were used had a range between 0.18-106.2 J/cm2. Where
no measurement took place (e.g due to excessive temperature increase) no bar exists.
Figure 3.1.5: Mean log CFU/mL L. innocua on MRD after treatment at the same dosages at
shortest distance with 3 different light equipments: NUV-vis (■), Continuous UV (■) and High
Intensity Light Pulses (■).
At low dosages (0.18, 0.36, and 0.72 J/cm2) the difference between the two
microorganisms, when the three light technologies were used, were all significant (p <
0.05, n=354). When 1.44 J/cm2 was used, the log 10 (CFU/mL) reduction at NUV-vis
light and continuous UV light for both microorganisms was significant (p <0.05, n=72),
whereas when comparisons with HILP light were performed, the differences between the
susceptibility of the tested microorganisms did not differ (p >0.05, n=192). When 2.832
J/cm2 was implemented in both continuous UV light technology and HILP, the
disinfection efficiency of E. coli and L. innocua did not differ significantly (p = 0.306
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and p = 0.116, respectively). Finally, at higher dosages, the correlations between log 10
(CFU/mL) reduction of NUV-vis and HILP, as the two organisms are concerned, were
all significant (p < 0.05). Generally, it was observed that in all light technologies (NUVvis, continuous UV, and HILP) a significant correlation (p < 0.05, n=600) between doses
and microorganisms log reduction existed.
The temperature increase for three light equipments and at different distances from the
lamp are shown in figures 3.1.6, 3.1.7, 3.1.8.
Figure 3.1.6: Mean Temperature increase
(ΔΤ ᵒC) for NUV-Vis light technology at
distances:
3 cm (▲), 12 cm (■) and 23 cm (●)
Figure 3.1.7: Mean Temperature increase
(ΔΤ ᵒC) for UV light technology at
distances:
6.5 cm (▲), 17 cm (■) and 28.5 cm (●)
Figure 3.1.8: Mean Temperature increase (ΔΤ ᵒC) for HILP light technology at distances: 2.5 cm
(▲), 8 cm (■), 11.5 cm (●) and 14 cm (♦).
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Temperatures remained below 50°C for all disinfection treatments used in the study.
However, it must be stated that HILP exhibited the greater temperature increases,
followed by NUV-Vis and Continuous UV light. For this reason, during HILP
treatments, samples were placed in an iced bath to minimize heating caused by the
infrared portion of the HILP light unit.
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3.2 Food Disinfection
3.2.1 Bacteria Disinfection
3.2.1.1 Lettuce
The first vegetable selected for food disinfection was lettuce. Its disinfection efficiency
against four bacteria was tested, after immersing whole lettuce leaves in 50 ppm and 200
ppm NaOCl solutions.
Figure 3.2.1.1.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E. coli, S.
aureus, S. Enteritidis, L. innocua inoculated on fresh romaine lettuce.
The inactivation effect of NaOCl against E. coli, S. aureus, S. Enteritidis and L. innocua
increased with increasing treatment time and concentration. Significant reductions
(p<0.05, n=48) were observed at 3-min treatment time when pairwise t-test was used,
with both NaOCl 50 ppm solution and NaOCl 200 ppm solution. For instance, treatment
time of 3-min and 200 ppm NaOCl solution reduced microorganisms significantly when
tested with pairwise t-test (p<0.05, n=48) by 1.92, 1.82, 1.96 and 2.01 log 10 , for E. coli,
S. aureus, S. Enteritidis and L. innocua respectively. Lower concentration of NaOCl (50
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ppm) reduced the above populations by 1.64±0.11 1.45±0.27, 0.92±0.21 and 1.63±0.14
log 10 , at the same treatment time. However, no significant differences (p>0.05, n=48)
were observed with pairwise t-test, for microbe populations when the treatment time
increased from 3 to 5-min. On the other hand, when the treatment time was increased
from 1 to 3-min, significant differences when pairwise t-test was used (p<0.05, n=48)
were observed for all the microorganisms, with the exception (p=0.199) of S. Enteritidis
which was treated with NaOCl 50 ppm.
Lettuce was then treated with two non-thermal, alternative disinfection technologies
Ultrasound and UV for treatment times ranging from 1 to 60-min and the results are
illustrated in figure 3.2.1.1.2.
Figure 3.2.1.1.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S. Enteritidis, L.
innocua inoculated on fresh romaine lettuce.
Treatment with US significantly reduced the numbers of all microorganisms inoculated
on lettuce. The reduction was found to be significant with anova test after 30-min of
treatment (p< 0.05, n=240). The maximum reductions of E. coli, S. aureus, S. Enteritidis
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and L. innocua observed on lettuce after US treatment, were 2.30 ± 0.34, 1.71 ± 0.20,
5.72 ± 0.05 and 2.95 ± 0.57 log 10 CFU/g.
Treatments with UV reduced significantly the concentrations of all types of
microorganisms (p<0.05, n=240). The reduction of four bacterial types depended on the
different time intervals as well as on the bacterial type concerned. Significant reduction
of all bacteria was achieved after 20-min (p<0.05). The reduction was finally about 1–
1.7 log 10 after 45-min treatment with UV in all different types of microorganisms. Both
Salmonella and E. coli in lettuce showed similar reductions when treated for the same
period with UV.
Combined technologies including alternative and conventional technologies in different
treatment times were also examined. The treatment times for alternative technologies
were in the range of 1-30-min. All the treatments were followed by 3 minute immersion
of lettuce in NaOCl solutions of 50 and 200 ppm concentration. The results for four
bacteria are illustrated in figure 3.2.1.1.3.
Figure 3.2.1.1.3: Disinfection Efficiency of combined alternative and conventional disinfection
technologies (US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200
ppm) on E. coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh romaine lettuce.
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All the combined disinfection treatments were effective after 1 minute treatment time
with non-thermal technology followed by 3-min treatment with NaOCl. For instance, E.
coli, S. aureus and L. innocua populations were significantly reduced (p<0.05), after 1
minute treatment of Ultrasound technology followed by 3-min of NaOCl 50 ppm,
whereas S. Enteritidis was not significantly reduced (p=0.029) after the same time, but
20-min treatment time needed for significant inactivation rate (p=0.04). However, when
200 ppm NaOCl immersion followed, the disinfection efficiency was significant
(p<0.05, n=336) at all treatment times, when tested with pairwise t-test.
Generally, US+NaOCl was more effective than UV+NaOCl. L. innocua was reduced by
1.83±0.32, 2.41±0.34, 1.78±0.08 and 1.95±0.11 log 10 , when US+NaOCl 50 ppm,
US+NaOCl 200 ppm, UV+NaOCl 50 ppm and UV+NaOCl 200 ppm were used
respectively. US+NaOCl 200ppm was efficient and reduced by 2.71, 2.33, 2.94 and 3.34
log 10 CFU/g E. coli, S. aureus, S. Enteritidis and L. innocua respectively, after 30-min
treatment, plus 3 minute treatment with conventional technology.
Finally, combinations of alternative non-thermal technologies were tested. The results
are observed in figure 3.2.1.1.4. The maximum treatment time for the combined
treatments tested was 30-min.
Figure 3.2.1.1.4: Disinfection Efficiency of combined alternative disinfection technologies on E.
coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh romaine lettuce.
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Combinations of alternative disinfection technologies did not show an additive effect.
However, when tested with pairwise t-test, they resulted in significant log reductions
(p<0.05, n=120) of all microorganisms. The greater log reductions were obvious after
10-min UV treatment followed by 20-min of US where 0.67±0.07, 0.35±0.01, 0.87±0.29
and 1.22±0.29 log 10 reduction for E. coli, S. aureus, S. Enteritidis and L. innocua were
found respectively. The lowest log reduction was presented for S. aureus, regardless the
combined disinfection method that was used.
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3.2.1.2 Strawberry
The fruit that dominates the Mediterranean diet, prior inoculated with bacteria cocktail
was tested with a series of conventional and alternative disinfection technologies.
Firstly, the results of the immersion in NaOCl 50 ppm and NaOCl 200 ppm are observed
in figure 3.2.1.2.1.
Figure 3.2.1.2.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E. coli, S.
aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries.
The disinfection efficiency of NaOCl was dependent on the concentration and the
treatment time. Longer treatment times, resulted in greater log reduction of all
microorganisms.
After 1 minute immersion in both NaOCl 50 ppm and NaOCl 200 ppm, no significant
reduction with pairwise t-test (p>0.05, n=48) was observed for all microorganisms.
However, at 3-min treatment time with NaOCl 200 ppm, significant population
reductions for E. coli (p=0.017), S. aureus (p=0.015), S. Enteritidis (p=0.008) and L.
innocua (p=0.07) were recorded. NaOCl 200 ppm and 3-min treatment time resulted in
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1.58±0.35, 1.29±0.28, 1.66±0.26 and 1.48±0.21 log 10 reductions of E. coli, S. aureus, S.
Enteritidis and L. innocua respectively. Whereas after 5-min treatment of strawberries
with 200 ppm NaOCl resulted in 1.91±0.30, 1.44±0.12, 1.75±0.26 and 1.52±0.21 log 10
of the above microorganisms respectively.
Significant differences were recorded with the use of pairwise t-test, when the treatment
time was enhanced from 1 to 3-min (p<0.05, n=48). Whereas, when the time was
increased from 3 to 5-min, the disinfection remained constant and the differences were
not significant (p>0.05, n=48).
Both Ultrasound and UV disinfection technologies were used for strawberry disinfection
and the results are recorded in figure 3.2.1.2.2.
Figure 3.2.1.2.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S. Enteritidis, L.
innocua inoculated on fresh strawberries.
In strawberries, the most significant reduction occurred after 10-min treatment with UV
(1.2 J/cm2) depending on the microorganism (p<0.05, n=48), according to pairwise ttest. E. coli and S. Enteritidis were reduced significantly after 10-min (p=0.05 and
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p=0.037 respectively). S. aureus reduced significantly (p=0.037) after 5-min treatment
with UV and L. innocua after 20-min UV treatment (p=0.038).
Treatment with US significantly reduced the numbers of all microorganisms on
strawberries (p<0.05, n=240). The maximum reductions of E. coli, S. aureus, S.
Enteritidis and L. innocua on strawberries were 3.04 ± 0.72, 2.41 ± 0.59, 5.52 ± 0.13
and 6.12 ±0.04 log 10 , respectively and were observed after the longest exposure times to
US technology. L. innocua was reduced significantly (p=0.05) after 5-min treatment
with US. However, S. aureus and S. Enteritidis needed 20-min to be reduced
significantly (p=0.037). It must be stated that no viable (<0.22 log 10 CFU/g) Salmonella
or Listeria cells were observed after 30-min treatment time with US.
Combinations of alternative (US and UV) with immersion in NaOCl 200 ppm for 3-min
were also selected for strawberries disinfection. The results are shown in figure
3.2.1.2.3.
Figure 3.2.1.2.3: Disinfection Efficiency of combined alternative and conventional technologies
(US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm) on E.
coli, S. aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries.
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Combined alternative and conventional treatments were used for strawberry disinfection.
All microorganisms inoculated in strawberry were reduced when combined technologies
were used. E. coli was reduced significantly (p<0.05, n=168) by 1.91±0.11, 2.36±0.22,
1.96±0.26 and 2.08±0.35 log 10 when the longest combined treatments US+NaOCl 50
ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm, were used. The
highest reductions among all microorganisms observed by S. Enteritidis where it was
reduced significantly (p<0.05, n=168) by 2.93±0.35, 3.50±0.39, 2.20±0.25 and
2.40±0.21 log 10 after longest exposure to combined treatments of US+NaOCl 50 ppm,
US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm.
In general terms, combinations of UV followed by NaOCl resulted in less disinfection
efficiency compared to US followed by NaOCl. However, all microbe populations were
significantly reduced (p<0.05, n=672), with all the combined treatments that were
selected.
Finally, combinations of alternative technologies were used for testing the disinfection
efficiency of microbial populations in strawberries and are shown in figure 3.2.1.2.4.
Figure 3.2.1.2.4: Disinfection Efficiency of combined alternative technologies on E. coli, S.
aureus, S. Enteritidis, L. innocua inoculated on fresh strawberries.
E. coli is more resistant to combined alternative technologies showing decreased log 10
reductions from 0.09±0.05 to 0.58±0.10. However, L. innocua population decreased by
0.64±0.03, 1.37±0.11, 1.83±0.15 and 1.47±0.07 log 10 when the above combined
alternative technologies were used. Moreover, S. aureus and S. Enteritidis were reduced
by 0.64±0.08, 0.57±0.07, 1.18±0.06, 0.90±0.06 log 10 and 0.62±0.06, 0.74±0.03,
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1.10±0.2, 0.82±0.04 log 10, respectively. All the populations reductions that were
achieved were statistically significant (p<0.05, n=120) when tested with pairwise t-test.
3.2.1.3 Cherry tomatoes
Cherry tomatoes are very common vegetables consumed raw in an everyday basis in a
Mediterranean diet. The disinfection methods used for cherry tomatoes are shown in
figures 3.2.1.3.1 – 3.2.1.3.4.
Figure 3.2.1.3.1: Disinfection Efficiency of NaOCl 50 ppm and NaOCl 200 ppm on E. coli, S.
aureus, S. Enteritidis, L. innocua inoculated on fresh cherry tomatoes.
Treatment with NaOCl, when tested with pairwise t-test, significantly (p<0.05, n=48),
reduced all microorganisms from the first minute of treatment. Moreover, it is obvious
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that the degree of disinfection was not enhanced significantly after 3-min treatment.
However, a greater reduction (p<0.05, n=48) was observed at 3-min treatment with 200
ppm NaOCl compared to 3-min treatment of NaOCL 50 ppm. The reduction from 3 to 5min was not significant (p>0.05, n=48).
For instance, 50 ppm NaOCl reduced E. coli, S. aureus, S. Enteritidis and L. innocua by
2.87±0.35, 2.18±2.25, 2.86±2.67 and 2.03±2.33 log 10 respectively, whereas 200ppm
NaOCl reduced the above microorganisms by 3.68±0.56, 2.98±2.67, 3.07±2.83 and
2.29±2.53 log 10 , respectively.
Then, US and UV were tested as fas as their disinfection efficiency is concerned and the
results are shown in figure 3.2.1.3.2
Figure 3.2.1.3.2: Disinfection Efficiency of US and UV on E. coli, S. aureus, S. Enteritidis, L.
innocua inoculated on fresh cherry tomatoes.
Treatments with US led to reductions of 0.64 - 3.16 log 10 CFU/g in the population of E.
coli, 1.06-2.62 log 10 CFU/g in the population of S. aureus, 1.23-3.29 log 10 CFU/g in the
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population of S. Enteritidis and 0.76-3.16 log 10 CFU/g in the population of L. innocua
(p<0.05, n=240). The effectiveness of the 37 kHz ultrasound bath treatement was
increased as the treatment time increased from 1-60-min. Significant reduction of all
bacteria was achieved after 10-min (p<0.05). The reduction was finally about 3.29 log 10
for S. Enteritidis followed by E. coli after the longest exposure time, with an average log
reduction of 3.16 log 10 (p<0.05). All microorganisms were significantly reduced
(p<0.05) from the first minute of treatment with US, except E. coli which needed 10-min
treatment (p=0.009), in order to be reduced significantly.
Treatments with UV, reduced the populations of all microorganisms, artificially
inoculated in cherry tomatoes. More specifically, UV treatments evaluated in this study
promoted the reduction of 0.82 - 2.39 log 10 CFU/g in the population of E. coli, 0.98-2.05
log 10 CFU/g in the population of S. aureus, 1.11-2.62 log 10 CFU/g in the population of
S. Enteritidis and 0.84-2.56 log 10 CFU/g in the population of L. innocua (p<0.05). The
reduction of four bacterial types depended not only on the different time intervals (1-60min) but also on the bacterial type. At 10-min treatment time significant reduction of all
bacteria was achieved (p< 0.05). The increase in contact time from 10 to 60-min of
treatment reduced contamination even further (p=0.04). S. Enteritidis achieved the
greatest reduction among all bacteria with an average of 2.62 log 10 CFU/g at 60-min
treatment time (p<0.05). All microorganisms were significantly reduced (p<0.05) from
the first minute of treatment with UV, except S. Enteritidis which needed 10-min
(p=0.024), in order to be reduced significantly.
Combined technologies of US and UV followed by immersion in NaOCl 50ppm and
NaOCl 200ppm are illustrated in figure 3.2.1.3.3.
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Figure 3.2.1.3.3: Disinfection Efficiency of combined alternative and conventional technologies
(US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm) on E.
coli, S. aureus, S. Enteritidis, L.innocua inoculated on fresh cherry tomatoes.
The combined alternative followed by conventional treatments, reduced significantly
(p<0.05, n=672) all microorganisms. When US followed by 3-min immersion of cherry
tomatoes in NaOCl 50 ppm solution was used, reductions of 3.63±0.28, 3.26±0.25,
3.63±0.03 and 3.39±0.05 log 10 were achieved for E. coli, S. aureus, S. Enteritidis and L.
innocua respectively. However, when US was combined with 3-min immersion in
NaOCl 200 ppm 0.50-1 log 10 further significant reductions (p<0.05) were observed for
all microorganisms. More precisely, the aforementioned microorganisms were reduced
by 4.78±1, 3.63±0.13, 4.27±0.41 and 3.84±0.28 log 10 respectively.
Furthermore, when UV was combined with NaOCl 50 ppm, the above microorganisms
were reduced by 3±0.08, 2.50±0.19, 3.39±0.36 and 2.86±0.03 log 10 respectively.
Whereas, when it was combined with NaOCl 200 ppm, the above microorganisms were
reduced significantly (p<0.05) by 3.94±0.22, 2.89±0.04, 3.9±0.55 and 3.28 ±0.29 log 10
respectively.
Combinations of alternative technologies were tested for treatment times of 10-30-min.
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Figure 3.2.1.3.4: Disinfection Efficiency of combined alternative technologies on E. coli, S.
aureus, S. Enteritidis, L. innocua inoculated on fresh cherry tomatoes.
Treatment with UV for 10min followed by US 20-min significantly reduced, when
tested with pairwise t-test, the numbers of all microorganisms (p<0.05, n=48) on cherry
tomatoes. The reductions achieved were 3.06±0.18, 2.63±0.20, 3.70±0.05 and 2.73±0.02
log 10 for E. coli, S. aureus, S. Enteritidis and L. innocua respectively.
Finally a more generalized statistical analysis was carried out. For instance, Tukey HSD
test was used in conjunction with an ANOVA to find disinfection technologies that are
significantly different from each other.
For lettuce, no significant differences were found between alternative disinfection
methods (p>0.05, n=480). However, when compared with conventional as well as with
combined alternative and conventional, the differences were significant (p<0.05, n=192
and n=672 respectively). The above was observed for all bacteria in lettuce except S.
Enteritidis. At the case of S. Enteritidis, a significant difference (p<0.05) between
Ultrasound and UV was found.
For strawberry, the results obtained for S. Enteritidis and L. innocua did not differ
significantly (p>0.05) between different disinfection methods. The only difference for
the aforementioned microorganisms (p=0.046, p=0.001 respectively) was found between
Ultrasound and UV technology. For E. coli and S. aureus there was a difference between
US and UV (p<0.05, n=240 for both microorganisms), as well as between alternative
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Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
disinfection methods and combinations of alternative followed by conventional
disinfection methods (p<0.05).
For cherry tomatoes, a greater difference between disinfection methods for
microorganisms was observed. E. coli and S. Enteritidis exhibited the same inactivation
pattern (p>0.05, n=240) when treated with US and UV, whereas they differed
significantly (p<0.05, n=432) when treated with the rest of disinfection methods. Gram
positive microorganisms susceptibility was the same for UV and US as well as for UV
and combinations of alternative disinfection technologies (p>0.05), whereas differed for
the other disinfection technologies.
Finally, for each disinfection methods used, Tukey HSD tests were performed to
determine difference between microorganisms. Generally, a variation was observed
among bacteria’s susceptibility to different disinfection methods. For lettuce, the four
bacteria did not exhibit any difference (p>0.05, n=180) when conventional treatments
were used. For US treatment S. Enteritidis differed significantly from the other three
bacteria (p<0.05). Moreover, for UV treatment, gram negative bacteria susceptibility
was the same (p=0.993) whereas a difference was observed for gram positive bacteria
(p=0.004).
For strawberry, the four bacteria exhibited the same behavior (p>0.05) when
strawberries were treated with conventional treatments, US and US followed by NaOCl
immersion. S. aureus in UV followed by NaOCl 50 ppm immersion treatments exhibited
a different behavior compared to the other three bacteria. When UV treatments were
conducted, the only difference observed was between E. coli and S. Enteritidis
(p=0.004), whereas the rest of microorganisms had a similar behavior (p>0.05).
Finally, for cherry tomatoes, a similar reduction of gram negative (p=0.878) and gram
positive (p=0.903) bacteria is observed when US treatment was followed by NaOCl
treatments. However, when UV followed by NaOCl treatments, the microorganisms
behave differently (p<0.05) between each other. When cherry tomatoes were treated
with combined alternative treatments, S. Enteritidis reduction was significantly (p<0.05)
different from the other three bacteria.
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Results
3.2.2 Adenovirus Disinfection
Figure 3.2.2.1: Standard Curve based on the entire hexon region of Ad35 cloned into pBR322
The construction of the standard curve is based on the use of the entire hexon region of
Ad41 cloned into pBR322 (kindly donated by A. Allard, University of Umeå). High
efficiency E. coli JM109 competent cells (Promega, L2001) were added to 1µl of
pBR322 plasmid containing the entire Ad41 hexon gene sequence following the
supplier instructions. The efficiency of these cells has been reported to be 2.2x108
CFU/µg. 100 µl of transformed E. coli JM109 were plated into a LB agar plate
containing ampicilline 100µg/ml. Consequently, 500µl of transformed bacteria were
aliquoted into a 1.5 ml tube and centrifuge 10-min at 5000xg. The supernatant was
discarded, pellet was resuspended in 1ml of LB 15% glycerol and kept frozen at -80ºC
for further production of standard. The other 400µl of transformed cells were kept at 4ºC
for further production of larger amounts of DNA. Afterwards, some of the colonies
growing were checked on LB plates by conventional PCR (using primers AdR and AdF
at 55ºC of annealing temperature) to contain the target DNA. Then colonies were
inoculated directly into PCR tubes by using sterile toothpicks. The target DNA was
obtained by using the QIAGEN Plasmid Midi kit (Cat. No. 12143) and the transformed
cells kept at 4ºC by following manufacturer’s instructions. Then dilution of the obtained
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DNA into Tris-EDTA pH 8 (10 mM Tris and 0.1 mM for EDTA) and quantification by
spectrophotometry several replicates of the DNA followed. Approximately 10μg of
DNA was linearized with BamHI restriction enzyme. Then the DNA was purified with
the QIAGEN QIAquick PCR Purification kit (Cat. No. 28104). The purity of linearised
plasmid was checked in an agarose gel 1%. If a band corresponding to undigested
plasmid was present more enzyme was added and the digestion was let to take place for
an additional 2 hours. Several replicates of the DNA were quantified by
spectrophotometry, then the DNA was serially diluted in order to obtain dilutions where
102 to 108 molecules per 10 µl are present.
Page 157
Results
Figure 3.2.2.2: HAdV Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and
Cherry tomatoes (light grey bars) and single step conventional Disinfection Treatments.
In the experimental conditions evaluated, HAdV was reduced when the samples were
immersed in sodium hypochlorite solutions and significant inactivation (p<0.05, n=20)
was observed as the treatment time of immersion to sodium hypochlorite 200 ppm was
enhanced (from 3 to 10-min). It must be stated that after the longest exposure time,
lettuce and strawberry achieved 4.95 and 5.02 log 10 GC/g reduction, whereas cherry
tomatoes achieved 3.76 log 10 GC/g reduction. Moreover, the treatment time of 3-min
was sufficient for inactivating HAdV as far as cherry tomatoes are concerned. An
additional 0.79 log 10 reduction was achieved for another 2-min immersion in sodium
hypochlorite solution. For strawberry and lettuce the treatment time plays an important
role in inactivation efficiency. The difference between 3 and 5-min was not found to be
statistically significant (p>0.05). However, when 10-min treatment time was
implemented, the inactivation rate was significantly enhanced (p<0.05). For instance, in
strawberries a double inactivation log 10 GC/g of HAdV was achieved. Whereas, in
lettuce an additional 1.23 log 10 reduction GC/g of HAdV was reported when the
treatment time was increased from 3 to 10-min.
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Figure 3.2.2.3: HAdV Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and
Cherry tomatoes (light grey bars) and single step Alternative Disinfection Treatments
In all foods, as expected, higher UV doses resulted in a greater decrease of viral growth
in ‘romaine’ lettuce, in strawberry pieces and in cherry tomatoes. UV was less effective
at reducing viral populations in lettuce. It was observed, that when the time was doubled
(from 30 to 60-min), the mean reduction of HAdV was also doubled for strawberry
(from -1.26 to -3.98 log 10 GC/g) and cherry tomatoes (from -0.92 to -2.22 log 10 GC/g).
Treatment with US was less effective (p>0.05, n=44) compared to UV. After the longest
exposure time, lettuce exhibited the greatest reduction (-1.79 log 10 GC/g) compared to
other fresh produces. However, the treatment time also played an important role, as far
as virus reduction is concerned.
Page 159
Results
Figure 3.2.2.4: HAdV Log Reduction of Lettuce (black bars), Strawberry (dark grey bars) and
Cherry tomatoes (light grey bars) and combined Disinfection Treatments.
A synergistic effect was observed when UV and US were followed by immersion in
sodium hypochlorite solutions, however, no additive effect was observed. The synergy
was enhanced further, when UV was followed by sodium hypochlorite (p<0.05, n=40),
rather than when US followed by sodium hypochlorite. Moreover, the sequential
treatment of alternative methods exhibited more promising results compared to the
combination of an alternative and a conventional treatment, in strawberries and cherry
tomatoes. In all cases the sequential application of two alternative technologies
depended on the time used for each method.
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3.2.3 High and Low Initial Load Disinfection Treatments
LETTUCE /
MICROORGANISMS
E.coli
S.aureus
S .enteritidis
L.innocua
DISINFECTION TREATMENTS
Initial
Concentration
UV
(30min)
UV
(60min)
UV+NaOCL
(33 min)
US
(30min)
US
(60min)
US+NaOCl
(33 min)
NaOCl
(3 min)
2,16E+08
1,68E+06
1,55E+04
6,36E+02
1,35E+07
1,32E+05
5,55E+03
8,18E+02
3,25E+06
2,88E+05
4,55E+03
2,73E+02
4,49E+07
1,35E+05
2,00E+04
2,73E+02
3,86+07
8,64E+05
8,98E+03
1,64E+02
1,86E+06
6,36E+04
8,18E+02
4,55E+02
7,09E+05
1,10E+04
1,18E+03
9,09E+01
7,27E+05
2,00E+04
6,64E+03
1,82E+02
1,81E+06
3,41E+03
1,33E+03
7,27E+01
7,64E+05
9,73E+04
1,82E+02
9,09E+01
7,27E+04
6,27E+03
7,27E+02
0,00E+00
1,73E+05
4,55E+04
2,00E+03
0,00E+00
2,83E+05
1,00E+04
2,73E+02
0,00E+00
4,36E+05
1,73E+03
9,09E+02
9,09E+01
6,36E+04
5,45E+03
9,09E+02
0,00E+00
1,09E+04
2,73E+02
9,09E+01
0,00E+00
1,50E+07
4,41E+05
9,23E+03
3,64E+01
1,20E+06
2,73E+04
1,09E+03
9,09E+01
2,00E+04
1,18E+03
4,55E+02
0,00E+00
5,09E+05
2,73E+03
3,64E+02
9,09E+01
1,45E+06
1,98E+04
4,00E+02
9,09E+00
1,91E+05
1,18E+04
9,09E+01
0,00E+00
6,36E+02
9,09E+01
9,09E+01
0,00E+00
3,64E+04
9,09E+02
9,09E+01
0,00E+00
7,36E+04
2,73E+02
0,00E+00
0,00E+00
7,27E+04
8,18E+02
5,45E+02
0,00E+00
2,27E+04
9,09E+02
9,09E+01
0,00E+00
6,36E+03
3,64E+02
0,00E+00
0,00E+00
6,85E+05
5,45E+03
3,64E+01
0,00E+00
5,45E+05
2,73E+02
1,82E+02
0,00E+00
1,35E+05
5,00E+03
8,18E+02
0,00E+00
3,00E+04
9,09E+01
0,00E+00
0,00E+00
Table 3.2.3.1: High and Low Inocula (Log 10 CFU/g) on lettuce and disinfection with selected
treatments.
STRAWBERRY /
MICROORGANISMS
DISINFECTION TREATMENTS
Initial
Concentration
E.coli
S.aureus
S. enteritidis
L.innocua
9,64E+07
1,30E+05
9,45E+02
3,73E+07
2,80E+05
3,73E+03
3,46E+06
2,55E+04
8,18E+02
1,11E+07
1,53E+05
1,64E+03
UV
(30min)
1,96E+06
3,64E+04
2,73E+01
8,18E+06
2,25E+04
9,09E+01
7,79E+04
2,36E+03
4,55E+02
1,22E+05
3,18E+03
2,73E+02
UV
(60min)
UV+NaOCL
(33min)
US
(30min)
US
(60min)
US+NaOCl
(33 min)
NaOCl
(3min)
1,10E+06
2,21E+04
9,09E+00
2,45E+06
1,73E+04
0,00E+00
1,19E+04
8,18E+02
0,00E+00
9,85E+04
6,36E+02
0,00E+00
1,48E+05
1,00E+03
0,00E+00
7,09E+05
1,09E+03
0,00E+00
1,73E+03
5,45E+02
0,00E+00
3,10E+05
9,09E+02
0,00E+00
1,91E+04
2,27E+03
1,82E+01
5,22E+06
1,70E+04
0,00E+00
8,00E+03
6,36E+02
9,09E+01
1,85E+04
4,00E+03
9,09E+01
3,64E+03
1,82E+02
0,00E+00
8,91E+05
1,55E+03
0,00E+00
9,09E+02
3,64E+02
0,00E+00
3,09E+03
1,82E+02
0,00E+00
8,64E+04
4,55E+02
0,00E+00
4,36E+05
3,64E+02
0,00E+00
7,27E+02
1,82E+02
0,00E+00
3,19E+04
2,73E+02
0,00E+00
1,35E+05
2,45E+03
0,00E+00
1,31E+06
1,82E+03
0,00E+00
4,91E+03
8,18E+02
0,00E+00
1,01E+05
2,09E+03
0,00E+00
Table 3.2.3.2: High and Low Inocula (Log 10 CFU/g) on strawberries and disinfection with
selected
treatments.
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Results
TOMATOES /
MICROORGANISMS
E.coli
S.aureus
S. enteritidis
L.innocua
5LSLNC9/TLON TR9ATa9NTS
Initial
Concentration
UV
(30min)
UV
(60min)
UV+NaOCL
(33 min)
US
(30min)
US
(60min)
US+NaOCl
(33 min)
NaOCl
(3min)
1,28E+07
4,01E+05
1,55E+03
6,43E+06
4,91E+04
5,45E+02
1,87E+07
1,39E+05
9,09E+02
1,36E+06
6,27E+04
8,18E+02
1,57E+05
3,01E+04
7,27E+01
1,72E+04
6,64E+03
9,09E+01
9,64E+04
3,09E+03
2,73E+02
1,07E+05
8,91E+03
1,82E+02
6,91E+04
7,82E+02
0,00E+00
9,09E+03
7,27E+02
0,00E+00
4,57E+04
2,09E+03
0,00E+00
1,50E+04
1,91E+03
0,00E+00
2,45E+03
1,18E+02
0,00E+00
3,18E+03
1,82E+02
0,00E+00
2,62E+04
1,91E+03
0,00E+00
3,27E+03
4,55E+02
0,00E+00
3,00E+05
1,34E+03
9,09E+00
1,50E+04
1,91E+03
0,00E+00
4,25E+04
1,73E+03
1,82E+02
5,77E+04
5,45E+03
0,00E+00
1,45E+03
2,09E+02
0,00E+00
1,82E+03
4,55E+02
0,00E+00
2,09E+03
7,27E+02
0,00E+00
4,45E+03
9,09E+02
0,00E+00
7,27E+01
1,82E+01
0,00E+00
1,45E+03
9,09E+01
0,00E+00
2,18E+03
5,45E+02
0,00E+00
8,18E+02
9,09E+01
0,00E+00
3,45E+03
1,73E+02
0,00E+00
1,30E+04
8,18E+02
0,00E+00
5,30E+04
9,09E+02
0,00E+00
2,52E+04
7,27E+02
0,00E+00
Table 3.2.3.3: High and Low Inocula (Log 10 CFU/g) on cherry tomatoes and disinfection with
selected treatments.
Adenovirus Tissue Culture Infectivity Assay and Lettuce
Treatments
Control
UV 30-min
UV 60-min
UV+NaOCl 33-min
US 30-min
US 60-min
US+NaOCl 33-min
NaOCl 3-min
Page 162
PCR (GC/mL)
5,04E+08
6,28E+05
1,42E+02
6,12E+03
1,31E+07
1,03E+06
3,02E+05
5,74E+02
Culture Assay
(PFU/mL)
109
106
103
103
107
107
106
103
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Figure 3.2.2.5: The results of Real Time PCR were also evaluated with cell cultures observed
under an Epifluorescence microscope.
Page 163
Results
3.2.4 Storage Conditions
The three fruits and vegetables prior inoculated with the cocktail of bacteria, and treated
with selected conventional and alternative treatments were stored for 15 days at 6°C.
Lettuce
Figure 3.2.4.1: E. coli, S. aureus, S. Enteritidis and L. innocua populations inoculated on
romaine lettuce before and after selected disinfection treatments during storage for 15 days at
6°C.
(control),
(UV+NaOCl treated),
(UV20+US10 treated).
Page 164
(UV treated),
(US treated),
(US+NaOCl treated),
(NaOCl treated),
(UV10+US20 treated),
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Populations of E. coli, S. aureus, S. Enteritidis and L. innocua inoculated on control
samples, increased during the storage period in romaine lettuce. More precisely, they
were increased by 0.95, 0.10, 1.11 and 1.73 log 10 respectively.
E. coli and S. Enteritidis in treated lettuces showed a similar behavior during storage
period and were steadily increased from day 0 to day 15. E. coli in UV- and US- treated
lettuces increased 0.74 and 1.62 log 10 respectively. Whereas, S. Enteritidis in the
aforementioned treated lettuces, increased 2.72 and 3.04 log 10 .
Populations of S. aureus and L. innocua in treated lettuces were generally decreased
from day 0 to day 3 and from day 3 onwards an increase was recorded. In UV- and UStreated lettuces after storage of 15 days a decrease of 0.15 and 0.72 log 10 was recorded
for S. aureus but an increase of 1.46 and 1.82 log 10 was observed for L. innocua
respectively.
Page 165
Results
Strawberry
Figure 3.2.4.2: E. coli, S. aureus, S. Enteritidis and L. innocua populations inoculated on
strawberries before and after selected disinfection treatments during storage for 15 days at 6°C.
(control),
(UV+NaOCl treated),
(UV treated),
(US treated),
(US+NaOCl treated),
(NaOCl treated),
(UV10+US20 treated),
(UV20+US10 treated).
From day 0 and throughout storage an increase on growth of E. coli and S. aureus was
observed (p<0.05) after 15 days of storage. They were increased from 7.20 to 8.34 log 10
and from 7.44 to 8.16 log 10 respectively. However, S. Enteritidis and L. innocua reduced
by 1.44 and 3.14 log 10 respectively.
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Birmpa Angeliki
All treated samples collected at storage day 3, exhibited a reduction in their populations.
However, at storage day 7 and 15, an increase was observed in some treated samples.
For example, E. coli, S. aureus and S. Enteritidis in US-treated samples showed an
increase of 0.64, 0.74 and 1.12 log 10 respectively after storage for 15 days. Moreover, an
increase of the same microorganisms (1.01, 0.67 and 1.33) in combined US+NaOCL
treated samples was observed after 15 days of storage.
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Results
Cherry Tomatoes
Figure 3.2.4.3: Populations of E. coli, S. aureus, S. Enteritidis and L. innocua inoculated on
cherry tomatoes before and after selected disinfection treatments during storage for 15 days at
6°C.
(control),
(UV+NaOCl treated),
(UV treated),
(US treated),
(US+NaOCl treated),
(NaOCl treated),
(UV10+US20 treated),
(UV20+US10 treated).
At refrigerated conditions, populations of all microorganisms increased steadily until
day 15, reaching control samples final populations of 8.05, 7.46, 6.65 and 7.75 log 10
CFU/g for E. coli, S. aureus, S. Enteritidis and L. innocua respectively. Moreover, all
treated cherry tomatoes samples significantly (p<0.05) increased their populations.
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Birmpa Angeliki
3.3 Food Quality parameters
3.3.1 Color
Lettuce
Physical
Quality
Control
Parameters
Time (min)
0
59.51±0.72
L*
-19.32±1.40
a*
31.25±0.21
b*
ΔΕ*
36.75±0.57
C*
45.46±0.63
WI
NaOCl 50 ppm
1
57.75±1.33
-18.31±0.80
27.48±1.39
4.74±1.33
33.02±2.52
46.37±1.79
3
56.21±3.19
-19.09±1.12
26.03±1.68
7.03±0.44
32.29±1.81
45.58±3.40
NaOCl 200 ppm
5
51.84±2.43
-18.47±1.42
24.42±1.14
10.69±1.88
30.62±2.76
42.89±1.77
1
53.73±1.18
-19.30±0.62
28.74±1.47
6.75±1.26
34.62±1.52
42.19±0.45
3
52.73±0.44*
-19.38±0.19
27.63±1.05*
7.99±0.48
33.75±0.97*
41.92±0.86*
5
52.83±0.98
-18.29±0.17
26.24±0.57
8.69±2.58
31.98±1.14
42.97±1.47
Page 169
Results
Parameters Control
Time (min)
Ultrasound Treatment
0
1
3
5
10
L*
59.71±0.72
58.82±0.65
57.82±0
57.94±0.65
57.57±0.49*
54.47±0.27* 54.86±0.52* 51.35±0.49* 47.54±2.92*
a*
-19.32±1.40
-16.47±22.63
-19.35±0
-18.50±2.88
-20.31±0.62
-21.49±0.60
-20.67±0.51
b*
31.11±0.22
30.46±0.29
30.07±0.58
30.61±1.92
28.79±0.74
28.89±1.12
28.97±0.94* 27.21±0.81* 27.79±1.07*
1.71±0.37
2.48±0.58
2.80±2.21
3.53±0.60
6.24±0.77
5.56±1.13
9.29±1.17
12.86±3.01
36±1.12
35.60±0.50
33.50±1.91
34.70±1.98
ΔΕ*
C*
36.64±0.64
36.21±0.17
35.76±0.49
35.81±2.80
35.24±0.45
WI
45.54±0.76
45.16±0.57
44.70±0.32
44.72±1.36
44.84±0.31
Control
Time (min)
20
30
45
-19.51±2.28
60
-20.75±1.99
41.95±0.77* 42.51±0.27* 40.91±0.82* 37.11±3.51*
Ultraviolet Light Treatment
0
1
3
5
10
20
30
L*
60.50±0.98
60.07±0.60
59.23±0.18
58.21±0.29
57.93±0.41
58.26±0.12
56.28±1*
a*
-19.18±0.70
-21.05±0.65
-20.23±0.02
-20.65±0.13
-19.09±1.01
-19.71±1.64
-20.60±2.15
-21.34±2
-21.42±1.65
b*
32.71±0.95
30.98±0.05
29.13±1.09
28.61±1.59
31.24±0.23
29.63±0.27
27.13±1.75
22.79±1.61
26.82±3.14
3.13±0.82
4.33±1.52
5.41±2.14
3.94±1.53
4.13±2.73
7.54±4.13
15.67±1.56
16.59±2.09
ΔΕ*
45
60
48.95±0.96* 45.91±1.60*
C*
37.92±1.17
37.46±0.32
35.47±0.91
35.31±1.22
36.61±0.60
35.60±0.75
34.14±0.28* 31.29±0.25* 34.40±2.15*
WI
45.19±0.11
45.24±0.22
45.94±0.72
45.28±0.57
44.22±0.33
45.14±0.44
44.53±0.96
Page 170
40.13±0.90* 35.89±2.50*
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Physical Quality Parameters
Time (min)
Control
US+NaOCl 50ppm
0
1
3
5
10
20
30
L*
59.71±0.72
54.61±0.04
53.73±0.38
53.16±1.95*
53.68±1.64*
51.81±1.08*
48.46±3.83*
a*
-19.32±1.40
-18.76±0.04
-18.51±0.70
-19.58±0.67
-19.14±1.12
-18.17±0.81
-17.95±0,.58
b*
31.12±0.21
27.43±0.55
26.46±1.51
26.81±2.38
26.43±1.88
25.07±1.33*
24.25±2.50*
6.46±0.20
7.72±1.37
8.10±1.56
8.03±0.93
10.15±1.60
13.32±4.58
ΔΕ*
C*
36.65±0.64
33.23±0.46
32.29±1.64
33.22±1.95
32.64±1.86
30.96±1.51
30.19±2.35
WI
45.53±0.75
43.75±0.25
43.56±0.64
42.55±2.12
43.32±2.01
42,.70±0.60*
40.18±2.20*
US+NaOCl 200ppm
Time (min)
0
1
3
5
10
20
30
L*
59.71±0.72
54.01±0.53
54.37±0.37
54.82±0.14*
54.16±0.92*
53.31±2.19*
52.80±2.33*
a*
-19.32±1.40
-19.77±0.29
-20.29±0.90
-20.78±0.82
-19.64±0.49
-20.05±0.98
-20.02±1.05
b*
31.12±0.21
27.45±0.87
29.61±1.18
29.75±0.73
26.85±0.44*
27.62±0.58*
26.79±1.32*
6.97±0.22
5.74±0.87
5.37±0.69
7.07±0.49
7.35±1.64
8.36±1.05
ΔΕ*
C*
36.65±0.64
33.83±0.78
35.90±1.48
36.29±1.00
33.27±0.30*
34.14±0.62*
33.45±1.57*
WI
45.53±0.75
42.91±0.81
41.93±0.65
42.04±0.53
43.35±0.60
42.14±1.63*
42.15±2.80*
Page 171
Results
Physical Quality Parameters
Time (min)
Control
UV+NaOCl 50ppm
0
1
3
5
10
20
30
L*
59.71±0.72
58.83±0.84
57.42±0.86*
55.27±1.27*
53.48±1.54*
52.34±4.01*
49.59±0.57*
a*
-19.32±1.40
-20.36±0.69
-19.61±0.73
-19.91±1.06
-22.33±3.07
-21.78±0.66
-21.88±0.58
b*
31.12±0.21
30.73±0.46
29.83±0.43
29.32±1.01
29.99±0.57
27.02±3.34
27.67±0.11*
1.46±0.36
3.21±0.86
5.28±1.34
7.11±2.05
8.75±5,.44
10.92±0.46
ΔΕ*
C*
36,.65±0.64
36.86±0.68
35.71±0.59
35.44±1.37
37.43±2.32
34.75±2.70
35.28±0.32
WI
45.53±0.75
44.74±1.05
44.42±0.58
42.91±0.15
40.27±2.29
40.90±1.81
38.47±0.55*
UV+NaOCl 200ppm
Time (min)
0
1
3
5
10
20
30
L*
59.71±0.72
57.20±1.49
54.86±0.25*
53.75±0.38*
50.98±0.73*
46.07±2.56*
43.91±2.08*
a*
-19.32±1.40
-19.49±1.30
-19.38±1.17
-20.17±1.86
-20.96±2.19
-20.30±0.40
-20.86±2.24
b*
31.12±0.21
28.44±1.73
27.43±0.26*
27.14±0.81*
23.38±1.31*
22.50±0.98*
23.18±0.58*
4.23±0.26
6.13±0.43
7.25±0,.78
11.84±1.00
16.22±2.10
17.88±1.75
ΔΕ*
C*
36.65±0.64
34.47±2.14
33.59±0.84
33.83±1.58
31.44±1,.52
30.31±0.76*
31.23±1.07*
WI
45.53±0.75
45.04±2.50
43.73±0.42
42.69±1.25
41.75±0.90
38.12±2.08
35.80±2.29
Page 172
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Physical
Quality
Parameters
Time (min)
Control
UV5+US5
UV10+US10
UV10+US20
UV20+US10
L*
59.08±0.45
54.62±0.48*
53.44±0.47*
52.87±0.15*
54.10±1.29*
a*
-19.85±1.45
-19.01±0.73
-19.49±0.55
-20.54±0.79
-21.11±0.69
b*
31.12±0.21
29.56±1.81
26.32±0.99*
26.95±0.65*
27.59±0.85*
5.13±0,.92
7.50±0.65
7.56±0.27
6.29±1.07
ΔΕ*
C*
36.93±0.59
35.15±1.78
32.76±0.48*
33.89±0.73
34.75±0.32
WI
44.88±0.48
42.59±0.92
43.07±0.58
41.95±0.42*
42.42±0.84*
Table 3.3.1.1: Values are average ± standard deviation of at least three experiments and represent the color parameters of romaine lettuce after each
processing time with each disinfection method: NaOCl 50 ppm, NaOCl 200 ppm, UV: Ultraviolet irradiation (254 nm), US: Ultrasound Treatment
(Frequency: 37 kHz, Power: 30 W/L), US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200ppm, UV+US. *: Asterisks
within different treatment methods indicate significant differences (p<0.05).
Page 173
Results
Strawberries
Physical
Quality
Control
Parameters
Time (min)
0
NaOCl 50 ppm
1
NaOCl 200 ppm
3
5
1
3
5
L*
42.02±0.55 39.29±0.51
37.95±2.11
37.85±2.47
39.32±0.46
38.52±1.61
37.85±2.39
a*
29.19±2.15 27.09±0.26
26.90±0.71
26.68±1.71
27.52±1.15
27.67±0.64
26.83±1.51
b*
21.52±1.39 18.34±0.40 16.67±2.38* 15.21±2.73* 18.17±0.62 16.71±2.32* 15.57±3.70*
ΔΕ*
C*
Page 174
5.21±1.12
7.08±3.50
8.10±4.19
5.01±1.69
6.65±3.29
8.09±4.89
36.32±1.08 32.72±0.01
31.68±1.82
30.76±2.53
32.98±1.30
32.36±1.44
31.10±2.92
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Physical
Quality
Parameters
Time (min)
Control
Ultrasound Treatment
0
1
3
5
10
20
30
45
60
L*
41.06±0.56
40.86±0.38
40.47±0.25
40.19±0.43
39.38±0.99
38.83±0.77
38.97±1.18
36.74±0.94
35.98±0.94*
a*
27.17±0.37
26.94±0.63
27.07±0.54
26.58±0.55
26.85±0.64
26.30±0.77
25.82±0,57
25.87±0.60
25.74±0.83
b*
20.99±2.16
20.72±2.46
20.10±2.68
19.65±2.60
19.92±4.65
19.33±4.01
18.48±2.61
14.54±1.02
15.21±1.05*
0.58±0.18
1.27±0.67
1.82±0.70
2.66±1.84
3.17±1.35
3.58±1.09
8.14±2.02
8.06±2.09
34.03±1,45
33.75±1,76
33.11±1,34
20.41±4.66*
19.82±4.01*
18.98±2.61*
15.03±1.02*
15.70±1.05*
ΔΕ*
C*
34.37±1,15
Control
Time (min)
Ultraviolet Light Treatment
0
1
3
5
10
20
30
45
60
L*
41.36±0.68
40.65±0.53
40.63±0.32
40.57±0.29
40.37±0.01
40.30±0.12
40.27±0.27
38.31±0.13*
37.79±0.15*
a*
28.09±0.96
23.29±5.23
23.93±5.24
22.82±4.34
21.36±5.15*
19.48±3.00*
18.89±2.26*
15.27±0.32*
15.52±0.40*
b*
19.90±1.47
19.02±1.69
17.72±0.49
18.77±1.14
19.88±1.76
19.33±1.61
19.49±1.72
18.33±2.07
18.30±1.87
4.98±4.35
5.10±3.92
5.48±3.47
6.84±5.02
8.71±2.90
9.28±1.66
13.31±0.59
13.18±1.32
19.51±1.69
18.21±0.49
19.26±1.14
20.21±2.05
19.82±1.61
19.98±1.72
18.82±2.07
18.80±1.87
ΔΕ*
C*
34.44±1.19
Page 175
Results
Physical Quality
Control
Parameters
Time (min)
US+NaOCl 50ppm
0
1
3
5
10
20
30
L*
43.66±0.50
42.94±0.72
40.67±2.76
39.16±1
39.55±0.82
39.96±0.18
39.94±1.03
a*
30.64±0.42
30.27±1.05
28.43±1.54
28.27±1.15
26.92±0.33
26.77±3.05
28,.84±0.70
b*
23.30±0.47
22.49±1.52
20.03±2.38
20.73±0.64
20.53±1.19
19.74±0.46
20.09±0.58
1.86±1.61
5.44±2.58
5.85±0.44
6.28±0.64
6.75±1.87
5.36±0.60
22.98±1.52
20.52±2.38*
21.23±0.64*
21.03±1.19*
20.24±0.46*
20.58±0.58*
ΔΕ*
C*
38.49±0.49
US+NaOCl 200ppm
Time (min)
0
1
3
5
10
20
30
L*
44.02±1.31
43.05±1.01
41.45±1.31
40.32±0.56
40.42±0.32
40.45±0.37
40.21±0.52
a*
24.64±5.88
22.73±3.95*
22.59±3.37*
23.17±4.85*
23.42±4.51*
21.80±4.31*
20.87±4.47*
b*
24.88±2.03
21.31±1.18
20.05±0.80
20.20±1.40
20.75±0.70
19.67±0.39
19.13±0.48
5.08±1.45
6.56±1.13
6.36±2.32
5.91±2.11
7.28±1.37
8.06±0.75
21.81±1.18*
19.87±1.01*
20.70±1.40*
21.25±0.70*
20.16±0.39*
19.62±0.48*
ΔΕ*
C*
Page 176
35.30±3.03
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Physical Quality
Parameters
Control
Time (min)
0
1
3
5
10
20
30
L*
41.86±1.33
41.09±0.37
40.41±1.28
39.78±1.25
39.31±0.45
38.92±0.23
38.06±1.52
a*
28.34±1.93
27.01±2.47
27.33±3.09
27.49±2,86
27.81±2.57
27.20±2.09
25.40±1.70
b*
21.67±2.40
20.62±0.84
19.02±0.90
19.15±0.96
18.37±0.88
16.85±2.17*
16.38±1.42*
2.52±1.91
3.90±2.02
4.00±0.57
4.48±1.81
6.60±1.55
7.93±1.62
21.12±0.84*
19.52±0.90*
19.64±0.96*
18.87±0.88*
17.35±2.17*
16.88±1.42*
ΔΕ*
C*
35.72±2.42
UV+NaOCl 50ppm
UV+NaOCl 200ppm
Time (min)
0
1
3
5
10
20
30
L*
41.86±1.33
41.74±1.52
40.73±1.52
38.62±2.27
38.16±1.93
39.55±0.44
40.19±1.59
a*
28.34±1.93
26.74±1.50
26.22±1,.20
25.86±0.28
24.49±2.19
23.88±3.01
25.13±2.69
b*
21.67±2.40
19.76±1.07
19.36±0.78
17.96±1.09
18.24±3.46
18.95±2.05
19.00±2.78
2.95±1.69
3.85±3.00
6.14±2.77
6.61±1.02
5.82±1.31
5.06±0.69
20.26±1.07*
19.85±0.78*
18.46±1.09*
18.73±3.46*
19.45±2.05*
19.49±2.78*
ΔΕ*
C*
35.72±2.42
Page 177
Results
Physical
Quality
Parameters
Time (min)
control
UV5+US5
UV10+US10 UV10+US20 UV20+US10
L*
38.23±0.29
35.55±0.12
33.95±0.86*
34.86±0.04*
36.12±0.76*
a*
26.91±0.84
25.74±0.15
22.76±0.76
25.21±1.78
25.20±1.33
b*
19.60±1.51 19.60±0.26*
15.01±0.29*
13.02±0.44*
17.07±1.16*
4.61±0.52
7.84±0.75
7.98±0.89
7.60 ±1.45
26.05±7.72
23.15±6.33
24.08±8.93
26.38±8.78
ΔΕ*
C*
33.32±0.40
Table 3.3.1.2: Values are average ± standard deviation of at least three experiments and represent the color parameters of strawberries after each processing time
with each disinfection method: NaOCl 50 ppm, NaOCl 200 ppm, UV: Ultraviolet irradiation (254 nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30
W/L), US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm, UV+US. *: Asterisks within different treatment methods indicate
significant differences (p<0.05).
Page 178
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Cherry Tomatoes
Physical
Quality
Control
Parameters
Time (min)
NaOCl 50 ppm
NaOCl 200 ppm
0
1
3
5
1
3
5
L*
41.47±1.08
39.12±0.90
38.34±0.15
38.11±0.41
39.65±0.26
39.90±0.10
38.68±0.09
a*
19.34±2.97
16.96±3.10
14.54±0.60
14.64±1.03
16.02±0.50
15.70±0.61
15.29±0.15
b*
18.86±1.20
17.07±0.53
15.65±0.74
15.42±1.15
19.20±0.43
19.13±0.62
17.71±0.08
4.13±1.70
7.04±0.77
7.11±0.52
4.31±2.10
4.33±1.89
5.58±1.90
19.74±2.94
25.01±0.23
24.75±0.48
23.40±0.12
ΔΕ*
C*
23.54±4.82
21.12±2.70
19.54±2.33
TCI
220.97±25.20
223.68±14
219.90±4.74 228.23±0.50 203.47±5.44 200.82±6.88 210.12±1.39
Page 179
Results
Physical
Quality
Control
Parameters
Time (min)
Ultrasound Treatment
0
1
3
5
10
20
30
45
60
L*
42.27±0.31
41.27±0.02
41.26±0.01
41.21±0.09
41.31±0.45
37.87±0.41*
36.31±0.27*
35.48±0.38*
35.41±0.66*
a*
19.33±2.96
15.69±0.11
15.67±0.08
15.79±0.06
15.18±0.25
12.00±0.15*
10.72±0.19*
9.76±0.08*
9.09±1.51*
b*
21.07±2.63
19.70±0.44
19.48±0.55
19.59±0.45
19.51±0.44
15.01±0.36
16.01±0.41
16.54±0.96
16±1.16
4.12±3.45
4.19±3.54
4.08±3.50
4.71±3.60
10.50±3.64
11.77±3.76
12.72±3.92
13.51±3.13
19.21±0.83
18.46±0.35
ΔΕ*
C*
28.59±3.94
25.19±0.39
25.00±0.41
25.16±0.38
24.72±0.50
19.22±0.37
19.27±0.45
TCI
207.69±2.24
193.98±2.21
195.15±3.65
195.52±2.09
191.09±0.58
202.96±0.35
184.74±0.38
Control
Time (min)
170.89±7.76* 165.85±8.04*
Ultraviolet Light Treatment
0
1
3
5
10
20
30
45
60
L*
42.27±0.31
41.56±0.28
40.45±0.11
40.67±0.69*
40.42±0.09*
40.34±0.10*
41.27±0.27*
38.31±0.13*
37.79±0.15*
a*
19.33±2.96
17.30±0.62
16.52±0.13
16.74±0.03
17.36±0.23
15.87±0.09
15.89±0.84
15.27±0.32
15.52±0.40
b*
21.07±2.63
19.59±1.31
18.25±1.49
17.59±1.59
20.61±0.51
20.20±0.26
18.82±0.91
17.99±1.29
17.64±1.90*
2.83±2.72
4.52±2.79
4.85±2.72
3.45±2.58
4.37±3.32
4.26±2.67
6.45±1.90
6.66±2.87
ΔΕ*
C*
28.59±3.94
26.14±1.36
24.63±1.18
24.30±1.17
26.94±0.52
25.69±0.15
25.64±1.23
23.61±1.19
23.52±1.18
TCI
207.69±2.24
205.47±3.96
211.19±8.70
216.39±9.69
202.70±9.69
194.56±1.94
200.84±0.82
209.28±6.44
215.14±6.37
Page 180
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Physical
Quality
Parameters
Control
Time (min)
0
1
3
5
10
20
30
L*
41.82±0.20
40.33±0.04
39.77±0.26
37.97±0,.03*
37.29±0.13*
36.11±0.02*
35.77±0.46*
a*
19.34±0.34
17.21±0,.08
16.22±0.34
15.32±2.18
14.84±2.98
14.18±1.70
12.84±0.57
b*
20.97±0.26
18.96±0.35
16.66±0.05
14.77±1.01
14.59±1.44
14.44±0.54
13,.91±0.69
3.49±3.57
5.82±4.15
8.68±5.28
9.48±6.28
10.30±4.62
11.42±4.71
ΔΕ*
US+NaOCl 50ppm
C*
28.53±3.99
25.61±0.30
23.25±0.22
21.30±2.04
20.83±3.11
20.26±1.48
18.93±0.86
TCI
209,49±3.14
211.68±1.73
221.15±1.96
232.71±13.96
232.00±13.55
232.55±11.39
226.77±3.47
US+NaOCl 200ppm
Time (min)
0
1
3
5
10
20
30
L*
41.82±0.20
40.21±0.06
37.51±1.24
36.77±0.70*
36.42±0.16*
36.85±1.07*
35.74±3.47*
a*
19.34±0.34
14.85±2.27
13.86±2.70
13.20±2.76
12.23±2.65
12.66±2.16
12.24±4.22
b*
20.97±0.26
17.58±0.52
15.22±0.55
15.08±0.19
14.08±1.96
14.05±2.45
13.79±4.83
5.98±5.35
9.23±4.19
10.33±4.84
11.55±5.97
11.11±6.82
12.05±6.34
ΔΕ*
C*
28.53±3.99
23.04±1.88
20.61±2.62
20.13±1.89
18.71±2.86
18.95±3.53
18.48±3.53
TCI
209.49±3.14
202.49±14.05
218.30±16.64
214.41±25.65
215.19±23.75
220.03±17.88
222.05±21.59
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Physical
Quality
Parameters
Control
Time (min)
0
1
3
5
10
20
30
L*
41.82±0.20
40.41±0.47
40.02±0.44
40.45±0.32
40.35±0.42*
40.29±0.12*
39.00±1.07*
a*
19.34±0.34
17.40±0.69
16.63±0.17
16.39±0.79
15.99±0.50
16.22±1.00
16.37±1.06
b*
20.97±0.26
18.90±0.82
18.41±0.47
18.51±0,.40
18.60±0.30
18.33±0.53
18.07±0.34
3.55±3.10
4.36±4.33
4.29±3.38
4.53±4.12
4.47±4.89
5.27±4.75
ΔΕ*
UV+NaOCl 50ppm
C*
28.53±3.99
25.69±1.07
24.80±0.46
24.73±0.58
24.53±0.53
24.48±1.04
24.38±0.84
TCI
209.49±3.14
213.16±1.70
211.96±2.86
208.38±6.07
205.23±1.93
208.70±4.22
214.80±4.56
UV+NaOCl 200ppm
Time (min)
0
1
3
5
10
20
30
L*
41.82±0.20
40.95±0.02
39.78±0.51
39.73±1.04
39.59±1.21*
39.22±1.13*
38.91±0.06*
a*
19.34±0.34
18.12±0.60
14.25±0.21
14.71±0.64
14.82±0.59
14.57±0.48
14.70±0.65
b*
20.97±0,.26
18.80±0.02
18.69±0.77*
18.93±0,.65*
19.25±0.42*
18.65±0.61*
18.56±0.13*
3.13±3.37
6.08±3.46
5.79±3.46
5.64±3.46
6.05±2.85
6.17±3.31
ΔΕ*
C*
28.53±3.99
26.11±0.42
23.50±0.71
23.99±0.29
24.30±0.11
23.67±0.76
23.68±0.30
TCI
209.49±3.14
216.80±3.66
192.34±3.28
194.77±11.45
193.97±9.64
196.71±4.26
198.95±6.31
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Physical
Quality
Parameters
Time (min)
control
UV5+US5
UV10+US10
UV10+US20
UV20+US10
L*
41.82±0.20
37.22±0.17*
37.32±0.58*
37.80±0.52*
36.92±0.11*
a*
19.34±0.34
11.59±0.34
10.72±0.66
12.96±0.15
11.40±0.35
b*
18.86±1.20
12.08±0.32*
11.57±1.05*
14.86±1.08*
13.55±1.18*
12.92±3.39
13.75±2.98
10.11±4.27
12.26±4.26
ΔΕ*
C*
23.54±4..82
16.74±0.44
15.78±1.19
19.72±0.85
17.73±0,.67
TCI
209.49±3.14
227.00±2.00
222.70±4.47
213.90±7.23
212.06±14.88
Table 3.3.1.3: Values are average ± standard deviation of at least three experiments and represent the color parameters of cherry tomatoes after each processing time
with each disinfection method: NaOCl 50 ppm, NaOCl 200 ppm, UV: Ultraviolet irradiation (254 nm), US: Ultrasound Treatment (Frequency: 37 kHz, Power: 30
W/L), US+NaOCl 50 ppm, US+NaOCl 200 ppm, UV+NaOCl 50 ppm, UV+NaOCl 200 ppm, UV+US. *: Asterisks within different treatment methods indicate
significant differences (p<0.05).
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Color was expressed in terms of L*, a* and b* values. The chromameter describes color
in three coordinates. More specifically, L* value indicated luminosity from 0 (black,
level of darkness) to 100 (white, level of light), a* indicated chromaticity on a green (60, negative number) to red (+60, positive number), and b* value was responsible for
chromaticity on a blue (-60, negative) to yellow (+60, positive) scale respectively.
When lettuce samples were treated with US for 45 and 60-min, some parts of the leafy
structure of lettuce lost their green color giving final values of ΔΕ* 9.29±1.17 and
12.86±3.01 respectively. Treatment with NaOCl 50 ppm for 7-min resulted in a
ΔΕ*=10.69±1.88. For UV treatments of 45 and 60-min the ΔΕ* observed were
15.67±1.56 and 16.59±2.09 respectively. Combined treatment of US 30min followed by
immersion in NaOCl 50 ppm for 3-min resulted in ΔΕ*: 13.32± 4.58. Whereas,
combined treatment of UV 20 and 30-min followed by immersion in 200 ppm NaOCl
has as a result an increase in ΔΕ* of 16.22±2.10 and 17.88±1.75 respectively.
As far as strawberry modifications are concerned, the highest net change of color was
observed for treatment with UV for 45 and 60-min respectively where an increase of
13.31±0.59 and 13.18±1.32 were observed.
Comparing the color of treated cherry tomatoes, the highest net change of color (ΔΕ*)
for lettuce was observed when the samples were treated with US at longest time intervals
(after 20-min). Combined treatments of US followed by NaOCl 50ppm and 200 ppm
exhibited a change (ΔΕ*>10) after 20-min and 10-min treatment respectively.
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3.3.2 Physicochemical Parameters
Lettuce
Figure 3.3.2.1: TAC of Romaine Lettuce before and after conventional, alternative and
combined disinfection technologies.
No significant differences (p>0.05, n=44) were observed as far as Total Antioxidant
Capacity (TAC) is concerned when conventional treatments at different treatment times
were used. However, when alternative disinfection treatments were used, an increase in
TAC concentration was obvious from the first-min of treatment, however the increase
was significant after 45-min with US (p=0.049) and at 60-min treatment with UV
(p=0.004). The increases after longest treatment times with UV and US reached 731 and
273 μmol Fe2+/g respectively. When combined alternative technologies were used, a
significant increase of 463 and 366 μmol Fe2+/g was detected at 20-min UV followed by
10-min US (p=0.005) and at 10-min UV followed by 20-min US (p=0.013),
respectively. Finally, significant increases (p<0.05, n=156) were observed when
combinations of alternative and conventional treatments occurred.
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Figure 3.3.2.2: TPC of Romaine Lettuce before and after conventional, alternative and combined
disinfection technologies.
Total Phenolic Content (TPC) concentration remained constant or was slightly decreased
when lettuce was immersed in NaOCL solutions. However, TPC increased by 3.76 and
3.01 mg gallic acid / g lettuce when UV and US alternative disinfection technologies
were used. However, the differences among different treatment times, were statistically
significant, after 45 min US treatment (p=0.033) and 30 min UV treatment (p=0.025).
The highest concentrations of TPC were detected in UV20+US10-, US+NaOCL50 -,
UV+NaOCl200 - and UV+NaOCl 50 - treated lettuces and were 22.18, 22, 22.25 and
22.65 mg gallic acid / lettuce respectively.
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Figure 3.3.2.3: AA of Romaine Lettuce before and after conventional, alternative and combined
disinfection technologies.
The Ascorbic Acid (AA) content of lettuces did not exhibit any significant changes
during different treatments. However, AA was slightly decreased (p>0.05, n=132) when
treatments of more than 30-min for US, UV and combinations of UV+US occurred.
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Results
Strawberries
Figure 3.3.2.4: TAC of Strawberries before and after conventional, alternative and combined
disinfection technologies.
When the strawberries are immersed in sodium hypochlorite solutions a slight but not
statistically significant increase is observed (p>0.05, n=44).
However, the mean
observed increases of UV and US treatment are after the longest treatment times, 309
and 223 μmol Fe2+/g respectively. US led to a significant (p<0.05) increase from 5
minute treatment, whereas UV led to a significant increase (p<0.05) from 3 minute
treatment. Moreover, the combined alternative treatments also led to an increase
(p<0.05, n=36) in TAC of strawberries. The higher content of total antioxidants is
obvious after the first-min of treatments.
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Figure 3.3.2.5: TPC of Strawberries before and after conventional, alternative and combined
disinfection technologies.
The values obtained in this study demonstrate the positive effect of alternative and
combined alternative technologies on the TPC of strawberries. However, the results
obtained by conventional treatments showed the negative or no effect of sodium
hypochlorite on the phenolic content of strawberries. UV and US led to significant
increases (p<0.05, n=108) in phenolic content after 30- min treatment.
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Results
Figure 3.3.2.6: AA of Strawberries before and after conventional, alternative and combined
disinfection technologies.
In all treated samples the AA content was decreased. However, a slight difference was
observed between UV and US treated samples, where the decrease was higher for US ,
US+NaOCl 50 ppm and US+NaOCl 200ppm treated samples (-0.18, -0.16, -0.17
respectively) after the longest treatment times (60-min, 33-min, 33-min), compared to
UV and UV+NaOCL treated samples (-0.12, -0.14, -0.1) at the same treatment times
respectively. UV and US decreased the AA content significantly (p<0.05, n=108) after
3-min treatment, whereas all combinations of alternative disinfection technologies were
statistically significant (p<0.05, n=36).
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Cherry Tomatoes
Figure 3.3.2.7: TAC of Cherry Tomatoes before and after conventional, alternative and
combined disinfection technologies.
It was observed that the TAC remained constant or slightly decreased when conventional
treatments were used. However, when alternative disinfection treatments were used, UV
resulted in greater values compared to US. In both technologies the increase was
significant (p<0.05, n=108) from the first minute of treatment. After the longest
exposure time (60-min) of UV, 155 μmol Fe2+ /g increase of antioxidants was observed,
whereas 115 μmol Fe2+ /g, after 60-min treatment with US. Moreover, the combined
effect of UV+US did not exhibit an additive effect, compared to single treatments,
however the increase was significant (p<0.05, n=36) when tested with pairwise t-test.
Furthermore, when combined alternative and conventional technologies were used, the
effect of sodium hypochlorite did not affect the final value of TAC on cherry tomatoes
(p>0.05, n=156).
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Results
Figure 3.3.2.8: TPC of Cherry Tomatoes before and after conventional, alternative and combined
disinfection technologies.
The values for TPC of cherry tomatoes slightly increased (p>0.05, n=44) after
conventional technologies, whereas a higher increase (p<0.05, n=108) is observed after
Ultrasound and UV technology, with increase of TPC by 3,07 mg gallic acid/g and 2,31
mg gallic acid/g respectively. The increase was significant from the first minute of US
treatment (p=0.003) and first minute of UV treatment (p=0.034). When combined
technologies were used, a statistical increase (p<0.05, n=180) was also observed, with
the higher increase (2,48 mg gallic acid/g) observed when US+NaOCl 200ppm was
implemented.
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Figure 3.3.2.9: AA of Cherry Tomatoes before and after conventional, alternative and combined
disinfection technologies.
The concentration of AA was generally remained constant or was slightly decreased.
However, no statistically significant differences (p>0.05, n=310) observed before and
after the use of different disinfection technologies, as far as their ascorbic acid content is
concerned.
Finally a more generalized statistical analysis was carried out. For instance, Tukey HSD
test was used in conjunction with an ANOVA to find disinfection technologies that are
significantly different from each other for each physicochemical parameter.
TAC did not differ significantly (p>0.05, n=620) when all disinfection methods were
used for strawberry and cherry tomatoes. However, differences between conventional
and other technologies (p<0.05) were recorded for lettuce.
TPC was different (p<0.05, n=310) when lettuce was immersed in NaOCl solutions
compared to other methods. However, the differences were not significant (p>0.05) for
the rest of disinfection technologies. As far as strawberry is concerned, TPC observed
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Results
when US method was used, was different compared to conventional methods
(p=0.0001), as well as compared to UV method followed by immersion in NaOCl 50
ppm solution (p=0.003). For cherry tomatoes, difference in phenolic content was
significant between US and NaOCl 50 ppm (p=0.002) and NaOCl 200 ppm (p=0.24),
and between US and combined UV followed by NaOCl 50 ppm (p=0.003) and UV
followed by NaOCl 200 ppm (p=0.001).
Differences in AA content among different technologies were observed in cherry
tomatoes. For instance, AA content between US and NaOCl 50 ppm and 200 ppm was
significant (p=0.0001 and p=0.019 respectively). Whereas in strawberries significant
differences were detected only between US and US followed by NaOCl 200 ppm
(p=0.043). No significant differences (p>0.05, n=310) were found in AA content of
lettuce when treated with different disinfection technologies.
The Pearson correlation was finally used to examine any correlation between
physicochemical values irrespectively of the disinfection method used. The relationship
between TAC and TPC values was found to be positive and near to linear for strawberry
(r=0.738, n=620) and cherry tomatoes (r=0.792, n=620) but a weak correlation (r=0.505,
n=620) between the two aforementioned values was found for lettuce.
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3.4 A user-friendly theoretical mathematical model for the
prediction of food safety in a food production chain
Three experts evaluated the effect of nine critical points to the final produce
(C10=lettuce).
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10 (OUTPUT)
C1
-
M
-
-
-
VS
M
M
W
-VS
C2
VS
-
M
M
-
VS
VS
VS
M
-VS
C3
-
M
-
-
M
-
-
-
-
-W
C4
-
M
-
-
-
-
-
-
-
-
C5
-
-
M
-
-
-
-
-
-
W
C6
-
M
-
-
-
-
W
-
-
W
C7
-
M
-
-
-
W
-
W
-
W
C8
-
M
-
-
-
-
W
-
-
W
C9
-
-
-
-
-
-
-
-
-
-W
C10(OUTPUT)
-
-
-
-
-
-
-
-
-
-
C1
C2
C3
C4
C5
C6
C1
-
VS -
C2
VS -
C3
-M
C4
-
C5
C7
C8
C9
C10 (OUTPUT)
-W -
VS VS -M W
-VS
-
W
-
S
S
M
M
S
W
-
-
-S
-
-
-
-M -M
-
-
-
-
-
-
-
-W -
-W W
-S
-
-
-
-
-
-S
-M
C6
-
-
-
-
W
-
M
M
-S
-M
C7
-
-
-
-
-
M
-
W
S
-M
C8
-
-
-
-
-
M
W
-
-M -W
C9
-
-
-
-
-
-
M
-M -
-W
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Results
C10(OUTPUT)
-
-
-
-
-
-
-
-
-
-
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10 (OUTPUT)
C1
-
S
-
-
-
VS
VS
-M
W
-VS
C2
VS
-
W
W
-
VS
VS
S
M
S
C3
-W
M
-
-
-M
-
-
-
-W
-M
C4
-
W
-
-
-
-
-
-
-W
-
C5
-W
W
W
-
-
-
-
-
-
W
C6
-
W
-
-
-
-
W
W
-M
W
C7
-
W
-
-
-
W
-
W
W
W
C8
-
W
-
-
-
W
W
-
-W
W
C9
-
-
-
-
-
-
W
-M
-
-W
C10(OUTPUT)
-
-
-
-
-
-
-
-
-
-
Table 3.4.1: Evaluation of three experts, where W: Weak, M: Medium, S: Strong, VS: Very
Strong
The concepts that were selected to be tested during the lettuce production procedure
were extracted from questionnaires that were filled from experts. The methodology
described extracts the knowledge from experts and exploits their experience of the
process. Each expert based on his/her experience knows the main factors that contribute
to the decision. Experts described the existing relationship firstly as “negative” or
“positive” and secondly, as a degree of influence using a linguistic variable, such as
“low”, “medium”, “high” etc.
More specifically, the causal interrelationships among concepts are declared using the
variable influence which is interpreted as a linguistic variable taking values in the
universe of discourse U = [-1, 1]. Its term set T (influence) is suggested to be comprised
of nine variables. Using nine linguistic variables, an expert can describe the influence of
one concept on another in detail and can discern it between different degrees. The nine
variables used here are: T (influence) = {negatively very strong, negatively strong,
negatively medium, negatively weak, zero, positively weak, positively medium,
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positively strong, positively very strong}. With this method the purpose was to diagnose
and predict the effect of different factors during the lettuce production chain in their
contribution to a final safe fresh lettuce.
Figure 3.4.1: The FCM Model
Figure 3.4.2: Fuzzy Cognitive Map.
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FCMs are a combination of methods of fuzzy logic and neural networks. It is a flexible
computational method, which is able to consider situations in which human reasoning
process includes fuzzy and uncertain descriptions. FCMs are fuzzy-graph structures for
representing causal reasoning. Their fuzziness allows hazy degrees of causality between
causal objects (concepts). The effect and the interrelationships between the nodes should
be calculated, in order to create a FCM. Each node is a concept, a main feature of the
system. Each interrelationship between the nodes represents a cause-effect relationship
that exists between concepts and determines the manner that one concept influences on
the value of the interconnected concepts.
W ij are the weights among concepts and they take their values in the universe of
discourse U = [-1, 1]. Each expert described the interconnections using linguistic
variables, which with a defuzzification method are transformed to a numerical weight
W ij , belonging to the interval [-1, 1].
•
W ij >0 positive causality, which means that when the value of concept C i
is increased the value of the concept C j is also increased.
•
W ij <0 negative causality, which means that when the value of concept C i
is increased the value of the concept C j is decreased.
•
W ij =0 no relationship between the concepts.
Generally, the value of each concept at every simulation step is calculated, computing
the influence of the interconnected concepts to the specific concept, by applying the
following calculation rule:
N
Ai ( k +1) f (k2 Ai ( k ) + k1 ∑ Aj ( k )W ji )
=
j ≠i
j =1
where A i (k+1) is the value of the concept C i at the iteration step k+1, A i (k) is the value of
the concept C j at the iteration step k, W jj is the weight of interconnection from concept
C i to concept C j and f is the sigmoid function. “k 1 ” expresses the influence of the
interconnected concepts in the configuration of the new value of the concept A i and k 2
represents the proportion of the contribution of the previous value of the concept in the
computation of the new value.
The sigmoid function f belongs to the family of squeezing functions, and the following
function is usually used to describe it:
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f =
1
1 + e−λ x
This is the unipolar sigmoid function, in which λ>0 determines the steepness of the
continuous function f(x).
As mentioned above, each expert estimated each weight Wij between nodes i and j,
according to his/her experience. In order to be sure about experts’ reliability, an
algorithm was used to calculate both the weights of each interconnection and the
credibility of experts. Each expert constructed his/her own weight matrix. Each weight
Wij was collected and then they were compared according to the algorithm that
followed.
The above variables were converted into numerical values with a defuzzification
method. The Center of Area (COA) defuzzification method is one of the most commonly
used defuzzification techniques. In this method, the fuzzy logic controller first calculates
the area under the scaled membership functions and within the range of the output
variable. The fuzzy logic controller then uses the following equation to calculate the
geometric center of this area.
where S is the support set of the membership function of the output μ(y).
After COA defuzzification method the final weight matrix was presented.
Figure 3.4.3: Subsequent values of concepts till convergence of 1st case.
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Results
In the 1st Case, the experts decided as initial values of the inputs the following: C1, C2,
C3, C5, C6, C7: very strong, C4, C8, C9: strong
The initial values for the concepts after COA defuzzyfication method were:
A(0)= [1 1 1 0.75 1 1 1 0.75 0.75]
The iterative procedure is being terminated when the values of concepts C i have no
difference between the latest two iterations. Considering λ=1 for the unipolar sigmoid
function and after N=9 iteration steps the system reaches an equilibrium point, where the
values do not change any more from their previous ones (figure 3.4.3). The calculated
value of the decision concept was C 10 =0.951, which corresponds to the 95.1% of the
output. Consequently the lettuce could be safe for consumption with 95.1% certainty.
Figure 3.4.4: Subsequent values of concepts till convergence of 2nd case.
In the 2nd Case, the representation of the concepts till their convergence were illustrated
(figure 3.4.4). The experts decided as initial values of the inputs the following: C1, C2,
C3, C5, C6, C7: strong, C4, C8: medium, C9:weak
The value of the decision concept was C 10 =0,818 which corresponds to the 81.8% of the
output. It needed 10 iteration steps in order to reach to an equilibrium point. It can be
concluded that the lettuce was also safe with 81.8% certainty for consumption in this
case.
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Figure 3.4.5: Subsequent values of concepts till convergence of 3rd case.
In the 3rd Case, the experts decided as initial values of the inputs the following: C1, C3,
C4: medium, C5, C6, C7: strong, C2, C8: weak, C9: zero
The output is C 10 =0.43, which corresponds to the 43% of the output (figure 3.4.5). This
means that the lettuce was not guaranteed (with 43% certainty) for human consumption.
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Results
3.5 Assessment of disinfection technologies based on infectivity
doses
Taking all the results into consideration, tables have been constructed which include the
initial microorganism load, the best disinfection value produced with each disinfection
technology and the infectious dose that is recorded in literature for each microorganism
(tables 3.5.1, 3.5.2, 3.5.3).
Data on pathogen-specific relative infectivity were collected from the U.S. Food and
Drug Administration’s (FDA), Public Health Agency of Canada (PHAC) and the
European pathogen fact sheets. Given that relative infectivity cannot be expressed as a
single numerical input but is, instead, a range of concentrations that depend on factors
such as individual susceptibility and pathogen species, strain, or subtype, a worst-case
scenario approach was taken whereby the relative infectivity categorization reflected the
lower end (highest risk) of this range.
UV+NaOCl
LETTUCE
E. coli
S. aureus
Control
10⁸
10⁸
UV
(60min)
106(**)
5
10 (**)
4
(30+3min,
200ppm)
105(*)
5
10 (**)
4
US
(60min)
105(*)
5
10 (**)
0
US+NaOCl
(30+3 min,
200ppm)
NaOCl
(200 ppm,
3min)
104(*)
105(*)
5
10 (**)
3
6
10 (**)
4
UV+US
(30min)
107(***)
INFECT
IOUS
DOSE
10 (***)
10⁶
105-106
7
S. Enteritidis
10⁶
10 (***)
10 (***)
10 (*)
10 (**)
10 (***)
105(***)
102-103
L. innocua
10⁷
105 (***)
104(***)
104(***)
103(**)
105(***)
106(***)
102-103
HAdV35
10⁸
102(**)
103(***)
106(***)
105(***)
102(**)
104(***)
101-102
Table 3.5.1: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different
disinfection methods at the longest exposure times and infectious doses for each microorganism
inoculated in lettuce. Stars show the severity of infection: low infectivity (*), medium infectivity
(**), high infectivity (***).
The combined technology of US followed by NaOCl was found to be the best
disinfection technology for reducing bacteria in lettuce. Moreover, UV and NaOCl
exhibited promising results in disinfecting lettuce from HAdV35. The combined
technology of UV+US recorded to have low disinfection efficiency for all
microorganisms, compared to the other technologies.
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STRAWBERRY
E. coli
S. aureus
Control
UV
(60min)
UV+NaOC
l (30+3
min,
200ppm)
US
(60min)
US+NaOCl
(30+3 min,
200ppm)
NaOCl
(200 ppm,
3min)
UV+US
(30min)
INFEC
TIOUS
DOSE
10⁷
106 (**)
105 (*)
104 (*)
104 (*)
106(**)
107(***)
10⁶
10⁷
5
10 (**)
4
10 (*)
104(***)
5
10 (**)
4
5
6
10 (*)
10 (**)
10 (**)
105-106
103(**)
100 (*)
102(**)
104(***)
105(***)
102-103
S. Enteritidis
10⁶
L. innocua
10⁷
104(***)
104(***)
100 (*)
104(***)
105(***)
104(***)
102-103
HAdV35
10⁷
102(**)
105(***)
105(***)
106(***)
103(***)
103(***)
101-102
Table 3.5.2: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different
disinfection methods at the longest exposure times and infectious doses for each microorganism
inoculated in strawberries. Stars show the severity of infection: low infectivity (*), medium
infectivity (**), high infectivity (***).
US seemed to be the most promising disinfection technology for reducing bacteria
population in strawberries, followed by US+NaOCl technology. However, UV was
recorded as the best option for reducing HAdV35 in strawberries.
Control
UV
(60min)
UV+NaO
Cl (30+3
min,
200ppm)
US
(60min)
US+NaO
Cl (30+3
min,
200ppm)
NaOCl
(200 ppm,
3min)
UV+US
(30min)
INFEC
TIOUS
DOSE
E. coli
10⁷
104(*)
103(*)
103(*)
102(*)
103(*)
104(*)
10⁶
S. aureus
10⁶
104(*)
103(*)
103(*)
102(*)
104(*)
104(*)
105-106
S. Enteritidis
10⁷
104(***)
104(***)
103(**)
103(**)
104(***)
103(**)
102-103
L. innocua
10⁶
104(***)
103(**)
103(**)
102(*)
104(***)
104(***)
102-103
HAdV35
10⁷
103(***)
104(***)
104(***)
104(***)
102(**)
104(***)
101-102
CHERRY
TOMATOES
Table 3.5.3: Values (CFU/g for bacteria and PFU/g for HAdV35) obtained with different
disinfection methods at the longest exposure times and infectious doses for each microorganism
inoculated in cherry tomatoes. Stars show the severity of infection: low infectivity (*), medium
infectivity (**), high infectivity (***).
The best disinfection results were recorded for cherry tomatoes, where the majority of
the disinfection methods exhibited promising results in reducing populations. However,
NaOCl seemed to be the only method that could reduce HAdV35. On the contrary, E.
coli and S. aureus were adequately reduced with all the disinfection methods. In general
terms US+NaOCl seemed to be a valuable disinfection method as it reduced the four
bacteria.
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Discussion
Chapter 4. DISCUSSION
Four approaches of experiments were conducted in order to evaluate the efficiency of
different disinfection technologies on different fresh produces. The final scope was to
ensure food safety and public health. For this reason, hurdle approaches (such as UV,
US, NaOCl, and combined disinfection treatments) were used in order to determine their
individual as well as potential synergistic/additive mode of action against foodborne
bacteria and viruses.
In recent years, consumer demand for safe, and natural products without chemical
residues has been of great importance. It is well-known that processing of vegetables and
fruits promotes a faster physiological deterioration, changes and microbial degradation
of the product even when only slight processing operations can be used, which may
result in degradation of their color, texture and flavour (Martin-Belloso, 2007). While
conventional food-processing methods extend the shelf-life of fruit and vegetables, the
minimal processing to which fresh RTE fruit and vegetables are subjected renders
products highly perishable, requiring chilled storage to ensure their reasonable shelf-life
(Martin-Diana et al., 2008). The vast majority of fresh minimally processed produce
manufacturers use chlorine in washing and decontamination procedures (Seymour,
1999). There is controversy about the formation of carcinogenic chlorinated compounds
in water (chloramines and trihalomethanes), calling into question the use of chlorine
(Wei et al., 1995). As a consequence, alternative, non-thermal treatments gain more and
more ground as promising technologies for food disinfection (Martin-Diana et al., 2008).
The first experimental approach of the present thesis evaluated the effectiveness of three
non-thermal light technologies (NUV-Vis, continuous UV, and HILP) on their ability to
inactivate two pathogens on a liquid matrix. The scope was to select the most effective
light technology for use in fresh produce companies. The second experimental approach
involved the use of non-thermal technologies (UV and US) as well as conventional
sodium hypochlorite (NaOCl) solutions, in order to evaluate the disinfection efficiency
of three RTE produces (romaine lettuce, strawberry and cherry tomatoes). The series of
these disinfection technologies included also combinations of the above technologies.
More precisely, UV+US, UV+NaOCl and US+NaOCl combined technologies were
used. RTE produces were inoculated with different concentrations of bacteria and virus
commonly found in many foodborne diseases and their disinfection efficiency with
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selected disinfection technologies was tested. Furthermore, with the aim of investigating
how the above pathogens survive during refrigerated storage, the three fresh produces
inoculated with the cocktail of the above four microorganisms were treated with selected
disinfection technologies and were kept in refrigerated conditions for 15 days. The
microbial load of romaine lettuce, strawberry and cherry tomatoes was recorded after 3,
7 and 15 days of storage at 6˚C.
In the third experimental approach, the quality and the physicochemical characteristics
of the above fresh RTE produces were tested before and after the use of disinfection
technologies, in order to extract conclusions about their nutritional properties in
combination with the disinfection technology used.
The fourth approach was a computerized model, which was proposed, in order to obtain
a risk assessment software tool to ensure food safety and public health.
Finally, conclusions based on infectivity doses for each pathogen and the results
obtained from the present study, were exported, with the final scope to assure public
health.
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Discussion
4.1 In Vitro Experiments with 3 Light Technologies
The initial aim of this study was to test the relative susceptibility of two bacteria (one
gram negative and one gram positive) using three different light techniques. Then, a
determination of the effectiveness of three light equipments on inactivation efficiency of
selected types of bacteria when different dosages were implemented, followed.
The current study demonstrated that both E. coli and L. innocua are susceptible to all
three light technologies investigated. Previous studies have investigated the lethal effects
of high-intensity ultraviolet 405 nm light on Escherichia, Salmonella, Shigella, Listeria,
and Mycobacteria as well as on Saccharomyces cerevisiae, Candida albicans, and
spores of Aspergillus niger (Murdoch et al., 2012, Murdoch et al., 2013). As the
mechanism of inactivation by visible light is believed to be through the production of
ROS, the susceptibility of both E. coli and L. innocua to ROS may play an important
role in the inactivation of these organisms by NUV-vis light of 405 nm. The mode of
action is based on the stimulation of endogenous microbial porphyrin molecules to
produce oxidizing reactive oxygen species (ROS), predominantly singlet oxygen (1O 2 )
that damages cells leading to microbial death (Maclean et al., 2008a). Specifically, 405
nm light has been shown to be capable of inactivating a range of predominantly
nosocomial pathogens and also Gram-negative food-related pathogens (Enwemeka et al.,
2008). When NUV-vis light was implemented, L. innocua proved to be the most readily
inactivated organism compared to E. coli (p < 0.05). Murdoch et al. (2012) found that L.
monocytogenes was most readily inactivated in suspension, whereas S. enterica was
most resistant. They concluded that 395±5 nm light inactivates diverse types of bacteria
in liquids and on surfaces, in addition to the safety advantages of this visible (non-UV
wavelength) light. Furthermore, it has been reported (Murdoch et al., 2013) that fungal
organisms may be somewhat more resistant to 405 nm light than bacteria.
The results obtained in this study are consistent with other studies which have reported
that Gram-positive species, in general, were more susceptible to 405 nm light
inactivation than Gram-negative species (Maclean et al., 2009). The prokaryotic bacteria
also exhibit considerable variability in susceptibility achieving 5-log 10 order reductions,
with doses as low as 18 J/cm2 with Campylobacter jejuni was tested (Murdoch et al.,
2010). When doses around 50–300 J/cm2 were implemented, Gram-positive species
were generally more susceptible than Gram-negatives (Maclean et al., 2009).
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Microbial inactivation by 405nm light exposure has been found to be dose-dependent
(Murdoch et al., 2012), and in applications where more rapid inactivation is desirable,
the use of a much higher power light source, would significantly reduce the exposure
times required for effective treatment. It must be emphasized that at the highest dosage
(36 J/cm2), 1.37 log 10 CFU/mL reduction was achieved for E. coli and a greater log
reduction (2.74 log 10 CFU/mL) was achieved for L. innocua. Our results are in
accordance with another study (Murdoch et al., 2012), where L. monocytogenes was
completely inactivated at an average dosage of 128 J/cm2, whereas a 2.18 log 10
reduction was achieved for E. coli at 192 J/cm2 dosage.
In the present study it was shown that, in order to achieve 2.66 log 10 CFU/mL reductions
for E. coli and 3.04 log 10 CFU/mL for L. innocua, respectively, a dosage of 2.832 J/cm2
with continuous UV equipment was needed. However, the samples were not treated
further due to the temperature arise (>50°C). Our results are not in agreement with other
studies (Schenk et al., 2011) where better reductions (7.2 log 10 CFU/mL reduction and
4.6 log 10 CFU/mL reduction for E. coli and L. innocua, respectively, at 1.2 kJ/cm2) were
achieved, perhaps due to different E. coli and L. innocua strains that were used. UV light
creates mutated bases that compromise cell functionality, but bacteria have developed
DNA repair mechanisms to restore DNA structure and functionality (Friedberg et al.,
2006). This phenomenon is reflected in the shape of the inactivation curves of our
experiment (Gayán et al., 2013). The killing effects of HILP are caused by the rich and
broad-spectrum UV content, the short duration, and the high peak power of the pulsed
light produced by the multiplication of the flash power manifold (Cheigh et al., 2013,
Gómez-López et al., 2007). Other researchers found that a significant reduction of 3.6
log 10 CFU/mL for E. coli K12 and 2.7 log 10 CFU/mL for L. innocua (p = 0.001) was
achieved with HILP technology (3.3 J/cm2) (Muňoz et al., 2012). Our results are similar
to another study (Muňoz et al., 2012) where 2.57 log 10 CFU/mL reduction for E. coli
and 2.14 log 10 CFU/mL reduction for L. innocua were achieved when 2.832 J/cm2
dosage was implemented. Other researchers (Chun et al., 2010, Krishnamurthy et al.,
2007) have also studied the application of HILP in a continuous system. Moreover,
Huang and Chen (2014) studied pulsed light in fresh produce. It is known that the
surface structure of fresh produce is usually complex and bacterial cells may lodge in
surface irregularities or crevices, such as calyx. As a consequence, the efficacy of HILP
can be reduced by preventing the highly directional, coherent PL beam from reaching its
target (Lagunas-Solar et al., 2006). Therefore, Huang and Chen (2014) impose the
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Discussion
importance of selecting the representative inoculation site in a microbial challenge
approach study.
Moreover, although HILP treatment is considered “non-thermal”, this is valid for
treatments of short durations. For longer treatment periods, the temperature of sample
increased to a level high enough to cause thermal inactivation of microorganisms.
Hence, it should be taken into account that somehow the temperature must be kept low.
In the study of Bialka and Demirci (2007), the HIPL-treated blueberries had a cooked
appearance and lost structural integrity when samples were treated at a high fluence
level. Moreover, in the study of Bialka and Demirci (2008), the maximum temperature
of 80° C was reported after treatment with HILP at fluence level of 72 J/cm2. Darker cutapple surfaces treated with HILP were observed in the study of Gomez et al. (2012).
This can be attributed to temperature increase during treatment. In another study of
Ramos-Villarroel et al. (2011), the use of 12 J/cm2 pulsed light resulted in a browning
and softening of fresh-cut avocado. Whereas, Fine and Gervais (2004) reported a
modification of color in pulsed treated pepper and flour, due to oxidation that might
have been caused by pulsed light.
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4.2 Food Disinfection
The Mediterranean diet is based largely on plant-based foods. The diet is built on the
cooking and food habits of people living in Mediterranean areas such as Greece, Spain,
and Italy. A special emphasis on fruits and vegetables is given when the Mediterranean
diet is followed, which should be included in the daily eating plan. The trick of the
Mediterranean diet is to eat vegetables that are steamed, grilled or raw. People, that like
the Mediterranean diet, have been found to have a lower risk of heart disease. Fruits and
vegetables can become contaminated with pathogenic microorganisms while growing in
fields, orchards, vineyards, or greenhouses, or during harvesting, post-harvest handling,
processing, distribution, and preparation in food service or home settings. Manure used
as a fertilizer or soil amendment, as well as in irrigation water, represent potential
sources of pathogens that can contaminate fruits and vegetables. E. coli O157:H7 and
Salmonella are carried by animals and shed in their feces (Beuchat et al., 2001). Despite
considerable progress made in improving the safety of fresh fruits and vegetables,
frequent foodborne outbreaks continue to occur. Among the major etiological agents
responsible for outbreaks in RTE fruits and vegetables are E. coli, Staphylococcus spp.,
Salmonella spp, and Listeria spp. Once attached, bacteria can survive on RTE produces
during postharvest storage (Sapers and Jones, 2006), and are capable of growing to
populations exceeding 107 CFU/g, provided that appropriate conditions exist (Wei et al.,
1995, Zhuang et al., 1995). Consumers are becoming more demanding and are looking
for safe food products with high quality retention. Thus, due to absence of processing for
the majority of these foods, disinfection remains one of the critical aspects in food
industries. For this reason, a number of washing and sanitizing agents have been
approved for fruits and vegetables disinfection. Food industries have currently started
using emerging, non-thermal technologies in food processing such as Ultraviolet
radiation, Ultrasound, pulsed light, cold plasma, ultrasounds and novel packaging
practices (Ramos et al., 2013). When microorganisms are stressed, through the
implementation of the above treatments, an adaptive response may follow which can
increase the organisms’ tolerance to the same or to a different type of stress. Many
bacteria react to stress by inducing the synthesis of various proteins (Jones and Inouye,
1994). Buchanan and Edelson (1999a), reported a cross protective effect of acid shock
and acid adaptation of enterohaemorrhagic E. coli (EHEC) against heat or other stresses
but also observed that the determination of survival of EHEC in acidic foods should
consider the strain and its ability to induce stress responses. The resistance or adaptation
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Discussion
of microorganisms to acid conditions can have implications for food safety (Buchanan
and Edelson, 1999a).
The objective of inoculating RTE produces with cocktail microorganisms was to
simulate real conditions that can occur during the food production chain of the lettuce,
the strawberry, and cherry tomatoes since all bacteria are considered important for the
food industry. E. coli O157:H7, Salmonella spp., and L. monocytogenes are the main
pathogens implicated in several foodborne outbreaks related to fresh produce (Griffin
and Tauxe, 1991, Sagong et al., 2011). Moreover, S. aureus is important as it concerns
the contamination of food by food handling (Oliveira et al., 2011). There are few studies
that have examined the effect of non-thermal technologies in the disinfection of one or
two pathogens (Alexandre et al., 2011, Bermúdez-Aguirre and Barbosa-Cánovas, 2013,
Bialka et al., 2008, Oliveira et al., 2011, São José and Dantas Vanetti, 2012,
Syamaladevi et al., 2012, Yaun et al., 2004).
4.2.1 Bacteria Disinfection
All the RTE foods were washed and then left to dry. Spot inoculation was the method
used to inoculate the bacteria and viruses on romaine lettuce and strawberries, as it is
more consistent and produces more reproducible results for the inoculation of a known
number of pathogen cells on fresh produce surfaces than the dipping inoculation method
(Beuchat et al., 2001). However, cherry tomatoes were dipped inoculated. Topography
of fruits and vegetables is a critical parameter for the adhesion of bacterial cells (Wang
et al., 2009). Studies have shown that E. coli O157:H7 was better attached to coarse,
porous or injured surfaces of green peppers, than to those without injuries (Wang et al.,
2009). Moreover, it has been shown that smooth surfaces (apple) are easy for bacterial
removal, but when the roughness is higher with some deep valleys (oranges and
avocados), bacteria is not totally exposed to the mechanical forces of washing. In
products with high roughness with valleys and big cavities (cantaloupe) bacteria are well
protected from mechanical forces and disinfection agents (Wang et al., 2009). Motility
of microorganisms facilitates pathogen entry into wounds, stomata and other existing
fruit surface openings (Deering et al., 2012). Internalization through the naturally
existing opening is considered as one of the major route of pathogens to entry the plant
tissue (Deering et al., 2012). Incidences of internalization depend on concentration of
bacteria, their location on the plant, age, integrity and stages of plant development, as
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well as indigenous agonistic/antagonistic bacteria present on plant have been reported
(Erickson, 2012).
Initial average populations for all microorganisms were about 7 log 10 CFU/g of fresh
produces (7.87, 7.96, 6.53, 7.52 log 10 CFU/g for E. coli, S. aureus, S. Enteritidis, L.
innocua respectively, for lettuce 7.85, 7.14, 6.14, 7.08 log 10 CFU/g for strawberry and
7.38, 6.58, 7.42, 7.11 log 10 CFU/g for cherry tomatoes). The numbers of E. coli, S.
aureus, S. Enteritidis and L. innocua recovered from lettuce, strawberry and tomatoes
samples were similar irrespectively the bacteria were inoculated separately or as a
cocktail, which demonstrated that all bacteria retained similar attachment. The bacterial
attachment is in accordance with other studies (Yang et al., 2003). Other studies have
also selected cocktail inoculums due to their simultaneous prevalence of all these strains
in vegetable and fruits (Bialka et al., 2008, Mahmoud, 2010, Ölmez and Temur, 2010,
Sagong et al., 2011, Yaun et al., 2004).
Variations between initial populations of bacteria to different produces were apparent,
which is in accordance with other researchers (Ziuzina et al., 2014), where with SEM
images they confirmed that larger populations of L. monocytogenes adherent cells in
addition to clusters of cells, were present. Despite the irregular nature of strawberry
surface, which would probably facilitate bacterial attachment, Ziuzina et al. (2014)
found that E. coli populations visualized by SEM on strawberry surface were still less
dense by comparison with L. monocytogenes. However, in the present study the
attachment of S. Enteritidis population was less compared to all other bacteria, which
could be explained by the fact that S. Enteritidis interacts with naturally existing
indigenous epiphytic bacteria. Depending on the types of epiphyte present, the survival
of pathogens can be either enhanced or inhibited (Erickson, 2012). For example, Cooley
et al. (2006) demonstrated that Enterobacter asburiae isolated from lettuce inhibited
colonization of E. coli, whereas another epiphyte Wausteria paucula had the opposite
effect, enhancing E. coli survival.
A common practice for food disinfection in food industry is the rinsing of fresh produce
with tap water. However, removal of bacteria from fresh produce with rinsing with tap
water is not effective since cells are very well attached and only some disinfectant agents
can reach the cells and inactivate them. For instance, E. coli cells are attached to lettuce
leaves with EspA filaments, which are the same filaments used to attach to human and
bovine cells, following a similar molecular mechanism as the one used to colonize to the
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Discussion
mammalian intestine. Generally, E. coli is characterized by having a strong and intimate
attachment to the host cell membrane. It can destroy the microvilli of the bacteria at the
bonding site.
According to studies, chlorine remains the most popular disinfection method for fresh
produce contamination (Gil et al., 2009, Issa-Zacharia et al., 2010, Rico et al., 2007,
Sapers, 2001). Chlorination is applicable to fruits and vegetables, using a postharvest
process with flumes, water dump tanks, and spray washers. This postharvest treatment
has been applied to various fruits and vegetables such as tomatoes, citrus, apples, pears
and peppers (Yoon, 2014).
Among its advantages, it can be concluded that it is an easy method in an industrial
setting, the contact time with the food is short and it is cost effective (Goodburn et al.,
2013). In our study, chlorine was delivered as sodium hypochlorite solution. It is
currently the most common sanitizer used in the fresh-cut produce industry. According
to WHO, in order to have an effective disinfection, chlorine needs to be used in
concentrations of 50 to 200ppm, at pH<8 and to be in contact with the produce for not
less than one minute (WHO, 2008). The experiments conducted in this study used
sodium hypochlorite solutions at pH: 6.5 at a low (50ppm) and a high (200 ppm)
concentration.
When sodium hypochlorite was used for lettuce disinfection, it was obvious that when 1
and 3-min treatment was used, NaOCl 200ppm exhibited better results (p<0.05), as far
as E. coli, S. aureus, S. Enteritidis and L. innocua is concerned. However, the
disinfection efficiency between two concentrations of NaOCl was statistically significant
(p<0.05) only for S. Enteritidis when 5-min treatment time was used. The main
challenge in leafy vegetables, such as romaine lettuce, is that bacteria migrate easily to
some points of difficult access for the sanitizers and thus protects the microorganisms
(Bermúdez-Aguirre and Barbosa-Cánovas, 2013). Published data indicated that
population reductions on produce surfaces with chlorine are within the range of 1–2
log 10 units (Sapers et al., 2001). In the study of Bermúdez-Aguirre and BarbosaCánovas, 2013, the highest inactivation for romaine lettuce was 3.5 log 10 reductions and
was achieved after 15-min in contact with the NaOCl solution of 100 ppm. They
concluded that 2-3 log 10 reduction of E. coli was achieved for lettuce, whereas 5-6 log 10
E. coli reduction for tomatoes. In another study, where iceberg lettuce was used, the
vegetable was washed for 1-min with 100ppm chlorine solution and inactivation of 1.4
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log 10 for coliforms occured (Allende et al., 2008). Chlorinated water is by far the most
common disinfectant method for washing produce. However, generally, it is believed to
have minimal effectiveness in sanitizing the vegetables, with less than 2-3 log 10
reduction (Chang and Schneider, 2012, Park et al., 2008). In another study with lettuce
leaves, it was reported that 1.79, 2.48 and 0.33 log 10 reductions were achieved for
Salmonella, E. coli 0157:H7 and aerobic mesophilic population respectively, when
lettuce leaves were immersed in 200 ppm chlorine for 10-min (WHO, 2008). Other
researchers studied the difference between 100 ppm chlorine solutions of temperatures
47°C and 4°C, and concluded that higher temperature resulted in greater microbial
reduction (Delaquis et al., 1999). Zhang and Faber (1996), using 200 ppm chlorine for
10-min at 4°C and 22°C found log 10 reductions of L. monocytogenes of 1.3 and 1.7 on
lettuce and 0.9 and 1.2 on cabbage, respectively. Lang et al (2004) demonstrated a 1.42
log 10 reduction of E. coli 0157:H7 when lettuce was immersed in 200 ppm chlorine
solution for 5-min. Bae et al. (2006) reported that 200 mg/L of chlorine, reduced
populations of E. coli, S. aureus, L. monocytogenes and S. Typhimurium to 2.08–2.66
log 10 . Iturriaga et al (2010) has also found a 4-5 log 10 reduction for S. Montevideo
achieved by chlorine (200-100 ppm) on tomatoes. Ge et al. (2013) recorded the
disinfection of S. Typhumurium in lettuce which resulted in 0.92 log 10 reduction. LopezGalvez (2009) clearly indicated the difference between rinsing with water and chlorine
disinfection, showing that 2 log 10 reduction was achieved for lettuce when chlorine was
used, compared to rinsing with water. Ölmez et al (2009) found a greater log 10 reduction
for E. coli (3.7 log 10 ) inoculated in lettuce when 100 ppm chlorine was used and the
temperature was 10 °C. Similar results have been exhibited by many researchers (Baur et
al., 2004, Mahmoud et al., 2010, Pereira et al., 2013, Weissinger et al., 2000). Mahmoud
et al. (2010) has studied the effect of chlorine dioxide gas to inactivate E. coli (2.5
log 10 ), L. monocytogenes (3 log 10 ), S. enterica (2.7 log 10 ) on strawberries. Pereira et al.
(2013) support that chlorine is a promising disinfection method for effectively reducing
pathogens in fresh produces. Baur et al. (2004) investigated the effectiveness of cold and
warm (50 °C) chlorinated water, as well as warm water without chlorine, for prewashing
trimmed, cored iceberg lettuce. The use of warm water resulted in a significant log
reduction of initial population of total aerobic bacteria, pseudomonads and
enterobacteriaceae. However, this was not an issue in this study as only non-thermal
technologies were selected, due to the fact that temperature more than 50 °C, can cause
more stress to the organisms as it is above their normal growth temperature.
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Discussion
Chlorine has been widely used as a sanitizer in commercial produce wash (Ahvenainen,
1996, Beuchat, 1998), and hypochlorous acid is one of the most efficacious form of
chlorine. However, studies have shown that chlorine used at concentrations between 50200 mg/L permitted by the FDA lacked efficacy in inactivation of human pathogens and
spoilage microorganisms (Beuchat, 1998, Zhang and Farber, 1996). The technical
challenge in a commercial fresh-cut wash operation has been how to maintain a
relatively stable level of hypochlorous acid as this weak acid reacts quickly with organic
materials present in wash solution (Gil et al., 2009). However, the reaction of chlorine
with organic residues can result in the formation of potentially mutagenic or
carcinogenic reaction products. This is a cause for concern since some restrictions in the
use of chlorine might eventually be implemented by regulatory agencies. Increasing
public health concerns regarding the possible formation of chlorinated organic
compounds has resulted in a demand for alternatives to chlorine (Singh et al., 2002).
Undesirable effects such as an unpleasant odor, softening of lettuce tissue and a
browning reaction may occur when foods are treated with high chlorine concentrations
(Kim et al., 2008). Chlorination of fruits and vegetables leads to side reactions between
active chlorine compounds (Cl 2 , HOCl, ClO-) and natural organic material, resulting in
the production of chlorinated disinfection by-products (DBPs) such as trihalomethane,
which is a carcinogenic substance (Chang et al., 2000). These reports clearly show the
need for alternative disinfection methods in order to exceed the apparent population
reduction “ceiling” of 1–2 log units. Thus, more effective, environmental friendly
methods must be used from industries (Sapers et al., 2001).
Many studies have been published, explaining the increase of use of alternative
disinfection techniques, trying to totally replace the conventional ones in the food
industry (Alexandre et al., 2012, Allende and Artés, 2003, Bermúdez-Aguirre and
Barbosa-Cánovas, 2013, Sagong et al., 2011, Syamaladevi et al.,2012).
Short-wave UV radiation (200–280 nm) –UV light– is considered the most germicidal
region of the UV spectrum for a great variety of microorganisms, having greater effect at
wavelengths between 250 nm and 260 nm (Kowalski, 2009). At these wavelengths, UV
photons are produced, which are mostly absorbed by thymine and cytosine nitrogenous
bases of the deoxyribonucleic acid (DNA). The result is the formation of cross-linking
photoproducts that interrupt the transcription and replication of DNA, thus leading to
cell death (López-Malo and Palou, 2005). To cope with DNA damage, bacteria have
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independent or dark repair systems. Photorepair is carried out by photolyase enzymes
which reverse DNA damages using the energy of visible light (Sinha and Hader, 2002).
UV light sensitivity varies significantly among different types of microorganisms. Most
authors agree that Gram-negative bacteria are more sensitive than are gram-positives,
followed by yeasts, bacterial spores, molds, viruses and protozoa (López-Malo and
Palou, 2005). These variations in UV resistance have been attributed to several intrinsic
microbial factors, including the cell wall thickness, cell size, pigment production,
composition, size and conformation of the genetic material, and cell DNA repair ability
(López-Malo and Palou, 2005, Tran and Farid, 2004). In addition, the physiological state
of cells, such as their growth phase, also determine their microbial sensitivity to UV
radiation (Bucheli-Witschel et al., 2010, Wassmann et al., 2011). Ultraviolet (UV) light
is one of the most promising technologies, due to its ability to inactivate a wide range of
spoilage and pathogenic microorganisms, its minimal loss of the nutritional and sensorial
quality of foods and its low energy consumption compared with other non-thermal
technologies (Guerrero-Beltrán and Barbosa-Cánovas, 2004). Short wave ultraviolet
(UVC, 254 nm) irradiation, is a process that has gained increasing attention after the
USFDA approved its use in 1999 as an alternative to thermal pasteurization of fresh
juice products (Flores-Cervantes et al., 2013, US FDA, 2000,)
Both Salmonella and E. coli in lettuce showed similar log 10 reductions when treated for
the same period with UV, which is in accordance with the study of Yaun et al. (2004).
Also, when UV dosages (7.56 kJ/m2) have been used in other fruit surfaces (peaches and
pears), better reductions of E. coli (up to 2.91 and 3.70 log 10 CFU/g for peaches and
pears respectively) have been achieved (Syamaladevi et al., 2012). In addition,
reductions of E. coli and Salmonella in strawberries were achieved up to 2.5 log 10
reduction, with dosages up to 64.4 J/cm2 of pulsed UV light (Bialka et al., 2008). The
low log reduction recorded in our experiment was due to the UV lamp choice. The
choice of a low dosage UV lamp technology was made in order to evaluate UV
technology for disinfection taking into consideration that the food should not be affected
in its color and quality in general. In agreement to other findings (Allende and Artés,
2003, Bermúdez-Aguirre and Barbosa-Cánovas, 2013, Yaun et al., 2004), our work
showed that higher UV doses resulted in a greater decrease of bacterial growth in
‘romaine’ lettuce, strawberry pieces and cherry tomatoes. The present results are in
accordance with Bermúdez-Aguirre and Barbosa-Cánovas (2013) who showed that
inactivation at short working distance and higher fluence (1.6 mW/cm2) was higher from
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the first-min of treatment. They found that after 60-min of treatment the inactivation
achieved for E. coli was about 2.8 log 10 when samples were closer to the radiation
source. The doses needed to reduce E. coli O157:H7, L. monocytogenes, S. enterica and
S. flexneri, by 5 log 10 reduction, on the surface of roma tomatoes were less than those
needed on other produces (Mahmoud, 2010). Sanitizers can achieve better contact with
smoother surfaces than with rough surfaces (Koseki et al., 2004). The germicidal effect
of UV light in fresh-cut fruit and vegetables with rough surfaces (strawberry and lettuce)
is usually within 1 and 2 log cycles, whereas for cherry tomatoes the reduction is greater.
Moreover, it has been stated that treatment of tomatoes with short wavelength ultraviolet
light has been shown to have a number of benefits. These include delayed senescence, as
manifested by the maintenance of both firm texture and green pigmentation, and
induction of resistance against phytopathogens such as Rhizopus stolonifer and Botrytis
cinerea (Barka et al., 2000, Obande, 2011, Stevens et al., 2004).
The efficacy of surface disinfection by UV on food surfaces is influenced by several
factors including: UV dose, UV dose rate, exposure time, surface characteristics, initial
bacterial inoculum level and bacterial type (Otto et al., 2011). Despite the known limited
ability of UV light to penetrate rough food surfaces, this study demonstrated that UV
light has the potential to reduce bacterial contamination on food surfaces such as lettuce
and strawberry surface and therefore has the potential to be used as post lethality
treatment to control pathogens in ready to eat foods. However, it has been shown that
UV was less effective at reducing populations of all bacterial types in strawberries when
compared to lettuce. To predict UV disinfection rates on food surfaces, more kinetic
inactivation data need to be obtained for pathogen and spoilage microorganisms, taking
into account interactions between microorganisms and surface materials, such as
shielding effects from incident UV and their dependency on surface structure or
topography. Considering the bacteria inoculated into both lettuce and strawberry, more
bacteria could have colonized deeper inside the strawberry, which could have reduced
the chance of bacterial exposure to the UV light. Therefore the internalized bacteria in
strawberry were possibly less affected by the UV-C light. It is known that UV-C light
can not penetrate deeply into the fresh produce (Morgan, 1989). Hadjok et al. (2008)
showed that UV (37.8 mJ/cm2) combined with 1.5% H 2 O 2 continuous spraying at 50 °C
achieved a 2.84 log 10 reduction of the internalized S. Montevideo in iceberg lettuce. Ge
et al. (2013) showed that UV irradiation with higher fluencies (150, 450, 900 mJ/cm2)
can significantly reduce the internalized S. Typhimurium in iceberg lettuce. Mahmoud
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(2010) studied the effect of E. coli O157:H7, L. monocytogenes, S. enterica and S.
flexneri on whole roma tomatoes by X-ray doses. Bermúdez-Aguirre and BarbosaCánovas (2013) studied the effectiveness of UV only on E. coli, artificially inoculated
on grape tomatoes. Other researchers have evaluated the survival of S. Enteritidis
inoculated on tomatoes using disinfection methods such as ozone, gamma-irradiation,
chlorine dioxide, pulsed UV (Aguiló-Aguayo et al., 2013, Daş et al., 2006, Todoriki et
al., 2009, Trinetta et al., 2010).
However, the mechanism of UV against internalized bacteria in the plant has not been
clearly illustrated and needs further exploration. Moreover, it has been shown that more
irregular and complicated surfaces are less decontaminated (Luksiene et al., 2012). Since
UV light has limited penetration and depth, plant morphological characteristics such as
roughness and presence of wounds on fruit surfaces impact microbial inactivation.
Understanding these influences is needed if this technology is to be commercialized
(Schenk et al., 2008). However, limited information is available on the influence of fruit
surface properties on the efficacy of UV for surface decontamination.
Furthermore, the interactions encountered between indigenous microorganisms derived
from the natural flora of foods and foodborne pathogens found in either the planktonic
and biofilm states have been studied for Salmonella spp., L. monocytogenes and E. coli
O157:H7 (Al-Zeyara et al., 2011, Møller et al., 2013 ). Indigenous microorganisms on
fresh produce alone can form biofilms (Liu et al., 2013) and shows antagonistic effect
with S. enterica on baby carrot (Liao, 2007). This could be an explanation of low
disinfection efficiency achieved when UV was used. Biofilm is common phenomenon
for fresh produce and difficult to eradicate using disinfectants (Jahid and Ha, 2012). The
efficacy of a disinfectant toward fresh produce microorganisms depends on the ability of
them to attach to plant tissues and form biofilms on the surface, stomata, trichomes, and
cutting edges of the plant material (Jahid and Ha, 2012, Olaimat and Holley, 2012). The
attachment and formation of biofilms on lettuce by Salmonella spp. have been reported
previously (Kroupitski et al., 2009, Patel and Sharma, 2010). S. Typhimurium (ST) and
E. coli O157:H7 are internalized through natural openings such as stomata, lenticels, and
lateral roots (Deering et al., 2012, Jahid, 2014).
From a practical point of view, the UV chamber plays an important role in food
disinfection. UV treatment is a simple and inexpensive method of processing which
leaves no residues behind and may prove worthy for use in post-harvest situations to
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improve safety and to maintain quality of fresh ready to eat produces. It has been stated
that the specific location of pathogens on produce surface influences the effectiveness of
UV lamps, thus different levels of reduction can be achieved (Mudkoparnahyaya et al.,
2014). One of the major concerns for the produce industry is limited shelf life. Several
studies have indicated that significant improvement of shelf life for fruits and vegetables
can be achieved by UV treatment due to inactivation of spoilage organisms and delayed
ripening process (Arvanitoyannis et al., 2009).
US is based on cavitation, which enhances the mechanical removal of attached or
entrapped bacteria on the surfaces of fresh produce by displacing or loosening particles
through a shearing or scrubbing action (Seymoor et al., 2002). The results demonstrated
that the reduction was greater as the treatment time was increased. São José and Vanetti
(2012) studied the effect of ultrasound (45 kHz, 25 °C) on cherry tomatoes. The
reduction of the total viable count, yeast and mold count, and inoculated S. enterica
typhimurium that adhered to the surface of the tomatoes was evaluated. Treatments for
30-min with ultrasound alone, led to reductions of 1.73 log 10 for S. enterica (São José
and Vanetti, 2012). In the study of Bilek and Turantas (2013) ultrasound treatment (37
kHz, 25 °C) removed an average of 1.60 log 10 CFU/g of the Salmonella population in
cherry tomatoes after 30-min and the increase in contact time from 30 to 60-min of
treatment reduced contamination even further. Similar findings were observed in the
present study. In this study when US was used, better disinfection efficacy was
presented in strawberries than lettuce for the majority of microorganisms. This may be
attributed to different food surface properties such as hydrophobicity, electric charge,
and roughness that can influence the adhesion and distribution of bacterial cells on food
surface (Araujo et al., 2010). The survival of microorganisms depends upon several
other factors such as type of strain, initial inoculums level, surface characteristics, and
growth conditions (Guerrero-Beltrán and Barbosa-Cánovas, 2004). Microbial reduction
by ultrasound is very important from the stand point of green decontamination and the
hurdle concept of inhibition and elimination methods for food preservation technologies
in fruits and vegetables (Bilek and Turantas, 2013). The lack of effectiveness that might
be caused by the ultrasonication system is correlated with the operational procedures
used in those disinfection treatments. Some key factors play an important role when
applying ultrasound to a produce. For instance, dissolved gas in a washing solution is
known to decrease the cavitation activity in a cleaning operation (Awad et al., 2012),
and, therefore, degassing is essential for any ultrasonic cleaning applications. Moreover,
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the acoustic field distribution in an ultrasonic treatment chamber or tank is not uniform,
mainly due to a standing wave formation. The nonuniform ultrasound field distribution
and hence the nonuniform cavitation will result in variations in microbial inactivation
activities at different locations in a washing tank. Consequently, during a wash
treatment, those produce lettuce leaves that have received a good dose of ultrasound
treatment and thus have a low microbial count would be easily cross-contaminated by
neighboring leaves that have received less ultrasound treatment due to blockage of
produce leaves to ultrasound propagation in the wash liquid and hence have a high
microbial population due to an un-even acoustic field distribution. A good understanding
of the underlining principles of power ultrasound, as well as a good design in wash
system and operation procedure is absolutely a prerequisite for fully utilizing the power
of ultrasound in produce decontamination applications. In addition, the efficacy of
ultrasound is also affected by ultrasound frequency, power level, the size and shape of
the ultrasonic bath, the depth, volume, temperature and nature of the liquid, and
treatment time (Zhou et al., 2009). Zhou (2010) has shown that the 75 kHz ultrasound
treatment was significantly less effective compared to two other frequencies (p < 0.05).
The acoustic power density was 79.41 W/L for 25 kHz, 68.95 W/L for 45 kHz and 33.64
W/L for 75 kHz under the maximum power level. Mason and Lorimer (2002)
mentioned, an increase in the gas content of a liquid can lead to a lowering of both the
cavitational threshold and the intensity of the shock wave from the collapse of the
bubble because of the increased number of nuclei (or weak spots) present in the liquid
and the greater “cushioning” effect in the microbubble. Therefore, a degassing step is
necessary in any practical ultrasonic cleaning application in order to remove the gases
and enhance the effectiveness of the process.
Finally, combined disinfection technologies were used. As reported by Seymour et al.
(2002), the combination of ultrasound with water or chlorinated water enhanced the
removal of S. Typhimurium attached to iceberg lettuce by 1 log 10 CFU/g compared to
water or chlorinated water alone. However, data (Nastou et al., 2012) demonstrated that
in an ultrasound-assisted wash, the decay of chlorine is further accelerated. Therefore,
precautions must be taken to regularly monitor chlorine concentration during ultrasound
and chlorine combined treatment. For this reason, in the present study the sodium
hypochlorite immersions followed the UV or US treatments. These observed differences
are likely to be due to differences in the microenvironment (topography, presence of
stomata, chemical composition) in which the bacteria find themselves on the different
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surfaces, which could either affect the washing processes directly (such as the presence
of crevices and fissures, a surface of low wettability) or indirectly by influencing the
physiological state of the bacteria (Nastou et al., 2012). Ajlouni et al. (2006)
demonstrated that washing Cos lettuce in combined treatments with ultrasound (40kHz)
with various sanitizers at different concentrations reduced the microbiological
populations by 1 to 2.5 log 10 CFU/g immediately after washing, but there was little
effect of ultrasonication alone on Cos lettuce regarding total or psychrophilic counts (p >
0.05), even during storage at 10°C (Zhou, 2010). However, they observed that after
long-time ultrasonication (20 min) significant (p < 0.05) damage to the quality of Cos
lettuce tissues was caused. Huang et al. (2006) reported that the treatment of 170-kHz
ultrasonication resulted in 2.97 log 10 reduction in Salmonella and 2.26 log 10 reduction in
E. coli O157:H7 on inoculated lettuce, while using combined ClO 2 and ultrasonication
to treat inoculated apples with Salmonella and E. coli resulted in bacterial reductions of
3.12 to 4.25 and 2.24 to 3.87 log 10 , respectively (Zhou, 2010). Other researchers, have
demonstrated that ultrasound in combination with 1% calcium hydroxide enhanced the
decontamination efficacy on alfalfa seeds inoculated with S. enterica and E. coli
O157:H7 (Scouten and Beuchat, 2002). Contradictory results regarding ultrasound
disinfection treatments have been proposed by many researchers. Dehghani et al. (2005)
investigated the impact of sonication as a disinfection method for determining the
effectiveness of ultrasound on the E. coli inactivation. Ugarte-Romero et al. (2006)
achieved a 5 log 10 reduction of E. coli with power ultrasound in apple cider. D’Amico et
al. (2006) studied the inactivation of microorganisms in milk and apple cider and
demonstrated that ultrasound technology was a promising processing alternative for the
reduction of microorganisms in liquid foods. However, Pagan et al. (1999) found that
ultrasonic treatment (20 kHz) at ambient temperature was not very effective against L.
monocytogenes. It has been reported that Gram-negative were more sensitive than Grampositive bacteria (Patil, 2010). Lee et al. (2009) concluded that the combination of lethal
factors (heat and/or sonication, with and without pressurisation) could significantly
shorten the treatment time needed to achieve a 5-log 10 reduction in the survival count of
E. coli K12. Wang et al. (2010) investigated that, Alicyclobacilli had a higher resistance
to ultrasonic treatments in apple juice than in buffer solutions, indicating that resistance
to ultrasound depends on their environment. Adekunte et al. (2010) indicated sonication
alone at moderate temperatures can achieve the desired 5 log 10 reductions in yeast cells
(Patil, 2010). Page (2003) studied the effect of combined technologies on inactivation of
B. subtilis spores. No cumulative inactivation was presented, when treatment of UV was
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followed by free chlorine, implying that the effects of the two processes were just
additive with no occurrence of synergy. In another study, the combined treatments of
ultrasound and organic acids were employed to inhibit pathogens on organic fresh
lettuce. More precisely, the combined treatment of ultrasound and organic acid for 5-min
achieved an additional 0.8 to 1.0 log 10 reduction of the three pathogens compared to
organic acid treatment alone and the combined treatment of ultrasound and 2.0% organic
acid (2.67 log 10 CFU/g) was the most effective in reducing the number of pathogens
(Sagong et al., 2011).
In the study of Yoon et al. (2014) reported that the combined chlorine–ionizing radiation
disinfection treatments result in synergistic benefits in regard to reducing the numbers of
natural microflora. Similarly, in this study, the combined methods result in a greater
reduction than when the single methods are used alone.
4.2.2 Adenovirus Disinfection
Even though no foodborne outbreak with HAdV has been documented so far, potential
viral transmission by foods is possible as they have been already detected in raw
vegetables (Cheong et al., 2009), and are believed to be possible indicators for HAV and
noroviruses. Different strategies have been developed to eliminate HAdV, mostly based
on inactivation of virus by UV technologies (Baxter et al., 2007, Thurston-Enriquez et
al., 2003b), ozone (Thurston-Enriquez et al., 2005), or chemical disinfectants such as
free chlorine (Baxter et al., 2007, Cromeans et al., 2010, Thurston-Enriquez et al.,
2003a), monochloramine (Baxter et al., 2007, Cromeans et al., 2010, Sirikanchana et al.,
2008) or by combination of these technologies (Shin and Lee, 2010).
In this study, lettuce and strawberry achieved better reductions compared to cherry
tomatoes, when immersed in sodium hypochlorite solutions. For instance after the
longest exposure time, lettuce and strawberry achieved 4.95 and 5.02 log 10 GC/g
reduction, whereas cherry tomatoes achieved 3.76 log 10 GC/g reduction. Baert et al
(2009) concluded that 200 ppm chlorine was able to render an additional 1 log 10
reduction of MNV-1 present on lettuce, compared to washing with tap water. On the
contrary, Gulati et al (2001) found that the same treatment of strawberries and lettuce
did not result in an additional reduction of FCV compared to tap water. Baert et al.
(2009) reported a significant lower decline by chlorination in the case MNV-1 lysate
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when it was directly spotted onto lettuce than when the inoculum was ten-fold diluted in
tap water. Less free chlorine was available in the first case showing that chlorine reacted
with the organic matter originating from the inoculum suspension. These observations
are in accordance with the present study and can explain the low disinfection of cherry
tomatoes by NaOCl solutions. Moreover, the use of only 1-2 pieces to determine the
efficacy of sanitizers is indicative, but the efficiency of these sanitizers could change
dramatically when introduced in an industrial process. Therefore, the produce/treatment
ratio is important in order to evaluate conditions that are observed in fresh produce
industries. In another study of Casteel et al. (2008), 1.7 log 10 reductions of both MS2
and HAV were observed on strawberries, tomatoes and lettuce treated with 20 ppm
chlorine. Inactivation rates of MS2 on lettuce differed significantly between studies
(Casteel et al., 2008, Dawson et al., 2005) and the present study. Factors such as
produce:treatment solution ratio, the presence of organic matter, inoculation method and
produce type are reported to influence the efficacy of chlorination towards bacterial
pathogens as well as viruses (Beuchat et al., 2004, Francis and O'Beirne, 2002, Lang et
al., 2004). Butot et al. (2007) found a significantly higher inactivation of FCV after
treatment of berries blueberries, strawberries, raspberries, basil and parsley with 200
ppm chlorine, suggesting the influence of produce type (Baert et al., 2009). Other factors
play an important role on the effectiveness of disinfection strategies on viruses. For
example, pH and water activity of foods can significantly influence the inactivation of
microorganisms by High Hydrostatic Pressure (HHP) (Patterson, 2005). Low pH, which
is characteristic for several fruits (e.g. berries) and vegetables (e.g. tomato), and pressure
can act synergistically leading to enhanced microbial inactivation (Patterson, 2005).
However, protection effect of lower pH in murine norovirus (MNV-1) was also
previously observed (Lou et al., 2011). Furthermore, some food components such as
proteins, lipids, carbohydrates or cations can confer a protective effect (Patterson, 2005).
Sucrose, which is an important nutrient of berries has been linked to virus-borne
outbreaks (Maunula et al., 2009), was recognized to protect feline calicivirus (FCV)
when treated with HHP (Kovac et al., 2012).
High chlorine levels would be required to achieve a 2 to 3 log 10 reduction of viruses on
fresh produce. The application of higher concentrations (more than 200 ppm) is limited
due to sensorial aspects. According to other studies (Duizer et al., 2004, Gulati et al.,
2001), it seems useless to increase the efficacy of chlorination since they showed that a
contact time beyond 10-min made little difference in antiviral activity towards FCV. In
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the present study, the treatment time of 3-min was sufficient for inactivating HAdV as
far as cherry tomatoes are concerned. An additional 0.79 log 10 reduction was achieved
for another 2-min immersion in sodium hypochlorite solution. For strawberry and lettuce
the treatment time plays an important role in inactivation efficiency. The difference
between 3 and 5-min was not found to be statistically significant (p>0.05). However,
when 10-min treatment time was implemented, the inactivation rate was significantly
enhanced (p<0.05). For instance, in strawberries a double inactivation log 10 GC/g of
HAdV was achieved. Whereas, in lettuce an additional 1.23 log 10 reduction GC/g of
HAdV was reported when the treatment time was increased from 3 to 10-min.
Viruses were likely sheltered from UV light by the strawberry matrix. UV inactivation
of micro-organisms is probably due to the absorption of UV by nucleic acids causing
dimerization of thymine in DNA or uracil in RNA (Nuanualsuwan and Cliver, 2003a,
Sommer et al., 2001). At higher doses (≥1000 mW●s/cm2) UV light can also affect the
capsid proteins. The combined effect of size/type of the virion and nucleic acids are
thought to be factors determining the resistance/sensitivity of viruses towards UV
(Sommer et al., 2001). A dose of 1 J/cm2 (corresponding with 1 W●s/cm2) reduced HAV
and poliovirus by 5.7 and 6.7 log 10 in PBS (Roberts and Hope, 2003). Fino and Kniel,
(2008) concluded that the UV light treatment on lettuce at a dose of 40 mW s/cm2
achieved 4.3, 4.0 and 3.5 log 10 reduction of respectively HAV, aichivirus and FCV.
They also studied strawberries which were found to have a lower disinfection efficiency
compared to lettuce. This is in accordance with the present study where the inactivation
of strawberries compared to lettuce was greater in all treatments, with the only exception
of UV at 60-min treatment time.
Bidawid et al. (2000a) found that 3 kGy of γ-irradiation was needed in order to achieve 1
log 10 reduction of HAV on lettuce or strawberries. However, UV was less effective at
reducing viral populations in lettuce. It was observed, that when the time was doubled
(from 30 to 60-min), the mean reduction of HAdV was also doubled for strawberry
(from -1.26 to -3.98 log 10 Genome Copies/g) and cherry tomatoes (from -0.92 to -2.22
log 10 GC/g). Moreover, it has been shown that more irregular and complicated surfaces,
such as lettuce are less decontaminated (Luksiene et al., 2012). Since UV light has
limited penetration and depth, plant morphological characteristics such as roughness and
presence of wounds on fruit surfaces impact microbial inactivation (Schenk et al. 2008).
Meng and Gerba (1996) found 3 log 10 inactivation of adenovirus type 40 at a UV dose
of 90 mJ/cm2 and 4 log 10 reduction at 120 mJ/cm2. Whereas, Thurston-Enriquez et al.
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(2003a) found that Ad40 requires over 150 mJ/cm2 for 3 log 10 and over 200 mJ/cm2 for
4 log 10 inactivation. Ad1, Ad2, and Ad6 require 120 mJ/cm2 for 3 log 10 inactivation
(Nwachuku et al., 2005). Variation between studies can occur as a result of viral
preparation methods and complexity of the adenovirus capsid.
Treatment with US was less effective (p<0.05) compared to UV. After the longest
exposure time, lettuce exhibited the greatest reduction (1.79 log 10 GC/g) compared to
other fresh produces. However, the treatment time also played an important role, as far
as virus reduction is concerned. Reduction of viruses by US is mainly due to the physical
phenomenon called cavitation (Alegria et al., 2009, Piyasena et al., 2003, Seymour et al.,
2002). Chrysikopoulos et el (2013) showed that the bacteriophages X174 and MS2,
which were used as model viruses, were inactivated adequately when relatively high US
frequencies (i.e., 582, 862, and 1142 kHz) were used.
The synergistic or additive effect of disinfectants has been investigated in some studies
(Cho et al., 2011, Chrysikopoulos et al., 2013, Lotierzo et al., 2005) by carefully
selecting the primary and secondary disinfectants and avoiding long contact times and
high concentrations. However, the mechanism of action by which the combination of
two disinfectants affects the disinfection action is still not clear. For instance, the
sequential application of ozone (or ozone/H 2 O 2 ) followed by free chlorine (Cho et al.,
2006) was shown to achieve a higher level of inactivation of Bacillus subtilis spores than
the sum of the inactivation level achieved with individual ozone (or ozone/ H 2 O 2 ) and
free chlorine application. This enhanced inactivation is referred to as a synergism, which
is beneficial since it leads to a reduction in the amount of disinfectant and reaction time
as well as a potential decrease in the formation of disinfection by-products (Rennecker et
al., 2000).
However, there are few reports in the literature regarding the synergism involved in
sequential disinfection processes employing UV or US followed by free chlorine (Cho et
al., 2011, Chrysikopoulos et al., 2013). Chrysikopoulos et al. (2013) found that, for the
case of MS2 US+UV was more effective than US alone. For the case of X174, US and
UV did not provide any synergistic effects, on the contrary, the inactivation of X174 was
hindered. Therefore, the combined use of US and UV should be employed only on
specific cases.
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In this study, a synergistic effect was observed when UV and US were followed by
immersion in sodium hypochlorite solutions, however, no additive effect was observed.
The synergy was enhanced more when UV was followed by sodium hypochlorite, rather
than when US followed by sodium hypochlorite (p<0.05). Moreover, the sequential
treatment of alternative methods exhibited more promising results compared to the
combination of an alternative and a conventional treatment, in strawberries and cherry
tomatoes. In all cases the sequential application of two alternative technologies
depended on the time used for each method.
It could be concluded, that there is a need for alternative sanitizers to be used for fresh
RTE fruits and vegetables not only for the organic food sector but also for the
conventional food processors (Ölmez et al., 2009). Thus, non-thermal technologies,
alone or in combination, could offer attractive benefits to food disinfection.
Several researchers reported that microorganisms can attach in inaccessible sites like the
stomata of leaves of leafy vegetables (Ells and Hansen, 2006, Itoh et al., 1998). Koseki
et al. (2001) stated that microorganisms on the surface of lettuce leaves were easily
disinfected, but bacteria inside the biofilms or cellular tissue, such as stomata could not
be disinfected (Ölmez and Temur, 2010). This could probably explain the lower
reductions of virus observed in lettuce, when combined technologies were used,
compared to other fresh produces.
In order to assure the results obtained by PCR, tissue culture assays for lettuce followed.
Plaque-based assays were selected in order to determine virus concentration in terms of
infectious dose. Viral plaque assays determine the number of plaque forming units (pfu)
in a virus sample, which is one measure of virus quantity. Specifically, a confluent
monolayer of host cells is infected with the virus at varying dilutions and covered with
DMEM medium. A viral plaque was formed when a virus infects a cell within the fixed
cell monolayer. Then, the virus infected cell lysed and spread the infection to adjacent
cells where the infection-to-lysis cycle was repeated. The infected cell area created
plaque (an area of infection surrounded by uninfected cells) was seen with an optical
microscope as well as visually (figure 3.2.2.5).
4.2.3 High and Low Initial Load Disinfection Treatments
In order to assure that the disinfection efficiency was independent of the high bacteria
inoculum that was selected throughout the experiments, selected experiments with
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conventional, alternative and combined technologies were conducted with different
initial bacteria inoculum. It was observed that in all RTE produces the bacteria
populations were under the limit of detection (<0.22 log 10 CFU/g), when three effective
treatments (NaOCl 3-min, US+NaOCl 33-min, US 60-min) and low initial bacteria
inocula were selected. In cherry tomatoes, the disinfection efficiency was even better,
and bacteria were shown to be under the limit of detection (<0.22 log 10 CFU/g), with
almost all disinfection treatments, since a low initial microbial load was inoculated. In
another study where cold plasma was used as a disinfection technology, different initial
concentrations complicated the comparison of the bacterial sensitivity to the ACP
treatments (Zuzina et al., 2014). In the study conducted by Fernandez et al. (2012) the
increase of concentration of S. Typhimurium from 5 to 8 log 10 CFU/filter reduced the
inactivation efficiency of ACP.
4.2.4 Storage Conditions
To examine the shelf life of the treated foodstuffs and test for possible microbial
reactivation after disinfection treatments, the foods after treatments were stored in fridge
(6°C) for 15 days.
During storage, the microflora on shredded iceberg lettuce leaves gradually increased for
untreated and treated samples. However, treated samples maintained microbial
populations significantly at a lower level compared to the untreated control. Mahmoud et
al. (2010) have studied the treatment with 2.0 kGy X-ray, and concluded that the
population of mesophilic, psychrotrophic, and yeast and mold was maintained under the
detectable limit for 12, 20, and 9 days storage, respectively. These results are in
agreement with another study Zhang et al. (2006) who reported that aerobic mesophilic
bacteria on fresh-cut lettuce irradiated with 1.0 kGy gamma rays were reduced by 2.4
log 10 CFU. The limited shelf life of fresh processed leafy lettuce is one of the greatest
problems faced by commercial marketers (Mahmoud et al., 2010). Oliveira et al., (2010)
studied the effect of modified atmosphere packaging on survival of E. coli, Salmonella
and Listeria on lettuce. In another study of De Oliveira et al. (2012), significant
differences on the behavior of L. monocytogenes on shredded organic lettuce were
observed throughout storage period (p<0.05) depending on the initial load of background
microbiota. Populations of L. monocytogenes on the ‘high’ counts increased slightly
from 3.8 to 5.0 log 10 CFU /g after 8 days. Numbers on the ‘medium’ mesophilic counts
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were similar with levels ranging from 3.8 to 5.3 log 10 CFU/g. On the contrary, when the
initial mesophilic count was ‘low’, populations of L. monocytogenes increased rapidly
with significant differences between other treatments during storage period, reaching
final population of 6.1 log 10 CFU/g. Our results are in accordance with Koseki and
Isobe, 2005 where an increase was observed in all microorganisms after storage at
refrigerated conditions. The increases were obvious after the 3rd day of storage (Koseki
and Isobe, 2005). Finally, Mahmoud et al., (2010) studied the effect of mesophilic
bacterial counts on untreated and irradiated treated lettuces after a total time of 30 days
storage period.
As far as strawberries is concerned, the effect of disinfection treatments total mesophiles
and yeasts and moulds, has also been studied by Alexandre et al. (2012). More precisely,
the impact of ozone, ultrasound, UV radiation, NaOCl, H 2 O 2 was studied. Strawberries
washed with hydrogen peroxide solutions (before storage) had the highest total
mesophiles reduction: 2.26 ± 0.38 and 1.59 ± 0.41 log 10 unit reductions, for H 2 O 2 at 5%
and 1%, respectively. During storage at refrigerated temperature, these two washing
treatments resulted in strawberries with lower microbial loads, when compared to the
results obtained with the remaining treatments. At the end of refrigerated storage,
samples pre-treated with ozone presented lower microbial loads than samples
ultrasonicated or treated with UV radiation. Throughout refrigerated storage,
strawberries pre-washed with all sanitizer solutions and with ozone presented lower
microbial loads (p<0.05) than untreated, ultrasonicated, UV irradiated or water-washed
samples. In the present study an increase in E. coli, S. aureus, S. Enteritidis and L.
innocua was obvious after the 7th day for all treatments. Miguel-Pintado et al. (2013),
studied the effect of HPP on storage of tomatoes and found that at day 30, fruits
submitted to HPP showed lower microbial counts while non-treated presented microbial
spoilage (Miguel-Pintado et al., 2013) Mukhopadhya et al. (2014) studied the effect of
UV on mold and yeast population of tomatoes, during storage for 21 days at 5°C. A
slight increase was observed from day 14th to day 21th. However, when the effect of UV
studied for total aerobic count on tomatoes, where the initial loads were higher, an
increase was observed for days 7, 14 and then the microbes decreased until day 21th.
Similar results were observed in the present study. Moreover, the effect of storage on
tomatoes was also studied by Aguiló-Aguayo et al. (2013), were pulsed light was used
for disinfection of tomatoes, was similar to our results. A steadily increase in total
aerobic mesophilic bacteria was observed from day 0 to day 15, with the control sample
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to have the highest values and treated samples to have lower. Finally, the study of Daş et
al. (2006), suggested that the storage plays an important role on the survival of S.
Enteritidis on tomatoes. All the treatments that were implemented in RTE produce in
this study, exhibited similar reaction, for all the microorganisms. The only difference
observed in S. Enteritidis inoculated on lettuce and strawberries and treated with
combined disinfection technologies. For example, Salmonella population in lettuce
showed a reduction from day 7 to day 15, when combined treatments were used
(UV+NaOCl, US+NaOCl), whereas all the other microorganisms were increased in all
the treatments. Moreover, Salmonella population in strawberries when treated with
UV+NaOCl and stored, remained constant from day 7 to day 15, compared to the
increase that was observed for all microorganisms treated with all the other disinfection
technologies.
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4.3 Food Quality parameters
4.3.1 Color
Color is one of the most important attributes affecting consumer perception of quality.
Moreover, it plays a key role in food preference and acceptability and may even
influence taste thresholds and sweetness perception (Clydesdale, 1993). When
comparing the color of the outer leaves of lettuces, mini romaine had more greenish
(higher hue) but less intense (lower chroma) color than romaine for instance. Tiwari et
al. (2009) reported a slight increase (1–2%) in the anthocyanin content of sonicated
strawberry juice at lower amplitude levels and treatment times. The color parameter
related to browning (Castaner et al., 1999) and to the breakdown of chlorophyll (Bolin
and Huxsoll, 1991) is a* value. A significant increase in a*, indicates a shift from
greenness to redness. Samples treated with chlorine showed higher levels of potential
browning (Martin-Diana et al., 2008). In the present study, the highest net change of
color (ΔΕ) for lettuce was observed when the samples were treated with US at longest
time intervals, which indicated that a significant non-enzymatic browning reaction was
present (Cao et al., 2010). Similar results were observed in another study for UV treated
lettuce (Bermúdez-Aguirre and Barbosa-Cánovas, 2013). Strawberry color is a mix of
red and yellow. Thus, Hunter a* and b* values or some combination of a* and b* may
be considered as the physical parameters describing visual color degradation (Rodrigo et
al., 2007). Both a* and b* values showed significant differences from 45-min of
treatments (p<0.05) with both non-thermal disinfection methods (UV, US). It was
obvious that C* value, which shows the degree of saturation, purity and intensity of
color changed significantly (p<0.05) for the strawberry samples treated with UV for 45 –
min and 60-min as well as for strawberry samples treated with ultrasound at the same
times, compared to the control sample. The color of the surrounding liquid was also
changed after 60-min of Ultrasound treatment and a slightly pink color was obvious
(data not shown). Moreover, the texture and the general appearance were modified after
45-min of Ultrasound treatment, whereas there was no obvious modification of the
appearance after 45-min of UV treatment for lettuces and strawberries. The decrease of
C* value may be attributed to enzymatic oxidation of anthocyanins leading to losses of
color brilliancy (Holzwarth et al., 2012). In another study, thermosonicated strawberry
samples retained their color (Alexandre et al., 2011). Aday et al. (2013) reported that
changes in L* values in US treated strawberries were not significantly important.
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However, 90 W treatment had an adverse effect on the anthocyanins' stability. Moreover
they reported that the bright color of strawberry was preserved when 30 W and 60 W
treatments were implemented for 5 and 10-min. In our study, the treated samples with 30
W/L ultrasonication, retained their bright color (L*, a*, b*, C*) for treatments of 10, 20
and 30-min , which is in accordance with the study of Aday et al. (2013). AguilóAguayo et al. (2013) studied the effect of Pulsed Light on physical characteristics (color,
firmness and fruit weight) as well as on nutritional composition of tomatoes. More
precisely, they concluded that PL treatments did not affect the color of tomatoes, by
observing the luminosity and hue values before and after treatments. Luksiene et al.
(2012) also reported that no difference in color of tomatoes was observed when PL
treatments were used for tomatoes. Bialka and Demirci (2008) also did not report any
significant difference in the skin color of strawberries when were treated with PL.
However, non-thermal PL technology resulted in a loss of firmness and loss of weight
for tomato samples, according to the study of Aguiló-Aguayo et al. (2013). In this study
no significant differences in net color of cherry tomatoes were observed, when treated
with UV and US which is in accordance with the aforementioned studies. It was noted
that the color readings in general have relatively large standard errors, which can be
attributed, in part, to the heterogenous composition of different tissues in lettuce samples
(Baur et al., 2004).
When sodium hypochlorite immersions were used, the color was degraded as the
treatment time was increased. The highest net color differences were observed in lettuce,
after the longest exposure time in NaOCl 200ppm, and the lowest differences were
observed in cherry tomatoes, compared to lettuce and strawberries. The chlorinated
samples showed higher levels of the red parameter (an indicator of browning), which is
similar to the findings of Martin-Diana et al., (2007). Low levels of luminosity indicate
high levels of browning since darkness is related to browning (Chen et al., 2010, MartinDiana et al., 2005,). However, ClO 2 has been proven to be effective in inhibiting
enzymatic browning of fruit and vegetables (Chen et al., 2010).
As far as combined disinfection technologies are concerned, the non-thermal combined
treatments (UV+US) exhibited slight differences in color of all RTE produces.
UV+NaOCl was a more severe treatment regarding the quality retention of lettuces,
whereas US+NaOCl was more severe for cherry tomatoes. For strawberries, all
combined treatments resulted in same differences in color. Total Color difference was
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also observed for lettuce after combined treatment with chlorine and US in the study of
Salgado et al., (2014).
4.3.2 Physicochemical Parameters
It is well known that fruits and vegetables are essential components of the human diet.
There is considerable evidence of the health and nutritional benefits associated with their
consumption (Abadias et al., 2008). Due to their high nutritional value (presence of high
levels of micronutrients and fibers), their consumption is recommended by many
organizations (WHO, FAO, USDA and EFSA) to reduce the risk of cardiovascular
diseases and cancer (Allende et al., 2006a, Ragaert et al., 2004). Among the primary
metabolites found in vegetables, soluble sugars and organic acids are important
components, and both greatly contribute to their flavor characteristics and nutritional
value. Moreover, carbohydrates promote ascorbic acid stability, and thus enhance the
vitamin content (Lopez et al., 2014). Moreover, consumers demand healthy, fresh-like
and easy to prepare products, as a consequence of their lifestyle changes. Thus,
minimally processed, RTE fresh-like fruits and vegetables have been developed
(Allende et al., 2006a, Allende et al., 2006b, Froder et al., 2007, Tournas, 2005). RTE
fruits and vegetables constitute a suitable meal as they provide a great variety of
nutrients, minerals, and vitamins and they do not need preparation in order to be
consumed (Froder et al., 2007). Furthermore, the freshness, economic handling and
attractive presentation of these types of foods are factors that enhance their marketing
(Little and Gillespie, 2008).
Lettuce in one of the most consumed vegetables in many countries. Romaine lettuce is
an important dietary leafy vegetable, which contains appreciable amounts of watersoluble antioxidant compounds such as vitamin C, phenolic compounds and lipidsoluble antioxidants, such as lutein and tocopherols (Rice-Evans et al., 1995, Rice-Evans
et al., 1996, Szeto et al., 2002). Moreover, it is believed that lettuce consumption is
correlated with an improved lipoprotein profile and antioxidant status, leading to a
prevention of lipid peroxidation in tissues thus having a cardiovascular protective effect
(Nicolle et al., 2004). Many antioxidants and phenolic components have been detected
in lettuce (Heimler et al., 2007). However, the concentrations of flavonoids and phenolic
acids in lettuce are sensitive to environmental conditions (Liu et al., 2007). Differences
observed in the nutritional composition of the studied romaine lettuces between the
experiments of this study, could be explained in part by differences in the head structure
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and size. Opener lettuce heads such as those of romaine and mini-romaine lettuces have
a higher photosynthetic area, which would contribute to increasing chlorophylls and
sugars as well as other related metabolites (Lopez et al., 2014, Mou, 2009).
Antioxidants deactivate radicals by two major mechanisms, single electron transfer
(SET) and hydrogen atom transfer (HAT) (Prior et al., 2005). FRAP is a SET-based
method, ABTS assay utilizes both HAT and SET mechanisms. The response of the
different antioxidants to these assays depends on their ability to quench free radicals by
hydrogen donation and/or their ability to transfer one electron to reduce any compound.
According to Gil et al. (2000), the major organic acids present in lettuce, malic and citric
acids, do not show antioxidant capacity when they are evaluated with the FRAP assay.
This may explain the different results observed when different methods are used. Lopez
et al. (2014) have found that mini- romaine type lettuces, show higher antioxidant
capacity with the ABTS method due to their high organic acids content, whereas
romaine type showed higher antioxidant capacity with the FRAP method, probably due
to their higher phenolic compound content.
The initial values of TAC, TPC and AA in this study are similar or slightly lower than
values from other studies (Tiveron et al., 2012, Zhan et al., 2013). This can be attributed
to many factors such as cultivating conditions of the produce, the extraction conditions
as well as differences in varieties of the lettuce produce.
Moreover, TAC, TPC and AA greatly can vary among different leaf positions. For
instance, outer leaves exhibit significantly higher TAC, than middle and inner leaves in
both red and green color lettuce where the differences were more distinct particularly in
red color cultivars (Ozgen and Sekerci, 2011). It has been demonstrated that outer leaves
have the highest phytonutrient content and antioxidant properties, compared to inner
leaves (Ozgen and Sekerci, 2011). Gobbo-Neto and Lopes (2007), reported that several
factors such as seasonality, temperature, water availability, soil nutrients, pollution, and
pathogen attack can affect the content of secondary metabolites in vegetables, such as
phenolic compounds. In a study of Tiveron et al. (2012), they evaluated the phenolic
content and antioxidant activity of a great variety of vegetables commonly consumed in
Brazil and they found that the highest ability to reduce Fe3+ to Fe2+ was found in lettuce
(447.1 μmol Fe2+/g), watercress (277.4 μmol Fe2+/g), and spinach (273.3 μmol Fe2+/g).
The above results are similar to the findings of this study, where 319 μmol Fe2+/g was
the average initial concentration of TAC in lettuce. The phenolic content found in lettuce
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in other studies was 16.9 mg GA/g DW lettuce, which was different from findings of
Chu et al (2002) and Llorach et al (2008) which found lower and higher TPC in lettuces
respectively.
The issue nowadays is to increase the shelf-life of food products by preventing loss of
sensory and nutritional quality at the same time. Thus, many industries tend to add
antioxidants (Ponce et al., 2004). So, disinfection technologies should not alter the
nutritional properties of lettuce. In the present study, TAC was enhanced when nonthermal disinfection technologies were used. The increase was more intense when UV
was used. It is worth mentioning that after 30 and 60-min treatment with UV the
concentration of TAC was increased by 500 and 829 μmol Fe2+/g respectively, whereas
545 and 674 μmol Fe2+/g was the TAC increase when US treatment for 30 and 60–min
respectively was achieved. Karaca and Velioglou (2014) also studied the effect of
disinfection treatments on different minimally processed fruit and vegetables. The
enhancing effect of UV on the total phenolic compounds of fruits and vegetables has
been well studied over the last few years. Scientific evidence shows that the DNAdamaging effect of UV light induces the accumulation of UV-absorbing flavonoids and
other phenolics compounds, predominantly in the epidermal tissues of fruit (Bravo et al.,
2012).
Kenny and O’Beirne (2009) reported decreased levels of ascorbic acid in lettuce treated
with chlorine, compared to tap-rinsed lettuce, which is in accordance with the results of
the present study. Ascorbic acid degradation reactions are often responsible for
important quality changes that occur during the storage of foods, limiting shelf-life.
When products are treated with strong oxidizers, loss of antioxidative compounds like
ascorbic acid can be induced. The results of this study indicated ascorbic acid
degradation in lettuce after disinfection treatments, which are in accordance with studies
on rice leaves (Imai and Kobori, 2008) and strawberries (Allende et al., 2007). Phenolics
are believed to extend shelf life and increase the stress tolerance of plants, leading to
lower postharvest losses (Hodges and Forney, 2003). Such a property is based on their
ability to scavenge reactive oxygen species that are known to be involved in leaf
senescence and in the plant antioxidant defense system. Phenolic acids may occur in
multiple conjugated forms with sugars, acids and other phenolic compounds (Robbins,
2003). In the present study TPC was increased when all emerging non-thermal
technologies were used. This increase in the concentration of phenolic compounds can
be attributed to the wounding stress from the treatment (Martin-Diana et al., 2008).
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Discussion
Finally, lettuce is a relatively poor source of vitamin C compared with other vegetables
(Bahorun et al., 2004). In another study, vitamin C (AA plus DHAA) showed an average
concentration of 78 mg/g FW (Nicolle et al., 2004). Generally, differences in vitamin C
concentration have been found among lettuce cultivars (Lopez et al., 2014). AA content
of romaine lettuce in this study was found to be 0.09 mg/g FW. Generally it remained
constant throughout non-thermal disinfection technologies, whereas it was decreased
when immersion in NaOCl solutions followed.
Anthocyanins can be found in many fruits and vegetables, and they are largely
responsible for the red color of ripe strawberries (Allende et al., 2007, Ayala-Zavala et
al., 2004). Moreover, ascorbic acid (AA) has long been considered an important
nutritional component of strawberries. In the present study, the AA content of
strawberries was 53 mg/100 g FW. In Selva strawberries the initial AA content was
found to be 86.4 for organic and 71.2 mg/100 g FW for conventional strawberries,
respectively (Crecente Campo et al., 2012). It is known that strawberries have an
antioxidant capacity up to 10-fold greater than that of other fruits (Szeto et al., 2002).
Although the literature suggests that the exposure of plant foods to stressful situations
could modulate the synthesis of such defense substances as polyphenols and
anthocyanins, an increased concentration of these substances was presented in our study,
which is in accordance with other studies (Crecente Campo et al., 2012). Different
studies have shown that the TPC varies depending on the variety of the strawberry
studied. Meyers et al. (2003) indicated that Earliglow, Evangeline, and Annapolis
varieties had the highest free phenolic contents, averaging 273 mg gallic acid/100 g FW,
whereas the lowest content was measured in Mesabi, Jewel, and Allstar, averaging 202
mg gallic acid/100 g FW. Wang and Lin (2000) reported TPC values of the juice from
strawberry fruits (cv Allstar) at different stages of maturity, which was found to be 256
and 103 mg of gallic acid equiv. (GAE) per 100 g fw for green and ripe fruit,
respectively. The TPC also varies with the attachment of the stem (Crecente Campo et
al., 2012).
Erkan et al. (2008) studied the effect of UV on antioxidant capacity of strawberries
fruits. UV enhanced the total phenolic content of strawberry, which is in accordance
with the present study. Moreover, the phenolic content of strawberries increased during
the 15 day storage period. However, this increase was relatively lower in control fruit
when compared to illuminated fruit (Erkan et al., 2008). Moreover, UV treatment has
been shown to cause a significant increase in the antioxidant capacity of peppers
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(Vicente et al., 2005) and blueberry fruits (Perkins-Veazie et al., 2008). Fernandez et al.
(2012) observed a difference among phenolic compounds and anthocyanins in
strawberry samples, and depended on different agricultural practices. Also, the radical
scavenging ability and the reducing capacity assayed by the DPPH and FRAP methods,
respectively, were higher in strawberries of organic farming (Gündüz and Özdemir,
2014). In another study of Li et al. (2014) the effect of UV on antioxidant capacity and
anthocyanins in strawberries was studied. The effect of alternative disinfection
technology such as high hydrostatic pressure on antioxidant activity, total phenolic
compounds, vitamin C and color of strawberry have been recorded in many studies
(Nunez Mancilla, 2013, Patras et al., 2009).
The lower or higher values of AA of cherry tomatoes compared to other studies, can be
attributed to the fact that often it has been oxidized to dehydroascorbic acid before
measurement (Szeto et al., 2002). Moreover, some of the differences may be owing to
differences in seasonality or the variety tested, or can also be related to geographical
factors (Szeto et al., 2002). The initial values of TAC, TPC and AA (5.97 μmol Fe2+/g,
1.19 mg/g and 0.25 mg/g FW respectively) for cherry tomatoes of this study are in
accordance with Hanson et al., (2004) where 0.87-1.53 mg GA/g and 0.18-0.34 mg
AA/g were observed for TPC and AA respectively.
The UV radiation had an enhancing effect on total phenolic compounds as well as on
antioxidant capacity of tomatoes which is in accordance with the study of Bravo et al.
(2012) and Alothman et al. (2009). However, other technologies such as gaseous ozone
treatments, have been reported to reduce TAC and TPC (Alothman et al., 2009). On the
other hand, antioxidants status and phenolic contents were not affected in tomatoes
exposed to ozone concentrations up to 1.0 μmol● mol−1 (Karaca and Velioglu, 2014).
A loss of phenolic-related antioxidant power in vegetables is likely to occur with
crushing, chopping or pureeing. Interestingly, disruption of the vegetable matrix has
been reported to increase the bioavailability of folate and lutein, but not of b-carotene or
AA (van het Hof, 1999). However, cherry tomatoes are consumed whole, as a
consequence no loss of nutritional substances is observed. The weight loss and the skin
wrinkling of UV -treated tomatoes observed in another study, indicated a reduction of
the water content in the fruit, which could be attributed to the increase in the respiratory
rate of samples due to stress from the UV treatments (Aguiló-Aguayo et al., 2013).
Microstructure changes on the tomato surfaces after UV disinfection are responsible for
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Discussion
partial dehydration of cherry tomatoes in the present study (data not shown). The
firmness of tomato was not affected by the UV doses during post treatment storage and
also there was no consistent change occurred in tomato color during storage due to UV
treatments, which is in accordance with Sagong et al., (2011). An increase in surface
temperature of RTE foods was observed when UV light at longest treatment times was
used. However, the temperature kept low throughout all treatments (<50° C). According
to Luksiene et al. (2012), heat generation is observed during light treatments and has
been shown to affect the cell walls of light- treated products during storage, but at high
fluences (Gómez et al., 2012). However, when the temperature is kept low, no problems
are recorded for RTE foods. The AA content was not significantly affected when all
disinfection treatments were used. These results are in accordance to Luksiene et al.
(2012) who observed that Pulsed Light (PL) did not affect the ascorbic acid content
neither in tomatoes nor in other fruit such as strawberries and fresh-cut mangoes.
In general terms, non-thermal disinfection technologies induce the accumulation of
phenolic compounds and flavonoids in fruits and vegetables as a defense mechanism
against irradiation. However, the increase in TAC and TPC can also be attributed to the
phenylalanine ammonialyase activity, which is one of the key enzymes in the synthesis
of phenolic compounds in plant tissues (Alothman et al., 2009). It has been found an
increase in the activity of phenylalanine ammonialyase in peaches and cabbage seeds
after UV exposure (Brown et al., 2001, Stevens et al., 1998). Moreover, it has been
established that various types of environmental stresses promote ethylene production of
fruit. Ethylene production increased in tomato leaves and peaches after irradiation with
UV-B and UV, respectively (Gonzalez-Aguilar et al., 2004). Ethylene is a well known
phytohormone which mediates biotic and abiotic stresses (Kohli et al., 2013), which is
important in the strawberry defence system (Derksen et al., 2013). Increase in TPC of
treated RTE fruits and vegetables can also be attributed to depolymerization and
dissolution of cell wall polysaccharides, which facilitated higher extractability (Bhat et
al., 2007). Increased TAC and TPC of US-treated RTE produces can also be attributed to
better extraction. Finally, Vitamin C content decreased with the increase in treatment
time of non-thermal treatments. Vitamin C is a heat-sensitive bioactive compound,
which in the presence of oxygen gets degraded by oxidative processes, which are
stimulated in the presence of light, oxygen, and enzymes like ascorbate oxidase and
peroxidase (Davey et al., 2000). While all treatments in the present study were carried
out directly in the presence of air, oxidation of vitamin C might have occurred
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contributing significantly to the observed slight reduction. Also, during extended
exposure to UV, and US mild heat might also have been generated, which has led to
decreased vitamin C contents. González-Aguilar et al., (2007) also reported the same
negative effect of UV irradiation on vitamin C content in mango “Tommy Atkins” fruits.
The correlation between frap and folin methods has been studied by many researchers
(Fu et al., 2011). Pantelidis et al. (2007) found a negative correlation between total
antioxidants and ascorbic acid in blackberries and raspberries. The present results
exhibited a negative correlation (r=-0.946, p>0.05) between TPC and AA in lettuce as
well as a negative correlation between TAC and TPC (r=-0.946, p>0.05), when Pearson
correlation analysis was done. However, a positive correlation was found in strawberries
fruits between TAC and TPC (r=0.853, p>0.05), and a negative between TAC and AA
(r=-0.853, p>0.05) whereas no correlation was found in the same parameters for cherry
tomatoes (r=0.355, p>0.05). However between TPC and AA in cherry tomatoes a
positive correlation was recorded (r=0.836, p>0.05). According to a study by Kalt et al.,
(1999), no direct correlation between AA content and TAC can be established
examining the antioxidant properties of small fruits. However, a strong correlation (r
=0.930–0.960, p<0.05) between total phenolics and antioxidant activity has been also
reported in stone fruits (Gil et al., 2002).
Tiveron et al. (2012) also recorded that phenolic compounds present in many vegetables
have no direct relationship with the antioxidant activity. From the above finding, it can
be concluded that different phenolic compounds and some non–phenolic compounds
present different antioxidant activity. As a consequence, it is not always guaranteed a
high antioxidant capacity, when certain phenolic compounds exist in RTE foods.
Moreover, differences in final results of TAC and TPC, can be attributed to differences
among methodologies. Furthermore, the chemical composition of vegetables plays also
an important role. For this reason, more than one method is suitable for the analysis of in
vitro antioxidant activity of RTE foods (Tiveron et al., 2012).
Results indicate that, in terms of antioxidant capacity, phenolic content and AA, a single
serving of some fruits and vegetables is worth several servings of others. Furthermore,
results show a rapid loss of antioxidants following fragmentation of some vegetables,
and this is prevented by mild acidification. While we do not yet know if increased
antioxidant intake is directly beneficial to human health, there is nevertheless a strong
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Discussion
inverse relationship between dietary antioxidants and mortality, as reported by Khaw et
al. (2001).
4.4 A user-friendly theoretical mathematical model for the
prediction of food safety in a food production chain
In the present study, a computerised food decision support system was described. Soft
computing is a set of computing techniques, such as Fuzzy Logic (FL), Artificial Neural
Networks (ANNs), and Genetic Algorithms (GAs). These computing techniques, unlike
hard computing, which refers to a huge set of conventional techniques such as stochastic
and statistical methods, offer somewhat “inexact” solutions of very complex problems
through modelling and analysis with a tolerance of imprecision, uncertainty, partial
truth, and approximation (Huang et al., 2010).
FCMs were used, which are a combination of methods of fuzzy logic and neural
networks. FL is a form of multi-valued logic derived from fuzzy set theory to deal with
reasoning that is approximate, rather than precise. In contrast to yes/no or 0/1 binary
logic (crisp), FL provides a set of membership values inclusively between 0 and 1 to
indicate the degree of truth (fuzzy). ANNs provide a way to emulate biological neurons
to solve complex problems in the same manner as the human brain (Huang et al., 2010).
The computation of the weights was undertaken by experts and their cooperation
decided the weights. Three experts who assessed and evaluated the relationships
between each critical control point by using linguistic variables. Experts participated in
the European FP7 project VITAL. During the project (3,5 years period) the experts filled
in background information questionnaires from a vertical production enterprise located
in Western Peloponnesus, which produces lettuces for the Greek market and also exports
to a few EU countries, performed fact finding missions, and participated in monthly
sampling campaigns. Thus, the data used to feed the presenting model were “real-world”
data.
As mentioned above, each expert estimated each weight Wij between nodes i and j,
according to his/her experience. In order to be sure about experts’ reliability, an
algorithm was used to calculate both the weights of each interconnection and the
credibility of experts. Each expert constructed his/her own weight matrix. Each weight
Wij was collected and then they were compared according to the algorithm that
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followed. First of all, if the number of the weights with the same sign is less than pi*N,
where N is the number of experts (N=3), then it was not clear if there was positive or
negative causality between the nodes and the experts should redefine their weights.
Otherwise, the process continues and the proposed weights were used to decide the
weight eventually. Every expert who defined a weight that abstains from the average
weight that the rest of experts have proposed was penalized, and his reliability was
reduced. Moreover, his specific weight was not taken into consideration. The same
procedure followed for each one element in the matrix separately. The use of this
algorithm gives credence to the method. The experts should be sure for their decisions in
order to retain their level of confidence high.
The above variables were converted into numerical values with a defuzzification
method. The Center of Area (COA) defuzzification method was one of the most
commonly used defuzzification techniques. In this method, the fuzzy logic controller
first calculated the area under the scaled membership functions and within the range of
the output variable. The fuzzy logic controller then used the following equation to
calculate the geometric center of this area.
where S is the support set of the membership function of the output μ(y).
After COA defuzzification method the final weight matrix was ready.
It was considered that k1=k2=1 and λ=1.
In the 1st case, the experts decided as initial values of the inputs the following: C1, C2,
C3, C5, C6, C7: very strong, C4, C8, C9: strong
Initial values for the concepts after COA defuzzyfication method were:
A(0)= [1 1 1 0.75 1 1 1 0.75 0.75]
The iterative procedure was terminated when the values of concepts C i had no difference
between the latest two iterations. Considering λ=1 for the unipolar sigmoid function and
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Discussion
after N=9 iteration steps, the system reached an equilibrium point, where the values did
not change any more from their previous ones.
The calculated value of the decision concept is C 10 =0.951, which corresponded to the
95.1% of the output. Consequently the lettuce was safe for consumption.
In the 2nd case, the experts decided as initial values of the inputs the following: C1, C2,
C3, C5, C6, C7: strong, C4, C8: medium, C9:weak
The value of the decision concept was C 10 =0,818 which corresponded to the 81.8% of
the output. It needed 10 iteration steps in order to reach to an equilibrium point. The
lettuce was also safe in this case.
In the 3rd case, the experts decided as initial values of the inputs the following: C1, C3,
C4: medium, C5, C6, C7: strong, C2, C8: weak, C9: zero
The output was C 10 =0.43, which corresponded to the 43% of the output. This means that
lettuce was not appropriate for consumption.
Undoubtedly, the 1st case includes the most promising results. With other words, there is
a 95.1% certainty that the lettuce which will be consumed in this case is safe. At the 2nd
case, the lettuce is also safe for consumption, but with less confidence. Finally, the
results of the 3rd case, indicate that the lettuce is not safe for consumption, thus, it must
be withdrown and not enter the market.
Computer-aided engineering (CAE) tools, where physical reality is replaced by its
equivalent computer model, and which allows implementation of ‘‘what if’’ scenarios
more quickly, can go a long way to increasing the efficiency and competitiveness of
food product, process and equipment design. However, CAE tools that are customized to
food processing and integrate several disciplines (e.g. engineering, food science, food
technology, etc.) need to be appropriately developed. CAE tools can improve safety and
quality, reduce costs and decrease ‘‘time to market’’ (Halder et al., 2011).
Perrot et al., used the fuzzy symbolic approach for an application to a support system at
a symbolic level to help the operators to evaluate the degree of cheese ripening during
manufacturing on the basis of sensory measurements achieved on-line by the operators
(Perrot et al., 2004).
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In the present study, nine concepts were selected as the most important ones concerning
the lettuce production. Experts can quantify the risks associated with the practices of
lettuce production from ‘farm-to-fork’. The present model should be able to accurately
describe the process by which contamination occurs and the impact to the endpoint of
interest: human health (Verhaelen et al., 2012).
Currently, uncertainty and ignorance about the hygienic effects of the individual
operations during production, processing, and handling limit the applicability of a
Decision Support System to specify HACCP criteria in a quantitative manner. The
usefulness of the DSS is expected to be more significant with continuous improvement
from collaboration of experts with different scientific background. Increased efficiency,
productivity, and competitiveness are among the benefits of the use of the mathematical
model. Moreover, it can offer cost effectiveness and high reliability. This could give to a
vegetable business a comparative advantage over other competitors (Groumpos and
Stylios, 2000, Nolan, 1997). One of the biggest challenges for DSS is the ex-ante
availability of real, relevant and representative data to base the decisions on complex
problems that can arise during the food production. Thus DSS can be a valuable and
easy tool that could be used on a daily basis not only from the industry itself but also
from food authorities that can easily control the quality standards that must be met by
foods provided for consumption in order to protect public health and to prevent
consumers from fraudulent practises. It is suggested that all companies involved in the
lettuce/leafy supply chains consider the recommendations contained within HACCP
guidelines to ensure the safe production and handling of lettuce/ leafy greens products
from field to fork (FDA, 2006). However, the use of software tools like Food Science
Decision Support Systems (DSS) using theories of Fuzzy Cognitive Maps, which have
not been yet widely used in Food Science, can be further explored and problems that can
arise during the food production chain can be studied in order to indicate the importance
of some critical control points during the food production in real time.
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Discussion
4.5 Assessment of disinfection technologies based on infectivity
doses
Fresh vegetables and fruits have come during the past decade to the forefront as
important vehicles of foodborne illnesses, accounting for 13% of reported outbreaks
between 1990 and 2005 with an identified food source (Tauxe et al., 2010). Salad
greens, lettuce, sprouts and melons were the leading vehicles of illness, with norovirus,
Salmonella and E. coli O157 being the most frequently identified pathogens. Although
increased consumption of fresh produce and better surveillance and detection of
foodborne outbreaks are contributing factors to the increased recognition of vegetables
and fruits as vehicles of illnesses, the increased occurrence of outbreaks associated with
fresh RTE produce is a common problem (Tauxe et al., 2010).
Information campaigns on how to handle and wash vegetables and fruits, are targeted to
increase public awareness and food safety knowledge by consumers. The crucial role of
“best practices” (whether GAP or GHP) to improve food safety is identified as the most
important control measure strategy to prevent contamination (Beuchat and Ryu, 1997,
Brackett, 1999, De Roever, 1998). Strategies for intervention to reduce foodborne
diseases include surveillance and monitoring, appropriate training for preventative
control and the adoption of food safety management systems and risk models. Finally,
critical is the role of companies and the disinfection methods that implement after
harvest of these produces.
Taking into account the best microbial reductions that occurred with the disinfection
technologies used in this study, and infectious doses of the tested microorganisms that
have been reported in the literature, conclusions regarding the effectiveness of the
aforementioned technologies and their impact on public health are presented.
The infective dose of ETEC for adults has been estimated to be at least 108 cells, but the
young, the elderly and the infirm may be susceptible to lower levels. Because of its high
infectious dose, analysis for ETEC is usually not performed unless high levels of E. coli
have been found in a food (at least 106 EIEC organisms are required to cause illness in
healthy adults). The infectious dose for O157:H7 is estimated to be 10 - 100 cells, but no
information is available for other EHEC serotypes (FDA, 2011). In this study almost all
disinfection technologies were capable of reducing the microbial load in three RTE
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produces with US+NaOCl, UV+NaOCl and US (60-min) being the most effective ones
for the three RTE produces.
The infectious dose of S. aureus has been reported to be at least 100,000 organisms in
humans (PHAC, 2011). Staphylococcal foodborne intoxication, in which major
symptoms are vomiting and diarrhoea, occurs after ingestion of thermostable
staphylococcal enterotoxins (SE) produced in food by enterotoxinogenic strains of
coagulase-positive staphylococci mainly S. aureus. SE are normally not only slightly,
inactivated during food processing, storage, distribution or during the preparation of the
food in the kitchen. Therefore, if enterotoxinogenic staphylococci are able to grow in
food to high numbers (more than 105 - 106 CFU/g or /mL) before they are killed there is
still a risk for intoxication with consumption. In this study, S. aureus was properly
reduced to acceptable levels with all disinfection technologies in cherry tomatoes,
combined technologies reduced the S. aureus to acceptable levels in strawberries,
whereas no disinfection technology assured the elimination of S. aureus to adequate
levels in lettuce.
Because of the high fatality rate and the low infective dose of Salmonella Enteritidis and
L. monocytogenes, there is a zero tolerance policy of pathogen in ready-to-eat food
products. The current international standards for Salmonella are lack of bacteria in 20 or
25 g. Thus detection of the pathogen is sufficient for routine tests by the food industry
(positive/negative results), but for research activities it is crucial not only to detect the
pathogen, but also to accurately quantify its levels. In large outbreaks infective dose was
often low and the ingestion of as little as 10 to 100 cells could result in illness (Blaser
and Newman, 1982). For instance, a nationwide outbreak of Salmonellosis in Germany,
which led to about 1000 cases, was traced to paprika and paprika-powdered potato chips.
The estimated infectious dose of salmonellae can vary, depending on the bacterial strain
ingested as well as on the immuno-competence of individuals. Data from outbreaks of
foodborne diseases indicate that infections can be caused by ingestion of 10-45 cells
(Kisluk et al., 2012, Lehmacher et al., 1995). The infectious dose of Salmonella
Enteritidis can be relatively small, 100 to 1,000 organisms and are enough to cause the
infection in some people. Food prepared from infected animals, insufficiently cooked
and food contaminated prior to consumption are the principal causes of infection (CDC,
2005). S. Enteritidis was reduced effectively in all RTE produces of this study only
when US (60-min) was used.
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Discussion
The last years, several well documented foodborne outbreaks and sporadic cases have
been described and L. monocytogenes has been isolated from a wide range of raw and
RTE meats, poultry, dairy products, seafoods and fruits and vegetables and from various
food processing environments (McLauchlin, 1996). Several more foodborne outbreaks
have been reported recently and the population that is most susceptible to listeriosis (the
elderly and immunocompromised), is increasing. There is a need for continued vigilance
and surveillance. The infectious dose of a foodborne pathogen depends on many
variables including the immune status of the host, the virulence and infectivity of the
pathogen, the type and amount of contaminated foods consumed, the concentration of
the pathogen in the food and the number of repetitive challenges. It was estimated that
low L. monocytogenes concentrations (approximately 1 CFU/g) were too frequent to be
responsible for listeriosis, whereas, the probability of exposure to a higher dose (> 1.000
CFU) was large enough to account for the observed rate of listeriosis (EU, 1999). Thus,
102-103 was the probable infectious dose correlated with fresh RTE produce. As a
consequence, L. innocua which has been selected as a representative bacteria of
pathogen L. monocytogenes, can be lowered or eliminated to acceptable levels in lettuce
and cherry tomatoes, when US+NaOCl (33-min) is selected as a combined method,
whereas US (60-min) is required for strawberry disinfection.
Viruses are often transmitted directly from person to person but epidemiological
investigations indicate that viral diseases can be transmitted by foods, particularly those
that receive little or no processing, such as shellfish, fresh fruit and vegetables and salad
items. The infective doses are not known but available evidence suggests that they are
very low. It has been estimated that NLVs have an infective dose of between 10 and 100
virus particles. Outbreaks associated with fresh produce and Hepatitis A virus have been
reported from several countries. Soft fruits, salads, strawberries and diced tomatoes have
all been implicated. Norwalk-like viruses have been associated with various items of
fresh produce including washed salads, frozen raspberries, coleslaw, green salads, potato
salad, and fresh cut fruits. Adenoviridae infectious dose is >150 plaque forming units
when given intranasally (PHAC, 2001). NaOCL was the only disinfection method that
exhibited promising results for reducing HAdV35 prior inoculated to three RTE
produces to acceptable levels, thus ensuring public health.
With the fresh produce being increasingly responsible for outbreaks of foodborne
illnesses, more effective food safety interventions are needed throughout the production,
processing and distribution of fresh vegetables and fruits. Therefore, many companies
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must focus on eco-friendly postharvest technologies that can contribute to replacing the
use of chemical fumigation techniques for crop quality preservation. Many alternative
treatments have been reported: UV treatments (Vicente et al., 2005), pulsed UV light
treatments (Xu and Wu, 2014), US treatments (Birmpa et al., 2013, Sagong et al., 2011),
ozone (Bermúdez-Aguirre and Barbosa-Cánovas, 2013, Bialka and Demirci, 2007) etc.
The last decade has witnessed several encouraging trends in the thinking about food
safety. Firstly, it has become clear that the responsibility for food safety is distributed
along the entire food chain of production, and is not only an issue of the final consumer.
Secondly, new strategies have been adopted such as HACCP, GHP, GMP, etc. Thirdly,
the implementation of new emerging, sustainable and environmentally friendly
disinfection technologies are important. There is a growing recognition that the problems
identified in the course of outbreak investigations are of concern to the entire food
industry and thus need open and public discussion. Increasingly, good epidemiologic
data are being used to guide action that protects the public health. Finally, new
surveillance tools are increasing the detection of widespread multi-jurisdictional
foodborne outbreaks that may be more common than previously recognized (Tauxe,
2002).
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Conclusions and Future Recommendations
Chapter 5. CONCLUSIONS AND FUTURE
RECOMMENDATIONS
5.1 Conclusions
The results of the present study showed that non-thermal as well as conventional and
combined disinfection treatments were effective in reducing microbial populations in
both liquid suspensions and food matrices.
Among light non-thermal disinfection treatments, HILP treatments were more effective
for the inactivation of both E. coli and L. innocua. Furthermore, this technology resulted
in more rapid and extensive inactivation than either continuous UVC and NUV-vis light
treatments. These observations associated with HILP may be attributable to the
comparatively higher penetration and emission power compared to continuous UV and
NUV-vis. Moreover it has a high peak power produced by the multiplication of the flash
power manifold, producing a light intensity at least 100 times greater than that of other
two light technologies during the same operating time. However, research must be
performed in real food matrixes, as it is known that HILP light generates off flavors. It
can be concluded that short treatment times for decontamination efficiency would be an
important factor related to productivity in food industry.
When UV and US treatments were used for food disinfection, both were good
alternatives to other preservative techniques that are currently being used by the produce
industry, due to their low cost, lack of extensive equipment and low energy
consumption. The effectiveness of these disinfection methods, were shown to be
influenced by the dose, the exposure time and the surface of the food product. US
reduced more effectively bacteria, whereas UV was more efficient in reducing HAdV.
Some changes in the color of produce can be controlled if the exposure time is kept as
low as possible, so as to inactivate effectively the microorganisms, but to still preserve
the quality of the product. Therefore UV and US may be of benefit to those with little
capital to invest as a means of ensuring product safety and quality. Combined treatments
are quite promising, if off-flavors as well as forming carcinogenic compounds from
NaOCl can be eliminated or kept at low levels.
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It should be noted that a comparison between these disinfection technologies is not a
straightforward procedure, as a successful comparison should be made in terms of
quality and nutritional aspects of the produces that are treated. Thus, non-thermal
technologies are sustainable and promising treatments, as they have been found to have a
positive effect on product quality.
Microbiological and molecular analyses can support quality management of food
product chains. However, the results of these analyses are time-consuming
(microbiological analyses) and cost-consuming (molecular assays). Moreover, the results
depend on the accuracy and calibration of the equipment. The proposed model based on
FCM’s provides to quality and product managers of a vegetable company, a total new
approach which is clear, simple, user friendly, real-time, easily accessible, fast, reliable,
and of low-cost. In addition, the aforementioned software tool can support the Food
Authorities to have on their desk a first evaluation of the products that they are going to
inspect.
Foodborne diseases are increasing recently, as the production of Fresh RTE food is also
increasing. The public perceives food safety as absolute, and the food industry has to
deal also with the ‘‘quality” of the produced foodstuffs. Everybody demand a zero-risk
food supply and claim that cost should not be a consideration, in order to ensure public
health. Thus, disinfection remains one of the most important aspects. Non-thermal
disinfection technologies are promising and sustainable as they can offer safe food to the
consumers, ensuring at the same time public health.
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Conclusions and Future Recommendations
5.2 Future Recommendations
The present study investigated E. coli, S. aureus, S. Enteritidis, L. innocua and HAdV
survival during different time intervals of various disinfection treatments.
The disinfection methods that were used in this study can reduce the microbial
populations on the surface of the produce by two to five log units. Higher reductions are
not achieved in practice due to the ability of microorganisms to attach strongly on the
surface of the produce, the presence of biofilms and due to embedding of the cells into
inaccessible nooks and crannies or areas such as stomata. Bacterial cells embedded
within a biofilm can withstand nutrient deprivation and pH changes, and are more
resistant to detachment and disinfectants than the individual cells. This in turn limits the
efficacy of disinfection treatments and introduces another challenge in assuring the
safety of the fresh produce. Therefore, treatments that are able to eliminate or
decompose biofilms should be further investigated. In green leaves such as in lettuce, for
instance, the leaf surface is covered by a waxy cuticle layer and thus the hydrophobic
interactions should be the main forces affecting the bacterial attachment. The use of
surfactants could help in the disruption of such interactions. It is clear that a better
understanding of the mechanisms involved in bacterial attachment and the biofilm
formation on the surface of fresh produce is necessary for improving the technology and
developing new intervention strategies. This can be achieved by exploring the
physiology and the morphology of bacteria, investigating the cell–cell and the cell–
surface interactions, the adhesion kinetics and biofilm formation, and the sensitivity of
bacteria to antimicrobial agents. Moreover, the host–pathogen interactions should be
studied on a per product basis, since the nature of the interactions is dependent on the
characteristic surface properties of a specific food product. Thus, SEM analysis of
untreated bacteria and viruses inoculated on produce surface is of importance, in order to
explore if a strong bacterial attachment in the form of clusters exists.
The research presented in this thesis provides comprehensive investigation into certain
disinfection technologies on certain strains of microorganisms. As a result it may be of
interest to consider the inclusion of multiple strains of the same organisms, as well as to
expand the studies in testing other important food related microorganisms.
The new technologies proposed must be better and cheaper than the existing ones to find
a place in the fresh RTE produce market. Moreover, since the disinfection efficacy is
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greatly affected by the quality parameters of water itself, it is also important to
understand the relation between the pH, temperature, and organic matter contents of
fresh produce and the efficacy of disinfection. The chemical cleanliness is also essential
for the food matrices, extremely in the case of conventional technologies and combined
treatments including NaOCl. Therefore, it is also necessary to test the chemical residues
left on the produce surfaces after disinfection operations.
The possibility that interaction of US and UV with different types of organic material
present in the food product could result in various radical production profiles should be
studied as this could also enhance understanding of how the process can be further
optimized. Thus, further research is required to ascertain the interaction of food
constituents with US and UV and the role of resulting compounds in the inactivation
process.
To reach a point where a holistic understanding of virus inactivation is in hand, a
number of matters will first need to be addressed. For example, the specific chemical
modifications that take place in the genome and capsid during disinfection and the
effects of these modifications on virus structure and function should be further
examined. Moreover, other virus strains should be used in order to investigate the
differences of disinfectant-induced modifications.
Applied US and UV treatments also did not change the quality (color) and the
physicochemical properties of RTE produces. Application of US and UV at higher doses
that effectively inactivates bacteria in shorter treatment times may change sensory
qualities of food. Hence, the possible impact of US and UV treatments on the sensory
quality of US- and UV- treated food matrices as well as upon the organoleptic and
structural properties of foods warrants further study.
Although, the efficiency obtained in this study is indicative, the efficiency of these
treatments should be further evaluated in industrial processes. Therefore, a
produce/treatment ratio equivalent to the fresh produce industry must be addressed using
individual pieces of RTE produce. Much research is needed to develop environmentally
friendly alternative processing and preservation methods for assuring the quality and
safety of fresh RTE products without contradicting with environmental protection,
consumer acceptability, sustainable use of resources, cost factors for the Produce
Company, and food regulatory provisions.
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Conclusions and Future Recommendations
Finally, the results obtained from the theoretical mathematical model in the present
study must be further correlated and validated with risk assessment models in order to
meet the gold standard criteria for quality risk assessment.
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References
References
Abadias, M., Usall, J., Anguera, M., Solson, C., Viñas, I. (2008). Microbiological
quality of fresh, minimally-processed fruit and vegetables, and sprouts from retail
establishments. International Journal of Food Microbiology , 123(1–2), 121–129.
Abadias, M., Alegre, I., Oliveira, M., Altisent, R., Viñas , I. (2012). Growth potential of
Escherichia coli O157:H7 on fresh-cut fruits (melon and pineapple) and vegetables
(carrot and escarole) stored under different conditions. Food Control, 27(1), 37–44.
Abdul-Raouf, U.M., Beuchat, L.R., Ammar, M.S. (1993). Survival and growth of
Escherichia coli O157:H7 on salad vegetables. Applied and Environmental
Microbiology, 59, 1999-2006.
Abuladze, T., Li, M., Menetrez, M.Y., Dean, T., Senecal, A., Sulakvelidze, A., (2008).
Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach,
broccoli, and ground beef by Escherichia coli O157:H7. Applied and Environmental
Microbiology, 74 (20), 6230–6238.
Ackers, M.L., Mahon, B.E., Leahy, E., Goode, B., Damrow, T., Hayes, P.S., Bibb, W.F.,
Rice, D.H., Barrett, T.J., Hutwagner, L., Griffin, P.M., Slutsker, L. (1998). An outbreak
of Escherichia coli O157:H7 infections associated with leaf lettuce consumption. Journal
of Infectious Diseases, 177, 1588-1593.
Aday, M.S., Temizkan, R., Büyükcan, M.B., Caner, C. (2013). An innovative technique
for extending shelf life of strawberry: ultrasound. LWT— Food Science and
Technology, 52 (2), 93–101.
Aday, M.S., Caner, C. (2014). Individual and combined effects of ultrasound, ozone and
chlorine dioxide on strawberry storage life. LWT - Food Science and Technology, 57,
344-351.
Adekunte, A., Tiwari, B.K., Scannell, A., Cullen, P.J., O’Donnell, C.P. (2010).
Modelling of yeast inactivation in sonicated tomato juice. International Journal of Food
Microbiology, 137(1):116-120.
Aguiló-Aguayo, I., Charles, F., Renard, C., Page, D., Carlin, F. (2013). Pulsed light
effects on surface decontamination, physical qualities andnutritional composition of
tomato fruit. Postharvest Biology and Technology, 86, 29–36.
Aguilar-Garcia, C., Gavin, G., Baragaño-Mosqueda, M., Hevia, P., Gavino, V.C. (2007).
Correlation of tocopherol, tocotrienol, γ-oryzanol and total polyphenol content in rice
bran with different antioxidant capacity assays. Food Chemistry, 102, 1228–1232.
Ahvenainen, R. (1996). New approaches in improving the shelf life of minimally
processed fruit and vegetables. Trends in Food Science & Technology, 7, 179-187.
Ajlouni, S., Sibrani, H., Premier, R., Tomkins, B. (2006). Ultrasonication and fresh
produce (Cos lettuce) preservation. Journal of Food Science, 71, 62-68.
Page 252
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Akbas, M.Y., Olmez, H. (2007). Effectiveness of organic acids, ozonated water and
chlorine dippings on microbial reduction and storage quality of fresh-cut iceberg lettuce.
Journal of the Science of Food and Agriculture, 87, 2609–2616.
Alegria, C., Pinheiro, J., Gonçalves, E.M., Fernandes, I., Moldão, M., Abreu, M. (2009).
Quality attributes of shredded carrot (Daucus carota L. cv. Nantes) as affected by
alternative decontamination processes to chlorine. Innovative Food Science and
Emerging Technologies, 10 (1), 61–69.
Alexandre, E., Santos-Pedro, D., Brandão, T., Silva, C. (2011). Study on
Thermosonication and Ultraviolet radiation processes as an alternative to blanching for
some fruits and vegetables. Food and Bioprocess Technology, 4, 1012–1019.
Alexandre, E., Brandão, T., Silva, C. (2012). Efficacy of non-thermal technologies and
sanitizer solutions on microbial load reduction and quality retention of strawberries.
Journal of Food Engineering, 108, 417–426.
Allen, C., Bent, A., Charkowski, A. (2009). Underexplored niches in research on plant
pathogenic bacteria. Plant Physiology, 150 (4), 1631–1637.
Allende, A., Artés, F. (2003). UV-C radiation as a novel technique for keeping quality of
fresh processed “Lollo Rosso” lettuce. Food Research International, 36, 739–746.
Allende, A., McEvoy, J.L., Luo, Y., Artes, F., Wang, C.Y. (2006a). Effectiveness of
two-sided UV-C treatments in inhibiting natural microflora and extending the shelf-life
of minimally processed ‘Red Oak Leaf’ lettuce. Food Microbiology, 23(3), 241–249.
Allende, A., Tomas-Barberan, F.A., Gil, M.I. (2006b). Minimal processing for healthy
traditional foods. Trends in Food Science & Technology, 17(9), 513–519.
Allende, A., Marin, A., Buendia, B., Tomas-Barberan, F., Gil, M.I. (2007). Impact of
combined postharvest treatments (UV-C light, gaseous O3, superatmospheric O2 and
high CO2) on health promoting compounds and shelf-life of strawberries. Postharvest
Biology Technology, 46, 201–211.
Allende, A., Selma, M.V., López-Gálvez, F., Villaescusa, R., Gil, M.I. (2008). Role of
commercial sanitizers and washing systems on epiphytic microorganisms and sensory
quality of fresh-cut escarole and lettuce. Postharvest Biology and Technology, 49, 155163.
Allwood, P.B., Malik, Y.S., Maherchandani, S., Vought, K., Johnson, L.A., Braymen,
C., Hedberg, C.W., Goyal, S.M. (2004). Occurrence of Escherichia coli, noroviruses,
and F-specific coliphages in fresh market-ready produce. Journal of Food Protection, 67,
2387–2390.
Alothman, M., Bhat, R., Karim, A.A. (2009). Antioxidant capacity and phenolic content
of selected tropical fruits from Malaysia, extracted with different solvents. Food
Chemistry, 115, 785–788.
Alvarado, A., Pacheco-delahaye, E., Hevia, P. (2001). Value of a tomato byproduct as a
source of dietary fiber in rats. Plant Foods food and Human Nutrition, 56, 335-348.
Page 253
References
Al-Zeyara, S. A., Jarvis, B., Mackey, B.M. (2011). The inhibitory effect of natural
microorganisms of food on growth of Listeria monocytogenes in enrichment broths.
International Journal of Food Microbiology, 145(1), 98–105.
Anderson, J.E., Beelman, R.R., Doores, S. (1996). Persistence of serological and
biological activities of staphylococcal enterotoxin A in canned mushrooms. Journal of
Food Protection, 59, 1292-1299.
Andrews, J.H., Harris, R.F. (2000). The ecology and biogeography of microorganisms
on plant surfaces. Annual Review Phytopathology, 38, 145–180.
Andrews-Polymenis, H.L., Santiviago, C.A., McClelland, M. (2009). Novel genetic
tools for studying food-borne Salmonella. Current Opinion Biotechnology, 20, 149-157.
Anonymous (2006). Leafy, green, healthful--but contaminated. The E. coli outbreak
from bagged spinach is a reminder that fruit and vegetables can harbor harmful bacteria.
Harvard Health Letter, 32, 4.
Araujo, E.A., Andrade, N.J., Silva, L.H.M., Carvalho, A.F., Silva, C.A., Ramos, A.M.,
(2010). Control of microbial adhesion as a strategy for food and bioprocess technology.
Food and Bioprocess Technology, 3, 321–332.
Argudín, M.A., Mendoza, M.C., Rodicio, M.R. (2010). Food Poisoning and
Staphylococcus aureus Enterotoxins. Toxins, 2: 1751–1773. ISSN 2072-6651.
Armon, R., Starosvetzky, J., Arbel, T., Green, M. (1997). Survival of Legionella
pneumophila and Salmonella Typhimurium in biofilm systems. Water Science and
Technology, 35: 293 - 300.
Artés, F. (2004). Refrigeration for preserving the quality and enhancing the safety of
plant foods. Bull. Int. Inst. Refrigeration LXXXIV (1), 5–25.
Artés, F., Allende, A. (2005). Minimal fresh processing of vegetables, fruits and
juices.In: Sun, D.W. (Ed.). Emerging Technologies in Food Processing, 26, 675–715.
Artés, F., Gómez, P., Aguayo, E., Escalona, V., Artés-Hernández, F. (2009). Sustainable
sanitation techniques for keeping quality and safety of fresh-cut plant commodities.
Postharvest Biology and Technology, 51, 287–296.
Artes-Hernández ,F., Rivera-Cabrera, F. Kader, A.A. (2007). Quality retention and
potential shelf-life of fresh-cut lemons as affected by cut type and temperature.
Postharvest Biology and Technology, 43, 245–254.
Arvanitoyannis, I.S., Stratakos, A.C., Tsarouhas, P. (2009). Irradiation applications in
vegetables and fruits: a review. Critical Reviews in Food Science and Nutrition, 49, 5,
427-462.
Arya, S.P., Mahajan, M., Jain, P. (2000). Non-spectrophotometric methods for the
determination of vitamin C. Analytica Chimica Acta, 417, 1-14.
Asao, T., Kumeda, Y., Kawai, T., Shibata, T., Oda, H., Haruki, K., Nakazawa, H.,
Kozaki, S. (2003). An extensive outbreak of staphylococcal food poisoning due to lowfat milk in Japan: estimation of entertoxin A in the incriminated milk and powdered
skim milk. Epidemiology and Infection, 130, 33-40.
Page 254
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Awad, T.S., Moharram, H.A., Shaltout, O.E., Asker, D., Youssef, M.M. (2012).
Applications of ultrasound in analysis, processing and quality control of food: a review.
Food Research International, 48, 410–427.
Ayala-Zavala, F.J., Wang, S.Y., Wang, C.Y., Gonzalez-Aguilar, G.A. (2004). Effect of
storage temperatures on antioxidants capacity and aroma compounds in strawberry fruit.
Food Science Technology, 37, 687–695.
Bachmann, R. (1975). Sterilization by intense ultraviolet radiation. The Brown Boveri
Review, 62, 206-209.
Bae, D.H., Yeon, J.H., Park, S.Y., Lee, D.H., Ha, S.D. (2006). Bactericidal effects of
CaO (scallop-shell powder) on foodborne pathogenic bacteria. Archives of Pharmacal
Research, 29, 298–301.
Baert, L., Uyttendaele, M., Vermeersch, M., Van Coillie, E., Debevere, J. (2008b).
Survival and transfer of murine norovirus 1, a surrogate for human noroviruses, during
the production process of deep-frozen onions and spinach. Journal of Food Protection,
71, 1590–1597.
Baert, L., Debevere, J., Uyttendaele, M. (2009). The efficacy of preservation methods to
inactivate foodborne viruses. International Journal of Food Microbiology, 131, 83–94.
Baert, L., Uyttendaele, M., Stals, A., Van Coillie, E., Dierick, K., Debevere, J.,
Botteldoorn, N. (2009b). Reported foodborne outbreaks due to noroviruses in Belgium:
The link between food and patient investigations in an international context.
Epidemiology and Infection, 137, 316–325.
Baert, L, Vandekinderen, I., Devlieghere, F., Van Coillie, E., Debevere, J., Uyttendaele,
M. (2009). Efficacy of sodium hypochlorite and peroxyacetic acid to reduce murine
norovirus 1, B40-8, Listeria monocytogenes, and Escherichia coli O157:H7 on shredded
iceberg lettuce and in residual wash water. Journal of Food Protection, 72(5), 1047-54.
Bahorun, T., Luximon-Ramma, A., Crozier, A., Aruoma, O.I. (2004). Total phenol,
flavonoid, proanthocyanidin and vitamin C levels and antioxidant activities of Mauritian
vegetables. Journal of the Science of Food and Agriculture, 84, 1553–1561.
Baird-Parker, A.C. (1962). An improved diagnostic and selective medium for isolating
coagulase-positive staphylococci. Journal of Applied Bacteriology, 25,12-19.
Barak, J.D, Liang, A., Narm, K.E. (2008). Differential Attachment to and Subsequent
Contamination of Agricultural Crops by Salmonella enterica. Applied Environmental
Microbiology, 74, 5568-5570.
Baranyi, J., Roberts, T.A. (1994). Review Paper: A dynamic approach to predicting
bacterial growth in food. International Journal of Food Microbiology, 23, 277-294.
Baranyi, J., Jones, A., Walker, C., Kaloti, A., Robinson, T.P., Mackey, B.M. (1996). A
combined model for growth and subsequent thermal inactivation of Brochothrix
thermosphacta. Applied Environmental Microbiology, 62, 1029–1035.
Barbosa-Cánovas, G.V., Góngora-Nieto, M.M., Swanson, B.G. (1998). Non thermal
electrical methods in food preservation. Food Science and Technology International, 4,
5, 363– 370.
Page 255
References
Barka, E.A., Kalantari, S., Makhlouf, J., Arul, J. (2000). Impact of UV-C irradiation on
the cell wall-degrading enzymes during ripening of tomato (Lycopersicon esculentum
Mill.) fruit. Journal Agriculture Food Chemistry, 48, 667–671.
Barrera M., Blenkinsop R., Warriner K. (2012). The effect of different processing
parameters on the efficacy of commercial post-harvest washing of minimally processed
spinach and shredded lettuce. Food Control, 25, 745-751.
Barth, M., Hankinson, T.R., Zhuang, H., Breidt, F. (2009). Microbiological Spoilage of
Fruits and Vegetables. Compendium of the Microbiological Spoilage of Foods and
Beverages, Food Microbiology and Food Safety, DOI 10.1007/978-1-4419-0826-1_6.
Baur, S., Klaiber, R., Hammes, W., Carle, R. (2004). Sensory and microbial quality of
shredded, packaged iceberg lettuce as affected by pre-washing procedures with
chlorinated and ozonated water. Innovative Food Science and Emerging Technologies,
5, 45-55.
Baxter, C.S., Hofmann, R., Templeton, M.R., Brown, M., Andrews, R.C. (2007).
Inactivation of adenovirus types 2, 5, and 41 in drinking water by UV light, free
chlorine, and monochloramine. Journal of Environmental Engineering, 133 (1), 95–103.
Bean, N.H., Griffin, P.M. (1990). Foodborne disease outbreaks in the United States,
1973-1987: pathogens, vehicles and trends. Journal of Food Protection, 53, 807-814.
Behling, R.G., Eifert, J., Erickson, M.C., Gurtler, J.B., Kornacki, J.L., Line, E., Radcliff,
R., Ryser, E.T., Stawick, B., Yan, Z. (2010). Selected Pathogens of Concern to Industrial
Food Processors: Infectious, Toxigenic, Toxico-Infectious, Selected Emerging
Pathogenic Bacteria (chapter 2). Principles of Microbiological Troubleshooting in the
Industrial. Food Processing Environment, Food Microbiology and Food Safety, DOI
10.1007/978-1-4419-5518-0_2,
Bell, K.Y., Cutter, C.N., Sumner, S.S. (1997). Reduction of foodborne microorganisms
on beef carcass tissue using acetic acid, sodium bicarbonate and hydrogen peroxide
spray washes. Food Microbiology, 14, 439–448.
Benech, R. O., Kheadr, E.E., Laridi, R., Lacroix, C., Fliss, I. (2002). Inhibition of
Listeria innocua in cheddar cheese by addition of nisin Z in liposomes of by in situ
production in mixed culture. Applied of Environmental Microbiology, 68(8), 36833690.
Benzie, I.F.F., Strain, J.J. (1996). The ferric reducing ability of plasma (FRAP) as a
measure of ‘‘antioxidant power’’: The FRAP assay. Analytical Biochemistry, 239(1),
70–76.
Bergelson, J.M., Cunningham, J.A., Droguett, G., Kurt-Jones, E.A., Krithivas, A., Hong,
J.S., Horwitz, M.S., Crowell, R.L., Finberg, R.A. (1997). Isolation of a common receptor
for coxsackie B viruses and adenoviruses 2 and 5. Science, 275, 1320-1323.
Bermúdez-Aguirre, D., Barbosa-Cánovas, G. (2013). Disinfection of selected vegetables
under nonthermal treatments: Chlorine, acid citric, ultraviolet light and ozone. Food
Control, 29, 82-90.
Page 256
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Berrang, M.E, Brackett, R.E, Beuchat, L.R. (1989). Growth of Listeria monocytogenes
on fresh vegetables stored under controlled atmosphere. Journal of Food Protection, 52,
702-705.
Berrang, M.E, Meinersmann, R.J., Smith, D.P., Zhuang, H. (2008). The effect of chilling
in cold air or ice water on the microbiological quality of broiler carcasses and the
population of Campylobacter. Poultry Science, 87, 992-998.
Bes-Rastrollo, M., Martínez-González, M.A., Sánchez-Villegas, A., Carmen de la
Arrillaga, F., Martínez, A. (2006). Association of fiber intake and fruit/vegetable
consumption with weight gain in a Mediterranean population. Nutrition, 22, 504 –511.
Beuchat, L.R. (1996). Listeria monocytogenes: Incidence on vegetables. Food Control,
7, 415, 223-228.
Beuchat, L.R., Ryu, J. H. (1997). Produce handling and processing practices. Emerging
Infectious Diseases, 3, 459-465.
Beuchat, L.R. (1998). Progress in conventional methods for detection and enumeration
of foodborne yeasts. Food Technology Biotechnology, 36, 267-272.
Beuchat, L.R., Farber, J.M., Garrett, E.H., Harris, L.J., Parish, M.E., Suslow, T.V.
(2001). Standardization of a method to determine the efficacy of sanitizers in
inactivating human pathogenic microorganisms on raw fruits and vegetables. Journal of
Food Protection, 64, 1079–1084.
Beuchat, L.R. (2002). Ecological factors influencing survival and growth of human
pathogens on raw fruits and vegetables. Microbes and Infection, 4, 413-423.
Beuchat, L.R., Adler, B.B., Lang, M.M. (2004). Efficacy of chlorine and a peroxyacetic
acid sanitizer in killing Listeria monocytogenes on iceberg and Romaine lettuce using
simulated commercial processing conditions. Journal of Food Protection, 67, 1238–
1242.
Bhat, R., Sridhar, K.R., Tomita-Yokotani, K. (2007). Effect of ionizing radiation on
antinutritional features of velvet bean seeds (Mucuna pruriens). Food Chemistry, 103,
860–866.
Bialka, K.L., Demirci, A. (2007). Decontamination of Escherichia coli O157:H7 and
Salmonella enterica on blueberries using ozone and pulsed UV-light. Journal of Food
Science, 72, 391–396.
Bialka, K., Demirci, A., Puri, V. (2008). Modeling the inactivation of Escherichia coli
O157:H7 and Salmonella enterica on raspberries and strawberries resulting from
exposure to ozone or pulsed UV-light. Journal of Food Engineering, 85, 444–449.
Bidawid, S., Farber, J.M. Sattar, S.A. (2000a). Contamination of foods by food handlers:
experiments on hepatitis A virus transfer to food and its interruption. Applied of
Environmental Microbiology, 66, 2759-63.
Bidawid, S., Farber, J.M., Sattar, S.A. (2000b). Contamination of foods by foodhandlers:
experiments on hepatitis A virus transfer to food and its interruption. Applied of
Environmental Microbiology, 66, 2759–2763.
Page 257
References
Bilek, S.E., Turantaş, F. (2013). Decontamination efficiency of high power ultrasound
in the fruit and vegetable industry, a review. International Journal of Food Microbiology,
166, 155–162.
Bintsis, T., Litopoulou-Tzanetaki, E., Robinson, R.K. (2000). Existing and potential
applications of ultraviolet light in the food industry - a critical review. Journal of the
Science of Food and Agriculture, 80, 637-645.
Birmpa, A., Sfika, V., Vantarakis, A. (2013). Ultraviolet light and ultrasound as nonthermal treatments for the inactivation of microorganisms in fresh ready-to-eat foods.
International Journal of Food Microbiology, 167, 96–102.
Biswas, S., Rolain, J. (2013). Use of MALDI-TOF mass spectrometry for identification
of bacteria that are difficult to culture. Journal of Microbiological Methods, 92, 14–24.
Blaser, M. J., Newman, L.S. (1982). A review of human salmonellosis: I. Infective dose.
Reviews of Infectious Diseases, 4(6), 1096-1106.
Bolin, H.R., Huxoll, C.C. (1991). Effect of preparation procedures and storage
parameters on quality retention of salad-cut lettuce. Journal of Food Science, 56,
416−418.
Bolyard, M., Fair, P. S. (1992). Occurrence of chlorate in hypochlorate solutions used
for drinking water disinfection. Environmental Science Technology, 26, 1663-1665.
Boon, C.S., Mc Clements, D.J., Weiss, J., Decker, E.A. (2010). Factors influencing the
chemical stability of carotenoids in foods. Critical Reviews in Food Science and
Nutrition, 50, 515-532.
Bowen, A., Fry, A., Richards, G., Beuchat, L. (2006). Infections associated with
cantaloupe consumption: A public health concern. Epidemiology and Infection, 134,
675–685.
Bozkurt, H., D’Souza, D.H., Davidson, P.M. (2014). A comparison of the thermal
inactivation kinetics of human norovirus surrogates and hepatitis A virus in buffered cell
culture medium, Food Microbiology, doi: 10.1016/j.fm.2014.04.002.
Brackett, R.E. (1999). Incidence, contributing factors, and control of bacterial pathogens
in produce. Postharvest Biology and Technology, 15, 305-311.
Bravo, S., García-Alonso, J., Martín-Pozuelo, G., Gómez, V., Santaella, M., NavarroGonzález, I., Periago, M.J. (2012). The influence of post-harvest UV-C hormesis on
lycopene, β-carotene, and phenolic content and antioxidant activity of breaker tomatoes.
Food Research International, 49, 296–302.
Brenner, F.W., Villar, R.G., Angulo, F.J., Tauxe, R., Swaminathan, B. (2000).
Salmonella Nomenclature. Journal of Clinical Microbiology, 38, 2465-2467.
Brondum, J., Egebo, M., Agerskov, C., Busk, H. (1998). Online pork carcass grading
with the autoform ultrasound system. Journal of Animal Science, 76, 1859–1868.
Brown, J.E., Lu, T.Y., Stevens, C., Khan, V.A., Lu, J.Y., Wilson, C.L. (2001). The
effect of low dose ultraviolet light-C seed treatment on induce resistance in cabbage to
black rot (Xanthomonas campestris pv. campestris). Crop Protection, 20, 873–883.
Page 258
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Brown, R.S., Rizzo, E.D. (2001). Methods for washing cores of cored lettuce heads.
United States Patent #6,196,237.
Buchanan, R.L., Edelson, S.G. (1999a). pH-dependent, stationary phase acid resistance
response of enterohemorrhagic Escherichia coli in the presence of various acidulants.
Journal of Food Protection, 62, 211–218.
Bucheli-Witschel, M., Bassin, C., Egli, T. (2010). UV-C inactivation in Escherichia coli
is affected by growth conditions preceding irradiation, in particular by the specific
growth rate. Journal of Applied Microbiology, doi:10.1111/j.1365-2672.2010.04802.x
Burnett, S.L., Beuchat, L.R. (2000). Human pathogens associated with raw produce and
unpasteurized juices, and difficulties in decontamination. Journal of Industrial
Microbiology and Biotechnology, 25, 281-287.
Butot, S., Putallaz, T., Sanchez, G. (2007). Procedure for rapid concentration and
detection of enteric viruses from berries and vegetables. Applied and Environmental
Microbiology, 73(1), 186–19.
Butz, P., Tauscher, B. (2002). Emerging technologies: chemical aspects. Food Research
International, 35 (2/3), 279– 284.
Buzby, J., Roberts, T. (1996). ERS updates US foodborne disease costs for seven
pathogens. Food Review, 19(3), 20–25.
CAC (2003). Recommended international code of practice, general principles of food
hygiene. CAC/RCP 1-1969, Rev. 4-2003 Codex alimentarius commission food hygiene
basic texts. Rome: Food and Agriculture Organization of the United Nations, World
Health Organization.
Calder, L., Simmons, G., Thornley, C., Taylor, P., Pritchard, K., Greening, G., Bishop, J.
(2003). An outbreak of hepatitis A associated with consumption of raw blueberries.
Epidemiology and Infection, 131, 745–751.
Calokerinos, A.C., Hadjiioannou, T.P. (1983). Direct potentiometric titration of
thiosulfate, thiourea and ascorbic acid with iodate using and iodide ion-selective
electrode. Microchemical Journal, 28(4), 464–469.
Canene-Adams, K., Campbell, J.K., Zaripheh, S., Jeffery, E.H., Erdman, J.W. (2005).
The tomato as a functional food. Journal of Nutrition, 135, 1226–1230.
Cao, S., Hu, Z., Pang, B., Wang, H., Xie, H., Wu, F. (2010). Effect of ultrasound
treatment on fruit decay and quality maintenance in strawberry after harvest. Food
Control, 21 (4), 529–532.
Capanoglu, E., Beekwilder, J., Boyacioglu, D., De Vos, R.C., Hall, R.D. (2010). The
effect of industrial food processing on potential by health-beneficial tomato antioxidants.
Critical Reviews in Food Science and Nutrition, 50, 919-930.
Casson, L.W., Bess, J.W. (2003). Conversion to On-Site Sodium Hypochlorite
Generation: Water and Wastewater Applications. Lewis Publishers, Boca Raton, FL.
Page 259
References
Castaner, M., Gil, M.I., Ruiz, M.V., Artes, F. (1999). Browning susceptibility of
minimally processed Baby and Romaine lettuces. European Food Research Technology,
209, 52−56.
Castaneda-Ramirez, C., Cortes-Rodriguez, V., de la Fuente-Salcido, N., Bideshi, D.K.,
del Rincon-Castro, M.C., Barboza-Corona, J.E. (2011). Isolation of Salmonella spp.
from lettuce and evaluation of its susceptibility to novel bacteriocins of Bacillus
thuringiensis and antibiotics. Journal of Food Protection, 74 (2), 274–278.
Casteel, M.J., Schmidt, C.E., Sobsey, M.D. (2008). Chlorine disinfection of produce to
inactivate hepatitis A virus and coliphage MS2. International Journal of Food
Microbiology, 125, 267–273.
CDC (Centers for Disease Control and Prevention) (1996). Surveillance for foodbornedisease outbreaks -- United States, 1988-1992. MMWR 45, 1-55.
CDC (Centers for Disease Control and Prevention) (1997). Hepatitis A associated with
consumption of frozen strawberries-Michigan. Journal American Medical Association,
277, 1271.
CDC (Centers for Disease Control and Prevention) (2000) Surveillance for foodborne
disease outbreaks -United States, 1993-1997. MMWR 49,1-51.
CDC (Centers for Disease Control and Prevention) (2005). Salmonella Enteridis.
http://www.cdc.gov/nczved/dfbmd/disease_listing/salmonellosis_gi.html
CDC (Centers for Disease Control and Prevention).(2012). Norovirus: Technical Fact
Sheet.
http://www.cdc.gov/ncidod/dvrd/revb/gastro/norovirus-factsheet.htm.
Page
accessed 11/11/13.
CDC (Centers for Disease Control and Prevention) (2013). Surveillance for Foodborne
Disease Outbreaks – United States, 1998-2008. L.Hannh Gould, Kelly A.Walsh,
Antonio R.Vieira, Karen Herman, Ian T. Williams, Aron J. Hall, Dana Cole.
Surveillance Summaries, 2013/62 (SS02),1-34.
CDC (2013). Surveillance for Foodborne Disease Outbreaks — United States, 2009–
2010. MMWR / Vol. 62 / No. 3.
Cerezo, A. B., Cuevas, E., Winterhalter, P., García, P., Troncoso, A. M. (2010).
Isolation, Identification and antioxidant activity of anthocyanin compounds in Camarosa
Strawberry. Food Chemistry, 123, 574-582.
Center for Food Safety and Applied Nutrition (CFSAN). (2001). Draft Assessment of
the Relative Risk to Public Health from Foodborne Listeria monocytogenes Among
Selected Categories of Ready-to-Eat Foods. Jan 2001. Available from:
<http://www.foodsafety.gov/~dms/lmrisksu.html> .
Chancellor, D.D, Tyagi, S., Bazaco, M.C., Bacvinskas, S., Chancellor, M.B. (2006).
Green onions: potential mechanism for hepatitis A contamination. Journal of Food
Protection, 69, 1468-1472.
Chang, C.Y, Hsiehm Y-H., Hsu, S.S., Hu, P.Y., Wang, K.H. (2000). The formation of
disinfection by-products in water treated with chlorine dioxide. Journal of Hazardous
Materials, B79, 89-102.
Page 260
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Chang, J. M., Fang, T. J. (2007). Survival of Escherichia coli O157:H7 and Salmonella
enterica serovars Typhimurium in iceberg lettuce and the antimicrobial effect of rice
vinegar against E. coli O157:H7. Food Microbiology, 24, 745–751.
Chang, A.S., Schneider, K.R. (2012). Evaluation of overhead spray-applied sanitizers for
the reduction of Salmonella on tomato surfaces. Journal of Food Science, 71(1), 65-69.
Chang, J.C., Ossoff, S.F., Lobe, D.C., Dorfman, M.H., Dumais, C.M., Qualls, R.G.,
Johnson. J.D. (1985). UV inactivation of pathogenic and indicator microorganisms.
Applied and Environmental Microbiology, 49, 1361-1365.
Chavant, P., Gaillard-Martinie, B., Talon, R., Hébraud, M., Bernardi, T. (2007). A new
device for rapid evaluation of biofilm formation potential by bacteria. Journal of
Microbiological Methods, 68(3), 605-612.
Cheigh, C.I., Hwang, H.J., Chung, M.S. (2013). Intense pulsed light (IPL) and UV-C
treatments for inactivating Listeria monocytogenes on solid medium and seafoods. Food
Research International, 54, 745–752.
Chen, Z., Zhu, C.,Zhang, Y.,Niu, D.,Du, J. (2010). Effects of aqueous chlorine dioxide
treatment on enzymatic browning and shelf-life of fresh-cut asparagus lettuce (Lactuca
sativa L.). Postharvest Biology and Technology, 58, 232–238.
Cheong, S., Lee, C., Song, S. W., Choi, W. C., Lee, C.H., Kim, S.J. (2009). Enteric
viruses in raw vegetables and groundwater used for irrigation in South Korea. Applied
Environmental Microbiology, 75, 7745–7751.
Cho, M., Kim, J.H., Yoon, J. (2006). Investigating synergism during sequential
inactivation of Bacillus subtilis spores with several disinfectants. Water Research, 40
(15), 2911-2920.
Cho M, Kim J, Kim JY, Yoon J, Kim JH. (2010). Mechanisms of Escherichia coli
inactivation by several disinfectants. Water Research, 44(11), 3410-3418.
Cho, M., Gandhi, V., Hwang, T.M., Lee, S., Kim, J.H. (2011). Investigating synergism
during sequential inactivation of MS-2 phage and Bacillus subtilis spores with
UV/H2O2 followed by free chlorine. Water Research, 45, 1063 -1070.
Chrysikopoulos, C., Manariotis, I., Syngouna V. (2013). Virus inactivation by high
frequency ultrasound in combination with visible light. Colloids and Surfaces B:
Biointerfaces, 107, 174– 179.
Chu, Y.F.,Sun, J., Wu, X., Liu, R.H. (2002). Antioxidant and antiproliferative activities
of common vegetables. Journal of Agriculture and Food Chemistry, 50, 6910–6916.
Chun, H.H., Kim, J.Y., Lee, B.D., Yu, D.J., Song, K.B. (2010). Effect ofUV-C
irradiation on the inactivation of inoculated pathogens and quality of chicken breasts
during storage, Food Control, 21, 3, 276–280.
Clark, C.G., Kruczkiewicz, P., Guan, C.,McCorrister, S.J., Chong, P.,Wylie, J., van
Caeseele, P., Tabor, H.A., Snarr, P., Gilmour, M.W., Taboada, E.N., Westmacott, G.R.
(2013). Evaluation of MALDI-TOF mass spectroscopy methods for determination of
Escherichia coli pathotypes. Journal of Microbiological Methods, 94, 180–191.
Page 261
References
Clément, A., Dorais, M., Vernon, M. (2008). Non destructive measurement of fresh
tomato lycopene content and other physicochemical characteristics using visible-NIR
Spectroscopy. Journal of Agricultural and Food Chemistry, 56, 9813-9818.
Cliver, D.O. (1994). Other foodbome viral diseases. In: Foodbome Disease Handbook,
Hui, Y. H., Gorham, J. R., Murrell, K. D., Cliver, D. O. (eds.) p. 137-143. New York,
Marcel Dekker.
Clydesdale, F.M. (1978). Colorimetry: Methodology and Applications. CRC Critical
Reviews in Food Science and Nutrition, Boca Raton, Fla., 243-301.
Clydesdale, F.M. (1993). Color as a factor in food choice. Critical Reviews in Food
Science and Nutrition, 33(1), 83–101.
Codex Alimentarius. (1999). Principles and guidelines for the conduct of
microbiological risk assessment. CAC/GL-30.
Considine, K.M. Kelly, A.L., Fitzgerald, G.F., Hill, C. and Sleator, R.D. (2008).
Highpressure processing e effects on microbial food safety and food quality. FEMS
Microbiology Letters, 281(1), 1-9.
Cooley, M.B., Chao, D., Mandrell, R.E. (2006). Escherichia coli O157:H7 survival and
growth on lettuce is altered by the presence of epiphytic bacteria. Journal of Food
Protection, 69, 2329-2335.
Costa, C., Antonucci, F., Pallottino, F., Aguzzi, J., Sun, D.W., Menesatti, P. (2011).
Shape analysis of agricultural products: a review of recent research advances and
potential application to computer vision. Food and Bioprocess Technology, 4, 673-692.
Covas, M.I. (2007). Olive oil and the cardiovascular system. Pharmacology Research,
55(3), 175-86.
Crawford-Miksza, L., Schnurr, D.P. (1996). Analysis of 15 adenovirus hexon proteins
reveals the location and structure of seven hypervariable regions containing serotypespecific residues. Journal Virology, 70, 1836–1844.
Crecente-Campo, J., Nunes-Damaceno, M., Romero-Rodríguez, M.A., Vázquez-Odériz.,
M.L. (2012). Original Research Article: Color, anthocyanin pigment, ascorbic acid and
total phenolic compound determination in organic versus conventional strawberries
(Fragaria ananassa Duch, cv Selva). Journal of Food Composition and Analysis, 28, 23–
30.
Cromeans, T.L., Kahler, A.M., Hill, V.R. (2010). Inactivation of adenoviruses,
enteroviruses, and murine norovirus in water by free chlorine and monochloramine.
Applied and Environmental Microbiology, 76 (4), 1028–1033.
Curtis G.D.W., Nichols W.W., Falla T.J. (1989) Letters in Applied Microbiology, 8,
169-172.
Dai, T., Gupta, A., Murray, C.K., Vrahas, M.S., Tegos, G.P., Hamblin, M.R. (2012).
Blue light for infectious diseases: Propionibacterium acnes, Helicobacter pylori, and
beyond? Drug Resistance Updates, 15, 223-236.
Page 262
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Dai, T., Gupta, A., Huang, Y., Yin, R., Murray, C.K., Vrahas, M.S., Sherwood, M.E.,
Tegos, G.P., Hamblin, M.R. (2013). Blue light rescues mice from potentially fatal
Pseudomonas aeruginosa burn infection: efficacy, safety, and mechanism of action.
Antimicrobial Agents and Chemotherapy, 57, 1238-1245.
D’Amico, D., Silk, T., Wu, J., Guo, M. (2006). Inactivation of microorganisms in milk
and apple cider treated with ultrasound. Journal of Food Protection, 69(3),556-563.
Daş, E., Gűrakan, C.G., Bayındırlı, A. (2006). Effect of controlled atmosphere storage,
modified atmosphere packaging and gaseous ozone treatment on the survival of
Salmonella Enteritidis on cherry tomatoes. Food Microbiology, 23, 430–438.
Davey, M.W., Van Montagu, M., Inze, D., Sanmartin, M., Kanellis, A., Smirnoff, N.,
Benzie, I.J.J., Strain, J.J., Favell, D., Fletcher, J. (2000). Plant L-ascorbic acid:
chemistry, function, metabolism, bioavailability and effects of processing. Journal of the
Science of Food and Agriculture, 80, 825–860.
Dawson, D.J., Paish, A., Staffell, L.M., Seymour, I.J., Appleton, H. (2005). Survival of
viruses on fresh produce, using MS2 as a surrogate for norovirus. Journal of Applied
Microbiology, 98, 203–209.
Deering, A.J., Mauer, L.J., Pruitt, R.E. (2012). Internalization of E. coli O157:H7 and
Salmonella spp. in plants: a review. Food Research International, 45, 567-575.
Dehghani, M. (2005). Effectiveness of ultrasound on the destruction of E. coli.
American Journal of Environmental Sciences, 1(3),187-189.
DeLange, R.J., Glazer, A.N. (1989). Phycoerythrin fluorescence-based assay for peroxy
radicals: a screen for biologically relevant protective agents. Analytical Biochemistry,
28, 300-306.
Delaquis, P., Stewart, J.S., Toivonen, P.M.A., Moyls, A.L. (1999). Effects of warm,
chlorinated water on microbial flora of shredded iceberg lettuce. Food Research
International, 32, 7-14.
Delaquis, P.J., Fukumoto, L.R., Toivonen, P.M.A., Cliff, M.A. (2004). Implications of
wash water chlornation and temperature for the microbiological and sensory properties
of fresh-cut iceberg lettuce. Postharvest Biology and Technology, 31, 81-91.
Dell’Erbaa, A., Falsanisia, D., Libertia, L., Notarnicolaa, M., Santoroa, D. (2007).
Disinfection by-products formation during wastewater disinfection with peracetic acid.
Desalination, 215, 177–186.
Denyer, S.P., Maillard, J.Y. (2002). Cellular impermeability and uptake of biocides and
antibiotics in Gram-negative bacteria. Journal of Applied Microbiology, 92, 35-45.
Denyer, S.P., Stewart, G.S.A.B. (1998). Mechanisms of action of disinfectants.
International Biodeterioration & Biodegradation, 41, 261-268.
De Oliveira, A.B.A., Ritter, A.C., Tondo, E.C., Cardoso, M.I. (2012). Comparison of
different washing and disinfection protocols used by food services in southern Brazil for
lettuce (Lactuca sativa). Food and Nutrition Sciences, 3,28-33.
Page 263
References
Derksen, H., Rampitsch, C., Daayf, F. (2013). Signaling cross-talk in plant diseasere
sistance. Plant Science, 207, 79–87.
De Roever, C. (1998). Microbiological safety evaluations and recommendations on fresh
produce. Food Control, 9, 321-347.
D’hallewin, G., Schirra, M., Pala, M., Ben-Yehoshua, S. (2000). Ultraviolet C
irradiation at 0.5kJ.m_2 reduces decay without causing damage or affecting postharvest
quality of Star Ruby grapefruit (C. paradisi Macf.). Journal of Agricultural and Food
Chemistry, 48, 4571–4575.
Ding, T., Rahman, S.M.E., Purev, U., Oh, Deog-Hwan. (2010). Modelling of
Escherichia coli O157:H7 growth at various storage temperatures on beef treated with
electrolyzed oxidizing water. Journal of Food Engineering, 97, 497–503.
Ding T., Iwahori J., Kasuga F., Wang J., Forghani F., Park M-S, Oh D-H. (2013). Risk
assessment for Listeria monocytogenes on lettuce from farm to table in Korea. Food
Control, 30, 190-199.
Directive 2000/13/EC of the European Parliament and of the Council on the
approximation of the laws of the Member States relating to the labelling, presentation
and advertising of foodstuffs. (2000). Official Journal of the European Communities.
Dominguez, A., Broner, S., Torner, N., Martinez, A., Jansa, J. M., Alvarez, J.,
Barrabeig, I., Cayla, J., Godoy, P., Minguell, S., Camps, N., Sala, M. R. (2010). Utility
of clinical epidemiological profiles in outbreaks of foodborne disease, Catalonia, 2002
through 2006. Journal of Food Protection, 73, 125–131.
Donnan, E.J., Fielding, J.E., Gregory, J.E., Lalor, K., Rowe, S., Goldsmith, P., Antoniou,
M., Fullerton, K.E., Knope, K., Copland, J.G., Bowden, D.S., Tracy, S.L., Hogg, G.G.,
Tan, A., Adamopoulos, J., Gaston, J., Vally, H. (2012). A multistate outbreak of
hepatitis a associated with semidried tomatoes in Australia, 2009. Clinical Infectious
Diseases, 54, 775–781.
Doyle, M. (1991). Escherichia coli O157:H7 and its significance in foods. International
Journal of Food Microbiology, 12, 289-302.
Doyle, M.P., Beuchat, L.R., Montville, T.J. (ed.). (2001). Food Microbiology:
Fundamentals and Frontiers. ASM Press, Washington, DC.
Doyle, M.P., Erickson, M.C. (2008). Summer meeting 2007 — the problems with fresh
produce: an overview. Journal of Applied Microbiology, 105, 317–330.
D'Souza, D. H., Sair, A., Williams, K., Papafragkou, E., Moore, C., Jaykus, L.A. (2006).
Persistence of caliciviruses on environmental surfaces and their transfer to food.
International Journal of Food Microbiology, 108(1), 84-91.
Dubois, E., Hennechart, C., Deboose`re, N., Merle, G., Legeay, O., Burger, C., et al.
(2006). Intra-laboratory validation of a concentration method adapted for the
enumeration of infectious F-specific RNA coliphage, enterovirus, and hepatitis A virus
from inoculated leaves of salad vegetables. International Journal of Food Microbiology,
108(2), 164–171.
Page 264
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Duizer, E., Bijkerk, P., Rockx, B., de Groot, A., Twisk, F., Koopmans, M. (2004).
Inactivation of caliciviruses. Applied and Environmental Microbiology, 70, 4538–4543.
Dumas, Y., Dadomo, M., Di Lucca, G., Grolier, P. (2003). Effects of environmental
factors and agricultural techniques on antioxidant content of tomatoes. Journal of the
Science of Food and Agriculture, 83, 369–382.
Dunn, J. (1995). Pulsed-light treatment of food and packaging. Food Technology, 49,
9, 95–98.
Dunn, C., Martin, W.J. (1971). Comparison of media for isolation of Salmonellae and
Shigellae from fecal specimens. Applied Microbiology, 22, 17-22.
Duthie, G.G., Duthie, S.J., Kyle, J.A.M. (2000). Plant polyphenols in cancer and heart
disease: implications as nutritional antioxidants. Nutrition Research Reviews, 13, 79–
106.
Dykes, G.A., Vegar, M., Vanderlinde., P.B. (2003). Quantification of Listeria spp.
contamination on shell and flesh of cooked black tiger prawns (Penaeus monodon).
Letters of Applied Microbiology, 37(4), 309-313.
Eastwood, M.A., Morris, E.R. (1992). Physical properties of dietary fibre that influence
physiological function. American Journal of Clinical Nutrition, 55, 436–442.
(EC) Commission Regulation No 2073/2005 on microbiological criteria of foods.
(2005). Official Journal of the European Union.
(EC) Regulation No 852/2004 of the European Parliaments and of the council on the
hygiene of foodstuffs. (2004). Official Journal of the European Union.
EFSA (European Food Safety Authorities) (2007). The community summary report on
trends and sources of zoonoses, zoonotic agents, antimicrobial resistance and foodborne
outbreaks in the European Union in 2006. The EFSA Journal 130.
Ells, T.C., Hansen, L.T. (2006). Strain and growth temperature influence of Listeria spp.
attachment to intact and cut cabbage. International Journal of Food Microbiology, 111,
34–42.
Elman, M., Lebzelter, J. (2004). Light therapy in the treatment of Acne vulgaris.
Dermatologic Surgery, 30, 2, 139–146.
Elmnasser, N., Guillou, S., Leroi, F., Orange, N., Bakhrouf, A., Federighi, M. (2007).
Pulsed-light system as a novel food decontamination technology: a review. Canadian
Journal of Microbiology, 53, 813-821.
Enwemeka, C.S., Williams, D., Hollosi, S., Yens, D., Enwemeka, S.K. (2008). Visible
405 nm SLD light photo-destroys methicillin-resistant Staphylococcus aureus (MRSA)
in vitro. Lasers in Surgery and Medicine, 40, 734-737.
EPA (U.S Environemental Protection Agency, National Primary Drinking Water
Regulations Interim Enhanced Surface Water Treatment. Final Rule, Federal Register.
1998a, 63 (241), 69478-69521.
Page 265
References
Erickson, M.C. (2012). Microbial ecology. Produce contamination. In: Gomez- Lopez,
V.M. (Ed.), Decontamination of Fresh and Minimally Processed Produce. WileyBlackwell Publishing, USA, 3-29.
Erkan, M., Wang, S.Y., Wang, C.Y. (2008). Effect of UV treatment on antioxidant
capacity, antioxidant enzyme activity and decay in strawberry fruit. Postharvest Biology
and Technology, 48, 163–171.
Ethelberg, S., Lisby, M., Bottiger, B., Schultz, A. C., Villif, A., Jensen, T., Olsen, K.E.,
Scheutz, F., Kjelso, C., and Muller, L. (2010). Outbreaks of gastroenteritis linked to
lettuce, Denmark, January 2010. Euro Surveillance, 15, 19484.
EU (1999). European Commision Health and Consumer Protection Directorate-General.
Opinion of the scientific committee on veterinary measures relating to public health on
Listeria Monocytogenes.
Eurovital. http://www.eurovital.org.
Evans, A.E., Ruidavets, J.B., McCrum, E.E., Cambou, J.P., McClean, R., Douste-Balzy,
P., McMaster, D., Bingham, A., Patterson, C.C., Richard, J.L., Mathewson, Z.M.,
Cambien, F. (1995). Dietary patterns, risk factors and ischaemic heart disease in Belfast
and Toulouse. Quarterly journal of medicine, 88, 469–77.
FAO/WHO (2008). Microbiological hazards in fresh leafy vegetables and herbs. Expert
Panel Report. Microbiological Risk Assessment Series, 14. WHO Press (138 pp).
FAOSTAT (2009). FAOSTAT Database on Agriculture,http://faostat.fao.org/.
FAOSTAT (2011). The FAO (Food and Agriculture Organization of the United Nations)
Statistical Database. http://faostat.fao.org.
Farber, J.M., Ross, W.H., Harwig, J. (1996). Health risk assessment of Listeria
monocytogenes in Canada. International Journal of Food Microbiology, 30, 145-156.
FDA (1995). Beverages: Bottled Water; Final rule, Food and Drug Admin. Federal
Regulations, 60, 57075-57130.
FDA (1998). Guidance for industry. Guide to Minimize Microbial Food Safety Hazards
for Fresh Fruits and Vegetables.
FDA (2001). Center for Food Safety and Applied Nutrition, Office of Plant and Dairy
Foods and Beverages. 2001 Jan 30. FDA survey of imported fresh produce. FY 1999
Field Assignment.
FDA (2006). (Commodity Specific Food Safety Guidelines for the Lettuce and Leafy
Greens
Supply
Chain
http://www.fda.gov/downloads/Food/FoodSafety/Product%20SpecificInformation/Fruits
VegetablesJuices/GuidanceComplianceRegulatoryInformation/UCM169008.pdf).
FDA
(2011).
BAM:
Diarrheagenic
Escherichia
coli.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070080.htm
Feldsine, P.T., Lienau, A.H., Forgey, R.L., Calhoon, R.D. (1997). Assurance polyclonal
enzyme immunoassay for detection of Listeria monocytogenes and related Listeria
Page 266
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
species in selected foods: collaborative study. Journal of AOAC International, 80(4),
775-790.
Fellows, P.J. (2000). Processing by Application of Heat. p. 229-240. In, Food Processing
Technologies - Principles and Practice CRC Press LLC, Boca Raton, FL.
Fernández, A., Shearer, N., Wilson, D.R., Thompson, A. (2012). Effect of microbial
loading on the efficiency of cold atmospheric gas plasma inactivation of Salmonella
enterica serovar Typhimurium. International Journal of Food Microbiology, 152(3), 175180.
Fernández, A., Noriega, E., Thompson, A. (2013). Inactivation of Salmonella enterica
serovar Typhimurium on fresh produce by cold atmospheric gas plasma technology.
Food Microbiology, 33(1), 24-29.
Feuerstein, O., Ginsburg, I., Dayan, E., Veler, D., and Weiss, E.I. (2005). Mechanism of
visible light phototoxicity on Porphyromonas gingivalis and Fusobacterium nucleatum.
Photochemistry and Photobiology, 81, 5, 1186–1189.
Fine, F., Gervais, P. (2004). Efficiency of pulsed UV light for microbial
decontamination of food powders. Journal of Food Protection, 67, 787-792.
PMID:15083732
Fino, V.R., Kniel, K.E. (2008). UV light inactivation of hepatitis A virus, Aichi virus,
and feline calicivirus on strawberries, green onions, and lettuce. Journal of Food
Protection, 71, 908–913.
Flomenberg, P. et al. (1994). Increasing incidence of adenovirus disease in bone marrow
transplant recipients. Journal of Infectious Diseases, 169, 775–781.
Flores-Cervantes, D.X., Palou, E., López-Malo, A. (2013). Efficacy of individual and
combined UVC light and food antimicrobial treatments to inactivate Aspergillus flavus
or A. niger spores in peach nectar. Innovative Food Science & Emerging Technologies,
20, 244–252.
Francis, G.A., O'Beirne, D. (2002). Effects of vegetable type and antimicrobial dipping
on survival and growth of Listeria innocua and E. coli. International Journal of Food
Science and Technology, 37, 711–718.
Frankel, E.N., Meyer, A.S. (2000). The problems using one-dimensional methods to
evaluate multifunctional food and biological antioxidants. Journal of the Science of Food
and Agriculture, 80, 1925–1941.
Fraser, J., Sperber, W. (1988). Rapid detection of Listeria in food and environmental
samples by esculin hydrolysis. Journal of Food Protection, 51, 762-765.
Friedberg, E.C., Walker, G.C., Siede, W., Wood, R.D., Schultz, R.A., and Ellenberger,
T. (2006). DNA Repair and Mutagenesis, ASN Press,Washington, DC, USA.
Froder, H., Martins, C.G., de Souza, K.L.O., Landgraf, M., Franco, B., Destro, M.T.
(2007). Minimally processed vegetable salads: Microbial quality evaluation. Journal of
Food Protection, 70(5), 1277–1280.
Page 267
References
Fu, L., Xu, B-T., Xu, X-R, Gan, R-Y., Zhang, Y., Xia, E-Q, Li, H-B. (2011).
Antioxidant capacities and total phenolic contents of 62 fruits. Food Chemistry, 129,
345–350.
Fueyo J.M., Martın M.C., Gonzalez-Hevia M.A., Mendoza M.C. (2001). Enterotoxin
production and DNA fingerprinting in Staphylococcus aureus isolated from human and
food samples. Relations between genetic types and enterotoxins. International Journal of
Food Microbiology, 67, 139–145.
Fukumoto, L.R., Toivonen, P.M.A., Delaquis, P.J. (2002). Effect of wash water
temperature and chlorination on phenolic metabolism and browning of stored iceberg
lettuce photosynthetic and vascular tissues Journal of the Science of Food and
Agriculture, 50, 4503-4511.
Furata, M., Yamaguchi, M., Tsukamoto, T., Yim, B., Stavarache, C.E., Hasiba, K.,
Maeda, Y. (2004). Inactivation of Escherichia coli by Ultrasonic Irradiation. Ultrasonics
Sonochemistry, 11, 57-60.
Gallimore, C.I., Pipkin, C., Shrimpton, H., Green, A.D., Pickford, Y., McCartney, C.,
Sutherland, G., Brown, D.W.G., Gray, J.J. (2005). Detection of multiple enteric virus
strains within a foodborne outbreak of gastroenteritis: An indication of the source of
contamination. Epidemiology and Infection, 133, 41–47.
Gandhi, M. and Chikindas, M.L. (2007). Listeria: A foodborne pathogen that knows
how to survive. International Journal of Food Microbiology, 113, 1-15.
Garayoa, R., Vitas, A.I., Díez-Leturia, M., García-Jalón, I. (2011). Food safety and the
contract catering companies: Food handlers, facilities and HACCP evaluation. Food
Control, 22, 2006-2012.
Garcia-Graells, C., Opstal, I.V., Vanmuyesen, S.C.M., Michiels, C.W. (2003). The
lactoperoxidase system increases efficacy of high-pressu re inactivation of foodborne
bacteria. International Journal of Food Microbiology, 81, 211-221.
Gayán, E., Àlvarez, I., Condón, S. (2013). Inactivation of bacterial spores by UV-C
light. Innovative Food Science & Emerging Technologies, 19, 140–145.
Ge, C., Bohrerova, Z., Lee, J. (2013). Inactivation of internalized Salmonella
Typhimurium in lettuce and green onion using ultraviolet C irradiation and chemical
sanitizers. Journal of Applied Microbiology, 114, 1415—1424.
Ge C., Lee C., Nangle Ed, Li J., Gardner D., Kleinhenz M., Lee J. (2014). Impact of
Phytopathogen Infection and Extreme Weather Stress on Internalization of Salmonella
Typhimurium in Lettuce. International Journal of Food Microbiology, 168–169, 24–31.
Geeraerd, A.H., Herremans, C.H., Van Impe, J.F. (2000). Structural model requirements
to describe microbial inactivation during a mild heat treatment. International Journal of
Food Microbiology, 10, 59(3), 185-209.
Giampieri, F., Tulipani, S., Alvarez-Suarez, J.M., Quiles, J.L., Mezzetti, B., Battino, M.
(2012). The strawberry: composition, nutritional quality, and impact on human health.
Nutrition, 28(1), 9-19.
Page 268
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Gil, M.I., Tomas-Barberan, F.A., Hess-Piercem B., Holcroft, D.M., Kader, A.A. (2000).
Antioxidant activity of pomegranate juice and its relationship with phenolic composition
and processing. Journal of Agriculture and Food Chemistry, 48, 4581-4589.
Gil, M.I., Selma, M.V., López-Galvez, F., Allende, A. (2009). Fresh-cut product
sanitation and wash water disinfection: Problems and solutions. International Journal of
Food Microbiology, 134, 37-45.
Gil, M.I., Tomäs-Barberän, F.A., Hess-Pierce, B., Kader, A.A. (2002). Antioxidant
capacities, phenolic compounds, carotenoids, and vitamin C contents of nectarine,
peach, and plum cultivars from California. Journal of the Science of Food and
Agriculture, 50, 4976–4982.
Gobbo-Neto, L. Lopes, N.P. (2007). Plantas medicinais: Fatores de influência no
conteúdo de metabólitos secundários. Quimica Nova, 30, 374–381.
Gomez, P.L., Salvatori, D.M., Garcia-Loredo, A., Alzamora, S.M., (2012). Pulsed light
treatment of cut apple: dose effect on color, structure, and microbiological stability.
Food and Bioprocess Technology, 5, 2311-2322.
Gómez-López, V.M., Ragaert, P., Debevere, J., Devlieghere, F. (2007). Pulsed light for
food decontamination: a review. Trends in Food Science and Technology, 18, 9, 464–
473.
Gomez-Sanchis, J., Molto, E., Camps-Vails, G., Gomez-Chova, L., Aleixos, N., Blasco,
J., (2008). Automatic correction of the effects of the light source on spherical objects.
An application to the analysis of hyperspectral images of citrus fruits. Journal of Food
Engineering, 85 (2), 191-200.
Gonzalez-Aguilar, G., Wang, C.Y., Buta, G.J. (2004). UV-C irradiation reduces breakdown and chilling injury of peaches during cold storage. Journal of the Science of Food
and Agriculture, 84, 415–422.
González-Aguilar, G.A., Zavaleta-Gatica, R., Tiznado-Hernández, M.E. (2007).
Improving postharvest quality of mango Haden by UV-C treatment. Postharvest Biology
and Technology, 45, 108–116.
Goodburn, C., Wallace, C. (2013). The microbiological efficacy of decontamination
methodologies for fresh produce: A review. Food Control, 32, 418-427.
Gordon, G., Luke, A. and Bubnis, B. (1995). Minimizing chlorate ion formation.
American Water Works Association, 87(6): 97-106.
Gorski, L., Palumbo, J.D., Mandrell R.E. (2003). Attachment of Listeria monocytogenes
to Radish Tissue Is Dependent upon Temperature and Flagellar Motility. Applied
Environmental Microbiology, 69, 258-266.
Gould, I.M. (2006). Costs of hospital-acquired methicillin-resistant Staphylococcus
aureus (MRSA) and its control. International Journal of Antimicrobial Agents, 28, 379384.
Gragg, S.E., Brashears, M.M. (2010). Reduction of Escherichia coli O157:H7 in Fresh
Spinach, using lactic acid bacteria and chlorine as a multihurdle intervention. Journal of
Food Protection, 73 (2), 358–361.
Page 269
References
Graves, L.M., Helsel, L.O., Steigerwalt, A.G., Morey, R.E., Daneshvar, M.I., Roof, S.E.,
Orsi, R.H., Fortes, E.D., Millilo, S.R., den Bakker, Henk C., Wiedmann, M.,
Swaminathan, B., Sauders, B.D. (2009). Listeria marthii sp. nov., isolated from the
natural environment, Finger Lakes National Forest. International Journal of Systematic
Evolution Microbiology, 60, 1280-1288.
Greber, U.F., Willetts, M., Webster, P., Helenius, A. (1993). Stepwise dismantling of
adenovirus 2 during entry into cells. Cell, 75, 477-486.
Greig, J.D., Todd, E. C., Bartleson, C.A., Michaels, B.S. (2007). Outbreaks where food
workers have been implicated in the spread of foodborne disease. Part 1. Description of
the problem, methods, and agents involved. Journal of Food Protection, 70, 1752–1761.
Griffin, P.M., Tauxe, R.V. (1991). The epidemiology of infections caused by
Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic
uremic syndrome. Epidemiology Review, 13, 60-98.
Groumpos, P.P, Stylios, C.D. (2000). Modeling supervisory control systems using fuzzy
cognitive maps. Chaos Solitons & Fractals, 113, 329–336.
Groumpos P.P. (2010). “Fuzzy Cognitive Maps: Basic Theories and their Application to
Complex Systems”, In: Glykas M. (ed) Fuzzy Cognitive Maps: Advances in Theory,
Methodologies, Tools and Applications, 247, 1-22, Springer-Verlag Berlin, Heidelberg.
Guerrero- Beltrán , J.A., Barbosa-Cánovas, G.V. (2004). Review: Advantages and
limitations on processing foods by UV light. Food Science and Technology
International, 10, 137-147.
Guerrero-Beltrán, J.A., Barbosa-Cánovas, G.V., & Swanson, B.G. (2005). High
hydrostatic pressure processing of fruit and vegetable products. Food Reviews
International, 21(4), 411-425.
Guffey, J.S., Wilborn, J., (2006). In vitro bactericidal effects of 405-nm and 470-nm
blue light. Photomedicine and Laser Surgery, 24, 684-688.
Gulati, B.R., Allwood, P.B., Hedberg, C.W., Goyal, S.M. (2001). Efficacy of commonly
used disinfectants for the inactivation of calicivirus on strawberry, lettuce, and a food
contact surface. Journal of Food Protection, 64, 1430–1434.
Gündüz, K., Özdemir, E. (2014). The effects of genotype and growing conditions on
antioxidant capacity, phenolic compounds, organic acid and individual sugars of
strawberry. Food Chemistry, 155, 298–303
Guven K., Mutlu M. B., Gulbandilar A., Cakir P. (2010). Occurrence and
characterization of Staphylococcus aureus isolated from meat and dairy products
consumed in Turkey. Journal of Food Safety, 30, 196-212.
Hadjok, C., Mittal, G.S., Warriner, (2008). Inactivation of human pathogens and
spoilage bacteria on the surface and internalized within fresh produce by using a
combination of ultraviolet light and hydrogen peroxide. Journal of Applied
Microbiology, 104, 1014–1024.
Page 270
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Häkkinen, S., Heinonen, M., Karenlampi, S., Mykkanen, H., Ruuskanen, J., Törrönen,
R. (1999). Screening of selected flavonoids and phenolic acids in 19 berries. Food
Research International, 32(5), 345-353.
Halder, A., Dhall, A., Datta, A., Glenn Black, D., Davidson, P.M., Li J.,Zivanovic, S.
(2011). A user-friendly general-purpose predictive software package for food safety.
Journal of Food Engineering, 104, 173–185.
Halpin, R.M, Duffy,L.,Cregenzán-Alberti, O, Lyng, J.G., Noci, F. (2014). The effect of
non-thermal processing technologies on microbial inactivation: An investigation into
sub-lethal injury of Escherichia coli and Pseudomonas fluorescens. Food Control, 41,
106-115.
Hancock, R.D., Mcdougall, G.J., Stewart, D. (2007). Berry fruit as "superfood": hope or
hype. Biologist, 54(2), 73-79.
Hannum, S.M. (2004). Potential impact of strawberries on human health: A review of
the science. Critical Reviews in Food Science and Nutrition, 44(1), 1-17.
Hanson P.M., Yang, R.Y., Wu, J., Chen, J.T., Ledesma, D., Tsou, S. (2004). Variation
for antioxidant activity and antioxidants in tomato. Journal of the American Society for
Horticultural Science, 129 (5), 704-711.
Hasler, C.M., Bloch, A.S., Thomson, C.A. (2004). Position of the American Dietetic
Association:functional foods. Journal of Americal Dietary Association, 104, 814–826.
Haughton, P., Gomez Grau, E., Lyng, J., Cronin, D. (2012). Susceptibility of
Campylobacter to high intensity near ultraviolet/visible 395±5 nm light and its
effectiveness for the decontamination of raw chicken and contact surfaces. International
Journal of Food Microbiology, 159, 267–273.
Hawkins, C.I., Pattison, D.I., Davies, M.J. (2003). Hypochlorite induced oxidation of
amino acids, peptides and proteins, Amino Acids, 25, 259-274.
HCDCP. (2012). http://www.keelpno.gr/en-us/home.aspx.
Heaton, J.C., Jones, K. (2008). Microbial contamination of fruit and vegetable and the
behavior of enteropathogens in the phyllosphere: a review. Journal of Applied
Microbiology, 104, 613-626.
Hedberg C.W., MacDonald K.L., Osterholm M.T. (1994). Changing epidemiology of
food-borne disease: a Minnesota perspective. Clinical and Infectious Diseases, 18, 671–
682.
Heimler, D., Isolani, L., Vignolini, P., Tombelli, S., Romani, A. (2007). Polyphenol
content and antioxidant activity some species of freshly consumed salads. Journal of
Agricultural and Food Chemistry, 55(5), 1724–1729.
Hellenic Statistical Authority (El.STAT.) (2014).
Hendriksen, R.S., Vieira, A.R., Karlsmose, S., Lo Fo Wong, D.M.A., Jensen, A.B.,
Wegener, H.C., Aarestrup, FM. (2011). Global Monitoring of Salmonella Serovar
Distribution from the World Health Organization Global Foodborne Infections Network
Page 271
References
Country Data Bank: Results of Quality Assured Laboratories from 2001 to 2007.
Foodborne Pathogens And Disease, 8, 8.
Hernandez, F., Monge, R., Jimenez, C., Taylor, L. (1997). Rotavirus and Hepatitis A
virus in market lettuce (Latuca sativa) in Costa Rica. International Journal of Food
Microbiology, 37, 221-223.
Hertog, M.G.L., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F.,
Giampaoli, S., Jansen, A., Menotti, A., Nedeljkovic, S. (1995). Flavonoid intake and
long-term risk of coronary heart disease and cancer in the seven countries study.
Archives of Internal Medicine, 155, 381–6.
Hewitt, J., Greening, G.E. (2004). Survival and persistence of norovirus, hepatitis A
virus, and feline calicivirus in marinated mussels. Journal of Food Protection, 67, 1743–
1750.
Himathongkham, S., Bahari, S., Riemann, H., Cliver, D. (1999). Survival of Escherichia
coli O157:H7 and Salmonella typhimurium in cow manure and cow manure slurry.
FEMS Microbiology Letters, 178, 251-257.
Hodges, D.M., Forney, C.F. (2003). Postharvest ascorbate metabolism in two cultivars
of spinach differing in their senescence rates. Journal of the American Society for
Horticultural Science, 128, 930–935.
Hoelzer, K., Pouillot, R., Gallagher, D., Silverman, M. B., Kause, J, Dennis, S. (2012).
Estimation of Listeria monocytogenes transfer coefficients and efficacy of bacterial
removal through cleaning and sanitation. International Journal of Food Microbiology,
157, 267-277.
Holzwarth, M., Korhummel, S., Kammerer, D., Carle, R. (2012). Thermal inactivation
of strawberry polyphenoloxidase and its impact on anthocyanin and color retention in
strawberry (Fragaria x ananassa Duch.) purées. Food Research Technology, 235, 1171–
1180.
Hsu et al. (2008). Evaluation of microbial inactivation and physicochemical properties
of pressurized tomato juice during refrigerated storage. LWT - Food Science and
Technology, 41(3), 367–375.
Hsu, L., Moraru, C.I. (2011). Quantifying and mapping the spatial distribution of fluence
inside a pulsed light treatment chamber and various liquid substrates. Journal of Food
Engineering, 103, 1, 84–91.
Huang, T.S, Xu, C. Walker, K., West, P., Zhang, S., Weese, J. (2006). Decontamination
efficacy of combined chlorine dioxide with ultrasonication on apples and lettuce. Journal
of Food Science, 71, 134-139.
Huang, Y., Lan, Y., Thomson, S., Fangc, A., Hoffmann W., Lacey, R. (2010).
Development of soft computing and applications in agricultural and biological
engineering. Computers and Electronics in Agriculture, 71, 107–127.
Huang., Chen, H. (2014). A novel water-assisted pulsed light processing for
decontamination of Blueberries, Food Microbiology, 40, 1-8.
Page 272
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Huang, D., Ou, B., Prior, R.L. (2005). The chemistry behind antioxidant capacity assays.
Journal of Agriculture and Food Chemistry, 53, 1841–1856.
Hung, H.-C., Joshipura, K.J., Jiang, R., Hu, F.B., Hunter, D., Smith-Warner, S.A.,
Colditz, G.A., Rosner, B., Spiegelman, D., Willett, W.C. (2004). Fruit and vegetable
intake and risk of major chronic disease. Journal of the National Cancer Institute, 96,
1577–1584.
Hunter, R.S., Harold, R. (1987). The measurement of appearance, 2nd ed. Wiley, New
York.
Imai, K., Kobori, K. (2008). Effects of the interaction between ozone and carbon dioxide
on gas exchange, ascorbic acid content, and visible leaf symptoms in rice leaves.
Photosynthetica, 46, 387–394.
Issa-Zacharia, A., Kamitano, Y., Muhimbula, H.S., Ndabikunze, B.K. (2010). A review
of microbiological safety of fruits and vegetables and the introduction of electrolyzed
water as an alternative to sodium hypochlorite solution. African Journal of Food Science
4, (13), 778-789.
Ito, K., Inoue, S., Hiraku, Y., Kawanishi, S. (2005). Mechanism of sit specific DNA
damage induced by ozone. Mutation Research/Genetic Toxicology and Environmental
Mutagenesis, 585 (1-2), 60-70.
Iturriaga, M.H., Escartin, E.F. (2010). Change in the effectiveness of chlorine treatments
during colonization of Salmonella Montevideo on tomatoes. Journal of Food Safety, 30,
300-306.
Jaeger, C.H., Lindow, S.E., Miller, S., Clark, E., Firestone, M.K. (1999). Mapping of
sugar and amino acid availability in soil around roots with bacterial sensors of sucrose
and tryptophan. Applied Environmental Microbiology, 65, 2685-2690.
Jahid, I.K., Ha, S.D. (2012). A review of microbial biofilms of produce: Future
challenge to food safety. Food Science and Biotechnology, 21(2), 299–316.
Jahid, I.K., Han, N.R., Srey, S., Ha, S.D. (2014). Competitive interactions inside mixedculture biofilms of Salmonella Typhimurium and cultivable indigenous microorganisms
on lettuce enhance microbial resistance of their sessile cells to ultraviolet C (UV-C)
irradiation. Food Research International, 55, 445–454.
Jeyamkondan, S., Jayas, D.S., Holley, R.A., (1999). Pulsed electric field processing of
foods: A review. Journal of Food Protection, 62 (9), 1088-1096.
Jin, P., Wang, S.Y., Wang,C.Y., Zheng, Y. (2011). Effect of cultural system and storage
temperature on antioxidant capacity and phenolic compounds in strawberries. Food
Chemistry, 124, 262–270.
Johansen, C., Falholt, P., Gram, L. (1997). Enzymatic removal and disinfection of
bacterial biofilms. Appied Environmental Microbiology, 63, 3724- 3728.
Johnston, L.M., Jaykus, L.A., Moll, D., Anciso, J., Mora, B., Moe, C.L. (2006). A field
study of the microbiological quality of fresh produce of domestic and Mexican origin.
International Journal of Food Microbiology, 112, 83-95.
Page 273
References
Jones, P.G., Inouye, M. (1994). The cold shock response- a hot topic. Molecular
Microbiology, 11, 811-818.
Jung, Y.S., Frank, J.F., Brackett, R.E. (2003). Evaluation of antibodies for
immunomagnetic separation combined with flow cytometry detection of Listeria
monocytogenes. Journal of Food Protection, 66(7), 1283-1287.
Juven, B.J., Pierson, M.D. (1996). Antibacterial effects of hydrogen peroxide and
methods for its detection and quantitation. Journal of Food Protection, 59, 1233–1241.
Kader, A.A., Saltveit, M.E. (2003). Respiration and gas exchange. In: Bartz, Brecht
(Eds.), Postharvest Physiology and Pathology of Vegetables, 2nd edition. Marcel Dekker,
Inc., New York, NY.
Kallner, A. (1986). Annals of the New York Academy of Sciences, 498, 418-423.
Kalt, W., Forney, C. F., Martin, A., Prior, R. L. (1999). Antioxidant capacity, vitamin C,
phenolics, and anthocyanins after fresh storage of small fruits. Journal of Agricultural
and Food Chemistry, 47, 4638–4644.
Kaper, J.B., Nataro, J.P, Mobley, H.L. (2004). Pathogenic Escherichia coli. Nature
Reviews Microbiology, 2(2), 123-40.
Karaca, H., Velioglu, Y.S. (2014). Effects of ozone treatments on microbial quality and
some chemical properties of lettuce, spinach, and parsley. Postharvest Biology and
Technology, 88, 46–53.
Katsikogianni, M., Missirlis, Y.F. (2004). Concise review of mechanisms of bacterial
adhesion to biomaterials and of techniques used in estimating bacteria material
interactions. European Cells and Materials, 8, 37–57.
Kawashima, L.M., Soares, V. (2003). Mineral profile of raw and cooked leafy
vegetables consumed in Southern Brazil. Journal of Food Composition and Analysis, 16,
605-611.
Kenny, O., O’Beirne, D. (2009). The effects of washing treatment on antioxidant
retention in ready-to-use iceberg lettuce. International Journal of Food Science and
Technology, 44, 1146–1156.
Khadre, M.A., Yousef, A.., Kim, J.G. (2001). Microbiological aspects of ozone
applications in food: A review. Journal of Food Science, 66(9), 1241-1252.
Khaw, K.T., Bingham, S., Welch, A., Luben, R., Wareham, N., Oakes, S., Day, N.
(2001). Relation between plasma ascorbic acid and mortality in men and women in
EPIC-Norfolk prospective study: a prospective population study. European Prospective
Investigation into Cancer and Nutrition. Lancet, 357, 657–663.
Kim, J.G., Yousef, A.E., Dave, S. (1999). Application of ozone for enhancing the
microbiological safety and quality of foods: A review. Journal of Food Protection, 62(9),
1071–1087.
Kim, J.G. (2008). Quality maintenance and food safety of fresh-cut produce in Korea.
The Asian and Australasian Journal of Plant Science Biotechnology, 2:1-6.
Page 274
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Kisluk, G., Hoover, D.G., Kneil, K.E., Yaron, S. (2012). Quantification of low and high
levels of Salmonella enterica serovar Typhimurium on leaves. LWT - Food Science and
Technology, 45, 36-42.
Kivits, A.G., van der Sman, F.J., Tijburg, L.B. (1997). Analysis of catechins from green
and black tea in humans: a specific and sensitive colorimetric assay of total catechins in
biological fluids. International Journal of Food Science and Nutrition, 48, 387-392.
Knekt, P., Isotupa, I., Rissanen, H., Heliövaara, M., Järvinen, R., Häkkinen, S., Aromaa,
A., Reunanen, A. (2000). Quercetin intake and the incidence of cerebrovascular disease.
European Journal of Clinical Nutrition, 54, 415–417.
Koc, K., Bhushan, B., Barthlott, W. (2008). Diversity of structure, morphology and
wetting of plant surfaces. Soft Matter, 4, 1943-1963.
Kohli, A., Sreenivasulu, N., Lakshmanan, P., Kumar, P.P. (2013). The
phytohormonecrosstalk paradigm takes center stage in understanding how plants
respond to abiotic stresses. Plant Cell Reports, 32, 945–957.
Kokkinos, P., Kozyra, I., Lazic, S., Bouwknegt, M., Rutjes S., Willems, K., Moloney,
R., de Roda Husman, A, M, Kaupke, A., Legaki, E., D’Agostino, M, Cook, N.,
Rzezutka, A., Petrovic, T., Vantarakis, A. (2012). Harmonised Investigation of the
Occurrence of Human Enteric Viruses in the Leafy Green Vegetable Supply Chain in
Three European Countries. Food and environmental virology, 4(4), 179-191.
Koopmans, M., von Bonsdor C-H., Vinje, J., de Medici, D., Monroe, S. (2002).
Foodborne viruses. FEMS Microbiology Reviews, 26, 187-205.
Koopmans, M., Vennema, H., Heersma, H., van Strien, E., van Duynhoven, Y., Brown,
D., Reacher, M., and Lopman, B. (2003). Early identification of common-source
foodborne virus outbreaks in Europe. Emerging Infectious Diseases, 9, 1136–1142.
Koopmans, M., Duizer, E. (2004). Foodborne viruses: an emerging problem.
International Journal of Food Microbiology, 90, 23– 41.
Koopmans, M. (2008). Progress in understanding norovirus epidemiology. Current
Opinion in Infectious Diseases, 21, 544–552.
Koponen, J.M., Happonen, A.M., Mattila, P.H., & Törrönen, A.R. (2007). Contents of
anthocyanins and ellagitannins in selected foods consumed in Finland. Journal of
Agricultural and Food Chemistry, 55(4), 1612-1619.
Koseki, S., Yoshida, K., Isobe, S., Itoh, K. (2001). Decontamination of lettuce using
acidic electrolyzed water. Journal of Food Protection, 64, 652-658.
Koseki, S., Yoshida, K., Kamitani, Y., Isobe, S., Itoh, K. (2004). Effect of mild heat pretreatment with alkaline electrolyzed water on the efficacy of acidic electrolyzed water
against Escherichia coli O157:H7 and Salmonella on Lettuce. Food Microbiology, 21,
559–566.
Koseki, S., Isobe, S. (2005). Growth of Listeria monocytogenes on iceberg lettuce and
solid media. International Journal of Food Microbiology, 101, 217 – 225.
Page 275
References
Kothary, M.H., Babu, U.S. (2007). Infective dose of foodborne pathogens in volunteers:
a review. DOI: 10.1111/j.1745-4565.2001.tb00307.x
Kovač, K., Diez-Valcarce, M., Raspor, P., Hernández, M., Rodríguez-Lázaro, D.,
(2012). Effect of high hydrostatic pressure processing on norovirus infectivity and
genome stability in strawberry puree and mineral water. International Journal of Food
Microbiology, 152 (1–2), 35–39.
Kowalski, W. (2009). Ultraviolet Germicidal Irradiation Handbook. Springer.
Krause, G., Terzagian, R., Hammond, R. (2001). Outbreak of Salmonella serotype
Anatum infection associated with unpasteurized orange juice. South Medicine Journal,
94, 1168-1172.
Krishnamurthy, K., Demirci, A., Irudayaraj, J.M. (2007). Inactivation of Staphylococcus
aureus in milk using flow-through pulsed UV light treatment system. Journal Food
Science, 72, 233–239.
Kroupitski,Y., Pinto, R., Brandl, M.T., Belausov, E., Sela S. (2009). Interactions of
Salmonella enterica with lettuce leaves. Journal of Applied Microbiology, 106, 18761885.
Kun, Y., Lule, U.S., Xiao-Lin, D. (2006). Lycopene: its properties and relationship to
human health. Food Reviews International, 22(4), 309–33.
Kurdziel, A.S., Wilkinson, N., Lanfton, S., and Cook, N. (2001). Survival of poliovirus
on soft fruit and salad vegetables. Journal of Food Protection, 64, 706-9.
Lagunas-Solar, M.C., Piña, C., MacDonald, J.D., Bolkan, L. (2006). Development of
pulsed UV light processes for surface fungal disinfection of fresh fruits. Journal of Food
Protection, 69, 2, 376-384.
Lambert, PA. (2002) Cellular impermeability and uptake of biocides and antibiotics in
Gram positive bacteria and mycobacteria. Journal of Applied Microbiology 92(Suppl.),
46-55.
Lampe, J.W., Peterson, S. (2002). Brassica, biotransformation and cancer risk: genetic
polymorphism alters the preventive effects of cruciferous vegetables. Journal of
Nutrition, 132, 2991–2994.
Lang, M.M., Harris, L.J., Beuchat, L.R. (2004). Survival and recovery of Escherichia
coli O157:H7, Salmonella, and Listeria monocytogenes on lettuce and parsley as
affected by method of inoculation, time between inoculation and analysis, and treatment
with chlorinated water. Journal of Food Protection, 67, 1092-1103.
Lauternborn, W., Ohl, C.D. (1997). Cavitation bubbles dynamics. Ultrasonics
Sonochemistry, 4, 67–75.
Lazcka, O., Del Campo, F.J., Munoz, F.X. (2007). Pathogen detection: a perspective of
traditional methods and biosensors. Biosensors and Bioelectronics, 22(7), 1205-1217.
Lee, S.K., Kader, A.A. (2000). Preharvest and postharvest factors influencing vitamin C
content in horticultural crops. Postharvest Biology and Technology, 20, 207–220.
Page 276
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Lee, M.T., Chen, B.H. (2002). Stability of lycopene during heating and illumination in a
model system. Food Chemistry, 78, 425-432.
Lee, J., Kentish, S., Ashokkumar, M. (2005). Effect of surfactants on the rate of growth
of an air bubble by rectified diffusion. The Journal of Physical Chemistry. B 109 (30),
14595–14598.
Lee, S.U., Joung, M, Yang, D.J., Park, S.H., Huh, S., Park, W.Y, Yu, J.R. (2008).
Pulsed-UV light inactivation of Cryptosporidium parvum. Parasitology Research, 102,
1293–1299.
Lee, H., Zhou, B., Liang, W., Feng, H., Martin, S.E. (2009). Inactivation of Escherichia
coli cells with sonication, manosonication, thermosonication and manothermosonication:
microbial responses and kinetics modelling. Journal of Food Engineering, 93 (3), 354364.
Leggett, H.C., Cornwallis, C.K., West, S.A. (2012). Mechanisms of Pathogenesis,
Infective Dose and Virulence in Human Parasites. PLoS Pathogens, 8(2), 1002512.
doi:10.1371/journal.ppat.1002512
Lehmacher, A., Bockemuhl, J., Aleksic, S. (1995). Nationwide outbreak of human
salmonellosis in Germany due to contaminated paprika and paprika-powdered potato
chips. Epidemiology and Infection, 115(3), 501-511.
Leistner, L. (2000). Basic aspects of food preservation by hurdle technology.
International Journal of Food Microbiology, 55, 181–86.
Leonardi, C., Ambrosino, P., Esposito, F., Fogliano, V. (2000a). Antioxidative activity
and carotenoid and tomatine contents in different typologies of fresh consumption
tomatoes. Journal of Agricultural and Food Chemistry, 48, 4723–4727.
Li, D., Luo, Z., Mou, W., Wang, Y., Ying, T., Mao, L. (2014). ABA and UV-C effects
on quality, antioxidant capacity andanthocyanin contents of strawberry fruit (Fragaria
ananassa Duch.). Postharvest Biology and Technology, 90, 56–62.
Liao, C.H. (2007). Inhibition of foodborne pathogens by native microflora recovered
from fresh peeled baby carrot and propagated in cultures. Journal of Food Science,
72(4), M134–M139.
Liltved, H., Landfald, B. (2000). Effects of high intensity light on ultraviolet-irradiated
and non-irradiated fish pathogenic bacteria. Water Research, 34(2): 481–486.
Lina, G., Bohach, G.A., Nair, S.P., Hiramatsu, K., Jouvin-Marche, E., Mariuzza, R.
(2004). Standard nomenclature for the superantigens expressed by Staphylococcus.
Journal of Infectious Diseases, 189, 2334-6.
Ling, W.H., Jones, P.J. (1995). Dietary phytosterols: a review of metabolism, benefits
and side effects. Life Sciences, 57,195–206.
Lipovsky, A., Nitzan, Y., Gedanken,A., Lubart, R. (2010). Visible light induced killing
of bacteria as a function of wavelength: implication for wound healing. Lasers in
Surgery and Medicine 42, 467-472.
Page 277
References
Little, C.L., Gillespie, I.A. (2008). Prepared salads and public health. Journal of Applied
Microbiology, 105(6), 1729–1743.
Liu, X., Ardo, S., Bunning, M., Parry, J., Zhou, K., Stushnoff, C., Stoniker, F., Yu, L.,
Kendall, P. (2007). Total phenolic content and DPPH radical scavenging activity of
lettuce (Lactuca sativa L.) grown in Colorado. Food Science and Technology, 40(3),
552–557.
Liu, N.T., Lefcourt, A.M., Nou, X., Shelton, D.R., Zhang, G., Lo, Y.M. (2013). Native
microflora in fresh-cut produce processing plants and their potentials for biofilm
formation. Journal of Food Protection, 76(5), 827–832.
Liu, T.Z., Chin, N., Kiser, M.D., Bigler, W.N. (1982). Specific spectrophotometry of
ascorbic-acid in serum or plasma by use of ascorbate oxidase. Clinical Chemistry,
28(11), 2225–2228.
Llorach, R., Martínez-Sánchez, A., Tomás-Barberán, F.A., Gil, M.I., Ferreres, F. (2008).
Characterisation of polyphenols and antioxidant properties of five lettuce varieties and
escarole. Food Chemistry, 108, 1028–1038.
Loff, M., Mare, L., de Kwaadsteniet M., Khan, W. (2014). 3M™Molecular Detection
system versus MALDI-TOF mass spectrometry and molecular techniques for the
identification of Escherichia coli 0157:H7, Salmonella spp. & Listeria spp. Journal of
Microbiological Methods, 101, 33–43.
López, A., Javier, G,A., Fenoll, J., Hellín, P., Flores, P. (2014).Chemical composition
and antioxidant capacity of lettuce: Comparative study of regular-sized (Romaine) and
baby-sized (Little Gem and Mini Romaine) types. Journal of Food Composition and
Analysis, 33, 39–48.
López-Gálvez F., Allende A., Selma M., Gil M. (2009). Prevention of Escherichia coli
cross-contamination by different commercial sanitizers during washing of fresh-cut
lettuce. International Journal of Food Microbiology, 133, 167–171.
López-Malo, A., Palou, E. (2005). Ultraviolet light and food preservation. Novel Food
Processing Technologies, CRC, Press, Boca Raton, F.F. 405-421.
Lotierzo, M., Féliers, C., Faure, N., Le Grand, L., Saby, S., Cervantes, P. (2005).
Synergistic effects of sequential treatment by UV irradiation and chemical disinfectant
for drinking water disinfection.
Lotito, S.B., Fraga, C.G. (1998). Catechin prevents human plasma oxidation. Free
Radical Biology and Medicine, 24, 435–41.
Lou, F.F., Lou, H., Neetoo, H.Q., Chen, J.R. Li. (2011). Inactivation of a human
norovirus surrogate by high-pressure processing: effectiveness mechanism, and potential
application in the fresh produce industry. Applied and Environment Microbiology, 77,
1862–1871.
Luksiene, Z., Gudelis, V., Buchovec, I., Raudeliuniene, J. (2007). Advanced highpower pulsed light device to decontaminate food from pathogens: effects on Salmonella
typhimurium viability in vitro. Journal of Applied Microbiology, 103, 1545–1552.
Page 278
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Luksiene, Z., Buchovec, I., Kairyte, K., Paskeviciute, E., Viskelis, P. (2012). Highpower light for microbial decontamination of some fruits and vegetables with different
surfaces. Journal of Food, Agriculture & Environment, 10 (3&4), 162–167.
Maas, J.L., Galletta, G.J. (1991). Ellagic acid, an anticarcinogen in fruits, especially in
strawberries: A review. HortScience, 26(1), 10–14.
Maclean, M., MacGregor, S.J., Anderson, J.G., Woolsey, G.A. (2009). Inactivation of
bacterial pathogens following exposure to light from a 405-nm LED array. Applied and
Environmental Microbiology, 75, 1932-1937.
Maclean, M., MacGregor, S.J., Anderson, J.G., Woolsey, G.A. (2008a). Highintensity
narrow-spectrum light inactivation and wavelength sensitivity of Staphylococcus aureus.
FEMS Microbiology Letters, 285, 227-232.
Maclean, M., MacGregor, S.J., Anderson, J.G., Woolsey, G.A. (2008b). “The role of
oxygen in the visible-light inactivation of Staphylococcus aureus,” Journal of
Photochemistry and Photobiology B, vol. 92, no. 3, pp. 180–184.
Maclean, M., MacGregor, S.J., Anderson, J.G., Woolsey, G.A., Coia, J.E., Hamilton, K.,
Taggar, I., Watson, S.B., Thakker, B., Gettinby, G. (2010). Environmental
decontamination of a hospital isolation room using high-intensity narrow-spectrum light.
Journal of Hospital Infection, 76, 247-251.
Mahmoud, B.S.M. (2010). Effects of X-ray irradiation on Escherichia coli O157:H7,
Listeria monocytogenes, Salmonella enterica and Shigella flexneri inoculated on
shredded iceberg lettuce. Food Microbiology, 27, 109–114.
Mahmoud, B.S.M., Bachman, G., Linton, R.H. (2010). Inactivation of Escherichia coli
O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on spinach
leaves by X-ray. Food Microbiology 27, 24–28.
Mahon, B.E., Ponka, A., Hall, W.N., Komatsu, K., Dietrich, S.E., Siitonen, A., Cage, G.,
Hayes, P.S., Lambert-Fair, M.A., Bean, N.H., Griffin, P.M., Slutsker, L. (1997). An
international outbreak of Salmonella infections caused by alfalfa sprouts grown from
contaminated seeds. Journal of Infectious Diseases, 175(4), 876-882.
Maillard, J.Y. (2002). Bacterial target site for biocide action. Journal of Applied
Microbiology, 92, 16S-27S.
Mangiapane, H., Thomson, J., Salter, A., Brown, S., Bell, G.D., White, D.A. (1992). The
inhibition of oxidation of low density lipoprotein by catechin, a naturally occurring
flavonoid. Biochemical Pharmacology, 43,445–50.
Mara, D., Sleigh, A. (2010). Estimation of norovirus infection risks to consumers of
wastewater-irrigated food crops eaten raw. Journal of Water Health, 8, 39–43.
Martin-Diana, A.B., Rico, D., Barry-Ryan, C., Frias, J.M., Mulcahy, J., Henehan,
G.T.M. (2005). Calcium lactate washing treatments for salad-cut Iceberg lettuce: effect
of temperature and concentration on quality retention parameters. Food Research
International, 38, 729–740.
Page 279
References
Martín-Diana, A.B., Rico, D., Barry-Ryan, C., Frías, J.M., Henehan, G.T.M., Barat,
J.M. (2007). Efficacy of steamer jet-injection as alternative to chlorine in fresh-cut
lettuce. Postharvest Biology and Technology, 45, 97–107.
Martín-Diana, A.B., Rico, D., Barry-Ryan, C. (2008). Green tea extract as a natural
antioxidant to extend the shelf-life of fresh-cut lettuce. Innovative Food Science and
Emerging Technologies, 9, 593–603.
Marquenie, D., Geeraerd, A.H., Lammertyn J., et al. (2003). Combinations of pulsed
white light and UV-C or mild heat treatment to inactivate conidia of Botrytis cinerea and
Monilia fructigena. International Journal of Food Microbiology, 85, 1-2, 185–196.
Mason, T.J., Lorimer, J.P. (2002). Applied Sonochemistry. The Uses of Power
Ultrasound in Chemistry and Processing. Wiley VCH, Weinheim.
Mattingly, J.A., Butman, B.T., Plank, M.C., Durham, R.J., Robison, B.J. (1988). Rapid
monoclonal antibody-based enzyme-linked immunosorbent-assay for detection of
Listeria in food-products. Journal of the Association of Official Analytical Chemists,
71(3), 679-681.
Mattison, K., Karthikeyan, K., Abebe, M., Malik, N., Sattar, S.A., Farber, J.M.,
Bidawid, S. (2007). Survival of calicivirus in foods and on surfaces: Experiments with
feline calicivirus as a surrogate for norovirus. Journal Food Protection, 70, 500–503.
Mattison, K., Harlow, J., Morton, V., Cook, A., Pollari, F., Bidawid, S., Farber, J.M.
(2010). Enteric viruses in ready-to-eat packaged leafy greens. Emerging Infectious
Diseases, 16, 1815–1817.
Maunula, L., Roivainen, M., Keranen, M., Makela, S., Soderberg, K., Summa, M., von
Bonsdorff, C.H., Lappalainen, M., Korhonen, T., Kuusi, M., and Niskanen, T. (2009).
Detection of human norovirus from frozen raspberries in a cluster of gastroenteritis
outbreaks. European Surveillance, 14.
Maynard, M., Gunnell, D., Emmett, P., Frankel, S., Smith, G.D. (2003). Fruit,
vegetables, and antioxidants in childhood and risk of adult cancer: the Boyd Orr cohort.
Journal of Epidemics and Community Health, 57, 218–225.
Mbithi, J.N., Springthorpe, S., Sattar, S.A. (1991). Effect of relative humidity and air
temperature on survival of hepatitis A virus on environmental surfaces. Applied
Environmental Microbiology, 57, 1394– 1399.
McClements, D.J. (1995). Advances in the application of ultrasound in food analysis and
processing. Trends in Food Science and Technology, 6, 293–299.
McDonnel, G. and Russell, A.D. (1999). Antiseptics and disinfectants: activity, action,
and resistance. Clinical Microbial Reviews, 12, 147-179.
McKellar, R.C., Delaquis, P. (2011). Development of a dynamic growth–death model
for Escherichia coli O157:H7 in minimally processed leafy green vegetables.
International Journal of Food Microbiology, 151, 7–14.
Page 280
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Mclauchlin, J. (1996). The relationship between Listeria and listeriosis. Food Control, 7,
187-193.
McMillan, N.S., Martin, S., Sobsey, M.D., Wait, D.A., Meriwether, R.A., MacCormack,
J.N. (1992) Outbreak of pharyngo-conjunctival fever at a summer camp – North
Carolina, 1991. MMWR 41, 342–347.
Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C., Griffin, P.
M., Tauxe, R.V. (1999). Food-related illness and death in the United States. Emerging
Infectious Diseases, 5, 607-625.
Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S.Y. (2006). Plant stomata
function in innate immunity against bacterial invasion. Cell, 126, 969-980.
Meng, Q.S., Gerba, C.P. (1996). Comparative inactivation of enteric adenoviruses,
poliovirus,and coliphage by ultraviolet irradiation. Water Research, 30, 2665–8.
Meyers, K.J., Watkins, C.B., Pritts, M.P., Liu, R.H. (2003). Antioxidant and
antiproliferative activities of strawberries. Journal of Agricultural and Food Chemistry,
51, 6887–6892.
Miguel-Pintado, C., Nogales, S., Fernández-León, A.M., Delgado-Adámez, J.,
Hernández, T., Lozano, M., Cañada-Cañada, F., Ramírez, R. (2013). Effect of
hydrostatic high pressure processing on nectarine halves pretreated with ascorbic acid
and calcium during refrigerated storage. LWT - Food Science and Technology, 54, 278284.
Miché, L., Balandreau, J. (2001). Effects of rice seed surface sterilization with
hypochlorite on inoculated Burkholderia vietnamiensis. Appl.Environ Microbiol 67,
3046-3052.
Michino, H., Araki, K., Minami, S., Takaya, S., Sakai, N., Miyazaki, M., Ono, A.,
Yanagawa, H. (1999). Massive outbreak of Escherichia coli O157:H7 infection in
schoolchildren in Sakai City, Japan, associated with consumption of white radish
sprouts. Americal Journal of Epidemiology, 150, 787-796.
Misra, N.N., Tiwari, B.K., Raghavarao, K.S.M.S.,Cullen, P.J. (2011). Non-thermal
plasma inactivation of food-borne pathogens. Food Engineering Reviews, 3(3-4), 159170.
Mitchell, A.,Hong, Y-J., Koh, E., Barrett, D., Bryant, D.E., Denison, R.F., Kafka, S.
(2007). Ten-Year Comparison of the Influence of Organic and Conventional Crop
Management Practices on the Content of Flavonoids in Tomatoes. Journal of Agriculture
and Food Chemistry, 55, 6154−6159.
Mohd. Adzahan, N., Benchamaporn, P. (2007). Potential of non-thermal processing for
food preservation in southeast asian countries. ASEAN Food Journal, 14(3), 141-152.
Møller, C.O., Ilg, Y., Aabo, S., Christensen, B.B., Dalgaard, P., Hansen, T.B. (2013).
Effect of natural microbiota on growth of Salmonella spp. in fresh pork—A predictive
microbiology approach. Food Microbiology, 34(2), 284–295.
Page 281
References
Montero, T.M., Molla, E.M., Esteban, R.M., Lopezandreu, F.J. (1996). Quality attributes
of strawberry during ripening. Scientia Horticulturae, 65(4), 239-250.
Moreno, Y., Ballesteros, L., García-Hernández, J., Santiago, P., González, A., Ferrús,
M.A. (2011). Specific detection of viable Listeria monocytogenes in spanish wastewater
treatment plants by fluorescent in situ hybridization and PCR. Water Research, 45,
4634–4640.
Morgan, R. (1989). UV ‘green’ light disinfection. Dairy Industries International, 54(11),
33–35.
Mou, B. (2009). Nutrient content of lettuce and its improvement. Current Nutrition and
Food Science, 5, 242–248.
Mukhopadhyaya, S., Ukuku, D.O., Juneja, V., Fan, X. (2014). Effects of UV-C
treatment on inactivation of Salmonella enterica and Escherichia coli O157:H7 on grape
tomato surface and stem scars, microbial loads, and quality. Food Control, 44, 110-117.
Müller. (2012). Genetic and phenotypic characteristics of importance for clonal success
and diversity in Salmonella. Thesis DTU.
Murchie, L.W., Cruz-Romero, M., Kerry, J.P., Linton, M., Patterson, M.F., Smiddy,
M.A., et al. (2005). High pressure processing of shellfish: a review of microbiological
and other quality aspects. Innovative Food Science & Emerging Technologies, 6, 257270.
Murdoch, L.E., MacLean, M., MacGregor, S.J., Anderson, J.G. (2010). Inactivation of
Campylobacter jejuni by exposure to high-intensity 405-nm visible light. Foodborne
Pathogens and Disease, 7, 10, 1211–1216.
Murdoch, L.E., MacLean, M., Endarko, E., MacGregor, S.J., Anderson, J.G. (2012).
Bactericidal effects of 405 nm light exposure demonstrated by inactivation of
Escherichia, Salmonella, Shigella, Listeria, and Mycobacterium species in liquid
suspensions and on exposed surfaces. TheScientificWorld Journal, vol. 2012, Article ID
137805, 8 pages.
Murdoch, L.E., McKenzie, K., Maclean, M., Macgregor, S.J., and Anderson, J.G.K.
(2013). Lethal effects of high-intensity violet 405- nm light on Saccharomyces
cerevisiae, Candida albicans, and on dormant and germinating spores of Aspergillus
niger. Fungal Biology, 117, 519–527.
Muňoz, A., Caminiti, I.M., Palgan, I., et al. (2012). Effects on Escherichia coli
inactivation and quality attributes in apple juice treated by combinations of pulsed light
and thermosonication. Food Research International, 45, 1, 299–305.
Nastou, A., Rhoades, J., Smirniotis, P., Makri, I., Kontominas, M., Likotrafiti, E.
(2012). Efficacy of household washing treatments for the control of Listeria
monocytogenes on salad vegetables. International Journal of Food Microbiology, 159,
247-253.
Nathan, R. (1997). American seeds suspected in Japanese food poisoning epidemic.
Journal of Natural Medicines, 7, 705-706.
Page 282
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Nauta, M.J. (2002). Modelling bacterial growth in quantitative microbiological risk
assessment: is it possible? International Journal of Food Microbiology, 73, 297– 304.
Nicolle, C., Cardinault, N., Gueux, E., Jaffrelo, L., Rock, E., Mazur, A., Amouroux, P.,
Rémésy, C. (2004). Health effect of vegetable-based diet: lettuce consumption improves
cholesterol metabolism and antioxidant status in the rat. Clinical Nutrition, 23, 605–614.
Nicorescu, I., Nguyen, B., Moreau-Ferret, M., Agoulon, A., Chevalier, S., and Orange,
N. (2013). Pulsed light inactivation of Bacillus subtilis vegetative cells in suspensions
and spices. Food Control, 31, 151–157.
Noah, A., Truswell, A.S. (2001). There are many Mediterranean diets. Asia Pacific
Journal of Clinical Nutrition, 10, 1:2-9.doi:10.1046/j.1440-6047.2001.00198.x.
Noci, F., Walkling-Ribeiro, M., Cronin, D.A., Morgan, D.J., Lyng, J.G. (2009). Effect of
thermosonication, pulsed electric field and their combination on inactivation of Listeria
innocua in milk. International Dairy Journal, 19(1), 30-35.
Noguchi, N., Tamura, M., Narui, K., Wakasugi, K., Sasatsu, M. (2002). Frequency and
genetic characterization of multidrug-resistant mutants of Staphylococcus aureus after
selection with individual antiseptics and fluoroquinolones. Biological and
Pharmaceutical Bulletin, 25, 1129-1132.
Nolan, J. (1997). A conceptual model for an intelligent fuzzy decision support system.
Heuristics, 10, 31-43.
Normanno, G., Firinu, A., Virgilio, S., Mula, G., Dambrosio, A., Poggiu, A., Decastelli,
L., Mioni, R., Scuota, S., Bolzoni, G., Di Giannatale, E., Salinetti, A.P., La Salandra, G.,
Batoli, M., Zuccon, F., Pirino, T., Sias, S., Parisi, A., Quaglia, N.C., Celano, G.V.
(2005). Coagulase-positive staphylococci and Staphylococcus aureus in food products
marketed in Italy. International Journal of Food Microbiology, 98, 73–79.
Normanno, G., La Salandra, G., Dambrosio, A., Quaglia, N.C., Corrente, M., Parisi, A.,
Santagada, G., Firinu, A., Crisetti, E., Celano, G.V. (2007). Occurrence, characterization
and antimicrobial resistance of enterotoxigenic Staphylococcus aureus isolated from
meat and dairy products. International Journal of Food Microbiology, 115, 290–296.
Nou, X., Luo, Y., Hollar, L., Yang, Y., Feng, H., Millner, P. (2011). Chlorine stabilizer
T-128 enhances efficacy of chlorine against cross-contamination by E. coli O157:H7 and
Salmonella in fresh-cut lettuce processing. Journal of Food Science, 76, 218-224.
Nuanualsuwan, S., Cliver, D. (2003). Infectivity of RNA from inactivated poliovirus.
Applied Environmental Microbiology, 69 (3), 1629− 1632.
Nuanualsuwan, S., Cliver, D. (2003a). Capsid functions of inactivated human
picornaviruses and feline calicivirus. Applied and Environmental Microbiology, 69,
350–357.
Nuñez-Mancilla, Y., Pérez-Won, M., Uribe, E., Vega-Gálvez, A., Di Scala, K. (2013).
Osmotic dehydration under high hydrostatic pressure: Effects on antioxidant activity,
total phenolics compounds, vitamin C and colour of strawberry (Fragaria vesca). LWT Food Science and Technology, 52, 151-156.
Page 283
References
Nwachuku, N., Gerba, C.P., Oswald, A., Mashadi, F. (2005). Comparative inactivation
of adenovirus serotype by UV light disinfection. Applied Environmental Microbiology,
71, 5633–6.
Obande, M., Tucker, G., Shama, G. (2011). Effect of preharvest UV-C treatment of
tomatoes (Solanum lycopersicon Mill.) on ripening and pathogen resistance. Postharvest
Biology and Technology, 62, 188–192.
Odriozola-Serrano, Isabel, Hernandez-Jover, Teresa, Martın-Belloso, Olga. (2007).
Comparative evaluation of UV-HPLC methods and reducing agents to determine
vitamin C in fruits. Food Chemistry, 105, 1151–1158.
Odriozola-Serrano, I., Soliva-Fortuny, R., Martín-Belloso, O. (2010). Changes in
bioactive composition of fresh-cut strawberries stored under superatmospheric oxygen,
low-oxygen or passive atmospheres. Journal of Food Composition and Analysis, 23 (1),
37–43.
Oh, S.K., Lee, N., Cho, y.S., Shin, D.B., Choi, S,y., Koo, M. (2007). Occurrence of
toxigenic Staphylococcus aureus in ready-to-eat food in Korea. Journal of Food
Protection, 70, 1153-1158.
Olaimat, A.N., and Holley, R.A. (2012). Factors influencing the microbial safety of fresh
produce: A review. Food Microbiology, 32(1), 1–19.
Olasupo, N.A., Fitzgerald, D.J., Narbad, A., Gasson, M.J. (2004). Inhibition of Bacillus
subtilis and Listeria innocua by nisin in combination with some naturally occurring
organic compounds. Journal of Food Protection, 67(3), 596-600.
Olivas, G.I., Barbosa-Canovas, G.V. (2005). Edible coatings of fresh-cut fruits. Critical
Reviews in Food Science and Nutrition, 45, 657-670.
Oliveira, M.A., Abeid Ribeiro, E.G., Morato Bergamini, A.M., Pereira De Martinis, E.C.
(2010). Quantification of Listeria monocytogenes in minimally processed leafy
vegetables using a combined method based on enrichment and 16S rRNA realtime PCR.
Food Microbiology, 27, 19–23.
Oliveira, M., Usall, J., Solsona, C., Alegre, I., Viñas, I., Abadias, M. (2011). Transfer of
Listeria innocua from contaminated compost and irrigation water to lettuce leaves. Food
Microbiology, 28, 590–596.
Ölmez, H., Kretzschmar, U. (2009). Potential alternative disinfection methods for
organic fresh-cut industry for minimizing water consumption and environmental impact,
a review. LWT - Food Science and Technology, 42, 686–693.
Ölmez, H., Temur, S.D. (2010). Effects of different sanitizing treatments on biofilms
and attachment of Escherichia coli and Listeria monocytogenes on green leaf lettuce.
LWT— Food Science and Technology, 43, 964–970.
Oms-Oliu, G., Martin-Belloso, O., Soliva-Fortuny, R. (2010). Pulsed light treatments for
food preservation. A review. Food Bioprocess Technology, 3, 13-23.
Oscar, T. P. (2011). Plenary lecture: innovative modeling approaches applicable to risk
assessments. Food Microbiology, 28, 777-781.
Page 284
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Otto, C., Zahn, S., Rost, F., Zahn, P., Jaros, D., Rohm, H. (2011). Physical methods for
cleaning and disinfection of surfaces. Food Engineering Reviews, 3, 171–188.
Ozgen, S. and Sekerci, S. (2011). Effect of leaf position on the distribution of
phytochemicals and antioxidant capacity among green and red lettuce cultivars.
Spanish Journal of Agricultural Research, 9, 801-809.
Pagan, R., Manas, P., Alvarez, I., Condon, S. (1999). Resistance of Listeria
monocytogenes to ultrasonic waves under pressure at sublethal (manosonication) and
lethal (manothermosonication) temperatures. Food Microbiology, 16, 139– 148.
Page M. (2003). Strategies for integrated control of biological agents by small utilities.
Master Thesis.
Papadimitriou, V., Sotiroudis, T., Xenakis, A., Sofikiti, N., V. Stavyiannoudaki, V.,
Chaniotakis, N. (2005). Oxidative stability and radical scavenging activity of extra
virgin olive oils: An electron paramagnetic resonance spectroscopy study. Analytica
Chimica Acta, 573, 453-458.
Parikh, Priti (2007). Efficacy of Ultraviolet Light and Antimicrobials to Reduce Listeria
monocytogenes in Chill Brines. phD Thesis. Blacksburg, Virginia
Park, E.J., Alexander, E., Taylor, G.A., Costa, R., Kang, D.H. (2008). Fate of foodborne
pathogens on green onions and tomatoes by electrolysed water. Letters in Applied
Microbiology, 46, 519-525.
Park, H.S, Park, J.Y., Yu, R. (2005). Relationship of obesity and visceral adiposity with
serum concentrations of CRP, TNF- and IL-6. Diabetes Research of Clinical Practice,
69, 29 –35.
Patel, J., Sharma, M. (2010). Differences in attachment of Salmonella enterica serovars
to cabbage and lettuce leaves. International Journal of Food Microbiology, 139(1–2),
41–47.
Patil, S. (2010). Efficacy of Ozone and Ultrasound for Microbial Reduction in Fruit
Juice. Doctoral Thesis. Dublin Institute of Technology.
Patterson, M.F. (2005). Microbiology of pressure-treated foods. Journal of Applied
Microbiology, 98, 1400-1409.
Patras, A., Brunton, N.B., Da Pieve, S., Butler, F. (2009). Impact of high pressure
processing on total antioxidant activity, phenolic, ascorbic acid, anthocyanin content and
colour of strawberry and blackberry purées. Innovative Food Science and Emerging
Technologies, 10, 308–313.
Pecson, B.M., Martin, L.V., Kohn, T. (2009). Quantitative PCR for determining the
infectivity of bacteriophage MS2 upon inactivation by heat, UV-B radiation, and singlet
oxygen: Advantages and limitations of an enzymatic treatment to reduce false-positive
results. Applied Environmental Microbiology, 75 (17), 5544−5554.
Pelayo-Zaldívar, C., Ebeler, S.E., Kader, A.A. (2005). Cultivar and harvest date effects
on flavor and other quality attributes of California strawberries. Journal of food quality,
28, 78-97.
Page 285
References
Peles, F., Wagner, M., Varga, L., Hein, I., Rieck, P., Gutser, K., Keresztúri, P., Kardos,
G., Turcsányi, I., Bér, B.,Szabóa, A. (2007). Characterization of Staphylococcus aureus
strains isolated from bovine milk in Hungary. International Journal of Food
Microbiology, 118, 186–193.
Pereira, C.I., Graça, J.A., Ogando, N.S., Gomes, A.M.P and Xavier, Malcata, F. (2009)
Bacterial dynamics in model cheese systems, aiming at safety and quality of
portuguese-style traditional ewe's cheeses. Journal of Food Protection, 72, 2243-2251.
Pereira, V.G., Marques, R., Marques, M., Benoliel, M.J., Barreto Crespo, M.T. (2013).
Free chlorine inactivation of fungi in drinking water sources. Water Research, 47, Issue
2, 517-523.
Pereira, R.N., Vicente, A.A (2010). Environmental impact of novel thermal and nonthermal technologies in food processing. Food Research International, 43, 1936–1943.
Pérez-Rodríguez, F., Posada-Izquierdo, G.D., Valero, A., García-Gimeno, R.M., Zurera,
G. (2013). Modelling survival kinetics of Staphylococcus aureus and Escherichia coli
O157:H7 on stainless steel surfaces soiled with different substrates under static
conditions of temperature and relative humidity. Food Microbiology, 33, 197-204.
Perkins-Veazie, P., Collins, J.K., Howard, L. (2008). Blueberry fruit response
topostharvest application of ultraviolet radiation. Postharvest Biology and Technology,
47,280–285.
Perrot, N., Agioux, L., Ioannou, I., Mauris, G., Corrieu, G., Trystram, G. (2004).
Decision support system design using the operator skill to control cheese ripeningapplication of the fuzzy symbolic approach. Journal of Food Engineering, 64, 321–333.
PHAC, (2011). Staphylococcus aureus. http://www.phac-aspc.gc.ca/lab-bio/res/psdsftss/staphylococcus-aureus-eng.php
PHAC, (2001). Adenovirus types 1, 2, 3, 4, 5 and 7 - Material Safety Data Sheets
(MSDS). Infectious Substances.
Piironen, V., Lindsay, D.G., Miettinen, T.A., Toivo, J., Lampi, A.M. (2000). Plant
sterols: biosynthesis, biological function and their importance to human nutrition.
Journal of Science and Food Agriculture, 80, 939–966.
Piyasena, P., Mohareb, E., McKellar, R.C. (2003). Inactivation of microbes using
ultrasound: a review. International Journal of Food Microbiology, 87, 207– 216.
Plaza, L., Sánchez-Moreno, C., De Ancos, B., Cano, M.P. (2006a). Carotenoid content
and antioxidant capacity of Mediterranean vegetable soup (gazpacho) treated by highpressure/temperature during refrigerated storage. European Food Research Technology,
223, 210–215.
Plaza, L., Sánchez-Moreno, C., Elez,-Martínez, P., De Ancos, B., Martín-Belloso, O.,
Cano, M.P. (2006b). Effect of refrigerated storage on vitamin C and antioxidant activity
of orange juice processed by high-pressure or pulsed electric field with regard to low
pasteurization. European Food Research Technology, 223, 487–493.
Page 286
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Ponce, A.G., Del Valle, C.E., Rural, S.I. (2004). Natural essential oils as reducing agents
of peroxidase activity in leafy vegetables. Food Science and Technology, 37(2), 199–
204.
Posada-Izquierdo, G.D, Pérez-Rodríguez, F., López-Gálvez, F., Allende, A., Selma,
M.V. Gil, M.I., Zurera, G. (2013). Modelling growth of Escherichia coli O157:H7 in
fresh-cut lettuce submitted to commercial process conditions: Chlorine washing and
modified atmosphere packaging. Food Microbiology, 33, 131-138.
Poysky, F.T., Paranjpye, R.N., Lashbrook, L.C., Peterson, M.E., Pelroy, G.A., Eklund,
M.W. (1993). Selective and differential medium for isolation of Listeria monocytogenes
from foods. Journal of Food Protection, 56(4), 326-329.
Prior, R.L., Cao, G. (2000). Antioxidant phytochemicals in fruits and vegetables: diet
and health implications. Hortscience, 35, 588–592.
Prior, R.L., Wu, X., Schaich, K. (2005). Standardized methods for the determination of
antioxidant capacity and phenolics in foods and dietary supplements. Journal of
Agriculture and Food Chemistry, 53, 4290–4302.
Proteggente, A.R., Pannala, A.S., Paganga, G., Van Buren, L., Wagner, E., Wiseman, S.,
et al. (2002). The antioxidant activity of regularly consumed fruit and vegetables reflects
their phenolic and vitamin C composition. Free Radical Research, 36(2), 217–233.
Quirós, A.R., Fernández-Arias, M., López-Hernández, J. (2009). A screening method for
the determination of ascorbic acid in fruit juices and soft drinks. Food Chemistry, 116,
509–512.
Raffo, A.,La Malfa, G., Fogliano, F., Maiani, G., Quaglia, G. (2006). Seasonal variations
in antioxidant components of cherry tomatoes (Lycopersicon esculentum cv. Naomi F1).
Journal of Food Composition and Analysis, 19, 11–19.
Ragaert, P., Verbeke, W., Devlieghere, F., Debevere, J. (2004). Consumer perception
and choice of minimally processed vegetables and packaged fruits. Food Quality and
Preference, 15(3), 259–270.
Rajkovic, A., Smigic, N., Devlieghere, F. (2010). Contemporary strategies in combating
microbial contamination in food chain. International Journal of Food Microbiology, 141,
S29–S42.
Rall, V.L.M., Vieira, F.P., Rall, R., Vietis, R.L., Fernandes, A., Candeias, J.M.G.,
Cardoso, K.F.G, Araujo, J.P. (2008) PCR detection of staphylococcal enterotoxin genes
in Staphylococcus aureus strains isolated from raw and pasteurized milk. Veterinary
Microbiology, 132, 408-413.
Ramos, B., Miller, F.A., Brandão, T.R.S. Teixeira, P. Silva, C.L.M. (2013). Fresh fruits
and vegetables. An overview on applied methodologies to improve its quality and safety.
Innovative Food Science and Emerging Technologies, 20, 1–15.
Ramos-Villarroel, A.Y., Martin-Belloso, O., Soliva-Fortuny, R. (2011). Bacterial
inactivation and quality changes in fresh-cut avocado treated with intense light pulses.
European Food Research Technology, 233, 395-402.
Page 287
References
Rangel, J.M., Sparling, P.H., Crowe, C., Griffin, P.M., Swerdlow, D.L. (2005)
Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982-2002.
Emerging Infectious Diseases, 11, 603-609.
RASSF.
http://ec.europa.eu/food/food/rapidalert/docs/rasff_annual_report_2010_en.pdf
(2010).
Ratkowsky, D.A., Olley, J., McMeekin, T.A., Ball, A., (1982). Relationship between
temperature and growth rate of bacterial cultures. Journal of Bacteriology, 149, 1 – 5.
Reboul, E., Borel, P., Mikail, C., Abou, L., Charbonnier, M., Caris-Veyrat, C., et al.
(2005). Enrichment of tomato paste with 6% tomato peel increases lycopene and betacarotene bioavailability in men. The Journal of Nutrition, 135, 790-794.
Reid, T.M.S., Robinson, H.G. (1987). Frozen raspberries and hepatitis. Americal
Epidemiology Infections, 98, 109-112.
Rekika, D., Khanizadeh, S., Deschenes, M. et al. (2005). Antioxidant capacity and
phenolic content of selected strawberry genotypes. HortScience, 40, 1777–1781.
Rennecker, J.L., Driedger, A.M., Rubin, S.A., Mariñas, B.J. (2000). Synergy in
sequential inactivation of Cryptosporidium parvum with ozone/free chlorine and
ozone/monochloramine. Water Research, 34 (17), 4121-4130.
Reyes, L.F., Villareal. J.E.Cisneros-Zevallos, L. (2007). The increase in antioxidant
capacity after wounding depends on the type of fruit or vegetable tissue. Food
Chemistry, 101, 1254-1262.
Rice, R.P., Rice, L.W., Tindall, H.D. (1987). Fruit and Vegetable Production in Africa.
Macmillan Publishers, U.K. 371.
Rice-Evans, C.A., Miller, N.J., Bolwell, P.G., Bramley, P.M., Pridham, J.B. (1995). The
relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radical
Research, 22, 4, 375.
Rice-Evans, C.A., Miller., N.J., Paganga, G. (1996). Structure-antioxidant activity
relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20
(7), 933-56.
Rico, D, Martin-Diana, A.B., Barat, J.M., Barry-Ryan, C. (2007). Extending and
measuring the quality of fresh-cut fruit and vegetables: A review. Trends in Food
Science & Technology, 18, 373-386.
Rimm, E.B., Kata,n M.B., Ascherio, A., Stampfer, M.J., Willett, W.C. (1996). Relation
between intake of flavonoids and risk for coronary heart disease in male health
professionals. Annual International Medicine, 125, 384–9.
Rivas, L., Fegan, N., Dykes, G.A. (2005). Physicochemical properties of Shiga toxigenic
Escherichia coli. Journal of Applied Microbiology, 99 (4), 716–727.
Robbins, R.J. (2003). Phenolic acids in foods: an overview of analytical methodology.
Journal of Agriculture and Food Chemistry, 51, 2866–2887.
Page 288
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Roberts, P., Hope, A. (2003). Virus inactivation by high intensity broad spectrum pulsed
light. Journal of Virological Methods, 110, 61–65.
Rodgers, S.L., Ryser, E.T. (2004). Reduction of microbial pathogens during apple cider
production using sodium hypochlorite, copper ion, and sonication. Journal of Food
Protection, 67(4), 766-771.
Rodrigo, D., Loey, A.V., Hendrickx, M. (2007). Combined thermal and high pressure
color degradation of tomato puree and strawberry juice. Journal of Food Engineering,
79, 553–560.
Romanazzi, G., Feliziani, E., Santini, M., Landi, L. (2013). Effectiveness of postharvest
treatment with chitosan and other resistance inducers in the control of storage decay of
strawberry. Postharvest Biology and Technology, 75, 24-27.
Rosenblum, L.S., Mirkin, I.R., Allen, D.T., Safford, S., Hadler, S.C. (1990). A
multifocal outbreak of hepatitis A traced to commercially distributed lettuce. American
Journal of Public Health, 80, 1075–1079.
Ross, A.V.I., Griffiths, M.W., Mittal, G.S., Deeth, H.C. (2003). Review: Combining
nonthermal technogies to control foodborne microorganisms. International Journal of
Food Microbiology, 89 (2-3), 125-138.
Ruiz-Cruz, S., Acedo-Felix, E., Diaz-cinco, M., Islas-Osuna, M.A., Gonzalez- Aguilar,
G.A. (2007). Efficacy of sanitizers in reducing Escherichia coli O157:H7, Salmonella
spp. and Listeria monocytogenes populations on fresh-cut carrots. Food Control, 18,
1383–1390.
Russell, A.D. (1983). Mechanism of action of chemical sporicidal and sporistatic agents.
International Journal of Pharmaceutics. 16, 127-140.
Russell, W.C. (2005). Adenoviruses. In Topley and Wilson's Microbiology and
Microbial Infections, 9th edn, pp. 439–447. Edited by B. W. J. Mahy & V. ter Meulen.
London: Hodder Arnold.
Rux, J.R., Burnett, R.M. (1999). Adenovirus capsid proteins. In Adenoviruses: Basic
Biology to Gene Therapy. edited by Prem Seth. Austin, TX: R.G.Landes Company, 516.
Sakr, M., Liu, S. (2014). A comprehensive review on applications of ohmic heating
(OH). Renewable and Sustainable Energy Reviews 39, 262–269.
Salgado, S.P., Pearlstein, A.J., Luo, Y., Feng, H. (2014). Quality of Iceberg (Lactuca
sativa L.) and Romaine (L. sativa L. var. longifolial) lettuce treated by combinations of
sanitizer, surfactant, and ultrasound. LWT - Food Science and Technology, 56, 261-268.
Saltveit, M.E. (2003). Fresh-cut vegetables, in: Bartz, J.A., Brecht, J.K. (Eds.),
Postharvest Physiology and Pathology of Vegetables, second ed., Revised and
Expanded. Marcel Dekker Inc., New York, pp. 755-779.
Sagong, H.G., Lee, S.Y., Chang, P.S., Heu, S., Ryu, S. (2011). Combined effect of
ultrasound and organic acids to reduce Escherichia coli O157:H7, Salmonella
Typhimurium and Listeria monocytogenes on organic fresh lettuce. International Journal
of Food Microbiology, 145, 287–292.
Page 289
References
Sánchez-Mata, M.C., Cámara-Hurtado, M., Dıéz-Marqués, C., Torija- Isasa, M. E.
(2000). Comparison of high-performance liquid chromatography and spectrofluorimetry
for vitamin C analysis of green beans (Phaseolus vulgaris L.). European Food Research
and Technology, 210(3), 220–225.
Sánchez-Moreno, C. (2002). Review: Methods used to evaluate the free radical
scavenging activity in foods and biological systems. Food Science and Technology
International, 8, 121–137.
Sánchez-Moreno, C., Plaza, L., De Ancos, B., Cano, M.P. (2006a). Impact of highpressure and traditional thermal processing of tomato puree on carotenoids, vitamin C
and antioxidant activity. Journal of Science in Food Agriculture, 86, 171–179.
Sánchez-Moreno, C., Plaza, L., De Ancos, B., Cano, M.P. (2006b). Nutritional
characterization of commercial traditional pasteurised tomato juices: carotenoids,
vitamin C and radical scavenging capacity. Food Chemistry, 98, 749–756.
São José, J.F.B., Dantas Vanetti, M.C. (2012). Effect of ultrasound and commercial
sanitizers in removing natural contaminants and Salmonella enterica Typhimurium on
cherry tomatoes. Food Control, 24, 95-99.
Sapers, G.M. (2001), Efficacy of Washing and Sanitizing Methods for Disinfection of
Fresh Fruit and Vegetable Products. Food Technology and Biotechnology, 39, (4), 305311.
Sapers, G.M., Jones, D.M. (2006). Improved Sanitizing Treatments for Fresh Tomatoes.
Journal of Food Science, 71, 252-256.
Sapers, G.M., Simmons, G.F. (1998). Hydrogen peroxide disinfection of minimally
processed fruits and vegetables. Food technology, 52, 48-52.
Sastry, S. K., Datta, A. K., and Worobo, R.W. (2000). Ultraviolet light. Journal of Food
Science, Supplementary, 65, 90-92.
Sattar, S.A., Springthorpe, V.S., Tetro, J.S., Vashon, R., and Keswick, B. (2002).
Hygienic hand antiseptics: should they not have activity and label claims against
viruses? American Journal of Infection Control, 30(6), 355-372.
Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M.A., Roy, S.L.,
Jones, J.L., Griffin, P.M. (2011). Foodborne illness acquired in the United States-Major
pathogens. Emerging Infectious Disease, 17(1), 7-15.
Scannell, A. G., Schwarz, G., Hill, C., Ross, R.P., Arendt, E.K. (2001). Pre-inoculation
enrichment procedure enhances the performance of bacteriocinogenic Lactococcus lactis
meat starter culture. International Journal of Food Microbiology, 64(1-2),151-159.
Schenk, M., Guerrero, S., Alzamora, S.M. (2008). Response of some microorganisms to
ultraviolet treatment on fresh-cut pear. Food and Bioprocess Technology, 1, 384–392.
Schenk, M., Raffellini, S., Guerrero, S., Blanco, G.A., Alzamora, S.M. (2011).
Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae by UVC light: study of cell injury by flow cytometry,” LWT—Food Science and Technology,
44, 1, 191–198.
Page 290
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Scott, D.B.M., Lesher, EC. (1963). Effect of ozone on survival and permeability of
Escherichia coli. Journal of Bacteriology, 85, 567-576.
Scouten, A.J., Beuchat, L.R. (2002). Combined effects of chemical, heat and ultrasound
treatments to kill Salmonella and Escherichia coli O157:H7 on alfalfa seeds. Journal of
Applied Microbiology, 92, 668-674.
Segerman, A., Arnberg, N., Erikson, A., Lindman, K., Wadell, G. (2003a). There are
two different species B adenovirus receptors: sBAR, common to species B1 and B2
adenoviruses, and sB2AR, exclusively used by species B2 adenoviruses. Journal of
Virology, 77, 1157–1162.
Seth, P. (1999a). Entry of adenovirus into cells. In Adenoviruses: Basic Biology to Gene
Therapy. edited by Prem Seth. Austin, TX: R.G.Landes Company, 31-38.
Seymour, I.J., Burfoot, D., Smith, R.L., Cox, L.A., Lockwood, A. (2002). Ultrasound
decontamination of minimally processed fruits and vegetables. International Journal of
Food Science and Technology, 37, 547–557.
Seo, K.H., Frank, J.F. (1999). Attachment of Escherichia coli O157:H7 to lettuce leaf
surface and bacterial viability in response to chlorine treatment as demonstrated by using
confocal scanning laser microscopy. Journal of Food Protection, 62(1), 3-9.
Shaghaghi, Masoomeh.Manzoori, Jamshid L., Jouyban Abolghasem. (2008).
Determination of total phenols in tea infusions, tomato and apple juice by terbium
sensitized fluorescence method as an alternative approach to the Folin–Ciocalteu
spectrophotometric method. Food Chemistry, 108, 695–701.
Shannon, K.E., Lee, D., Trevors, J.T., Beaudette, L.A. (2007). Application of real-time
quantitative PCR for the detection of selected bacterial pathogens during municipal
wastewater treatment. Science of Total Environment, 382, 121–129.
Sharma, M., Patel, J.R., Conway, W.S., Ferguson, S., Sulakvelidze, A. (2009).
Effectiveness of bacteriophages in reducing Escherichia coli O157: H7 on fresh-cut
cantaloupes and lettuce. Journal of Food Protection, 72 (7), 1481–1485.
Shin, G.A., Lee, J.K. (2010). Inactivation of human adenovirus by sequential
disinfection with an alternative ultraviolet technology and monochloramine. Canadian
Journal of Microbiology, 56 (7), 606–609.
Simon, J.A., Hudes, E.S., Tice, J.A. (2001). Relation of serum ascorbic acid to mortality
among US adults. American Journal of Clinical Nutrition, 20, 255–263.
Sinha, R.P., Häder, D.P. (2002). UV-induced DNA damage and repair: a review.
Photochemical and Photobiology Sciences, 2002,1, 225-236.
Singh, N., Singh, R.K, Bhunia, A.K., Stroshine, R.L. (2002). Effect of inoculation and
washing methods on the efficacy of different sanitizers against Escherichia coli
O157:H7 on lettuce. Food Microbiology, 19, 183-93.
Sirikanchana, K., Shisler, J.L., Mariñas, B.J. (2008). Inactivation kinetics of adenovirus
serotype 2 with monochloramine. Water Research, 42 (6–7), 1467–1474.
Page 291
References
Skerget, M., Kotnik, P., Hadolin, M., Hras, A.R., Simonic, M., Knez, Z. (2004).
Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their
antioxidant activities. Journal of Food Chemistry, 89(2), 191-198.
Skibola, C.F., Smith, M.T. (2000). Potential health impacts of excessive flavonoid
intake. Free Radical Biology and Medicine, 29, 375–383.
Smith, S., Dunbar, M., Tucker, D. and Schaffner, D.W. (2003) Efficacy of a commercial
produce wash on bacterial contamination of lettuce in a food service setting. Journal of
Food Protection, 66, 2359-2361.
Smith, D.S. (2005). Development of a positive selection strategy to investigate the
regulation of quorum sensing in Erwinia. PhD thesis, Department of Biochemistry,
University of Cambridge.
Sokmen, M., Angelova, M., Krumova, E., Pashova, S., Ivancheva, S., Sokmen, A.,
Serkedjieva, J. (2005). In vitro antioxidant activity of polyphenols extracts with antiviral
properties from Geranium sanguineum. Life Sciences, 76(25), 2981-2993.
Solomon, E.B., Pang, H.J., Matthews, K.R. (2003). Persistence of Escherichia coli
O157:H7 on lettuce plants following spray irrigation with contaminated water. Journal
of Food Protection, 66, 2198-2202.
Soon, J.M., Singh, H., Baines, R. (2011). Foodborne diseases in Malaysia: A review.
Food Control, 22, 823-830.
Sommer, R., Pribil, W., Appelt, S., Gehringer, P., Eschweiler, H., Leth, H., Cabaj, A.,
Haider, T. (2001). Inactivation of bacteriophages inwater bymeans of non-ionizing (UV253.7 nm) and ionizing (gamma) radiation: a comparative approach. Water Research,
35, 3109–3116.
Sommers, C. Kozempel, M., Fan, X., Radewonuk, E.R. (2002). Use of vacuum-steamvacuum and ionizing radiation to eliminate Listeria innocua from ham. Journal of Food
Protection, 65(12), 1981-1983.
Spanos, G.A., Wrolstad, R.E. (1990). Influence of processing and storage on the
phenolic composition of Thompson seedless grape juice. Journal of Agricultural &
Food Chemistry, 38, 1565–1571.
Stefani, S., Chung, D.R., Lindsay, J.A., Friedrich, A.W., Kearns, A.M.,Westh,H.,
Mackenzie, F.M. (2012). Meticillin-resistant Staphylococcus aureus (MRSA): global
epidemiology and harmonisation of typing methods. Int. J. Antimicrob. Agents 39:273282.
Stevens, C., Khan, V.A., Lu, J.Y., Wilson, C.L., Pusey, P.L., Igwegbe, E.C.K., Kabwe,
M., Mafolo, Y., Liu, J., Chalutz, E., Droby, S. (1997). Integration of ultraviolet (UVC)
light with yeast treatment for control of postharvest storage rots of fruits and vegetables.
Biological Control, 10, 98–103.
Stevens, C., Khan, V.A., Lu, J.Y., Wilson, C.L., Pusey, L.P., Kabwe, M.K. (1998). The
germicidal and hormetic effects of UV-C light on reducing brown rot disease and yeast
microflora of peaches. Crop Protection, 17, 129–134.
Page 292
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Stevens, C., Khan, V.A., Lu, J.Y., Chalutz, E., Droby, S., Kabwe, M.K., Haung, Z.,
Adeyeye, O., Pusey, P.L., Tang, A.Y.A. (1999). Induced resistance of sweet potato to
Fusarium root rot by UV-C hormesis. Crop Protection, 18, 463–470.
Stevens, C., Khan, V.A., Lu, J.Y., Wilson, C.L., Pusey, P.L., Kabwe, M.K., Igwegbe,
E.C.K., Chalutz, E., Droby, S. (1998). The germicidal and hormetic effect of UV-C light
on reducing brown rot disease and yeast microflora of peaches. Crop Protection, 17(1),
75–84.
Stevens, C., Liu, J., Khan, V.A., Lu, J.Y., Kabwe, M.K., Wilson, C.L., Igwegbe, E.C.K.,
Chalutz, E., Droby, S. (2004). The effects of low-dose ultraviolet light-C treatment on
polygalacturonase activity, delay and Rhizopus soft rot development of tomatoes. Crop
Protection, 23, 551–554.
Stine, S.W., Song, I., Choi, C.Y., Gerba, C.P. (2005). Effect of relative humidity on
preharvest survival of bacterial and viral pathogens on the surface of cantaloupe, lettuce,
and bell peppers. Journal of Food Protection, 68, 1352–1358.
Strauss, J.H., Strauss, E.G. (2002). Viruses and Human Disease. San Diego. Academic
Press, a division of Harcourt, Inc.
Suslick, K.S. (1989). The chemical effects of ultrasound. Scientific American, 80–87.
Syamaladevi, R., Lu, X., Sablani, S., Insan, S. K., Adhikari, A., Killinger, K., Rasco, B.,
Dhingra, A., Bandyopadhyay, A., Annapure, U. (2012). Inactivation of Escherichia coli
Population on Fruit Surfaces Using Ultraviolet-C Light: Influence of Fruit Surface
Characteristics. Food Bioprocess Technology. DOI 10.1007/s11947-012-0989-0.
Szeto, Y.T., Tomlinson, B., Benzie, I.F. (2002). Total antioxidant and ascorbic acid
content of fresh fruits and vegetables: implications for dietary planning and food
preservation. British Journal of Nutrition, 87(1), 55-9.
Takeshita, K., Shibato, J., Sameshima T. et al. (2003). Damage of yeast cells induced by
pulsed light irradiation. International Journal of Food Microbiology, 85, 1-2, 151–158.
Takeuchi, K., Frank, J.F. (2000). Penetration of Escherichia coli O157: H7 into lettuce
tissues as affected by inoculum size and temperature and the effect of chlorine treatment
on cell viability. Journal of Food Protection, 63 (4), 434–440.
Taormina, P.J., Rocelle, M., Clavero, S., Beuchat, L.R. (1998). Comparison of selective
agar media and enrichment broths for recovering heat-stressed Escherichia coli O157:H7
from ground beef. Food Microbiology, 1998, 15, 631-638.
Taormina, P. J., Beuchat, L. R., Slutsker, L.M. (1999). Infections associated with eating
seed sprouts: An international concern. Emerging Infectious Diseases, 5(5), 626-634.
Tauxe, R.V. (2002). Surveillance and investigation of foodborne diseases; roles for
public health in meeting objectives for food safety. Food Control, 13, 363–369.
Tauxe, R.V., Doyle, M.P., Kuchenmüller, T., Schlundt, J., Stein, C.E. (2010). Evolving
public health approaches to the global challenge of foodborne infections. International
Journal of Food Microbiology, 139, 16–28.
Page 293
References
Taylor, W.J. (1965). Isolation of Shigellae. I. Xylose lysine agars: new media for
isolation of enteric pathogens. American Journal of Clinical Pathology, 44, 471-475.
Taylor, W.J., Schelhart, D. (1967). Isolation of Shigellae. IV. Comparison of plating
media with stools. American Journal of Clinical Pathology, 48, 356-362.
Temple, N.J., Gladwin, K.K. (2003). Fruit, vegetables, and the prevention of cancer:
research challenges. Nutrition, 19, 467–470.
Teopfl, S., Mathys, A., Heinz, V., Knoor, D. (2006). Review: Potential of high
hydrostatic pressure and pulsed electric fields for energy efficient and environmentally
friendly food processing. Food Reviews International, 22, 405-423.
Teunis, P.F., Moe, C.L., Liu, P., Miller, S.E., Lindesmith, L., Baric, R.S., Le Pendu, J.,
Calderon, R.L. (2008). Norwalk virus: How infectious is it? Journal of Medical
Virology, 80, 1468–1476.
Thanomsub, B., Anupunpisit, V., Chanphetch, S., Watcharachaipong, T., Poonkhum, R.,
Srisukonth, C. (2002). Effects of ozone treatment on cell growth and ultrastructural
changes in bacteria. Journal of General and Applied Microbiology, 48, 193-199.
Thomas, D.Y., Jarraud, S., Lemercier, B., Cozon, G., Echasserieau, K., Etienne, J.,
Gougeon, M.L., Lina, G., Vandenesch, F. (2006). Staphylococcal enterotoxin-like toxins
U2 and V, two new staphylococcal superantigens arising from recombination within the
enterotoxin gene cluster. Infection and Immunity, 74, 4724–4734.
Thurston-Enriquez, J.A., Haas, C.N., Jacangelo, J., Gerba, C.P. (2003a). Chlorine
inactivation of adenovirus type 40 and feline calicivirus. Applied and Environmental
Microbiology, 69 (7), 3979–3985.
Thurston-Enriquez, J.A., Haas, C.N., Jacangelo, J., Riley, K., Gerba, C.P. (2003b).
Inactivation of feline calicivirus and adenovirus type 40 by UV radiation. Applied and
Environmental Microbiology, 69 (1), 577–582.
Thurston-Enriquez, J.A., Haas, C.N., Jacangelo, J., Gerba, C.P. (2005). Inactivation of
enteric adenovirus and feline calicivirus by ozone. Water Research, 39 (15), 3650–3656.
Thurston-Enriquez, J., Haas, C., Jacangelo, J., Gerba, C. (2005). Inactivation of enteric
adenovirus and feline calicivirus by chlorine dioxide. Applied and Environmental
Microbiology, 71 (6), 3100−3105.
Tiveron, A.P., Melo, P.S., Bergamaschi, K.B., Vieira, T.M.F., Regitano-d’Arce M.A.B.,
and Alencar, S.M (2012). Antioxidant Activity of Brazilian Vegetables and Its Relation
with Phenolic Composition. International Journal of Molecular Sciences, 13, 8943-8957.
Tiwari, B.K., O’Donnell, C.P., Patras, A., Brunton, N., Cullen, P.J. (2009). Effect of
ozone processing on anthocyanins and ascorbic acid degradation of strawberry juice.
Food Chemistry, 113 (4), 1119–1126.
Todar K. (2012) (http://textbookofbacteriology.net/salmonella_4.html)
Todd, E. (1990). Epidemiology of foodborne illness: North America. The Lancet, 336
(8718), 788-790.
Page 294
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Todd, E.C.D., Notermans, S. (2011). Surveillance of listeriosis and its causative
pathogen, Listeria monocytogenes. Food Control, 22, 1484-1490.
Todoriki S., Bari L., Kitta K., Ohba M., Ito Y., Tsujimoto Y., Kanamori N., Yano E.,
Moriyama T., Kawamura Y., Kawamoto S. (2009). Effect of gamma-irradiation on the
survival of Listeria monocytogenes and allergenicity of cherry tomatoes. Radiation
Physics and Chemistry ,78, 619–621.
Toole, G.A., Parker, M.L., Smith, A.C., Waldron, K.W. (2000). Mechanical roperties of
lettuce. Journal of Material Science, 35, 3553–3559.
Toor, R.K., Savage, G.P. (2005). Antioxidant activity in different fractions of tomatoes.
Food Research International, 38, 487-494.
Toropova, K., Basnak, G., Twarock, R., Stockley, P.G., Ranson, N.A. (2008). The
Three-dimensional Structure of Genomic RNA in Bacteriophage MS2: Implications for
Assembly. Journla of Molecular Biology, 375 (3), 824−836.
Torriani, S., Orsi, C., Vescovo, M. (1997). Potential of Lactobacillus casei, culture
permeate, and lactic acid to control microorganisms in ready-to-use vegetables. Journal
of Food Protection, 60, 1564–1567.
Tournas, V.H. (2005). Moulds and yeasts in fresh and minimally processed vegetables,
and sprouts. International Journal of Food Microbiology, 99(1), 71–77.
Trichopoulou, A., Naska, A., Antoniou, A., Friel, S., Trygg, K., Turrini, A. (2003).
Vegetable and fruit: the evidence in their favour and the public health perspective.
International Journal of Vitamin and Nutrition Research, 73, 63–69.
Trinetta, V., Morgan, M.T., Linton, R.H. (2010). Use of high-concentration-short-time
chlorine dioxide gas treatments for the inactivation of Salmonella enterica spp.
inoculated onto Roma tomatoes. Food Microbiology, 27, 1009–1015.
Tulipani, S., Romandini, S., Alvarez Suarez, J.M., Capocasa, F., Mezzetti, B., Busco, F.,
et al. (2008). Folate content in different strawberry genotypes and folate status in healthy
subjects after strawberry consumption. Biofactors, 34, 47–55.
Uesugi, A.R., Moraru, C.I. (2009). Reduction of Listeria on readyto- eat sausages after
exposure to a combination of pulsed light and nisin. Journal of Food Protection 72, no.
2, pp. 347–353.
Ugarte-Romero, E., Feng, H., Martin, S., Cadwallader, K., Robinson, S. (2006).
Inactivation of Escherichia coli with power ultrasound in apple cider. Journal of Food
Science, 71, E102-E108.
Urbanucci, A., Myrmel, M., Berg, I., von Bonsdorff, C.H., Maunula, L. (2009). Potential
internalisation of caliciviruses in lettuce. International Journal of Food Microbiology,
135, 175–178.
US FDA (2000). Irradiation in the production, processing and handling of food. Part
179. Fed. Regist. 65: 21 CFR (2000), pp. 71056–71058.
USFDA. (2004). FDA Guidance to Industry. Recommendations to Processors of Apple
Juice or Cider on the Use of Ozone for Pathogen Reduction Purposes.
Page 295
References
Valente, A., Sanches-Silva, A., Albuquerque, T.G., Costa, H.S. (2014). Development of
an orange juice in-house reference material and its application to guarantee the quality of
vitamin C determination in fruits, juices and fruit pulps. Food Chemistry, 154, 71–77.
van het Hof, K.H., Tijburg, L.M.B., Pietrzik, K., Weststrate, J.A. (1999). Influence of
feeding different vegetables on plasma levels of carotenoids, folate and vitamin C. Effect
of disruption of the vegetable matrix. British Journal of Nutrition, 82, 203–212.
Van Houdt, R., Michiels, C. (2005). Role of bacterial cell surface structures in
Escherichia coli biofilm formation. Research in Microbiology, 156, 626–633.
Van Impe, J.F., NicolaY, B.M., Martens, T., De Baerdemaeker, J., Vandewalle, J.
(1992). Dynamic mathematical model to predict microbial growth and inactivation
during food processing. Applied Environmental Microbiology, 58, 2901–2909.
Vandekinderen, I., Van Camp, J., De Meulenaer, B., Veramme, K., Denon, Q., Ragaert,
P., et al. (2007). The effect of the decontamination process on the microbial and
nutritional quality of fresh-cut vegetables. Acta Horticulturae, 746, 173–179.
Vardar, C., Ilhan, K., et al. (2012). "The application of various disinfectants by fogging
for decreasing postharvest diseases of strawberry." Postharvest Biology and
Technology, 66(0): 30-34.
Velusamy, V., Arshak, K., Korostynska, O., Oliwa, K., Adley, C. (2010). An overview
of foodborne pathogen detection: in the perspective of biosensors. Biotechnology
Advances, 28(2), 232-254.
Verhaelen, K, Bouwknegt, M, Carratalà, A, Lodder-Verschoor, F, Diez-Valcarce, M,
Wang, J., Membré, J. M., Ha, S. D., Bahk, G. J., Chung, M. S., Chun, H. S., et al.
(2012). Modelling the combined effect of temperature and relative humidity on
Escherichia coli O157:H7 on lettuce. Food Science and Biotechnology, 21, 859-865.
Vicente, A., Pineda, C., Lemoine, L., Civello, P.M., Martinez, G., Chaves, A. (2005).
UV-C treatments reduce decay, retain quality and alleviate chilling injury in pepper.
Postharvest Biology and Technology, 35, 69–78.
Vilhelmsson, O. (2000). Specific Solute effects and osmoadaptation in Staphylococcus
aureus. Thesis, The Pennsylvania State University.
Vinha, A., Alves, R.C., Barreira, S.V.P., Castro, A., Costa, A.S.G., Oliveira, B.P.P.
(2014). Effect of peel and seed removal on the nutritional value and antioxidant activity
of tomato (Lycopersicon esculentum L.) fruits. LWT - Food Science and Technology,
55, 197-202.
Wadell, G. (1984). Molecular epidemiology of adenoviruses. Current Topics in
Microbiology and Immunology. 110, 191-219.
Walkling-Ribeiro, M., Noci, F., Cronin, D.A., Lyng, J.G., Morgan, D.J. (2008).
Inactivation of Escherichia coli in a tropical fruit smoothie by a combination of heat and
pulsed electric fields. Journal of Food Science, 73(8), M395-M399.
Wang, S.Y., Lin, H.S. (2000). Antioxidant activity in fruits and leaves of blackberry,
raspberry, and strawberry varies with cultivar and developmental stage. Journal of
Agricultural and Food Chemistry, 48, 140–146.
Page 296
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Wang, S.Y, Zheng, W., Galletta, G.J. (2002). Cultural system affects fruit quality and
antioxidant capacity of strawberries. Journal of Agriculture and Food Chemistry, 50,
6534-6542.
Wang, H., Feng, H., Liang, W., Luo, Y., Malyarchuk, V. (2009). Effect of Surface
Roughness on Retention and Removal of Escherichia coli O157:H7 on Surfaces of
Selected Fruits. Journal of Food Science, 74, E8-E15.
Wang, J., Hu, X., Wang, Z. (2010). Kinetic models for the inactivation of
Alicyclobacillus acidiphilus DSM 14558 (T) and Alicyclobacillus acidoterrestris DSM
3922 (T) in apple juice by ultrasound. International Journal of Food Microbiology,
139(3),177- 181.
Wang, R.X., Nian, W.F., Wu, H.Y., Feng, H.Q., Zhang, K., Zhang, J., Zhu, W.D.,
Becker, K.H., Fang, J. (2012). Atmospheric-pressure cold plasma treatment of
contaminated fresh fruit and vegetable slices: inactivation and physiochemical properties
evaluation. The European Physical Journal D, 66(10), 1-7.
Warning, A., Datta, A. (2013). Interdisciplinary engineering approaches to study how
pathogenic bacteria interact with fresh produce. Review. Journal of Food Engineering
114, 426–448.
Warriner, K., Huber, A., Namvar, A., Fan, W., Dunfield, K. (2009). Recent advances in
the microbial safety of fresh fruits and vegetables. Advances in Food Nutrition Research,
57, 155-208.
Wassmann, M., Moeller, R., Reitz, G. et al. (2011). Growth phase-dependent UV-C
resistance of Bacillus subtilis: data from a short-term evolution experiment. Archives of
Microbiology, 193, 11, 823-832.
Wayner, D.D., Burton, G.W., Ingold, K.U., Locke, S.(1985). Quantitative measurement
of the TRAP of human blood plasma by controlled lipid peroxidation. FEBS Letters,
187, 33-37.
Wei, C.I., Huang, T.S., Kim, J.M., Lin, W.F., Tamplin, M.L., Bartz, J.A. (1995). Growth
and survival of Salmonella montevideo on tomatoes and disinfection with chlorinated
water. Journal of Food Protection, 58, 829−836.
Wei, J., Jin, Y., Sims, T., Kniel, K.E. (2010). Manure- and biosolids-resident murine
norovirus 1 attachment to and internalization by Romaine lettuce. Applied
Environmental Microbiology, 76, 578–583.
Weickert, Μ.Ο., Pfeifer, A.F. (2008). Metabolic effects of dietary fiber consumption
prevention of diabetes. Journal of Nutrition, 138, 439-442.
Weissinger, W.R., Chantarapanont, W., Beuchat, L.R. (2000). Survival and growth of
Salmonella baildon in shredded lettuce and diced tomatoes, and effectiveness of
chlorinated water as a sanitizer. International Journal of Food Microbiology, 62, 123–
131.
Wegener, H.C., Hald, T., Wong, D.L.F., Madsen, M., Korsgaard, H., Bager, F., GernerSmidt, P., Molbak, K. (2003). Salmonella control programs in Denmark. Emerging
Infectious Diseases, 9,774- 780.
Page 297
References
Wheeler, C., Vogt, T.M., Armstrong, G.L., Vaughan, G., Weltman, A., Nainan, O.V.,
Dato, V., Xia, G., Waller, K., Amon, J., Lee, T.M., Highbaugh-Battle, A., Hembree, C.,
Evenson, S., Ruta,M.A., Williams, I.T., Fiore, A.E., Bell, B.P. (2005). An outbreak of
hepatitis A associated with green onions. The New England Journal of Medicine, 353,
890–897.
Wheeler, D.L., Chappey, C., Lash, A.E., Leipe, D.D., Madden, T.L., Schuler, G.D.
Tatusova, T.A. Rapp, B.A. (2000). Database resources of the National Center for
Biotechnology Information. Nucleic Acids Research, 28, 10-14.
Wigginton, K.R., Kohn, T. (2012). Virus disinfection mechanisms: the role of virus
composition, structure, and function. Current Opinion in Virology, 2, 84–89.
Wijtzes, T., van’t Riet, K., Huis in’t Veld, J.H.J., Zwietering, M.H. (1998). A decision
support system for the prediction of microbial food safety and food quality. International
Journal of Food Microbiology, 42, 79–90.
WHO (2000). WHO, Resolution WHA 53.15 on Food Safety. Fifty- third Health
Assembly, Geneva, Switzerland.
WHO (2003). GEMS/Food regional diets: regional per capita consumption of raw and
semi-processed agricultural commodities. Geneva.
WHO (2008). Foodborne Disease Outbreaks: guidelines for investigation and control.
Willett, W.C., Sacks, F., Trichopoulou, A., Drescher, G., Ferro-Luzzi, A., Helsing, E.,
Trichopoulos, D. (1995). Mediterranean diet pyramid: a cultural model for healthy
eating. American Journal in Clinical Nutrition, 61(6 Suppl), 1402-1406.
Woodling, S.E., Moraru, C.I. (2007). Effect of spectral range in surface inactivation of
Listeria innocua using broad-spectrum pulsed light. Journal of Food Protection, 70, 4,
909– 916.
Wright, J.R., Sumner, S.S., Hackney, C.R., Pierson, M.D., Zoecklein, B.W. (2000).
Efficacy of ultraviolet light for reducing Escherichia coli O157: H7 in unpasteurized
apple cider. Journal of Food Protection, 63,563-567.
Xu, W., Wu, C. (2014). Decontamination of Salmonella enterica Typhimurium on green
onions using a new formula of sanitizer washing and pulsed UV light (PL). Food
Research International, 62, 280–285.
Yajima, H., Takao, M., Yasuhira, S., Zhao, J.H., Ishii, C., Inoue, H., Yasui, A. (1995). A
eukaryotic gene encoding an endonuclease that specifically repairs DNA damage by
ultraviolet light. The EMBO Journal, 14(10), 2393–2399.
Yang, R.F., Cheng, T.S., Shewfelt, R.L. (1990). The effect of high temperature and
ethylene treatment on the ripening of tomatoes. Journal of Plant Physiology, 136, 368372.
Yang, H., Summer, S., Li, Y. (2003). The effect of pH on inactivation of pathogenic
bacteria on fresh-cut lettuce by dipping treatment with electrolyzed water. Journal of
Food Science, 68, 3.
Page 298
Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
Yang, Y., Luo, Y., Millner, P., Turner, E., Feng, H. (2012). Assessment of Escherichia
coli O157:H7 transference from soil to iceberg lettuce via a contaminated field coring
harvesting knife. International Journal of Food Microbiology, 153, 345–350.
Yates, M.V., Malley, J., Rochelle, P., Hoffman, R. (2006). Effect of adenovirus
resistance on UV disinfection requirements; a report on the state of adenovirus science.
Journal of the American Water Works Association. 98(6), 93-106.
Yaun, B.S., Sumner, S.S., Eifert, J.D., Marcy, J.E. (2004). Inhibition of pathogens on
fresh produce by ultraviolet energy. International Journal of Food Microbiology, 90, 1–
8.
Yeni, F., Acar, S., Polat, Ö.G., Soyer, Y., Alpas, H. (2014). Rapid and standardized
methods for detection of foodborne pathogens and mycotoxins on fresh produce. Food
Control, 40, 359-367.
Yin, R., Dai, T., Avci1, P., Jorge, A.E.S., CMA de Melo, W., Vecchio, D., Huanh, Y-Y,
Guptal, A., Hamblin, M.R. (2013). Light based anti-infectives: ultraviolet C irradiation,
photodynamic therapy, blue light, and beyond. Current Opinion in Pharmacology,
13,731–762.
Yoon, M., Jung, K., YoollLee, K.Y., Jeong, Y., Lee, J.W, Park, H.J. (2014). Synergistic
effect of the combined treatment with gamma irradiation and sodium
dichloroisocyanurate to control gray mold (Botrytis cinerea) on paprika. Radiation
PhysicsandChemistry, 98, 103–108.
Ziuzina, D., Patil, S., Cullen, P.J., Keener, K.M., Bourke, P. (2014). Atmospheric cold
plasma inactivation of Escherichia coli, Salmonella enterica serovar Typhimurium and
Listeria monocytogenes inoculated on fresh produce. Food Microbiology, 42, 109-116.
Zhan, L., Hu, J., Ai, Z., Pang, L., Li, Y., Zhu, M. (2013). Light exposure during storage
preserving soluble sugar and L-ascorbic acid content of minimally processed romaine
lettuce (Lactuca sativa L.var. longifolia). Food Chemistry, 136, 273–278.
Zhang, S., Faber, J.M. (1996). The effects of various disinfectants against Listeria
monocytogenes on fresh-cut vegetables. Food Microbiology, 13, 311- 321.
Zhang, S., Iandolo, J., Stewart, C. (1998). The enterotoxin D plasmid of Staphylococcus
aureus encodes a second enterotoxin determinant (sej). FEMS Microbiology Letters,
168, 227–233.
Zhou, B., Feng, H., Luo, Y.G. (2009). Ultrasound enhanced sanitizer efficacy in
reduction of Escherichia coli O157:H7 population on spinach leaves. Journal of Food
Science, 74 (6), 308–313.
Zhou, Bin (2010). Investigation on factors imfluencing ultrasound-assisted surface
decontamination of fresh and fresh-cut vegetables. Dissertation submitted at University
of Illinois at Urbana-Champaign
Zhu, J.K. (2001). Plant salt tolerance, Trends Plant Science, 6, 66–71.
Zhuang, R.Y., Beuchat, L.R., Angulo, F.J. (1995). Fate of Salmonella Montevideo on
and in raw tomatoes as affected by temperature and treatment with chlorine. Applied
Environmental Microbiology, 61, 2127-2131.
Page 299
References
Internet Pages
www.Elma-ultrasonic.com
www.hielcher.com
www.xenoncorp.com
www.foodengineeringmag.com
www.intechopen.com
www.ibebvi.be
BOOKS
Barth, Margaret, Hankinson Thomas, R., Zhuang, Hong, and Breidt, Frederick. (2009).
Microbiological Spoilage of Fruits and Vegetables. W.H. Sperber, M.P. Doyle (eds.),
Compendium of the Microbiological Spoilage of Foods and Beverages, Food
Microbiology and Food Safety, DOI 10.1007/978-1-4419-0826-1_6, Springer
Science+Business Media.
Cullen, P.J, Tiwari, B, and Valdramidis, V. (2012). Novel Thermal and Non-Thermal
technologies for fluid foods. Elsevier Inc.
Koutchma, Tatiana, Forney, Larry, Moraru, Carmen (2009). Ultraviolet light in food
technology: principles and applications. Taylor & Francis Group, LLC.
Martín-Belloso, Olga., Soliva-Fortuny, Robert. (2011). Advances in Fresh-Cut Fruits
and Vegetables Processing. CRC Press, Taylor & Francis Group.
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Appendix
APPENDIX
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Birmpa Angeliki
PUBLICATIONS
1. Birmpa A., Sfika V. and Vantarakis A. (2013).Ultraviolet light and Ultrasound as
non-thermal treatments for the inactivation of microorganisms in fresh ready-to-eat
foods. International Journal of Food Microbiology 167: 96-102.
2. Birmpa Angeliki, Vantarakis Apostolos, Paparrodopoulos Spyros, Whyte Paul and
Lyng James. (2014). Efficacy of three light technologies for reducing microbial
populations in liquids”. BioMed Research International, Volume 2014, Article ID
673939, 9 pages.
Submitted for Publication
1. Angeliki Birmpa, Spyros Paparrodopoulos, Vasiliki Sfika, Apostolos Vantarakis.
Efficacy of non thermal technologies in the microbial inactivation of fresh ready-to-eat
cherry tomatoes (Submitted).
2. Angeliki Birmpa, Maria Bellou and Apostolos Vantarakis. Effect of non-thermal,
conventional and combined disinfection technologies on the stability of human
Adenoviruses as fecal contaminants on fresh ready to eat produce surfaces (Submitted to
Journal of Food Protection).
3. Angeliki Birmpa, Apostolos Vantarakis, Antigoni Anninou, Maria Bellou, Petros
Kokkinos, Peter P. Groumpos. A user-friendly mathematical model for the prediction of
food safety (Submitted to Journal of Food Processing and Technology).
4. A.Birmpa, J.Lyng, P.Whyte, S.Paparrodopoulos, E.Sazakli, M.Leotsinidis and
A.Vantarakis. The promising use of non-thermal green technologies and their effect on
the quality of foods (Submitted).
Page 303
Appendix
CONFERENCE ORAL PRESENTATIONS
International
1. A. Birmpa, P. Kokkinos, V. Sfika, A. Vantarakis: “Ultraviolet radiation and
ultrasonication as non-thermal treatments for the inactivation of microorganisms in fresh
ready-to-eat foods."23rd International ICFMH Symposium, FoodMicro2012, 37/9/2012. Global Issues in Food Microbiology, Istanbul, Turkey.
2. M. Bellou, A. Birmpa, P. Kokkinos, A. Vantarakis, “Viral Outbreaks Linked To Fresh
Produce Consumption the Last Two Decades: A Systematic Review” 24 th ECCMID
European Society of Clinical Microbiology and Infectious Diseases, 10-13 May 2014,
Barcelona, Spain.
3. Angeliki Birmpa, Michalis Leotsinidis, Eleni Sazakli, Tzina Tsichlia and Apostolos
Vantarakis. Effect of disinfection technologies on quality and nutritional properties of
lettuce, strawberries and cherry tomatoes. European Symposium on Food Safety, 7-9
May 2014, Budapest, Hungary.
4. Angeliki Birmpa, Michalis Leotsinidis, Eleni Sazakli, Spyros Paparrodopoulos, Paul
Whyte, James Lyng, Apostolos Vantarakis. The promising use of non thermal green
technologies and their effect on the quality of foods. 3rd International Iseki Food
Conference, 21-23 May 2014, Athens, Greece.
5. Angeliki Birmpa, Spyros Paparrodopoulos, Paul Whyte , James Lyng and Apostolos
Vantarakis. Efficacy of three light technologies for reducing microbial populations.
IAFP 2014, 3-6 August 2014, Indianapolis, Indiana, USA.
6. Angeliki Birmpa, Panagiotis Pitsos, Spyros Paparrodopoulos, Vasiliki Sfika,
Apostolos Vantarakis. Efficacy of non thermal technologies combined with chlorine for
reducing microbial populations in ready to eat products. IAFP 2014, 3-6 August 2014,
Indianapolis, Indiana, USA.
Greek
1. Πίτσος Παναγιώτης, Μαυρίδου Αθηνά, Μπίρμπα Αγγελική, Βανταράκης Απόστολος.
Επίδραση της χρήσης χλωρίου στην απολύμανση έτοιμων προς κατανάλωση τροφίμων.
ΠΕΤΙΕ, 5-7 Δεκεμβρίου 2013. Αθήνα.
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Non-thermal technologies for the disinfection of food and risk assessment for Public Health
Birmpa Angeliki
CONFERENCE POSTERS
International
1. A. Birmpa, D. Papadopoulou, A. Kokkinos, A. Vantarakis. “Evaluation of disinfectant
efficacy by ultraviolet light in a laboratory swimming pool model”. 5th International
Conference Swimming pool and Spa, 9- 12/4/2013 Rome, Italy.
2. P. Ziros, P. Kokkinos, A. Birmpa, N. Karagiannis, A. Vantarakis. “Real Time PCR
Molecular Monitoring of Zygosaccharomyces bailii During Soft Drinks Production to
Determine
Contamination
Sources”.
23rd
International
ICFMH
Symposium,
FoodMicro2012, Global Issues in Food Microbiology, 3- 7/9/2012. Istanbul, Turkey.
3. Birmpa Angeliki, Anninou Antigoni, Nikolaou Alexandra, Kokkinos Petros,
Groumpos Peter and Vantarakis Apostolos. “A Theoritical Mathematical Model for the
analysis of lettuce quality in a Food Production Chain using Fuzzy Cognitive Maps”.
International Conference on Predictive Modelling in Food (ICPMF8), 16-20 September
2013 Paris, France.
4. Angeliki Birmpa, Maria Tselepi and Apostolos Vantarakis. Effect of disinfection
technologies on Escherichia coli, Staphylococcus aureus, Salmonella enteritidis and
Listeria innocua inoculated on lettuce, strawberries and cherry tomatoes during a
refrigerated storage period. European Symposium on Food Safety, 7-9 May 2014,
Budapest, Hungary.
5. Angeliki Birmpa, Maria Bellou and Apostolos Vantarakis. Effect of non-thermal,
conventional and combined disinfection technologies on the stability of human
Adenoviruses as fecal contaminants on fresh ready to eat produce surfaces. ISFEV, 2-5
September 2014, Corfu, Greece.
Greek
1. Μπίρμπα Αγγελική, Κόκκινος Πέτρος, Σφήκα Βασιλική, Βανταράκης Απόστολος.
«Χρήση
εναλλακτικών
μη
θερμικών
τεχνολογιών
για
την
απολύμανση
μικροοργανισμών σε έτοιμα προς κατανάλωση τρόφιμα». 8ο Πανελλήνιο Συνέδριο
Βιοεπιστημόνων, Βιοεπιστήμες, Μοχλός ανάπτυξης της κοινωνίας, 18-20 Οκτωβρίου
2012, Συνεδριακό και Πολιτιστικό Κέντρο Πανεπιστημίου Πατρών.
Page 305