Download Montel

Ecological approaches for managing microbial
diversity to improve safety of traditional food.
Marie-Christine Montel,
Unité de Recherches fromagères
INRA Aurillac
Example of cheese
from experiments performed in WP2A of Truefood project
Improvement of microbial safety
WP2A Truefood project
Task 2A.1
Improvement microbial safety of milks through reduction of mastitis
and the use of antibiotics by feeding regimes and other management
practices
( ISS, INRA,)
Task 2A.2
Management of microbial diversity for inhibiting pathogenic bacteria
(L. monocytogenes, S. aureus) in traditional cheeses
( INRA, UL, TUM, DRI)
Task 2A.3
Improvement of environmental conditions governing cheese ripening
taking account process efficiency and cheese quality
(INRA, DRI)
Task 2A.4
Development of a bio-preservation (using lactic acid bacteria) for
inhibiting food pathogens on pork muscle tissues (ADIV)
Why an ecological approach?
Because
Cheeses and milk are microbial ecosystems
– Cheese is a system in which microbial populations
(living or biotic part ) live and have relationships between
them and with their surrounding environments ( abiotic
part) .
» Microbial populations interact each other in
synergy or antagonism and their life dpends on
nutriments, temperature, humididity, oxygen.
–The life of these microbes is determinant for the qualities
of cheeses.
Microbial safety
Pathogenic bacteria :
•
commercialised raw milk cheeses have to comply with EC regulation
•Listeria monocytogenes
•Salmonella
•Staphylococus aureus ( no enterotoxins production )
•Eschercihia coli
Opportunist pathogenic bacteria
Metabolite productions
•Enterotoxins
•Mycotoxins
•Biogenic amines
Above all,
great taste,
Benefits refer to
pleasure and happiness of consumers
who require authenticity , traceability, sensorial
properties, safety and health values.
Why to manage microbial diversity ?
To eliminate
pathogenic bacteria
EC regulation
for 4 species
Minimise
To preserve microbial
community:
during manufacturing
and ripening
Increase
Risks
Benefits
Microbial Safety
Gustative pleasure
Health
A challenge for industrial producing Traditional Fermented
products (Cheese, sausage…) for conciliating all these aspects
and meet all these requirements
Our behaviour in managment of microbial diversity
for microbial safety must have always in mind the
benefits of microbial diversity for cheeses
-Healthy aspects?
-Contribution in hurdle technology
-Diversity of their sensorial properties
Management of microbial diversity
Elimination of pathogenic micro-organisms and
maintain of microbial diversity having an interest
through all the process
control of raw material : microbial quality of
milk, meat...
 control of microbial dynamics during ripening
 Tools for tracking and monitoring microbial diversity
How to identify and monitor microbial
community
Molecular
Classical
Milk , cheeses
Picture depending on culture
media, cultavibility of strains
Identification by
phenotypic tests,
 genomic tests (speciesPCR,
16s or 23s DNAr sequencing)
Physical spectra ( FTIR)
PCR-SSCP,TGGE, TTGE,
DGGE, lH-PCR…
Monitor microbial dynamics
Picture biased by
•Dominant population detected
•DNA Extraction
•PCR amplification
•Coelution in the same peaks
Approaches get rich each other
What approaches for biopreservation of
traditional products ?
1. Selection of strains or metabolites (example : nisine)
2. Understanding microbial ecosystems having antagonist
activities
• Example of milk and raw milk cheese ecosystems
Selection of strains or inhibiting
metabolites
Validation at industrial scale
In vitro screening of inhibitory activity
Coculture in milk or other media
Identification of inhibiting
substances bacteriocins,
H 2 O2 ,
1
Strains Collections
Challenge tests in
experimental cheese
GAP here!!!!
Milks , cheeses
Detection
of bacteriocin genes
Test on agar well
Agar diffusion Bioluminescence
Selection of strain inhibiting
Time consuming and fastidious
Frequent fails due to gap between the
results obtained in vitro and those
obtained in cheeses
 Consortia with high antilisteria activities by
in vitro test but not at the surface of cheeses
Low number of strains selected for example
in Truefood project able to inhibit and without
effect on sensorial properties
Strategies by ecological approach
Rely on a general principle in ecology indicating that:
the whole is more than the sum of each individu as
many interactions - synergy, antagonism,
competition..- can occur
 Hypothesis that preservation of the microbial
community with the wholeness of its diversity is important
for the different functions -inhibition of pathogens,
production of aromatic compounds
Understanding microbial ecosystem having
antilisteria activity
Advice for milk production or cheese
ripening
proposal of consortium
Test of simplified microbial
consortia in experimental cheese
Screening of milks, or cheese surface
on experimental cheeses
Analysis of the most inhibitory
microbial consortia
Identification of microbial and
biochemical part of the ecosystem
Examples of microbial cheese
ecosystems with antilisteria activities
 Microbial consortia from Surfaces of cheeses
Smear cheeses: Munster
Farm Saint-nectaire cheeses
Microbial consortium from Raw milk from
Saint-Nectaire area
First example
consortia of microbes from the surface of
Saint-Nectaire
Study of Inhibition of Listeria monocytogenes at
the surface
Antilisteria activities of surfaces of SaintNectaire cheese microbial consortia
Comparison of inhibitory effect of consortia (34) selected from farm
Saint-Nectaire surface cheeses. (Trials on surface of non cooked pressed
0,5
SN15
SN11
SN26
SN19
SN13
SN9
SN22
SN30
SN24
SN25
SN23
SN12
SN29
SN6
SN8
SN34
SN20
SN28
SN16
SN32
SN2
SN17
SN1
SN35
SN31
SN27
SN3
SN14
SN33
SN18
SN10
SN5
SN4
SN7
cheeses ripened at 7°C during 28 days)
Inhibition
0
-0,5
No inhibition
-1
-1,5
-2
DLog -2,5
 Great diversity in the antilisteria activities
High inhibition
observed without relation with pH values
o 5.9<pH28d<8.4
 Selection of one consortium SN15
Stability over storage of the inhibiting effect of
SN15 consortia against Listeria monocytogenes
ΔLog
Lm (ufc/cm²)CFU/cm2
Delta Log
[L.monocytogenes]
T emps
de conservation
Time
(months) en mois
0
3
4
9
14
16
24
0,00
-0,50
-1,00
-1,50
-2,00
-2,50
-3,00
-3,50
ΔLog Lm = Log (Lm SN15) - Log (Lm control)
Microbial consortium SN15 still
inhibitory after 24 months
storage at -20°C
Identification of microbial consortium
(SN15) inhibiting L. monocytogenes
Identification by phenotypic test, RFLP and 16S
A=Lactic acid bacteria (6 species)
DNAr sequencing
D=Yeasts (4 species)
Candida sake
Yarrowia lipolytica
Debaryomyces hansenii
Geotrichum sp.
D
A
D
C
B
C=Gram negative bacteria (3 species)
Proteus vulgaris
Serratia proteomaculans
Pseudomonas fluorescens or syrinqae
Lb. casei
Lb. curvatus
Ln. mesenteroides
Carnobacterium mobile
Marinilactibacillus psychrotolerans
E. faecalis
B= Micrococcaceae/ Corynebacteriaceae
(7 species)
Arthrobacter nicotianae Arthrobacter
bergeri
Staphylococcus pulvereri
Staphylococcus xylosus
Brevibacterium linens or casei/
Brevibacterium antiquum
Brachybacterium
Reconstitution of complex consortium
SN15 from the surface
 Constitution of consortium with 19 strains (one strain by species)
inoculated at 2 Log /cm2 at the surface of cheeses
 Comparison of L. monocytogenes growth at the surface of
cheeses with the natural complex or reconstituted consortia
3 yeasts
Inhibition
3 Gram
negative
DLog
reconstituted
Complex
6 Lactic acid bacteria
D
7 Micrococcaceae/
Corynebacteriaceae
Complex more inhibitory
than the reconstituted
Study in progress for
understanding why?
Second example
microbial consortium from raw milk
Study Inhibition of L. monocytogenes
in the core of cheeses
Complex consortium selected in previous study : Millet al, 2006; Saubusse et al, 2007
Composition of Microbial consortium
from raw milk having antisteria activty
( Saubusse et al, 2007)
Great diversity : 29 microbial species
D=Yeasts (5 species)
Rhodosporium babjevae
Debaryomyces hansenii
Candida pseudointermedia
C. pararugosa
C. deformans
D
A
D
C
B
C=Gram negative bacteria (5 species)
Pseudomonas putida
Enterobacter amnigenus
Acinetobacter sp.
Chryseobacterium sp
Stenotrophomonas maltophilia
A=Lactic acid bacteria (9 species)
Lb. casei / Lb. farciminis
Lb. curvatus
Lb. plantarum
Ln. pseudomesenteroides
Ln. citreum
E. hirae
E. Faecalis
A. viridans
B= Micrococcaceae/ Corynebacteriaceae
(10 species)
Staph. saprophyticus/ Staph. equorum/
Staph haemolyticusCorynebacterium
casei/Coryne. flavescens
Arthrobacter nicotianae
Brevibacterium linens
Exiguobacterium sp.
Brachybacterium rhamnosus
Macrococcus caseolyticus
Simplification of reconstituted
consortia from raw milk
Lactic acid bacteria
Gram negative
A
B
A
C
A
B
Micrococcaceae/
Corynebacteriaceae
C
D
yeast
B
D
A
B
C
D
A
A
B
B
A
D
C
B
D
D
Inoculation in
pasteurised milk
with S. thermophilus (St)
Manufacturing
non coocked
pressed cheeses
Comparison of
L. monocytogenes
to a control with only St
thermophilus
Microbial and
biochemical analysis
Inhibition of L. monocytogenes with
simplified consortia in core of cheeses
1,5
ABCD
AB
AD
A
ABC
ABD
BDC
BD
B
1
0,5
A=Lactic acid bacteria
ΔLog Lm
0
B=Micrococcaceae/
Corynebacteriaceae
-0,5
-1
C=Gram negative
-1,5
D=yeast
-2
-2,5
ΔLog Lm = Log (Lm assay) - Log (Lm control)
Synergy between lactic acid bacteria and non lactic
acid bacteria in the inhibition
Loss of inhibition in simplified consortia without lactic
acid bacteria
Link between [L. monocytogenes] and
[organic acids]- [volatil compounds]- pH
5
4
3
1
AB
3-methylbutyric acid
0
pH
Butyric acid
-1
2-heptanol
2-butanol
2-pentanol
ABCD
2
Fact. 2 : 32,90%
Low count of
listeria
L lactate
D lactate
Acetic acid
-2
AD
ESTERS
-5
KETONES
ALDEHYDES
Hexanoic acid
-4
High count of
listeria
control
ALCOOLS
-3
Ethyl formiate
Ethyl butanoate
B
A
2-methyl-propanal
butanal
2-hexanone
4-methyl-2-pentanone
2,3-pentanedione
2,3-butanedione
2-butanone
-6
-7
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
Active
: 51,77%
HypothesisFact.
on 1the
role of acetic acid and D lactate in
the inhibition of L. monocytogenes in cheeses
 Important variation in volatiles profils according to
consortia inoculated
Inhibition by several hurdles during ripening
pH>5.3
Bacteriocins H202
pH
<8mg/g <2mg/g
Ac. lactic
L
Ac. lactic
D
<0.3mg/g
Ac. acetic
Alcohols
Ethyl esters
Besides these microbial factors, environnemental factors can also limit
the development of pathogenes
 Temperature, relative humidity during ripening
What are the populations involved in the
production ?
 Microbial dynamics studied by culture methods
associated with molecular tools
 Example of microbial balance in cheese with
consortium AB the most inhibitory
Lactobacillus
Nmax Log UFC/g
9
8
7
6
5
19 species inoculated still
present
Leuconostoc
Enteroccoccus
BUT
Corynebacteriaceae Difficulties to quantifye species
Micrococcaceae
of this group
In conclusion, what applications for
traditional fermented food?
 Arguments for maintaining microbial diversity as a thumb
for safety of fermented products ( ex cheese)
 High inhibitory potentialities of complex microbial
ecosytem from the surface of Cheese but application
still limited by difficuties to reconsitute it
 Further studies needed to understand why
 Scientific data to think about suitable balance between
microbial populations in milk
 Further studies to adapt milk production practices
In conclusion, what applications for
traditional fermented food?
 Proposal a simplified microbial consortium still complex
associating lactic acid bacteria ( 8 species) and non lactic
acid bacteria ( 12 species?) for inhibiting L. monocytogenes
in the core of cheese but its industrial use need some
improvments
 Optimise the preparation of the consortium
and insure its stability overtime
 Validate it use at industrial scale
WP6 of
Truefood
 Evaluate its effect on sensorial properties
 Develop rapid methods to quantifye non lactic acid
bacteria in cheese
Thank you for your attention and coming
instead of visiting the museum
 Partners of WP2A of Truefood project and
especially Cécile Callon for her helpful
contribution in this presentation
Financial support : European Commission under the 6th Framework
programme for RTD ( contrat N° Food CT-2006-016264)