Download Lactobacillus plantarum - UEF Electronic Publications

KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 220
KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 220
CARME PLUMED-FERRER
Lactobacillus plantarum
From Application to Protein Expression
Doctoral dissertation
To be presented by permission of the Faculty of Natural and Environmental Sciences
of the University of Kuopio for public examination in Auditorium L22,
Snellmania building, University of Kuopio,
on Saturday 27 th October 2007, at 12 noon
Department of Biosciences
Applied Biotechnology
Nutrition and Food Biotechnology
University of Kuopio
JOKA
KUOPIO 2007
Distributor :
Kuopio University Library
P.O. Box 1627
FI-70211 KUOPIO
FINLAND
Tel. +358 17 163 430
Fax +358 17 163 410
http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html
Series Editors:
Professor Pertti Pasanen, Ph.D.
Department of Environmental Science
Professor Jari Kaipio, Ph.D.
Department of Applied Physics
Author’s address:
Department of Biosciences
Applied Biotechnology
Nutrition and Food Biotechnology
University of Kuopio
P.O. Box 1627
FI-70211 KUOPIO
FINLAND
Tel. +358 17 163 573
Fax +358 17 163 322
E-mail: [email protected]
Supervisors:
Professor Atte von Wright, Ph.D.
Department of Biosciences
Applied Biotechnology
University of Kuopio
Professor Maria Halmekytö, Ph.D.
Department of Biosciences
Applied Biotechnology
University of Kuopio
Paula Hyvönen, D.V.M
Department of Biosciences
Applied Biotechnology
University of Kuopio
Reviewers:
Dr. Nuria Canibe, Ph.D.
Animal Health, Welfare and Nutrition
Faculty of Agricultural Sciences
Tjele, Denmark
Professor Pier Sandro Cocconcelli, Ph.D.
Instituto di Microbiologia,
Università Cattolica del Sacro Cuore
Piacenza, Italy
Opponent:
Dr. Arthur Ouwehand, Ph.D.
Danisco Finland Oy
Kantvik, Finland
ISBN 978-951-27-0698-3
ISBN 978-951-27-0793-5 (PDF)
ISSN 1235-0486
Kopijyvä
Kuopio 2007
Finland
Plumed-Ferrer, Carme. Lactobacillus plantarum: From application to protein expression. Kuopio
University Publications C. Natural and Environmental Sciences 220. 2007. 60 p.
ISBN 978-951-27-0698-3
ISBN 978-951-27-0793-5 (PDF)
ISSN 1235-0486
ABSTRACT
Lactic acid bacteria (LAB) are a heterogeneous group of organisms found in a wide range of
environmental niches due to their large adaptation capacity. They are responsible for the
fermentation of many fermented food and feed products because they can transform sugars into
organic acids and decrease the pH of the medium. This acidification prevents the growth of
pathogenic bacteria, making the fermented product safe for the consumer. However, a product
that has been fermented spontaneously has the disadvantage of being unpredictable and
uncontrollable. A solution to this problem is inoculation with a concentrated bacterial culture
(starter culture) in order to control the fermentation process.
Liquid feeding is a common system in Finland for feeding pigs of all ages. Although
feeding pigs with a liquid diet is not a new technique, much recent research has focused on
explaining the benefits because it can replace the use of antibiotics in animal production.
However, pig liquid feed is rapidly spoiled by pathogens such as Enterobacteriaceae unless the
lactic acid bacteria population dominates the fermentation. In order to solve this problem, we
looked for a lactic acid bacterium that could control the fermentation in this particular matrix.
For this purpose, the stability of the liquid feed was analysed and the lactic acid bacterial
population identified. A Lactobacillus plantarum strain was chosen as a potential starter culture
for this type of feed. Subsequently, two trials were conducted in order to check the growth
potential of this bacterium in liquid feed prepared in laboratory conditions and later in a
commercial farm with the purpose of obtaining real life results. The results suggested that
Lactobacillus plantarum REB1 had the potential to be used as starter culture in pig liquid feed,
providing a proper fermentation with high numbers of lactic acid bacteria, low pH, stable
numbers of yeasts and low Enterobacteriaceae.
Moreover, the extensive adaptation capacity of Lactobacillus plantarum and the wide
range of applications where this bacterium has been used makes research on this topic especially
interesting and challenging. For this reason, two strains of Lactobacillus plantarum from
different origin (REB1 and MLBPL1, the latter from vegetable material) were used in
proteomics studies in order to check metabolic differences between strains and between growth
conditions. The results show that the metabolisms of both strains are very similar. They were
found to ferment different carbohydrates simultaneously (hexoses and pentoses) with preferential
expression in the early-exponential phase, and to use carbohydrates differently in three media
even though there was always an excess of glucose content.
Overall, this study has identified a bacterial strain with potential applications, and provided
a better understanding of the metabolic capacity of this particular species and some clues about
their adaptation potential.
Universal Decimal Classification: 579.864, 636.084.422, 636.4, 577.124
National Library of Medicine Classification: QW 85, QW 142.5.A8, QU 34
CAB Thesaurus: lactic acid bacteria; Lactobacillus plantarum; pigs; pig feeding; wet feeding; fermentation; starters;
carbohydrate metabolism; bacterial proteins; electrophoresis; mass spectrometry.
ACKNOWLEDGEMENTS
This work was carried out in the Department of Biosciences, Applied Biotechnology, Nutrition
and Food Biotechnology at the University of Kuopio. I would like to express my gratitude to the
staff of Applied Biotechnology for providing a pleasant and comfortable work environment.
Specifically, my sincere gratitude:
To Professor Atte von Wright, my principal supervisor, for giving me the opportunity to start
working in the department and later, for making it possible to continue this work as part of my
Ph.D. studies; for introducing me to the world of microbiology and especially to lactic acid
bacteria, which turned out to be one of my passions; for his comments and support on this work
and for his time and advice during all these years.
To Professor Maria Halmekytö and Paula Hyvönen, my other supervisors, for their expert advice
in parts of my studies. I am especially grateful to Paula, for sharing her opinions and wise
suggestions, her extensive knowledge and "translation" on the pig project, for always having her
door open, and for making me feel always welcome in the department.
To Dr. Nuria Canibe and Professor Pier Sandro Cocconcelli, for kindly agreeing to review this
thesis.
To Mrs. Sari Pekkarinen and Mr. Olli-Matti Pekkarinen for their kindness, for always providing
me with feed samples, and for the opportunity to use their pig facility. To Pellonpaja Oy, for
making this pig project a reality and for their interest in my work.
To the former and current members of the laboratory, Marta, Kaisu K., Katariina, Riikka, Tiina,
Dina, Heidi, Anna-Liisa, Marianne, Jenni, Kaisu R., Ulla, Jouni, Mirja, Elvi, Eeva-Liisa,
Kristiina, Riitta, recently Elena, and many more students, with whom I have spent long hours
and shared memorable experiences. Thank you for making a pleasant working atmosphere in the
department and for your assistance and friendship.
To my dear friends in Finland, Leena, Stefanos, Dimitris, Maria, Darin, Christos, Elina, Thomas,
Alexis, Nektaria, Quoc, Vana, Alba, Rafael, Joan, Sandra, Eva, Vicente, Rebeca, Michael,
Suvikki, Sami, Mesi, Visa and many more, for being part of my life, for making Kuopio feel like
home, for their endless support during all these years and for listening.
To my dear friends in Spain, Marta, Maite, Àlex, Dario, Dídac, Maria, Miriam, Cristina, Carles,
Josep, Desirée, Aleix and many more, for keeping our friendship going and for their support
even from so far away.
To my parents, Santos and Teresa, for understanding my decision to come to Finland, for always
encouraging me to do what I want and where I want, for raising me to be strong enough to go
through Ph.D. studies. And to my brothers, Jordi and Enric, for always being there when I
needed them. Thank you so much!
To my many other relatives, specially my nephews Víctor and Álex, for making me feel part of a
big and continuously growing family even while living abroad.
Finally, to my closest family, Marc and Laia, for everything! Marc, thank you for your endless
support, for your love, for being an essential part of my life. Laia, thank you for your
unconditional love and for giving me the strength I needed to finish this thesis. Thank you!
For the financial support of this work, I would like to thank Pellonpaja Oy, CIMO, Tekes, the
University of Kuopio and the ABS Graduate School.
Kuopio, September 2007.
Carme Plumed-Ferrer
ABBREVIATIONS
2-DE
CcpA
CCR
CFU
cre
DE
ESI
FLF
GIT
HPr
ID
LAB
labFLF
labRFLF
MALDI
Mr
MRS
MS
NFLF
OD
OGU
PCA
PFGE
pI
PTM
PTS
rpm
SDS-PAGE
TOF
Two-dimensional electrophoresis
Global catabolite control protein A
Carbon catabolite repression
Colony-forming units
Catabolite responsive elements
Digestible energy
Electrospray ionization
Fermented liquid feed
Gastrointestinal tract
Phosphocarrier protein
Internal diameter
Lactic acid bacteria
Laboratory fermented liquid feed
75% replenished laboratory fermented liquid feed
Matrix-assisted laser desorption/ionization
Molecular mass
De Man, Rogosa and Sharpe
Mass Spectrometry
Non-fermented liquid feed
Optical density
oesophago-gastric ulcer
Principal component analysis
Pulsed field gel electrophoresis
Isoelectric point
Post-translational modifications
Phosphotransferase system
Revolutions per minute
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Time of flight
LIST OF ORIGINAL PUBLICATIONS
I. C. Plumed-Ferrer, M. Llopis, P. Hyvönen and A. von Wright. Characterization of the
microbial community and its changes in liquid piglet feed formulations. J Sci Food Agric
2004; 84:1315-1318.
II. C. Plumed-Ferrer, I. Kivelä, P Hyvönen and A. von Wright. Survival, growth and
persistence under farm conditions of a Lactobacillus plantarum strain inoculated into
liquid pig feed. J Appl Microbiol 2005; 99: 851-858.
III. C. Plumed-Ferrer, A. von Wright. Fermented pig liquid feed: nutritional, safety and
regulatory aspects. Revised manuscript submitted to J Appl Microbiol.
IV. K. M. Koistinen*, C. Plumed-Ferrer*, S. O. Kärenlampi and A. von Wright.
Comparison of growth phase-dependent cytosolic proteomes of two Lactobacillus
plantarum strains used in food and feed fermentations. FEMS Microbiol Lett 2007; 273:
12-21.
*
The authors contributed equally to this work.
V. C. Plumed-Ferrer, K. M. Koistinen, S. J. Lehesranta, S.O. Kärenlampi and A. von
Wright. Lactobacillus plantarum proteome in different growth media, a comparison of
two strains. Submitted to Appl Environ Microbiol.
CONTENTS
1.
INTRODUCTION .................................................................................................. 15
1.1
Lactic acid bacteria .................................................................................................... 15
1.2
Lactobacilli................................................................................................................. 15
1.3
Carbohydrate metabolism of lactic acid bacteria........................................................ 16
1.3.1
Glycolysis............................................................................................................... 16
1.3.2
Phosphoketolase pathway ....................................................................................... 16
1.3.3
Pyruvate metabolism............................................................................................... 19
1.3.4
Malolactic fermentation .......................................................................................... 19
1.3.5
Global control of sugar metabolism in LAB ............................................................ 20
1.4
Starter cultures........................................................................................................... 20
1.5
Lactobacillus plantarum ............................................................................................ 21
1.6
Pig feed ...................................................................................................................... 22
1.6.1
Background on pig nutrition and feed formulations ................................................. 22
1.6.2
Liquid feeding (Check study III for a review).......................................................... 23
1.6.2.1
Fermented liquid pig feed ............................................................................... 24
1.6.2.2
Pig growth performance.................................................................................. 25
1.7
Proteomics.................................................................................................................. 26
1.7.1
2-DE....................................................................................................................... 27
1.7.2
Mass Spectrometry ................................................................................................. 28
1.7.3
Proteomics latest developments and limitations ....................................................... 28
2.
AIMS OF THE STUDY .......................................................................................... 30
3.
MATERIALS AND METHODS ............................................................................ 31
3.1
Pig feed experiments .................................................................................................. 31
3.1.1
Bacterial strains ...................................................................................................... 31
3.1.2
Microbial analyses of liquid feed............................................................................. 31
3.1.3
Laboratory scale liquid feed fermentations .............................................................. 31
3.1.4
Farm scale experiments........................................................................................... 32
3.1.4.1
Commercial farm............................................................................................ 32
3.1.4.2
Liquid feed ..................................................................................................... 32
3.1.4.2.1 Non-Fermented Liquid Feed........................................................................ 33
3.1.4.2.2 Fermented Liquid Feed ............................................................................... 33
3.1.5
Pigs' performance.................................................................................................... 33
3.1.6
Statistical analysis................................................................................................... 33
3.2
Proteomic experiments ............................................................................................... 34
3.2.1
4.
Bacterial strains and their cultivation .......................................................................34
3.2.1.1
Growth phase-dependent proteome..................................................................34
3.2.1.2
Media- dependent proteome ............................................................................34
3.2.2
Extraction of soluble proteins...................................................................................34
3.2.3
Two-dimensional electrophoresis.............................................................................35
3.2.4
Gel image analysis and statistical analysis................................................................35
3.2.5
Mass spectrometric identification of proteins ...........................................................35
3.2.6
Fermentative sugars and fermentation end-products analyses ...................................35
RESULTS ............................................................................................................... 36
4.1
Pig feed experiments ...................................................................................................36
4.1.1
The stability of the microbial community in NFLF...................................................36
4.1.2
Characterization of the lactic acid bacteria community in liquid feed and isolation of a
Lactobacillus plantarum strain .............................................................................................36
4.1.3
Effect of the inoculation of Lactobacillus plantarum REB1 into liquid feed .............37
4.1.3.1
Inoculation of Lactobacillus plantarum REB1 in laboratory conditions............37
4.1.3.2
Inoculation of Lactobacillus plantarum REB1 in farm conditions ....................37
4.1.4
4.2
Animal performance ................................................................................................38
Proteomic experiments................................................................................................38
4.2.1
Protein expression patterns ......................................................................................39
4.2.1.1
Proteins differentially expressed between growth phases .................................40
4.2.1.2
Proteins differentially expressed between media ..............................................43
4.2.1.2.1 Fermentative sugars and fermentation end-products .....................................44
5.
DISCUSSION......................................................................................................... 47
5.1
Fermented liquid feed .................................................................................................47
5.1.1
Stability of pig liquid feed .......................................................................................47
5.1.2
Isolation of Lactobacillus plantarum REB1 .............................................................47
5.1.3
Inoculation of Lactobacillus plantarum REB1 in laboratory conditions ....................48
5.1.4
Inoculation of Lactobacillus plantarum REB1 in farm conditions ............................48
5.1.5
Pigs' health and performance....................................................................................48
5.2
Proteomic experiments................................................................................................49
5.2.1
Proteins differentially expressed between growth phases..........................................49
5.2.1.1
Carbohydrate metabolism ................................................................................50
5.2.1.2
Biosynthetic metabolism .................................................................................50
5.2.1.3
Stress response ................................................................................................50
5.2.2
Proteins differentially expressed between media ......................................................51
5.2.2.1
Carbohydrate metabolism ................................................................................51
5.2.2.2
Biosynthetic metabolism................................................................................. 52
5.2.2.3
Stress response ............................................................................................... 52
6.
SUMMARY AND CONCLUSIONS ...................................................................... 53
7.
REFERENCES ....................................................................................................... 53
Introduction
1. INTRODUCTION
1.1 Lactic acid bacteria
Lactic acid bacteria (LAB) are a group of Gram positive, low GC, catalase-negative, acid
tolerant, non-respiring, non-sporulating rod or cocci that synthesise lactic acid as the major
metabolic end-product during the fermentation of carbohydrates (Axelsson, 2004). These
bacteria are a heterogeneous group of organisms with a diverse metabolic capacity. For this
reason, they are highly adapted to a wide range of conditions making them extremely successful
in food and feed fermentations. LAB are responsible for the fermentation of, for example,
sauerkraut, sourdough, cassava, all fermented milks, pickled vegetables and silage (Gardner et
al., 2001; Manso et al., 2004; Eon et al., 2007).
LAB include species from around 20 different genera such as Aerococcus,
Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus,
Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weisella. Lactobacillus is the
largest of these genera, comprising around 80 recognized species (Axelsson, 2004; Makarova et
al., 2006).
LAB are heterotrophic and generally have complex nutritional requirements due to the lack
of some biosynthetic pathways. Most species have several requirements for amino acids and
vitamins. Because of this, LAB can only be found where these requirements can be met
(Charalampopoulos et al., 2002). In sum, they are a group of organisms that are diverse but
physiologically similar, specialized in nutrient-rich environments, limited in biosynthetic ability,
and with a metabolism aimed at acid production.
1.2 Lactobacilli
Lactobacilli are the largest genera in LAB. They are a very heterogeneous group, widespread in
nature and containing the most acid-tolerant species (Klaenhammer et al., 2002; Makarova et al.,
2006). Species such as Lactobacillus plantarum and L. casei can be found in a number of
different environments whereas other species such as L. sanfransiscensis and L. delbrueckii are
found only in certain habitats (Axelsson, 2004).
Lactobacilli can be divided into three groups according to the principal carbohydrate
pathway employed by the species (Axelsson, 2004).
•
Group I, obligate homofermentative: Lactobacilli in this group convert hexoses into
lactic acid via glycolysis, the Embden-Meyerhof pathway, but they are unable to ferment
pentoses or gluconate. It includes species such as L. acidophilus and L. delbrueckii.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
15
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
•
Group II, facultative heterofermentative: Lactobacilli in this group usually ferment
hexoses into lactic acid, but with some strains and under some conditions, hexoses can be
converted into a mixture of lactic acid, carbon dioxide and ethanol (or acetic acid in the
presence of an alternative electron acceptor). Pentoses are fermented into lactic acid and
acetic acid via the phosphoketolase pathway. The group includes species such as L.
plantarum and L. casei.
•
Group III, obligate heterofermentative: Lactobacilli in this group ferment hexoses into a
mixture of lactic acid, carbon dioxide and ethanol (or acetic acid). Pentoses are converted
to lactic acid and acetic acid. This group includes species such as L. brevis and L. reuteri.
1.3 Carbohydrate metabolism of lactic acid bacteria
The metabolism of the LAB is focused on the effective use of many different carbohydrates, the
fermentation of which provides energy (as ATP) for its use in mainly biosynthetic pathways
during cell division. Sugars (mono- and disaccharides) can be generally introduced into the cell
as free sugars or as sugar phosphates. Disaccharides are then hydrolysed to monosaccharides.
Monosaccharides will subsequently enter one of the two main fermentative pathways: glycolysis
or the phosphoketolase pathway (Axelsson, 2004).
1.3.1 Glycolysis
Glycolysis (or the Embden-Meyerhof-Parnas pathway) is one of the fermentative pathways of
LAB where hexoses such as glucose, fructose, galactose and mannose are oxidised to pyruvate,
and subsequently reduced to lactic acid. In glycolysis, when normal conditions such as an excess
of sugars and limited oxygen are present, one mol of hexoses is converted into two mol of lactic
acid. Two mol of ATP are consumed in the upper part of glycolysis (in the two phosphorylation
steps) whereas four mol of ATP are produced in the lower part of the pathway, when two mol of
glyceraldehyde-3-phosphate are converted into two mol of pyruvate. Pyruvate is then reduced to
lactate by a NAD+ dependent lactate dehydrogenase (Figure 1). This step allows the re-oxidation
of NADH, thus equilibrating the redox balance of the cell. This process is referred to as
homolactic fermentation (Axelsson, 2004).
1.3.2 Phosphoketolase pathway
The phosphoketolase pathway (or pentose phosphate pathway, or 6-phosphogluconate pathway)
is responsible for the fermentation of hexoses and also pentoses. In the phosphoketolase
pathway, hexoses and pentoses are transformed to pentose-5-phosphate, by decarboxylation in
the case of hexoses and by phosphorylation in the case of pentoses. The pentose-5-phosphate is
subsequently split to glyceraldehyde-3-phosphate and acetyl phosphate. Glyceraldehyde-3phosphate is incorporated into the lower part of glycolysis, and acetyl phosphate is reduced to
ethanol (Figure 2). This process is referred to as heterolactic fermentation (Axelsson, 2004).
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
16
Introduction
ATP ADP
(1) Galactose
(1) Galactose-1-P
(1) Glucose-1-P
(1) Fructose
Sack
ATP ADP
(1) Glucose
ATP
ADP
Glk
(1) Mannose (ext)
(1) Glucose-6-P
Pgi
Pmi
(1) Fructose-6-P
ATP
ADP
(1) Mannose-6-P
Pfk
(1) Fructose-1,6-DP
Fba
(1) Dihydroxy-acetone P
(1) Glyceraldehyde-3-P
NADH + H+
NAD+
GapB
(2) Glycerate-1,3-DP
ADP
ATP
(2) Glycerate-3-P
(2) Glycerate-2-P
(2) Phosphoenolpyruvate
ADP
ATP
Pyk
(2) Pyruvate
NADH + H+
NAD+
Ldh
(2) Lactate
Figure 1. Glycolysis (Embden-Meyerhof-Parmas pathway). Abbreviations: Fba, fructose
bisphosphate aldolase; GapB, glyceraldehyde-3-phosphate dehydrogenase; Glk, glucokinase; Ldh,
lactate dehydrogenase; Pfk, 6-phosphofructokinase; Pgi, glucose-6-phosphate isomerase; Pmi,
mannose-6-phosphate isomerase; Pyk, pyruvate kinase; Sack, fructokinase.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
17
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
Fructose
Sack
Glucose
Glk
ATP
ADP
ATP
ADP
Glucose-6-P
Fructose-6-P
Pmi
Mannose-6-P
NAD+
Pgm
Glucose-1-P
Gpd
NADH + H+
Galactose-1-P
ATP
ADP
Glucono-1,5-lactone-6-P
Galactose
Mannose (ext)
6-P-gluconate
NAD+
NADH + H+
Gnd
Ribulose-5-P
CO2
RpiA
RbsK
Ribose
Ribose-1-P
ADP ATP
Xylulose-5-P
Xpk
Glyceraldehyde-3-P
NADH + H+
NAD+
Acetyl phosphate
GapB
Glycerate-1,3-DP
ADP
ATP
Acetyl CoA
NADH + H+
NAD+
Glycerate-3-P
Glycerate-2-P
Acd
Acetaldehyde
NADH + H+
NAD+
Adh
Ethanol
Phosphoenolpyruvate
ADP
ATP
Pyk
Pyruvate
NADH + H+
NAD+
Ldh
Lactate
Figure 2. Phosphoketolase pathway (pentose phosphate or 6-phosphogluconate pathway).
Abbreviations: Acd, acetaldehyde dehydrogenase; Adh, alcohol dehydrogenase; GapB,
glyceraldehyde-3-phosphate dehydrogenase; Glk, glucokinase; Gnd, phosphogluconate
dehydrogenase; Gpd, Glucose-6-phosphate 1-dehydrogenase; Ldh, lactate dehydrogenase; Pgm,
phosphoglucomutase; Pmi, mannose-6-phosphate isomerase; Pyk, pyruvate kinase; RbsK, ribokinase;
RpiA, ribose 5-phosphate isomerase A; Sack, fructokinase; Xpk1, phosphoketolase.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
18
Introduction
1.3.3 Pyruvate metabolism
Under normal conditions pyruvate is used as an electron acceptor when reduced to lactate in
either homo- or herterolactic fermentation. This enables the cells to regenerate NAD+, a
molecule necessary for the continuation of the fermentation. However, under different
conditions, the bacteria have alternative ways of consuming pyruvate (Figure 3). The use of
these different pyruvate pathways will determine the final end-products of the fermentations
(Gänzle et al., 2007).
The growth conditions and the metabolic capacity of the bacterial strain will determine the
pathway by which pyruvate will be transformed. Thus, it has been reported that under an excess
of pyruvate and in the presence of oxygen, pyruvate can be transformed to acetolactate, and
subsequently to diacetyl and acetoin. Pyruvate can be reduced to acetyl-CoA, which can be used
for anabolic purposes such as lipid biosynthesis. This pathway can be expressed aerobically by
the enzyme pyruvate dehydrogenase or anaerobically by the pyruvate formate lyase. Acetyl CoA
can also be used as an electron acceptor and be reduced to ethanol, or as a precursor for ATP
formation, resulting in acetate. Acetate formation can also take place more directly from
pyruvate to acetyl phosphate and subsequently to acetate. This pathway has been reported to be
active under glucose limitation (Figure 3) (Axelsson, 2004).
2,3-Butanediol
NADH + H+
NAD+
Diacetyl
2 NADH + 2 H+
2 NAD+
NAD+
Formate
NADH + H+
Acetoin
Pfl
Pdh NADH + H+
Pox
Pyruvate
NADH + H+
NAD+
Ethanol
NAD+
Als
Acetolactate
Acetyl-CoA
Mae
Malate
Ldh
Acetyl-P
NADH + H+
NAD+
Lactate
ADP
ATP
Ack
Acetate
MleS
Figure 3. Pyruvate metabolism. Abbreviations: Ack, acetate kinase; Als, acetolactate synthase; Ldh,
lactate dehydrogenase; Mae, malic enzyme; MleS, malolactic enzyme; Pdh, pyruvate dehydrogenase;
Pfl, pyruvate-formate lyase; Pox, pyruvate oxidase.
1.3.4 Malolactic fermentation
L-malic acid is a common compound in fruits and plants. Two enzymes are responsible for the
degradation of this organic acid. The NAD+-dependent malic enzyme is responsible for the
decarboxylation of malate to pyruvate. Moreover, certain species of LAB possess the capacity to
convert malate directly to lactate by the so-called malolactic enzyme. This reaction is called
malolactic fermentation and it is the most common pathway for the dissipation of malate (Figure
3) (Axelsson, 2004).
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
19
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
The bacteria capable of metabolizing malate via malolactic fermentation show improved
growth characteristics when co-fermented with a carbohydrate such as glucose, compared with
the growth characteristic during the fermentation of glucose alone (Loubriere et al., 1992). The
increased growth rate of the malolactic bacteria when grown on malate-glucose medium is not
fully understood. However, it has been shown that malolactic fermentation generates some
energy advantage to the cells by increasing the internal pH even though the uptake of malate
requires energy (Passos et al., 2003).
1.3.5 Global control of sugar metabolism in LAB
When bacteria are exposed to different carbon sources, they choose the substrate that yields a
maximum profit for growth. To do so, they have developed a sophisticated mechanism that
allows them to sense the nutritional situation and modulate their metabolic capacities by a global
regulatory system called carbon catabolite repression (CCR). CCR in gram-positive bacteria is
involved in sensing the physiological state of the cell and regulating carbon consumption by the
mediation of a component of the phosphoenolpyruvate-dependent phosphotransferase system
(PTS), the phosphocarrier protein HPr, and the global catabolite control protein A (CcpA)
(Titgemeyer and Hillen, 2002).
In the presence of glucose, HPr is mainly histidine-phosphorylated (HPr-His-P), which
activates glucose PTS-dependent transport. When the levels of repressive sugars (other than
glucose) are high, HPr-Ser-P predominates, which interacts with CcpA, increasing the affinity to
bind to the catabolite responsive elements (cre) found in the promotor regions of many catabolic
operons, and mostly represses their transcription. Thus, the ratio of HPr-His-P/HPr-Ser-P
determines the utilization of a particular carbohydrate (Muscariello et al., 2001; Titgemeyer and
Hillen, 2002).
Any alteration of this carbohydrate regulatory system, such as mutations in the PTS
component, CcpA or Hpr, will change the fermentation of sugars and the subsequent formation
of end-products. For example, a ccpA mutant of Lactococcus lactis showed the production of
metabolites characteristic of a mixed-acid fermentation instead of the expected homolactic
fermentation (Luesink et al., 1998).
1.4 Starter cultures
LAB are used in the food and feed industry for their ability to ferment carbohydrates into mainly
lactic acid and, as a consequence, reduce the pH of the medium. This acidification is one of the
most desirable side-effects of LAB fermentations because it prevents the growth of undesirable
bacteria, such as most of the human and animal pathogens, and thus it prolongs the shelf life of
the food and feed (Weinberg et al., 2003; Siezen et al., 2004). Moreover, the acidity can also
change the texture of the medium by protein precipitation, as well as improve its flavour
(McFeeters, 2004; Klaenhammer et al., 2007).
Most fermented foods and feeds are still produced by spontaneous fermentation. This is
characterised by an initiation phase where the amount of LAB is low. During this phase, aerobic
organisms and facultatively anaerobic Enterobacteriaceae are mostly responsible for the
fermentation. When LAB and yeasts reach high amounts, the fermentation is called primary
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
20
Introduction
fermentation. The bacterial growth rate in this phase depends on many factors such as the
original microbial population, the physical and chemical properties of the product and the
environment. LAB are responsible for the production of organic acids that lower the pH of the
product, preventing the development of pathogens. However, the fermentation can also reach the
secondary fermentation which is represented by spoilage bacteria, yeasts and moulds, the
organisms responsible for the use of residual sugars and fermentative acids as substrates (Mäki,
2004).
The natural microflora inherent in foods and feeds is normally inefficient and its
fermentation is uncontrollable and unpredictable. Thus, spontaneous fermentation cannot be
relied to produce enough organic acids to reach a low pH that could prevent the growth of
pathogenic bacteria (Beal et al., 2005). A solution to this problem is the inoculation of a
concentrated culture of bacteria (starter culture) which can provide a more controlled and
predictable fermentation and ensure the safety of the product and, potentially, the safety of the
consumer (Mäki, 2004).
Starter cultures used in food and feed fermentations must possess appropriate and specific
characteristics depending on the qualities desired in the final fermentation (Buckenhüskes, 1993;
Hébert et al., 2000; G-Alegría et al., 2004). For example, when safety aspects are a concern, a
starter culture can be used with high potential for producing lactic acid or antimicrobials such as
bacteriocins (Mäki, 2004). When the aim is to change the texture and aroma, a starter culture
with potential producing, for example, diacetyl and acetoin (compounds associated with butter
aroma in fermented milk products) can be used (Axelsson, 2004).
1.5 Lactobacillus plantarum
Lactobacillus plantarum is a facultative heterofermentative LAB, metabolically very flexible and
versatile, encountered in many environmental niches, and with broad applications, e.g. as a
starter culture in vegetable (Salovaara, 2004) and meat (Ammor and Mayo, 2007) fermentations;
as probiotic for humans (Goossens et al., 2005) and animals (Demecková et al., 2002); and lately
as a delivery vehicle for therapeutic compounds (Pavan et al., 2000).
The complete genome of one L. plantarum strain has recently been sequenced
(Kleerebezem et al., 2003). This strain has been shown to possess the largest chromosome size
(3.3Mb) within LAB. It has a circular chromosome with 3052 potential protein-encoding genes
and more than 2500 predicted proteins with assigned biological function. The genome encodes
all enzymes required for the glycolysis and phosphoketolase pathway, as well as a number of
enzymes for a large pyruvate-dissipating potential. The sequence of L. plantarum strain WCFS1
also revealed a region in the chromosome containing genes involved in sugar transport and
utilization. This gene cluster has been called a "lifestyle adaptation region", suggested for use
when the bacteria need to adapt efficiently to environmental changes (Kleerebezem et al., 2003).
Moreover, microarray-based genotyping studies have shown that the gene categories
exceptionally conserved in L. plantarum WCFS1 were those involved in biosynthesis and
degradation of structural compounds such as proteins, lipids and DNA (Molenaar et al., 2005).
Lactobacillus plantarum has been much investigated due to its wide adaptation capacity
and its numerous applications. Research has been mainly focused on the bacterial responses to
stress factors such as heat shock (De Angelis et al., 2004), the presence of high concentrations of
lactic acid (Pieterse et al., 2005) and bile (Bron et al., 2006), as well as low pH and ethanol (GKuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
21
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
Alegría et al., 2004). However, new studies on the dynamics of the global metabolism of strains
of L. plantarum are emerging (Molenaar et al., 2005; Boekhorst et al., 2006; Cohen et al., 2006).
1.6 Pig feed
1.6.1 Background on pig nutrition and feed formulations
Pig production represents an important part of the food animal industry throughout the world.
Pork is a good source of energy, proteins, minerals and vitamins, and it is the most widely
consumed red meat in the world (NRC, 1998). Proper formulation of pig diet is fundamental to
efficient pork production. Moreover, feeding pigs a balanced diet is an essential part of the pig
profit equation. Since feed accounts for 55 to 75% of total costs, feeding and nutrition can make
a huge difference to piggery profits (NRC, 1998). The nutritional requirements of pigs vary
according to the climate, environment, and the sex, genotype, weight and age of the pigs.
Swine operation units may include the different stages of the pig life cycle, from farrowing
to finish units, or combinations of separate units, including nursery units, grower-finishing units
or breeding units. Each stage requires different diets, resulting in great differences in the volume
and nutrient composition (Brooks et al., 2003a).
The nutritional requirements of the pigs are basically represented by the digestible energy
(DE) and protein (amino acid) content of the feed. DE is used for the maintenance, growth and
reproduction of the pigs, and it represents only the energy in the diet that is digested and
available. Proteins are used for growth and especially for muscle tissue development. In order to
synthesise proteins, amino acids are required and each one is equally important. However, the
so-called essential amino acids assume greater importance for the diet formulation due to the
inability of the pigs to synthesise them (Brooks et al., 2003a).
Pigs need both energy and protein to prosper and the balance between them should be
corrected frequently as the pigs grow. This balance is expressed as a ratio (g lysine/MJ DE),
lysine being the first limiting amino acid of most cereal-based diets. The correct lysine to energy
ratio in the diet will optimise pig performance. If there is an excess of energy in the diet and it is
not balanced with lysine, pigs will convert the oversupply of energy into fat. On the other hand,
an excess of lysine would be a waste of protein, an expensive part of the diet (Brooks et al.,
2003b).
As pigs get older, they put on a greater amount of fat and less lean meat than younger pigs.
Thus, younger pigs need a diet higher in amino acids than older pigs so they can grow
proportionally more muscle tissue. Furthermore, as pigs get older, their need for lysine falls and
a greater proportion of the food energy is required for maintenance rather than growth.
Consequently, the rate of lean growth declines continuously with increasing live-weight of the
animal (Brooks et al., 2003a).
Only a limited number of pig units use more than two or three diets throughout the whole
growth of the pigs in order to accomplish the continuous adjustment of lysine to DE.
Consequently, grow-finish pigs on most production units are supplied with an excess of protein
and other nutrients, which will result in an excessive amount of excreted nutrient in faeces and
urine. In earlier times, this manure, which contains relatively high amounts of nitrogen,
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
22
Introduction
phosphorus, potassium and other nutrients, was an excellent fertilizer when applied to land.
However, during the past decade, the number of large pig units and the intensity of the
production have rapidly increased, leading to a potential surface and ground water contamination
and to an accumulation of minerals in the soil (NRC, 1998; Brooks et al., 2003a).
A possible solution to this problem involves modern computerized liquid feeding facilities,
which are capable of delivering diets of different nutrient balance to specific pens of pigs. This
can be pre-set onto the feeding computer from the first day and according to a feed growth curve.
The benefits of this feeding system (phase feeding) include a progressive reduction in diet costs,
a potential improvement in feed efficiency, and a decrease in nutrient excretion and reduction of
potential contaminants (Brooks et al., 2003a).
1.6.2 Liquid feeding (Check study III for a review)
Traditionally many pigs were fed a diet mixed with whey or water. With the increase in farm
size, there was a need for automatic systems and the pig industry switched to dry feeding
systems. However, recent developments in computer driven systems have led to a revival of
liquid feeding (MLC, 2003).
Liquid feeding can be defined and differentiated from other feeding systems because it
involves the use of a diet that is prepared from either liquid or dry components and mixed with
water. The final dry matter proportion is generally between 20 and 30%. These liquid diets are
thoroughly mixed in the pig unit and distributed to the pigs through a computerized system. If
the pig unit is rearing different pig ages and with different production objectives, the system
allows the manufacture of a range of diets to match their specific nutrient requirements (Brooks
et al., 2003a).
One reason for the installation of liquid feed delivery systems for pigs is mainly the readily
available liquid residues from food industries. The use of liquid co-products allows not only the
recycling of those products, which are an important environmental problem for the food industry,
but also the reduction of feed costs (Scholten et al., 1999). A nutritional problem related to the
use of co-products is the variability of their composition. However, these products can be used
efficiently and without causing any damage to pig performance if diets are reformulated
continuously to balance the changes that may occur in the composition between loads and during
farm storage (Brooks et al., 2003b).
There are other more practical advantages of liquid diets compared with dry feeds. The use
of liquid feed reduces the food waste, as dust, when handling and feeding. Consequently, there is
a reduction of dust in the atmosphere, improving the pigs’environment and health by reducing
respiratory problems (Russell et al., 1996). Liquid feed, compared with dry feed, also provides a
diet more palatable especially for weaning piglets, which have consumed very little solid food
and are unfamiliar with the idea of drinking water. Generally, the stress associated with the diet
change or the transfer to a different unit could be reduced by providing the pigs with a more
familiar and palatable diet such as a liquid diet (Brooks et al., 2001).
Some disadvantages associated with liquid diets compared to dry diets have been reported.
One example is the risk of ulceration in the oesophageal region of the stomach [oesophagogastric ulcer (OGU)] due to the relatively high level of undissociated volatile fatty acids entering
the squamous epithelial cells (Krakowka et al., 1998; Robertson et al., 2002). Another
disadvantage is related to the free amino acid levels, especially lysine, which appears to be lower
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
23
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
in liquid diets than in dry diets due to microbial amino acid decarboxylation during fermentation
(Niven et al., 2006; Canibe et al., 2007a, 2007b). However, it is not clear yet which microorganisms are mainly responsible for the amino acid degradation, although Niven et al. (2006)
reported to be due to the metabolism of E. coli.
Liquid feeding systems can distribute the feed to the pen trough either at a number of times
per day or ad libitum. Many restricted feeding systems rely on a feed curve to steadily increase
feed allowance per pig per day. This is calibrated taking into account the lysine to energy ratio of
the pigs. Ad libitum systems are becoming more popular because they have been suggested to
improve meat tenderness. This feeding system allows the pigs to determine their own daily
routines by their feed consumption behaviour. However, there are certain genotypes that may
result disadvantaged due to the excessive fat deposition in the finishing stage, as the pigs will
consume an excess of energy per day (MLC, 2003).
Liquid feed has been much investigated during the last decade because it has been
considered an alternative to the use of antibiotics as growth promoters in animal production
(Mikkelsen and Jensen, 1998; Canibe et al., 2007a). This research on liquid pig feeds has shown
a number of beneficial effects, such as (Kim et al., 2001; van Winsen et al., 2001b; Canibe and
Jensen, 2003):
•
•
•
•
•
•
increase in feed utilization and digestibility
improvement in growth performance of pigs in all ages
increase in lactic acid and volatile fatty acids in the stomach of pigs
decrease in pH in the stomach of pigs
decrease in Enterobacteriaceae along the gastrointestinal tract of pigs
improvement in villus:crypt ratio in the small intestine of pigs.
These beneficial effects are associated with the fermentation that spontaneously occurs
when feed is mixed with water (Beal et al., 2002). The fermentation is the result of the
proliferation of the microbial population inherent in the raw material of the feed. This microbial
population could be very variable from one feed formulation to another, but it basically contains
LAB and yeasts, but it might also contain some pathogenic microbes such as coliforms,
Salmonella and moulds.
There are a number of parameters that influence the establishment and development of the
bacterial fermentation in a liquid diet. Some of those parameters are:
•
•
•
•
•
the type and quantity of bacteria in the raw materials
the growth potential of those bacteria
the temperature of the liquid feed
the temperature of the water added to the feed
the pH of the liquid feed.
1.6.2.1 Fermented liquid pig feed
The liquid feed could be considered to be fermented or non-fermented, in relation to the time that
the ingredients spend in the water. Thus, non-fermented liquid feed (NFLF) is liquid feed that
has been prepared immediately before it is fed to the pig or the feed that is just mixed directly in
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
24
Introduction
the troughs. Fermented liquid feed (FLF) is defined here as feed in which fermentation has been
induced by:
•
•
•
steeping the feed in water for a certain time and at a certain temperature
backslopping
an starter culture such as a LAB inoculant.
However, NFLF may become spontaneously FLF if some feed remains in the tanks
(backslopping) or when the feed is served ad libitum (Beal et al., 2002).
A liquid diet is reported to be properly fermented when there are high numbers of LAB
dominating the fermentation. For their growth, LAB consume the complex carbohydrates from
the feed, converting them into mainly lactic acid as well as other organic acids. Consequently,
the pH of the medium decreases and the environment becomes hostile to contaminants such as
Enterobacteriaceae (Canibe et al., 2007a). However, many fermentation processes do not reach
high enough levels of LAB and organic acids, and therefore the pH is not low enough to prevent
the growth of pathogenic bacteria (Beal et al., 2005).
The time before the liquid feed reaches a proper fermentation is also very important
(Canibe and Jensen, 2003). During this time (first phase of fermentation), the number of LAB
and the concentration of organic acids are low and thus the pH is high. There is also the
possibility that a bloom of yeasts and Enterobacteriaceae may occur. The growth of
inappropriate yeasts is not desirable because they produce off-flavours and taints, making the
feed unpalatable to the pigs (Savard et al., 2002; Brooks et al., 2003).
In order to skip the first phase of fermentation, the addition of a concentrated LAB strain
as a starter culture to the liquid feed has been shown to be a very effective approach (Canibe et
al., 2001; van Winsen et al., 2001a, 2001b; Beal et al., 2002). The addition of an organic acid to
the feed in order to decrease the pH has also been shown to prevent the growth of
Enterobacteriaceae (Geary et al., 1999; Canibe et al., 2001) although it apparently has no effect
on yeasts (Canibe et al., 2007a). Moreover, adding an organic acid to the feed results to be a
more expensive approach (Geary et al., 1999).
1.6.2.2 Pig growth performance
It seems logical for weaning pigs to get use to a liquid diet rather than a dry diet. Liquid feed
provides the advantage of preventing dehydration due to the pigs’unknown separate eating and
drinking behaviour (Brooks et al., 2001). In addition, liquid feed has become very popular
because it might improve growth performance and influence the bacterial ecology of the
gastrointestinal tract (GIT) of the pigs (Russell et al., 1996; van Winsen et al., 2001b).
Many studies have demonstrated that both weaning and growing-finishing pigs perform
better with liquid feed than dry feed (Brooks et al., 2003; Russell et al., 1996; Kim et al., 2001;
van Winsen et al., 2001b). However, Lawlor et al. (2002) found no benefit and even a decrease
in daily weight gain in 27-day weaned pigs fed liquid feed compared with dry fed pigs, and there
is considerable variation between different studies for growing-finishing pigs (Brooks et al.,
2003; Canibe and Jensen, 2003). These studies mainly showed no differences in growth
performance between pigs fed dry or liquid diets.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
25
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
Feeding a pig with FLF has an effect on the GIT of the animals (Deprez et al., 1987;
Scholten et al., 2002): it has been reported to increase the LAB and yeasts population and
decrease the pH in the stomach of animals (Canibe and Jensen, 2003). However, an increase in
pH in the small intestine, probably due to the secretion of pancreatic juice stimulated by the high
levels of lactic acid in the feed, has been reported (Mikkelsen and Jensen, 1998). However, in
general it seems that feeding a fermented liquid diet reduces the Enterobaceriaceae in the entire
GIT even though the LAB population remain unaltered (Højberg et al., 2003).
1.7 Proteomics
Post-genomic tools and technologies have changed the experimental approaches by which
complex biological systems can be characterised. Proteomics has become one of the most
important techniques in functional genome characterization. Proteomics is a large-scale study of
the whole cell protein content. It is mainly a measurement of the presence and abundance of
proteins, but it can also give information such as the protein sequence, modification state,
molecular interaction, activity and structure. Proteins are normally the functional molecules of a
cell, and thus they reflect differences in the gene expression (Bendixen, 2005). Genomics and
transcriptomics cannot be directly related with the actual state of a cell because genes can be
present but not functional, and the number of mRNA copies does not always reflect the number
of functional proteins present. Thus, proteomics has the potential to provide information on the
protein expression and subsequently on the function of genes, with the ultimate goal of
explaining how the environment interacts in order to control cellular functions and form the
physiological traits of that cell (Morelli et al., 2004; Mullen et al., 2006)
Proteomics is a tool developed mainly on the basis of two-dimensional electrophoresis (2DE) coupled with mass spectrometry (MS) for the analysing of several hundreds of proteins
simultaneously (Figure 4) (Cash, 2000; Gygi et al., 2000; Champomier-Vergès, 2002).
Proteomics is not a new technique: it actually started in 1975 with the introduction of the first 2D gels, which were used for mapping proteins from Escherichia coli (O'Farrell 1975), mouse
(Klose 1975) and guinea pig (Scheele 1975). However, even though the proteins could be
separated and visualised, they could not be identified. The first major technology to appear for
the improvement of proteomics was the sequencing of proteins by Edman degradation. It
allowed the identification of proteins from 2-D gels and the generation of the first 2-D databases.
Protein identification was later improved by the development of MS technology, which increased
the sensitivity and accuracy of the protein identification results. But the biggest growth on
proteomics started right after the results from large-scale nucleotide sequencing projects.
Because of the amount of information that these projects provided, it was possible to reach a
level of protein identification that would have been difficult to acquire with the improvements
made in MS technology. Thus, protein identification relies basically on the existence of some
form of database for a given organism (Graves and Haystead, 2002).
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
26
Introduction
Figure 4. Proteomic schematic representation. A) Sample preparation, 2-dimensional electrophoresis and gel
analysis. B) Protein identification by mass spectrometric analysis (Encarnación et al., 2005).
Proteomics has been used for mapping (analogously to genome sequencing) by
characterizing and making a comprehensive database of the cellular proteome. However,
compared with genomics, proteomics is far more complicated not only due to the complex
variety of protein modification forms, but also because of the extra work required for the
proteome analysis of a cell that it is constantly changing its physiological state over time. In this
sense, every single cell has an infinite number of proteomes. The complexity behind global
proteomics analysis has made this technique evolve into so-called comparative proteomics. This
refers to comparative studies that are based at parallel quantitative protein expression patterns,
and focusing only on the proteins with deferential protein expression (Monteoliva and Albar
2004; Manso et al., 2005; Mullen et al., 2006).
1.7.1 2-DE
2-DE is an electrophoretic method for the analysis of complex protein mixtures extracted from
cells, tissues or other biological samples. The technique separates the proteins according to two
different properties and in two simple steps. In the first step, the proteins are separated by their
net charge or isoelectric point (pI) (first dimension), and in the second step, the proteins are
separated according to their molecular mass (Mr) (second dimension). One of the main strengths
of 2-DE is the ability to separate proteins with different post-tranlational modifications (PTM).
This is because many of those modifications confer a difference in charge or a difference in
mass. For example, a single phosphoprotein may appear in a 2-DE as multiple spots with
differences in pI (Graves and Haystead, 2002).
After 2-DE the protein spots are stained with e.g. coomassie blue, silver-staining or
fluorescent dyes. Once the 2-DE gels are scanned, the gels are analysed. There are a number of
software programs available for 2-DE gel image analyses, which allow the normalization of the
spots intensities, background subtraction and comparative quantification of the spots from two
samples (Görg et al., 2004). Thus, the proteins expression from two different samples can be
qualitatively and quantitatively compared.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
27
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
1.7.2 Mass Spectrometry
MS makes it possible to obtain information such as peptide mass or amino acid sequence. This
information is then used to identify the protein by searching nucleotide and protein databases. In
order to get this information, proteins go through a three step process:
•
•
•
Sample preparation, where spots are extracted from the gels and digested mainly with
trypsin. After digestion, the sample is purified to remove gel contaminants, and normally
concentrated.
Sample ionization, where peptides are charged (converted to ions by the addition or loss
of protons) by methods such as electrospray ioniztion (ESI) or matrix-assisted laser
desorption/ionization (MALDI).
Mass analysis, where the mass analyser of a mass spectrometer follow the conversion of
the peptides to ions. Examples of mass analyser are quadrupole, time of flight (TOF) and
ion trap.
Generally, most of the mass spectrometers consist of four basic elements: an ionization
source, one or more mass analysers, an ion mirror, and a detector. The names of the mass
spectrometers are derived from the names of the ionization source and the mass analysers. Thus,
some of the most common ones are quadrupole-TOF, MALDI-TOF, MALDI-QqTOF, and triple
quadrupole (Graves and Haystead, 2002).
1.7.3 Proteomics latest developments and limitations
Since proteomics started, new techniques have been developed and a number of improvements
have been made to the old ones. Thus, the introduction of immobilized pH gradients increased
considerably the reproducibility of 2-DE, the use of fluorescent dyes improved the sensitivity of
the spot detection, and specialized pH gradients allowed the resolution of more spots per gel.
Moreover, there has been an introduction of new and accurate mass spectrometers, and the large
amount of information that has been lately available in databases after the sequencing of many
new genomes (Görg et al., 2004) has led to a revival of proteomics.
However, there are a number of problems in proteomics basically due to the limitations of
this technique. For example, it is still a very laborious and time-consuming technique despite the
efforts to automate 2-DE. Another serious problem of 2-DE is that the analysis is constrained to
only a limited subset of the cellular proteins. Thus, proteomics excludes:
•
•
•
hydrophobic proteins such as membrane proteins
extremely acid or basic proteins (spots with pI below 3 or above 10, spots not well
represented) (Görg et al., 2000)
proteins larger than 200 kDa or smaller than 15 kDa (Görg et al., 2000; Guelfi et al.,
2006, abstract).
Another problem is the visibility of low-copy proteins. High abundance proteins can
dominate the gel image, making the low abundance proteins undetectable (Graves and Haystead,
2002). Proteomics also faces the problem that a single spot could be represented by two or more
proteins. Restringing the 2-DE pH ranges helps improve the separation of proteins with similar
migration and their subsequent quantification. However, the actual problem is the fact that
different proteins may have exactly the same pI and Mr, thus migrating to the same place. The
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
28
Introduction
overlapping of spots in 2-DE is a practical problem that has been frequently reported and no
immediate solution has been proposed to date (Pietrogrande et al., 2003).
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
29
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
2. AIMS OF THE STUDY
The overall aim of the present study was to find and characterize a lactic acid bacterium with
high potential abilities for use as a starter culture. Thereafter, the aims were expanded and the
isolated lactic acid bacterium was subsequently characterized at the protein level in order to
elucidate the mechanisms and abilities of this bacterium in actual fermentation processes.
The specific questions of each individual study were:
•
To study and characterize the microbial stability of a particular pig liquid feed. (I)
•
To characterize and identify the lactic acid population encountered in the pig liquid feed
samples, and to choose a strain for potential use as starter culture. (I, II)
•
To check the potential use of the bacterial strain as starter culture in liquid feed
formulated in laboratory conditions. (II)
•
To check the potential use of the bacterial strain as starter culture in liquid feed
formulated in a commercial farm. (II)
•
To analyse the different strategies used for feeding pigs a fermented liquid feed and its
effects on the feed and pigs. (III)
•
To reach a better understanding of the physiology and metabolic potential of
Lactobacillus plantarum during its growth in a standard medium. (IV)
•
To expand our knowledge of Lactobacillus plantarum by studying the metabolic
potential when fermenting different vegetable media. (V)
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
30
Materials and Methods
3. MATERIALS AND METHODS
3.1 Pig feed experiments
3.1.1 Bacterial strains
Lactobacillus plantarum REB1 was collected from the LAB isolated in liquid feed as shown in
study I. This bacterium was chosen for being the dominant strain in the feed analysed. A
rifampicin-resistant mutant from this strain was obtained, characterized, and used for the
fermentation of the liquid feed as described in study II.
3.1.2 Microbial analyses of liquid feed
The stability of the microbial community developed in liquid feed in farm conditions was
followed for three months as described in study I. The microbial groups analysed were total
aerobic bacteria, LAB, proteolytic bacteria, lipolytic bacteria, amylolytic bacteria, Bacillus
cereus, and yeasts and moulds. Randomly selected LAB colonies from MRS (De Man, Rogosa
and Sharpe) agar (Lab M, Lancashire, UK) plates were characterized using a biochemical
commercial test as described in study I. The dominating species were confirmed by PCR
amplifications, by sequencing the 16S rDNA-V1 region as described in study II. The bacteria
were further characterised at the strain level by pulsed field gel electrophoresis (PFGE) as
described in study II.
Lactobacillus plantarum REB1 was used to inoculate fresh liquid feed (study II). In order
to follow the strain inoculated in the liquid feed, selection of a rifampicin-resistant mutant was
carried out using rifampicin gradient plates as described in study II. The effects of the inoculated
strain on the endogenous microflora of the liquid feed (especially on the yeasts and
Enterobacteriaceae groups) were followed as described in study II. The microbial groups
analysed were LAB, L. plantarum REB1-RifR, yeasts and Enterobacteriaceae from NFLF and
FLF in laboratory and farm scale experiments, and from faecal samples (except for yeasts),
during the three-month period examined.
3.1.3 Laboratory scale liquid feed fermentations
These fermentations were done in order to confirm the growth potential of L. plantarum REB1RifR in liquid feed, to check its compatibility with other LAB populations, and to determine the
effects on feed concentrations of Enterobacteriaceae and yeasts. Two liquid feed samples were
prepared and inoculated with L. plantarum REB1 (8 log cfu/ml). The samples were designed as
laboratory feed (labFLF), and 75% replenished laboratory feed (labRFLF). In labRFLF, 75% of
the feed was replaced with fresh feed twice daily in order to simulate actual feeding conditions.
The monitoring of the microbial population lasted for seven days (study II). The feed
composition is shown in Table 1.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
31
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
3.1.4 Farm scale experiments
3.1.4.1 Commercial farm
The pig unit was located in Vehmersalmi, 50 km southeast of Kuopio, Finland. The farm was
adapted to the growing-finishing stages. The pigs arrived at the farm weighing approximately 25
kg and were sent to the slaughterhouse weighing approximately 80 kg, after a period of three
months. The farm contained 20 separate pens all located in the same shed. Ten pens were
separated from the other ten by a one-meter wide corridor. There were 13 pigs per pen with the
exception of one pen that was smaller and permitted only seven pigs. Thus, the total number of
pigs allowed was 254. The feed was prepared in two separate 1800 litre tanks and distributed to
the pigs through an automated feeding system. The tanks were located in a separate room. The
tanks and pipes were cleaned only before the start of each trial (studies I-II).
3.1.4.2 Liquid feed
The feed formulation is showed in Table 1. Dry feed was mixed with three parts of water. Feed
was prepared automatically four times per day during approximately the first month of the trial
(until the pigs weighed about 60 kg) and five times per day for the rest of the trial. The fresh feed
prepared was always mixed with older feed remaining in the tanks (backslopping). The feeding
regime started with 1.2 feed units per day, reaching 3 feed units per day at the end (one feed unit
correspond to 3.65 kg of feed). The feed amount increased progressively during the trial and
varied according to the appetite of the animals. The temperature of the feed varied between 14
and 23 C (studies I-II).
Table 1. Composition of the diets as fed basis (Polarfami Oy, Kuopio, Finland).
Ingredients
g/kg
Dry matter concentration (%)
Barley
690
86-100
Concentrated liquid whey (Polarfarmi Oy)
180
26-28
Soyabean meal (3% fat)
128
87
2
99
Mineral and Vitamin premix (Oy Feedmix Ab, Koskenkorva, Finland)
a
Nutrients
g/kg
Proteins
51.45
Fat
22.67
Fibre
11.75
Starch
152.12
Lycine
2.07
Methionine
0.64
Cystine
0.72
Threonine
1.43
0.25
Tryptophan
3.71
Minerals and Vitamins
a
Liquid feed dry matter: 27%
b
Minerals and Vitamins (in mg/kg or iu/kg): Ca, 700 mg; P, 1,970 mg; Mg, 510 mg; Na, 320 mg; Fe 22.36 mg; Cu,
3.51 mg; Zn, 12.95 mg; Se, 0.04 mg; vitamin A, 139.75 iu; vitamin D, 12.70 iu; vitamin E, 8.45 mg; vitamin B1,
1.41 mg; vitamin B2, 1.11 mg; vitamin B6, 1.25 mg; biotin, 0.03 mg.
b
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
32
Materials and Methods
3.1.4.2.1 Non-Fermented Liquid Feed
NFLF was moderately acidified with formic acid in order to prevent uncontrolled growth of
Enterobacteriaceae. Formic acid (76%) was added (0.9 l/metric tonne) twice per day during the
first week, and only when the pH of the feed exceeded 5 during the rest of the trial. NFLF was
mixed approximately one hour before feeding for practical reasons. From 10 to 15% of the older
feed remained in the tank at every refilling (studies I-II).
3.1.4.2.2 Fermented Liquid Feed
The fermentation in FLF was initiated by mixing the feed with feed pre-fermented by L.
plantarum REB1-RifR in laboratory conditions to give a final concentration of approximately 8
log cfu/ml liquid feed. The bacteria used to inoculate the feed were allowed to grow overnight in
liquid feed in smaller tanks before being added to the actual tank (Figure 5). FLF was reinoculation with L. plantarum REB1-RifR when counts dropped to 5-6 log cfu/ml.
x4
x2
x4
0.5 l
15 l
50 l
L. plantarum
REB1-RifR
Inoculated liquid feed
(overnight incubation)
Inoculated liquid feed
(overnight incubation)
In laboratory
1800 l
~8 log cfu/ml L. plantarum
REB1-RifR in liquid feed
In commercial farm
Figure 5. Steps followed to achieve a fermented liquid feed with approximately 8 log cfu/ml L. plantarum REB1RifR.
FLF was mixed immediately after feeding to enable it to ferment in the tank for about 3.5 –
5 h during the day and 10 h during the night. The amount of residual feed was approximately
50% during the first month of the trial, and progressively decreasing to approximately 25% at
end of the trial (study II).
3.1.5 Pigs' performance
Pigs were weighed three times during the growth period in study II. Performance was compared
between pigs fed with NFLF and FLF. Additionally, faecal samples were collected and analysed
at one or two week periods throughout the cycle.
3.1.6 Statistical analysis
All bacterial counts were log transformed. Simple linear regression analysis was performed in
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
33
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
order to examine the bacterial stability of the liquid feed, as described in study I. An
independent-sample t-test was used in order to analyse the differences between LAB and L.
plantarum REB1 counts in laboratory experiments, as well as for differences between pigs'
weights, as described in study II. All analyses were done using SPSS software (Chigago, IL,
USA).
3.2 Proteomic experiments
3.2.1 Bacterial strains and their cultivation
Two different strains of L. plantarum were used in proteomics experiments. L. plantarum strain
REB1 was isolated from a spontaneously fermented cereal-based feed (studies I-II), and L.
plantarum strain MLBPL1 from a spontaneously fermented white cabbage (MTT Agrifood
research Finland, Jokioinen, Finland). Two per cent of an overnight aerobic culture grown to
late-stationary phase in MRS broth (Lab M, Lancashire, UK) was used to inoculate the actual
samples (MRS broth in studies IV-V, and cucumber juice and pig liquid feed in study V). These
cultures were aerobically incubated at 30°C with slow agitation (150 rpm). Five independent
samples were made for each strain (five biological replicates). The bacteria were pelleted (3000
x g, 5 min, 4°C) and frozen immediately in liquid N2. The samples were stored at -70 °C (studies
IV-V).
3.2.1.1 Growth phase-dependent proteome
The medium used for the growth phase-dependent proteome was MRS broth, which is a
standard-rich medium. From each replicate, four samples were taken representing the following
growth phases: lag phase (OD660 = 0.1), early-exponential phase (OD660 = 0.7), late-exponential
phase (OD660 = 1.5), and early-stationary phase (OD660 = 2.0). The growth phases were
confirmed by calibrating the OD660 readings against CFU counts (study IV).
3.2.1.2 Media- dependent proteome
The three media used for the media-dependent proteome were cucumber juice, pig liquid feed
and MRS broth. Samples were taken after 8 hours of incubation, corresponding to the
exponential growth phase. In order to calculate the exact growth of each batch, the bacterial
counts of each sample at 8 hours were determined by plating in MRS plates. Pig liquid feed and
cucumber juice samples were subsequently filtrated before freezing (study V).
3.2.2 Extraction of soluble proteins
The extraction of the cytosolic proteins was done as described in studies IV-V. Briefly, the cells
were washed and lysed with lysozyme. After DNase treatment, samples were centrifuged and the
supernatants were collected. The proteins were subsequently precipitated with ice-cold acetone
with trichloroacetic acid. After an overnight incubation at -20°C, the proteins were washed with
acetone and the pellets dried and stored at -70°C.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
34
Materials and Methods
3.2.3 Two-dimensional electrophoresis
The proteins were solubilized and protein concentrations calculated as described in studies IV-V.
For isoelectric focusing, 24 cm IPG strips with a linear pH range of 4-7 (Amersham Biosciences,
Uppsala, Sweden) were used. The focusing was done in an Ettan IPGPhor system (Amersham
Biosciences). The strips were subsequently stored at -70°C. Prior to the SDS-PAGE, the strips
were equilibrated. The SDS-PAGE was run overnight in 19 x 23 cm gels in a Hoefer DALT
system (Amersham Biosciences). The gels were stained with SYPRO orange (study IV) and
ruby (study V) (Amersham Biosciences) following the manufacturer's instructions. Gel images
were acquired with an FLA-3000 fluorescent image analyzer (Fuji Photo Film Co. Ltd., Tokyo,
Japan).
3.2.4 Gel image analysis and statistical analysis
Image analysis was performed with PDQuest software (Bio-Rad, Hercules, CA, USA). Spot
intensities were normalised to the total intensity of valid spots to minimize errors caused by
differences in the amount of proteins and in staining intensity. Differences in spot intensities in
the growth-phase gels (study IV) were analysed statistically with Friedman's test using the SPSS
software package (SPSS, Chicago, IL, USA). Differences in spot intensities in the growth media
gels (study V) were analysed statistically with the nonparametric Kruskal-Wallis test and
principal component analysis (PCA) using SPSS software package. The differences in both
statistical tests were considered significant at the level of P<0.01.
3.2.5 Mass spectrometric identification of proteins
The SYPRO orange- or ruby-stained 2-DE gels were re-stained with silver using a mass
spectrometry-compatible method (Shevchenko et al., 1996). Tryptic digestion of the protein
spots excised from the gels and sample preparation were performed according to Koistinen et al.
(2002).
The tryptic peptides were analysed with one of the two mass spectrometry systems
described by Lehesranta et al. (2005). The peptides were identified against the L. plantarum
genome (Kleerebezem et al., 2003) or the complete proteome UniProtKB, NCBI non-redundant
protein database, or known proteins from Lactobacillae. Matches of MS/MS spectra against
sequences in the databases were verified manually.
3.2.6 Fermentative sugars and fermentation end-products analyses
All samples for the media-dependent proteome were additionally subjected to HPLC analysis in
order to detect the consumption of sugars and the production of organic acids. HPLC (Agilent
technologies, Santa Clara, California, USA) analyses were performed as described in study V.
Briefly, monosaccharides and organic acids were pre-treated with sulfuric acid, centrifuged and
filtrated. The samples were subsequently analysed with REZEX RHM-Monosaccharide H + (8%)
column (Phenomenex Inc., Torrance, California, USA) eluted with 0.4 ml/min of 0.5 mM
sulfuric acid. Disaccharides were centrifuged, filtrated and subsequently analysed with a
ZORBAX (250 x 4.6 mm ID) carbohydrate column (Agilent technologies) eluted with 1.4
ml/min of 80% acetonitrile.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
35
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
4. RESULTS
4.1 Pig feed experiments
4.1.1 The stability of the microbial community in NFLF
Pig liquid feed was examined for different bacterial groups in order to check the microbial
stability during a period of approximately three months (12 weeks). The results showed that in
just one week, there were already high numbers of total aerobic bacteria and LAB (approx. 7.5
log cfu/ml). Lipolytic, proteolytic and amylolytic bacteria as well as yeasts and moulds showed
numbers of around 5 log cfu/ml although proteolytic bacteria numbers were very low (2 log
cfu/ml) during the first week. Generally, all bacterial groups showed a tendency to elevated
counts towards the end of feeding period, and a high degree of stability, except for amylolytic
bacteria, the numbers of which were rather variable (Figure 6-7). Yeast and mould numbers
were also rather variable with high counts in some specific weeks (study I).
Figure 6. Microbial counts (log cfu/ml) in pig liquid
feed during a period of 3 months.
Figure 7. Microbial counts (log cfu/ml) in pig liquid
feed during a period of 3 months.
4.1.2 Characterization of the lactic acid bacteria community in liquid feed
and isolation of a Lactobacillus plantarum strain
Approximately one hundred colonies randomly selected from different MRS plates cultivated
with liquid feed were characterized. Lactobacilli accounted for 65% of the colonies, followed by
Leuconostocs with 21.6%. Pediococci (10.7%) and Lactococci (2.7%) accounted for the rest of
the colonies examined (study I).
Within the Lactobacilli group, several colonies were tentatively identified as L. plantarum.
The colonies identified were further characterised by PCR at the species level and PFGE at the
strain level. The results showed that all colonies identified as L. plantarum were the same strain.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
36
Results
This strain was noted as L. plantarum REB1 (studies I-II).
4.1.3 Effect of the inoculation of Lactobacillus plantarum REB1 into liquid
feed
4.1.3.1 Inoculation of Lactobacillus plantarum REB1 in laboratory conditions
Two samples of liquid feed (labFLF and labRFLF) were inoculated with L. plantarum REB1 (8
log cfu/ml) and allowed to ferment at room temperature for a week. Only in labRFLF, 75% of
feed was replaced twice daily with fresh liquid feed. The results showed that there were no
differences between L. plantarum REB1 and the total LAB counts, indicating that L. plantarum
REB1 dominated the fermentation of the feed (Figure 8-9). Moreover, the results showed the
growth potential of this bacterium in this medium, reaching numbers of approximately 10 log
cfu/ml in only 24 hours after the inoculation. Enterobacteriaceae were not detected in any of the
samples.
Figure 8. Bacterial counts and pH
measurements in laboratory fermented liquid
feed.
Figure 9. Bacterial counts and pH
measurements in laboratory fermented liquid
feed with 75% removed feed and replaces
with fresh feed twice a week.
Sample labRFLF showed fluctuating and slightly lower L. plantarum REB1 counts than in
labFLF. It also showed fluctuating and slightly higher pH. However, yeasts were more variable
in labFLF, whereas they were almost undetectable in labRFLF (Figure 8-9) (study II).
4.1.3.2 Inoculation of Lactobacillus plantarum REB1 in farm conditions
Lactobacillus plantarum REB1 was inoculated into the farm liquid feed. The bacterial
examinations were compared with the ones from the control liquid feed (non-inoculated). The
results showed that even though the LAB counts increased to the same levels in both feeds, in
NFLF they needed nine days to reach stable numbers, whereas LAB in FLF were high from the
first day. L. plantarum REB1 was inoculated five times during the 63-day trial, showing stability
for 11 days after the first inoculation and decreasing stability thereafter. The pH was
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
37
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
significantly lower in FLF than in NFLF, and Enterobacteriaceae were more frequent in NFLF
than in FLF. Moreover, yeasts were very unstable during the first 10 days in NFLF, with a peak
during the first three days, whereas yeasts remained very stable in FLF during the whole trial
(Figure 10-11) (study II).
Figure 10. Non-fermented liquid feed bacterial
counts and pH measurements.
Figure 11. Fermented liquid feed bacterial counts
and pH measurements.
4.1.4 Animal performance
There were no major health problems with the pigs fed either with NFLF or FLF. Some mild
diarrhoea cases were detected in both groups and one pig died from ulceration in FLF group. The
performance of the pigs, as indicated by final weight, showed no statistically significant
differences between the pigs fed with FLF or NFLF at any time of their growth (study II).
4.2 Proteomic experiments
The bacterial growth was followed in both experiments: the growth phase- (study IV) and the
media-dependent experiments (study V). The results showed a slight time-frame difference
between the growth curves of L. plantarum MLBPL1 and REB1, cultured in MRS broth (Figure
12). In REB1 strain, the lag-phase lasted one hour longer than in MLBPL1. The actual collecting
time points are presented in Figure 12.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
38
Results
Figure 12. Bacterial growth curves in MRS broth with the collecting times marked.
The bacterial counts in MRS broth, cucumber juice and liquid feed at the moment of
collection are shown in Figure 13. The results showed that the colony counts of both strains
were significantly (P<0.01 or P<0.05) higher in cucumber juice and liquid feed than in MRS.
Moreover, the cell counts in REB1 strain were slightly but not significantly higher than in the
MLBPL1 strain (P>0.01).
Figure 13. Bacterial growth (log cfu/ml) after 8 hours of
incubation. Abbreviations: MRS, MRS broth; Cu, cucumber
juice; Feed, pig liquid feed. Groups with different letters differ;
a, P<0.01; b, P<0.05.
4.2.1 Protein expression patterns
2-DE was performed in order to detect differential protein expressions between the two strains
and between different conditions (growth phases and media, study IV and V respectively). A
preliminary 2-DE with a pH gradient from 3 to 10 showed that most of the proteins were
distributed within the 4-7 pH range. This pH was therefore selected for the subsequent analysis
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
39
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
in order to improve sensitivity. Figure 14-16 shows representative 2-DE of L. plantarum grown
in MRS broth, cucumber juice and liquid feed.
Figure 14. 2-DE reference map of L. plantarum REB1
in MRS broth.
Figure 15. 2-DE reference map of L. plantarum
REB1 in cucumber juice.
Figure 16. 2-DE reference map of L. plantarum REB1
in pig liquid feed.
4.2.1.1 Proteins differentially expressed between growth phases
The number of spots quantified for REB1 and MLBPL1 strains were 775 and 748, respectively.
Of those, 60 (REB1) and 104 (MLBPL1) spots showed statistically significant (P<0.01)
differences between the growth phases. A total of 231 spots were successfully identified,
including 39 spots from REB1 and 64 from MLBPL1, showing significant differential
expression between growth phases, and some additional spots which did not show differences
but were chosen because of their high intensity. The proteins with differential expression pattern
between the growth phases are listed in Table 2. The expression patterns of all the identified
proteins are shown in the supplementary material of study IV.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
40
Results
Table 2. Identified proteins with statistically significant changes in their expression (P<0.01), their preferential
expression phase and functional classification.
Functional classification
Gene name
L
EE
LE
ES
Protein name
Glycolysis
pfk
6-phosphofructokinase
M
fba
fructose bisphosphate aldolase
M
gapB
glyceraldehyde-3-phosphate dehydrogenase
RM
gapB
glyceraldehyde-3-phosphate dehydrogenase
M
pyk
pyruvate kinase
M
Pyruvate metabolism
ack1
acetate kinase
M
M
ldhD
D-lactate dehydrogenase
RM
pox2
pyruvate oxidase
M
Phosphoketolase pathway
pgm
phosphoglucomutase
M
gnd2
phosphogluconate dehydrogenase
M
xpk1
phosphoketolase
R
rbsK2
ribokinase
RM
rpiA1
ribose-5-phosphate isomerase A
M
Carbohydrates metabolism
mtlD
mannitol-1-phosphate 5-dehydrogenase
M
Transport system
pts9AB
mannose PTS, EIIAB
RM
hpr
phosphocarrier protein HPr
R
Purine metabolism
apt
adenine phosphoribosyltransferase
M
M
purB
adenylosuccinate lyase
M
purA
adenylosuccinate synthase
RM
purH
bifunctional purine biosynthesis protein purH
M
fhs
formate-tetrahydrofolate ligase
R
purK1
phosphoribosylaminoimidazole carboxylase, ATPase subunit
M
purine nucleosidase
R
Pyrimidine metabolism
pyrB
aspartate carbamoyltransferase
M
pyrAA
carbamoyl-phosphate synthase, pyrimidine-specific small chain
M
pyrG
CTP synthase
M
pyrC
dihydroorotase
M
pyrD
dihydroorotate oxidase
RM
pyrE
orotate phosphoribosyltransferase
RM
Amino acid metabolism
cysK
cysteine synthese
M
dapA1
dihydrodipicolinate synthase
RM
RM
hicD3
L-2-hydroxyisocaproate dehydrogenase
R
R
metK
methionine adenosyltransferase
RM
R
gabD
succinate-semialdehyde dehydrogenase (NAD(P)+)
R
Peptidases and protein modifications
pepC1
cysteine aminopeptidase
RM
R
ppiB
peptidylprolyl isomerase
M
M
pepR1
prolyl aminopeptidase
R
DNA replication, recombination and repair
dnaN
DNA-directed DNA polymerase III, beta chain
M
M
gyrB
DNA gyrase, B subunit / DNA topoisomerase
R
recA
recombinase A
RM
RM
Transcription factors
greA2
transcription elongation factor
M
M
Translation factors
fusA2
elongation factor G
R
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
41
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
tsf
frr
elongation factor Ts
RM
RM
ribosome recycling factor
R
Aminoacyl tRNA synthetases
hisS
histidine-tRNA ligase
M
SerS2
serine-tRNA ligase
M
M
tyrS
tyrosine-tRNA ligase
M
Ribosomal proteins: synthesis and modification
gatA
glutamyl-tRNA amidotransferase, subunit A
M
M
R
gatB
glutamyl-tRNA amidotransferase, subunit B
M
M
rplJ
ribosomal protein L10
M
rpsA
ribosomal protein S1
RM
rpsB
ribosomal protein S2
M
ribosomal protein S30EA
M
M
Peptidoglycan biosynthesis
ddl
D-alanine-D-alanine ligase
RM
RM
Cell shape and cell division
divIVA
cell division initiation protein
M
M
mreB2
cell shape determining protein MreB
R
Stress proteins
clpC
ATP-dependent Clp protease, ATP-binding subunit ClpC
M
clpE
ATP-dependent Clp protease, ATP-binding subunit ClpE
RM
clpL
ATP-dependent Clp protease, ATP-binding subunit ClpL
R
groEL
chaperonin groEL
M
M
hsp1
small heat shock protein
RM
hsp3
small heat shock protein
RM
tpx
thiol peroxidase
M
M
Proteins with general function
typA
GTP-binding protein
R
GTP-binding protein
R
atpA
H(+)-transporting two-sector ATPase, alpha subunit
M
ndh1
NADH dehydrogenase
R
npr2
NADH peroxidase
M
nadE
NAD+ synthase (glutamine-hydrolysing)
M
oxidoreductase
R
oxidoreductase (putative)
M
Unknown function
hypothetical protein
R
RM
hypothetical protein
M
hypothetical protein
M
L, Lag phase; EE, Early-Exponential phase; LE, Late-Exponential phase; ES, Early-Stationary phase; M, L.
plantarum MLBPL1; R, L. plantarum REB1.
The overall expression patterns of the proteins identified from L plantarum REB1 and
MLBPL1 were strikingly similar. However, L. plantarum MLBPL1 showed more proteins with
growth phase-dependent expression (more strongly up- and down-regulated) than the REB1,
which had a similar pattern but fewer of them displayed differential expression during growth
(study IV supplementary material).
The proteins highly expressed in the lag phase were mainly proteins for carbohydrate
metabolism (e.g. D-lactate dehydrogenase, pyruvate oxydase, phosphoketolase, manose PTS),
the majority of proteins for the de novo synthesis of purines and stress proteins. In the earlyexponential phase, the proteins preferentially expressed were mostly glycolytic proteins but also
proteins for the phosphoketolase phathway (e.g. phosphogluconate dehydrogenase, ribokinase,
ribose-5-phosphate isomerase) as well as the phosphocarrier protein HPr.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
42
Results
Only a few proteins showed preferential expression in the late-exponential phase, mainly
proteins related with biosynthetic pathways (e.g. purine nucleosidase, H(+)-transporting twosector ATPase, alpha subunit). The transition from exponential phase to stationary phase was
represented by the collection of bacteria at the early-stationary phase. The proteins highly
expressed in this phase included pyruvate kinase, an isoform of glyceraldehyde-3-phosphate
dehydrogenase and proteins for the de novo synthesis of pyrimidines.
4.2.1.2 Proteins differentially expressed between media
The number of spots quantified for REB1 and MLBPL1 strains were 924 and 905, respectively.
Of those, 240 (REB1) and 209 (MLBPL1) spots showed statistically significant (P<0.01)
differences between the growth phases. A total of 232 spots were successfully identified,
including 136 spots from REB1 and 110 from MLBPL1, which showed statistically significant
differences (P<0.01) between growth phases, and some additional spots which did not show
differences but were chosen because of their high intensity. The expression patterns of all the
identified proteins are shown in the supplementary material of study V.
The expression patterns of the proteins identified from L. plantarum REB1 and MLBPL1
were remarkably similar. Unfortunately, in numerous cases it was not possible to identify the
same proteins from both strains. However, it seems that L. plantarum REB1 showed more
proteins with media-dependent expression (more strongly up- and down-regulated) than in
MLBPL1, which had similar protein patterns but with a more constant (not significantly
different) expression. Moreover, the spots representing a single protein (isoforms) were abundant
in both strains.
The proteins highly expressed in MRS broth were mostly glycolytic enzymes and some
proteins for pyruvate metabolism (Figure 17). The proteins for the de novo synthesis of purines
and pyrimidines were also highly expressed in this medium. The proteins with the lowest
expression include the key enzyme of the oxidative branch of the phosphoketolase pathway (i.e.
phosphogluconate dehydrogenase) as well as the enzyme phosphoglucomutase (study V
supplementary material).
In cucumber juice, the proteins for the carbohydrate metabolism were rather variable
(Figure 17). Thus, proteins such as D-lactate dehydrogenase, lactate oxydase and malolactic
enzyme were highly expressed in this medium, whereas proteins such as glucokinase, mannose
PTS EIIAB, phosphoenolpyruvate-protein phosphatase, glucose-6-phosphatase isomerase and 1phosphofructokinase showed the lowest expression. The proteins for the de novo synthesis of
purines and pyrimidines also showed high expression in this medium, as did some proteins for
amino acid and DNA metabolism (study V supplementary material).
Glycolytic enzymes and proteins for pyruvate metabolism generally showed low
expression in liquid feed (Figure 17). However, high expression of the two phosphotransferase
systems (PTS) identified was detected, as well as of proteins for the first steps of the degradation
of hexoses (i.e. glucokinase, glucose-6-phosphate isomerase, phosphoglucomutase and 1phosphofructokinase). The highest expression of phosphogluconate dehydrogenase was detected
in this medium, while the expression of proteins for the de novo synthesis of purines and
pyrimidines was limited in liquid feed (study V supplementary material).
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
43
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
Maltose
Sucrose
Mannitol
se
no
an
M
D-mannitol-1-P
PtsI
Pmi
D-mannose-6-P
Pts9AB
Pfk
R/ M
FruK
UDP-Glucose
NADH + H+
GapB
R/M
Gnd2
R/M
Nucleotide
sugar
metabolism
R/ M
Glycerate-1,3-bisP
RbsK2
NAD+
R/ M
Pmg9
NADH + H+
Ribose-5-P
Xylulose-5-P
Ribose
ADP
ATP
Xpk1
Diacetyl
Acetoin
Glycerate-2-P
R
Prs1
EnoA1
PEP
Pyk
ADP
NAD+
NADH + H+
M
Ribose-1-P
R/ M
R/ M
ATP
Malate
Pox2
Pyruvate
Mae
NADH + H+
CO2
R/M
MleS
M
M
Als
2-acetolactate
Pgm
R
Ribose
NAD+
RpiA
Rpe
Pgk
Glycerate-3-P
NADH + H+
Glucose
Ribulose-5-P
ADP
ATP
Galactose
6-P-gluconate
NADH + H+
2,3-Butanediol
M
Galactose 1-P
R/ M
Glyceraldehyde-3-P
NAD +
R
Lactose
TpiA
R/ M
Glucose-1-P
GalU
Glucono-1,5-lactone-6-P
NAD+
Dihydroxyacetone P
M
Pgm
Gpd
NADH + H+
M
R/M
Pts9AB
NAD +
Fructose-1,6-bisP
Fba
PtsI
R
Glucose-6-P
Pgi
M
R
ADP
M
Fructose-1-P
Fructose
Fructose-6-P
ATP
Glk
ADP
SacK
R
R/M
ATP
ATP
ADP
lu
co
se
Map
Glucose
Fructose
M
MtlD
R
Malate
G
R
R/M
NAD+
LdhL
NADH + H
NAD+
Lox
L-Lactate
LoxD
LdhD
RPRR
Acetyl-P
+
M
H2O2
CO2
R/M
M
Ack1
Acetate
Ribose-1,5-bisP
Nucleotide
metabolism
D-Lactate
Figure 17. Schematic representation of the carbohydrate pathways used by L. plantarum strains REB1 and
MLBPL1. The expression of the identified enzymes catalysing the different steps is indicated by white (MRS), grey
(cucumber), and black (feed) bars. The strain and statistical significance of each protein pattern is indicated by R
(REB1) or M (MLBPL1), and by black (P<0.01) or grey (P>0.01), study V. Abbreviations: : Ack1, acetate kinase;
Als, acetolactate synthase; EnoA1, enolase 1; Fba, fructose bisphosphate aldolase; FruK, 1-phosphofructokinase;
GalU, UTP--glucose-1-phosphate uridylyltransferase; GapB, glyceraldehyde-3-phosphate dehydrogenase; Glk,
glucokinase; Gnd2, phosphogluconate dehydrogenase; Gpd, Glucose-6-phosphate 1-dehydrogenase; LdhD, Dlactate dehydrogenase; LdhL1, L-lactate dehydrogenase; Lox, lactate oxidase; LoxD, lactate oxidase
(oxidoreductase); Mae, malic enzyme; Map, maltose phosphorylase; MleS, malolactic enzyme; MtlD, mannitol-1phosphate 5-dehydrogenase; Pfk, 6-phosphofructokinase; Pgi, glucose-6-phosphate isomerase; Pgk,
phosphoglycerate kinase; Pgm, phosphoglucomutase; Pmg9, phosphoglyceromutase 2; Pmi, mannose-6-phosphate
isomerase; Pox2, pyruvate oxidase; Prs1, ribose-phosphate pyrophosphokinase; Pts9AB, mannose PTS, EIIAB; PtsI,
phosphoenolpyruvate--protein phosphatase; Pyk, pyruvate kinase; RbsK2, ribokinase; Rpe, ribulose-phosphate 3epimerase; RpiA, ribose 5-phosphate isomerase A; Sack, fructokinase; TpiA, triosephosphate isomerase; Xpk1,
phosphoketolase.
4.2.1.2.1 Fermentative sugars and fermentation end-products
The sugar analyses confirmed that glucose was, as expected, the main sugar in MRS broth. The
results showed that for each mol of glucose consumed, two mols of lactic acid were produced in
a homofermentative pattern. No production of acetic acid or ethanol was detected in this
medium. The total hexose consumption at the moment of collection was approximately 50%
(Table 3).
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
44
Results
Table 3. Sugars, organic acids, ethanol and pH measurements from MRS broth cultures. The concentrations (mM)
are expressed as the mean of three replicates ± SD.
REB
MLBPL1
Glucose
Fructose
Lactose
Sucrose
Galactose
Total sugars
Total hexoses
Lactic acid
Acetic acid
Malic acid
Ethanol
pH
Hexose
utilization
ND, not detected.
0h
8h
Difference
0h
8h
Difference
79.62 ± 0.58
14.74 ± 0.17
2.63 ± 0.34
3.99 ± 2.05
ND
100.98
107.60
ND
82.83 ± 0.34
ND
ND
6.18
51.11 ± 0.49
ND
0.96 ± 0.10
1.75 ± 0.13
3.22 ± 0.09
57.04
59.75
82.62 ± 0.66
83.02 ± 0.82
ND
ND
4.56
-28.51
-14.74
-1.67
-2.24
3.22
-43.94
-47.85
82.62
0.19
80.87 ± 0.68
14.82 ± 0.06
3.05 ± 1.73
0.98 ± 1.70
ND
99.72
103.75
ND
84.46 ± 0.75
ND
ND
6.19
40.77 ± 1.07
ND
1.78 ± 0.20
1.14 ± 1.31
2.16 ± 0.02
45.85
48.77
109.72 ± 0.82
85.10 ± 0.75
ND
ND
4.38
-40.10
-14.82
-1.27
0.15
2.16
-53.87
-54.99
109.72
0.64
-1.62
44.47%
-1.81
53.00%
Cucumber juice analyses showed that there was high concentration of glucose and fructose
in this medium. Despite the high sugar concentration, the consumption of hexoses was very low.
The results showed mainly the production of lactic acid in this medium implying homolactic
fermentation. Additionally, cucumber juice contained malic acid which was completely
consumed. No ethanol was detected. The results showed a hexose consumption of only 10% in
this medium (Table 4).
Table 4. Sugars, organic acids, ethanol and pH measurements from cucumber juice cultures. The concentrations
(mM) are expressed as the mean of three replicates ± SD.
REB
MLBPL1
Glucose
Fructose
Lactose
Sucrose
Galactose
Total sugars
Total hexoses
Lactic acid
Acetic acid
Malic acid
Ethanol
pH
Hexose
utilization
ND, not detected.
0h
8h
Difference
0h
8h
Difference
167.14 ± 2.45
66.02 ± 0.92
ND
ND
3.57 ± 0.04
236.73
236.73
ND
33.43 ± 0.46
13.66 ± 0.08
ND
5.10
152.60 ± 1.17
63.25 ± 0.47
ND
ND
3.54 ± 0.02
219.39
219.39
41.87 ± 0.66
35.59 ± 0.44
ND
ND
4.18
-14.54
-2.77
168.23 ± 5.61
66.41 ± 2.25
ND
ND
3.59 ± 0.12
238.23
238.23
0.00
33.77 ± 1.14
13.66 ± 0.37
ND
5.09
146.25 ± 2.43
60.92 ± 1.02
ND
ND
3.42 ± 0.05
210.58
210.58
44.69 ± 0.63
34.61 ± 0.65
ND
ND
4.09
-21.98
-5.50
-0.03
-17.34
-17.34
41.87
2.16
-13.66
-0.92
7.32%
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
-0.17
-27.65
-27.65
44.69
0.84
-13.66
-1.00
11.61%
45
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
Liquid feed contained a more variable sugar composition, with lower concentrations than
the other two media. However, there was higher consumption of hexoses and significantly higher
production of lactic acid than in cucumber juice. Moreover, there was production of acetic acid
in this medium, indicating heterolactic fermentation. No ethanol production was detected. The
total hexose consumption in this medium was over 30% (Table 5).
Table 5. Sugars, organic acids, ethanol and pH measurements from pig liquid feed cultures. The concentrations
(mM) are expressed as the mean of three replicates ± SD.
REB
MLBPL1
Glucose
Fructose
Lactose
Sucrose
Galactose
Total sugars
Total hexoses
Lactic acid
Acetic acid
Malic acid
Ethanol
pH
Hexose
utilization
ND, not detected.
0h
8h
Difference
0h
8h
Difference
13.92 ± 2.60
13.66 ± 2.16
22.81 ± 0.63
11.44 ± 1.17
ND
61.83
96.07
6.65 ± 0.22
ND
7.92 ± 0.91
0.50 ± 0.10
5.28
14.53 ± 1.14
19.96 ± 1.40
15.10 ± 0.43
ND
ND
49.60
64.70
82.46 ± 1.81
8.11 ± 0.30
ND
0.47 ± 0.08
4.07
0.61
6.30
-7.71
-11.44
12.41 ± 2.39
12.16 ± 2.50
20.90 ± 1.90
9.40 ± 2.35
ND
54.87
85.18
6.17 ± 0.55
ND
6.98 ± 0.88
0.59 ± 0.07
5.28
11.59 ± 1.62
16.84 ± 1.93
13.97 ± 1.33
ND
ND
42.40
56.37
93.24 ± 3.74
8.82 ± 0.95
ND
0.65 ± 0.15
3.96
-0.81
4.68
-6.94
-9.40
-12.23
-31.38
75.82
8.11
-7.92
-0.03
-1.21
32.66%
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
-12.47
-28.81
87.07
8.82
-6.98
0.06
-1.32
33.83%
46
Discussion
5. DISCUSSION
5.1 Fermented liquid feed
Fermented liquid feed has been lately much investigated for its potential as an alternative to the
use of antibiotics in pig production (Mikkelsen and Jensen, 1998; Canibe et al., 2007a). The
fermentation process has been claimed to be the reason for the benefits associated with this type
of feeding. However, properly fermented liquid feed is not simple to produce. A number of
factors may lead to the growth of undesirable bacteria and consequently damage pigs' health
(check study III for a review). However, the addition of concentrated LAB inoculants as starter
culture has been reported to be a solution to the control of the fermentation that inevitably occurs
in a liquid diet (Canibe et al., 2001; van Winsen et al,. 2001a, 2001b; Beal et al., 2002).
Therefore, our purpose was to find a LAB strain which could dominate the fermentation in pig
liquid feed for its potential use as starter culture.
5.1.1 Stability of pig liquid feed
In order to accomplish our objectives, the microbial composition of a regular pig liquid feed was
characterized, as well as its stability during an extended time period, with special attention to the
LAB population. It is important to emphasize that the feed samples examined microbiologically
were obtained from an actual commercial farm, where no specialized sterilization treatments
were applied to the feed, feed tanks or pipes in order to eliminate the natural microflora. The
feed temperature varied considerably between day and night due to the fact that the feed tanks
were located in a separate room without heating and thus bacterial growth conditions were
inevitably affected. Organic acids (formic acid) were normally added to the feed when pH
exceeded 5. All these procedures are part of the normal routine in this specific farm. Moreover,
for practical reasons 10-15% of feed remained at each refilling, which could act as a starter
culture for the fresh feed.
In spite of all these typical uncontrolled on-farm conditions, the bacterial populations
examined were found to be considerably stable with even a slight increase in bacterial counts
over time. However, amylolytic bacteria and yeasts and moulds were relatively unstable. The
instability of yeasts is especially important because they can produce off flavours and taints, and
make the feed unpalatable to the pigs. The results also indicated that the feed was dominated by
LAB, especially Lactobacilli.
5.1.2 Isolation of Lactobacillus plantarum REB1
The LAB encountered in pig liquid feed were further characterized and a L. plantarum strain was
selected as a potential LAB inoculant. This strain was chosen because it was the predominant
strain within the LAB population, but also because L. plantarum has a long history of natural
occurrence and safe use in a variety of food and feed products, especially in vegetable and plant
material, and because it is one of the most acid-tolerant species (Morelli et al., 2004; de Vries et
al., 2006). Therefore, L. plantarum largely qualified to be used as starter culture for pig liquid
feed.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
47
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
5.1.3 Inoculation of Lactobacillus plantarum REB1 in laboratory conditions
Subsequently, a starter culture of L. plantarum REB1 was inoculated into pig liquid feed in
laboratory conditions in order to confirm the growth potential of this strain, and to check its
compatibility with the bacterial population in the feed. L. plantarum REB1 dominated the
fermentation in this particular feed, reaching remarkably high counts, decreasing considerably
the pH, preventing any growth of Enterobacteriaceae and reducing substantially the yeast
population. However, laboratory conditions are far from the situation that L. plantarum REB1
would have to confront in a commercial farm. This was first demonstrated by the replacement of
feed twice per day, simulating the actual feeding procedure. It resulted with one log daily
fluctuation in L. plantarum REB1 numbers and a corresponding effect on pH. These results
confirm that LAB needed time to develop and that the feeding frequencies of pigs can
considerably interfere with the fermentation of the feed. Interestingly, yeasts were almost
undetectable in the feed replacement experiment.
5.1.4 Inoculation of Lactobacillus plantarum REB1 in farm conditions
Once the growth potential of L. plantarum REB1 was confirmed, the next step was to face real
farm conditions. Thus, a starter culture of L. plantarum REB1 was inoculated into the pig liquid
feed (FLF). The standard feed without inoculum was used as control (NFLF). An important
difference between feeds was that NFLF had 10-15% of feed remaining in the tank at each
refilling whereas the remaining feed in FLF was intentionally much higher. Due to the tank
limitations, the residual feed in FLF was approximately 50% at the beginning of the trial,
decreasing progressively as the pigs' feed intake increased, until the amount of residual feed was
25%.
Lactobacillus plantarum REB1 inoculated in FLF provided a proper fermentation from the
first day (high LAB, low pH, low Enterobacteriaceae, stable yeasts). In comparison, the
fermentation in NFLF needed over a week to stabilise, some particularly high counts of
Enterobacteriaceae were detected, and yeasts were higher than in FLF. Moreover, formic acid
was added repeatedly in NFLF whereas there was no need for this in FLF. The results showed
that the inoculation of L. plantarum REB1 in this particular liquid feed has the potential to
improve feed sanitation, and consequently favourably influence pigs' health.
Although in laboratory conditions L. plantarum REB1 dominated the fermentation, the
competition with the endogenous LAB microflora in farm conditions soon led to a decrease in L.
plantarum REB1 counts. In this situation, the REB1 strain was re-inoculated up to five times.
These results demonstrated that the strain did not interfere with the development of a
spontaneous LAB-dominated microflora, but rather accelerated the process. Moreover, these
results might also mean that the re-inoculation of the strain after the early stages of the
fermentation might not be necessary because the developing LAB microflora could convey the
expected benefits of FLF.
5.1.5 Pigs' health and performance
Even though the experimental trial (study II) was focused on characterizing the bacterial
developments in the liquid feed, some additional measurements related to the pigs' health were
noted such as pigs' weight and general health status (diarrhoea and ulcerations).
It has been reported that growing pigs showed improved performance when fed with a
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
48
Discussion
liquid died, compared with pigs fed with a dry diet (Canibe and Jensen, 2003; Dung et al., 2005).
Our results showed no improvement in performance between pigs fed with NFLF and FLF. A
fermentation process eventually took place in NFLF, probably making the feed not sufficiently
different for the detection of an improvement in pigs' performance. Moreover, no differences in
diarrhoea cases were found between groups. Indeed, only a few mild diarrhoea cases were
detected during the whole trial, and randomly in both groups.
Ulceration is an important side effect when feeding FLF because the pH of the feed can be
remarkably low. In our experiment, the pH of FLF reached very low values, the lowest being
3.72. However, only one pig from this group died from ulceration. Thus, it can be stated that this
particular liquid feed did not increase the incidence of ulceration.
5.2 Proteomic experiments
Nowadays, modern genetic techniques are considered to be highly promising on achieving more
controlled fermentation processes. However, the effort in this direction may not be successful
unless there is a thorough understanding of the physiology and biochemistry of these organisms.
We are now in the genomic era, where many LAB genomes have been sequenced and many
more are underway. This research direction will allow us to access the genetic data of these
organisms with enormous potential to elucidate the physiological context (Morelli et al., 2004)
The metabolism of LAB may look relatively simple but these bacteria have a high
metabolic ability to adapt to a variety of niches and conditions. L. plantarum has been found in
many vegetable, cereal and silage fermentation as a member of spontaneously fermentation
microflora or as an intentionally added starter culture (Mäki, 2004, Salovaara, 2004). Other
applications of this bacterial species include meat fermentation (Ammor and Mayo, 2007), as
probiotics (Goossens et al., 2005; Demeckova et al., 2002) and as a delivery vehicle for
therapeutic compounds (Pavan et al., 2000). The extensive adaptation capacity of L. plantarum
to different environments and its wide range of applications make this bacterium especially
interesting and challenging. One of our goals was to reach a better understanding of the
physiology and metabolic potential of L. plantarum, and we expect that it will provide ideas
leading to eventual practical applications.
The complete genome of a strain of L. plantarum (strain WCFS1) has been sequenced and
published (Kleerebezem et al., 2003). This has provided very valuable information, the most
striking concerning its high potential to import and metabolize a wide range of carbohydrates
(Kleerebezem et al., 2003; Molenaar et al., 2005). Genomic analysis provides a static view of an
organism, whereas proteomics allows a more dynamic observation of the cellular events
occurring in, for example, food and feed fermentations. Proteomic analyses were thus chosen in
order to elucidate metabolic differences between L. plantarum strains and conditions. In
particular, our aims were to obtain a better understanding of the metabolic capacity of two strains
of vegetable and cereal origin during growth in a standard rich medium and to elucidate how
adaptation to different environments is reflected in the protein expression of these two strains.
5.2.1 Proteins differentially expressed between growth phases
Even though L. plantarum strain REB1 was isolated from cereal-based fermentation and strain
MLBPL1 from vegetable-based fermentation, the protein expression patterns showed that the
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
49
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
metabolic processes occurring through their growth were very similar in both strains when
grown in the same standard rich medium. Unfortunately, we did not detect any difference in the
expression pattern of the identified proteins from the lag phase that could explain the time-frame
difference between strains.
5.2.1.1 Carbohydrate metabolism
MRS broth is a standard, undefined rich medium where glucose is the main sugar. During the
catabolism of rapidly metabolizable sugars such as glucose, the expression of proteins involved
in the catabolism of other sugars is usually negatively regulated in Lactobacilli (Titgemeyer &
Hillen, 2002; Kleerebezem et al., 2003; Palmferldt et al., 2004). However, L. plantarum REB1
and MLBPL1 showed simultaneously expression of glycolytic proteins as well as enzymes for
the phosphoketolase pathway, both with preferential expression in the early-exponential phase.
These results might suggest that both strains have a carbohydrate metabolism generally primed
to use many carbohydrates in parallel, and that this trait could be related to the extended capacity
of L. plantarum to adapt to a wide variety of environmental niches.
The preferential expression of the proteins pyruvate oxidase and phosphoketolase in lag
phase might indicate that the two strains of L. plantarum examined here could create a pool of
acetyl phosphate in this phase, which could be converted to acetate later in the exponential phase
and gain an ATP molecule. Moreover, there was some indication of energy production in the
early-stationary phase, which is represented by the preferential expression of the enzyme
pyruvate kinase and an isoform of the key glycolytic enzyme glyceraldehyde-3-phosphate.
5.2.1.2 Biosynthetic metabolism
The expression of proteins associated with the de novo synthesis of purines and pyrimidines was
high during the lag phase in both strains, decreasing sharply thereafter. Moreover, the de novo
synthesis of pyrimidines increased suddenly again in the early-stationary phase. These results
suggest that the preferential expression of proteins for nucleotide synthesis in the lag phase
might indicate an accumulation of these compounds before the actual cell division. This was also
seen for Lactococcus lactis (Larsen et al., 2006). However, the high expression of the proteins
for the de novo synthesis of pyrimidines in the early-stationary phase suggests the exhaustion of
these compounds in this phase and the need to re-synthesise them.
The preferential expression of proteins involved in amino acid metabolism and DNA
replication, recombination and repair was mostly in the early- and late-exponential phases, and
occasionally remaining high still in the early-stationary phase. These results were expected due
to the requirements for cell division during these phases.
5.2.1.3 Stress response
Most of the proteins involved in the stress response in L. plantarum REB1 and MLBPL1 were
preferentially expressed in the lag phase. Stress proteins are normally expressed during the
stationary phase due to, for example, the exhaustion of carbohydrates for energy production or a
considerably decrease in the pH of the medium. Because the inoculated bacteria came from a
culture grown to the late-stationary phase, it is possible that the stress response observed was a
carryover effect from the previous culture. However, this is not likely to be the case in this
situation because the bacteria sampled had already been in the fresh medium for one hour (in
MLBPL1) or two hours (in REB1) (Figure 12). Thus, it is unknown what triggered the
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
50
Discussion
activation of a stress response during the lag phase.
5.2.2 Proteins differentially expressed between media
The differences in the expressions patterns of the identified proteins between L. plantarum REB1
and MLBPL1 grown in three different media sampled at the exponential phase were very small,
as was also seen in our previous study. Thus, it seems that within vegetable- and cereal-based
strains, the bacteria use the same metabolic strategies regardless of their origin.
5.2.2.1 Carbohydrate metabolism
Lactobacillus plantarum is a facultative heterofermentative bacterium, which means that this
bacterium ferments hexoses via glycolysis and pentoses via phosphoketolase pathway, leading to
homo- or heterolactic fermentation respectively (Axelsson, 2004). However, fermentation of
hexoses via the phosphoketolase pathway has been recently reported (Pieterse et al., 2005). The
results of our previous study also suggested the fermentation of hexoses via the phosphoketolase
pathway, indicated by the expression of the protein phosphogluconate dehydrogenase, the key
enzyme of the oxidative branch of the phosphoketolase pathway (study IV). This hypothesis has
been confirmed in study V, because the expression of phosphogluconate dehydrogenase was also
found in all three media with the highest expression in liquid feed. Moreover, the expressions of
proteins for carbohydrate metabolism were generally rather variable. The highest expression of
glycolytic proteins occurred in MRS both (as also was seen in study IV), whereas the lowest was
in liquid feed.
In addition to the proteomics experiments, the fermentation of sugars and the fermentative
organic acids obtained in all three media were analysed in order to confirm the results obtained
from the proteins expression patterns. In all three media, the fermentation of sugars was
homolactic. However, liquid feed showed some signs of heterolactic fermentation, indicated by
the production of acetate.
The total hexose consumption was surprisingly different in the three media. In cucumber
juice, the medium with the highest glucose content, the consumption of hexoses was roughly
10%, whereas it was 33% in liquid feed and 50% in MRS broth. Interestingly, the lowest
bacterial counts were observed in MRS broth. This discrepancy between sugar consumption and
growth may be a consequence of the descarboxylation of malate, a compound found in cucumber
and liquid feed but not in MRS broth. It is known that the co-fermentation of malate with another
carbohydrate such as glucose can increase the biomass yield and the lactic acid production of a
culture compare with the growth on glucose alone (Axelsson, 2004; Passos et al., 2003). Thus,
the malate consumed in cucumber juice and liquid feed might have provided a growth advantage
compared with the bacterial growth in MRS broth. These results are supported by the expression
of the malolactic enzyme, which showed the highest expression in cucumber juice, followed by
liquid feed.
The total hexose consumption also supported the preferential expression of the proteins
involved in the first steps of carbohydrate degradation, i.e. mannose PTS, phosphoenolpyruvateprotein phosphatase, glucokinase, glucose-6-phosphate isomerase, 1-phosphofructokinase. The
expression was high in MRS broth and liquid feed, but low in cucumber juice. Interestingly, the
main enzyme involved in the carbon catabolite repression system (CcpA) showed the same
expression pattern as the previous enzymes, suggesting that in both L. plantarum REB1 and
MLBPL1, this protein might be connected with their ability to adapt to different environments.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
51
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
5.2.2.2 Biosynthetic metabolism
The expression of proteins involved in purine and pyrimidine metabolisms seemed to reflect the
accessibility of these compounds in the media. The proteins from the de novo synthesis of
nucleotides in cucumber juice and MRS broth showed higher expression than in liquid feed.
Moreover, the high expression of ribokinase in MRS broth and cucumber juice might indicate
that the bacteria used riboses to obtain the pentose ring for the nucleotide synthesis in these
media.
The protein expressions of the identified enzymes for amino acid metabolism were
relatively variable. However, it generally showed a lower expression in liquid feed compared
with the other media. These results suggest that liquid feed contained large amounts of amino
acids and peptides, probably obtained from the whey component. However, the expressions of
proteins for DNA metabolism were very similar in all three media, as was the expression of
enzymes for protein synthesis.
5.2.2.3 Stress response
There are different stress conditions that activate stress responses, such as acidic and alkaline
stress, oxidative stress, heat and cold stress, osmotic stress, starvation, etc (van de Guchte et al.,
2002; Pieterse et al., 2005). In our previous study using MRS broth, a stress response was
detected in the lag phase, whereas in the exponential and early-stationary phases, the stress
proteins showed a lower and more constant expression. In this study, we were not studying the
responses to any stress condition but rather the mechanisms involved in adaptation to the
environment. This is shown by the identified stress proteins which were highly expressed but
mostly did not show significant differences between the media, indicating that no stress situation
was present in any case at the moment of cell collection.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
52
Summary and Conclusions
6. SUMMARY AND CONCLUSIONS
From the liquid feed studies presented here (studies I-III), we can make the following
conclusions:
•
The bacterial populations in the liquid feed were generally very stable in the long period
examined.
•
From the LAB population in liquid feed, a L. plantarum strain (REB1) was chosen
because it was the dominating strain in this particular feed. It was selected for use as
starter culture.
•
The addition of the starter culture let to a properly fermented liquid feed. The feed
contained a high LAB population from the first day, low pH, stable numbers of yeasts
and showed a decrease in pathogenic bacteria.
•
Even though L. plantarum REB1 dominated the fermentation in the liquid feed prepared
in laboratory conditions, it did not dominate the fermentation in the feed prepared in the
commercial farm. However, the starter culture accelerated the development of a total
LAB microflora.
These results demonstrate that the inoculation of L. plantarum REB1 into pig liquid feed can
benefit the fermentation of the feed microbiologically, providing a feed safe for the pigs and
eventually ensure the safety of the consumer.
From the proteomics studies presented here (studies IV-V), we can make the following
conclusions:
•
Two strains of L. plantarum of different origin were compared at the protein expression
level, showing remarkably similar results. However, L. plantarum MLBPL1 showed
more proteins with growth phase-dependent expression (more up- and down-regulated),
whereas L. plantarum REB1 showed more proteins with media-dependent expression.
•
Both strains have a very active metabolism during the lag phase and showed a difference
in activity between early- and late-exponential phases.
•
Both strains seem to ferment different carbohydrates simultaneously during the earlyexponential phase.
•
Both strains showed substantial differences in the expression of proteins for the
carbohydrate metabolism even though there was an excess of glucose in all the media.
Both L. plantarum strains have very similar biosynthetic behaviour in the three media beside the
requirements for essential components, suggesting that the environmental variability does not
affect the biosynthetic metabolism.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
53
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
7. REFERENCES
Ammor MS and Mayo B. Selection criteria for lactic acid bacteria to be used as functional starter
cultures in dry sausage production: An update. Meat Sci 2007; 76: 138-146.
Axelsson L. Lactic Acid Bacteria: Classification and Physiology. In S Salminen, A von Wright
and A Ouwehand (ed.), Lactic Acid Bacteria: Microbial and Functional Aspects, 3rd ed. Marcel
Dekker, New York, NY, 2004, p. 1-66.
Beal JD, Niven SJ, Campbell A and Brooks PH. The effect of temperature on the growth and
persistence of Salmonella in fermented liquid pig feed. Int J Food Microbiol 2002; 79: 99-104.
Beal JD, Niven SJ, Brooks PH and Gill BP. Variation in short chain fatty acid and ethanol
concentration resulting from the natural fermentation of wheat and barley for inclusion in liquid
diets for pigs. J Sci Food Agric 2005; 85: 433-440.
Bendixen E. The use of proteomics in meat science. Meat Sci 2005; 71: 138-149.
Boekhorst J, Wels M, Kleerebezem M and Siezen RJ. The predicted secretome of Lactobacillus
plantarum WCFS1 sheds light on interactions with its environment. Microbiology 2006; 152:
3175-3183.
Bron PA, Molenaar D, de Vos WM and Kleerebezem M. DNA micro-array-based identification
of bile-responsive genes in Lactobacillus plantarum. J Appl Microbiol 2006; 100: 728-738.
Brooks PH, Moran CA, Beal JD, Demecková V and Campbell A. Liquid feeding for the young
piglet. In MS Varley and J Wiseman (ed.), The Waner Pig: Nutrition and Management, CAB
International, University of Nottingham, UK, 2001, p. 153-178.
Brooks PH, Beal JD and Niven S. Liquid feeding of pigs I. Potential for reducing environmental
impact and for improving productivity. Anim Sci Pap Rep 2003a; 21: 7-22.
Brooks PH, Beal JD, Niven SJ and Demeckova V. Liquid feeding of pigs II. Potential for
improving pig health and food safety. Anim Sci Pap Rep 2003b; 21: 23-39.
Buckenhuskes HJ. Selection Criteria for Lactic-Acid Bacteria to be used as Starter Cultures for
various Food Commodities. FEMS Microbiol Rev 1993; 12: 253-272.
Canibe N, Miquel N, Miettinen H. and Jensen JJ. Addition of formic acid or starter cultures to
liquid feed. Effect on pH, microflora composition, organic acid concentration and ammonia
concentration. Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet 2001; 66: 431-434.
Canibe N and Jensen BB. Fermented and nonfermented liquid feed to growing pigs: effect on
aspects of gastrointestinal ecology and growth performance. J Anim Sci 2003; 81: 2019-2031.
Canibe N, Virtanen E and Jensen BB. Microbial and nutritional characteristics of pig liquid feed
during fermentation. Anim Feed Sci Technol 2007a; 134: 108-123.
Canibe N, Hojberg O, Badsberg JH and Jensen BB. Effect of feeding fermented liquid feed and
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
54
References
fermented grain to piglets on gastrointestinal ecology and growth performance. J Anim Sci
2007b; In press.
Cash P. Proteomics in medical microbiology. Electrophoresis 2000; 21: 1187-1201.
Champomier-Vergès MC, Maguin E, Mistou MY, Anglade P and Chich JF. Lactic acid bacteria
and proteomics: current knowledge and perspectives. J Chromatogr B 2002; 771: 329-342.
Charalampopoulos D, Pandiella SS and Webb C. Growth studies of potentially probiotic lactic
acid bacteria in cereal-based substrates. J Appl Microbiol 2002; 92: 851-859.
Cohen DP, Renes J, Bouwman FG, Zoetendal EG, Mariman E, de Vos WM and Vaughan EE.
Proteomic analysis of log to stationary growth phase Lactobacillus plantarum cells and a 2-DE
database. Proteomics 2006; 6: 6485-6493.
De Angelis M, Di Cagno R, Huet C, Crecchio C, Fox PF and Gobbetti M. Heat shock response
in Lactobacillus plantarum. Appl Environ Microbiol 2004; 70: 1336-1346.
De Vries MC, Vaughan EE, Kleerebezem M and de Vos WM. Lactobacillus plantarumsurvival, functional and potential probiotic properties in the human intestinal tract. Int Dairy J
2006; 16: 1018-1028.
Dung, N.N.X., Manh, L.H. and Ogle, B. Effects of fermented liquid feeds on the performance,
digestibility, nitrogen retention and plasma urea nitrogen (PUN) of growing-finishing pigs.
Livestock Research for Rural Development 2005; 17: 102.
Encarnación S, Hernández M, Martínez-Batallar G, Contreras S, Vargas MD and Mora J.
Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect
stimulons and regulons in bacteria with sequenced or partially sequenced genomes. Biol Proc
Online 2005; 117-135.
Eom HJ, Seo DM and Han NS. Selection of psychrotrophic Leuconostoc spp. producing highly
active dextransucrase from lactate fermented vegetables. Int J Food Microbiol 2007; 117: 61-67.
G-Alegría E, López I, Ruiz JI, Sáenz J, Fernández E, Zarazaga M, Dizy M, Torres C and RuizLarrea F. High tolerance of wild Lactobacillus plantarum and Oenococcus oeni strains to
lyophilisation and stress environmental conditions of acid pH and ethanol. FEMS Microbiol Lett
2004; 230: 53-61.
Gänzle MG, Vermeulen N and Vogel RF. Carbohydrate, peptide and lipid metabolism of lactic
acid bacteria in sourdough. Food Microbiol 2007; 24: 128-138.
Gardner NJ, Savard T, Obermeier P, Caldwell G and Champagne CP. Selection and
characterization of mixed starter cultures for lactic acid fermentation of carrot, cabbage, beet and
onion vegetable mixtures. Int J Food Microbiol 2001; 64: 261-275.
Geary TM, Brooks PH, Beal JD and Campbell A. Effect on weaner pig performance and diet
microbiology of feeding a liquid diet acidified to pH 4 with either lactic acid or through
fermentation with Pediococcus acidilactici. J Sci Food Agric 1999; 79: 633-640.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
55
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
Goossens D, Jonkers D, Stobberingh E and Stockbrugger R. The effect of the probiotic Lplantarum 299v on the faecal and mucosal bacterial flora of patients with inactive ulcerative
colitis. Eur J Gastroenterol Hepatol 2005; 17: A62-A62.
Görg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R and Weiss W. The current
state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000;
21: 1037-1053.
Görg A, Weiss W and Dunn MJ. Current two-dimensional electrophoresis technology for
proteomics. Proteomics 2004; 4: 3665-3685.
Graves PR and Haystead TAJ. Molecular biologist's guide to proteomics. Microbiol Mol Biol
Rev 2002; 66: 39.
Guelfi KJ, Casey TM, Giles JJ, Fournier PA and Arthur PG. A proteomic analysis of the acute
effects of high-intensity exercise on skeletal muscle proteins in fasted rats. Clin Exp Pharmacol
Physiol 2006; 33: 952-957.
Gygi SP, Corthals GL, Zhang Y, Rochon Y and Aebersold R. Evaluation of two-dimensional gel
electrophoresis-based proteome analysis technology. Proc Natl Acad Sci U S A 2000; 97: 93909395.
Hébert EM, Raya RR, Tailliez P and de Giori GS. Characterization of natural isolates of
Lactobacillus strains to be used as starter cultures in dairy fermentation. Int J Food Microbiol
2000; 59: 19-27.
Højberg O, Canibe N, Knudsen B and Jensen BB. Potential rates of fermentation in digesta from
the gastrointestinal tract of pigs: Effect of feeding fermented liquid feed. Appl Environ
Microbiol 2003; 69: 408-418.
Kim JH, Heo KN, Odle J, Han IK and Harrell RJ. Liquid diets accelerate the growth of earlyweaned pigs and effects are maintained to market weight. J Anim Sci 2001; 79: 427-434.
Klaenhammer T, Altermann E, Arigoni F, Bolotin A, Breidt F, Broadbent J, Cano R, Chaillou S,
Deutscher J, Gasson M, van de Guchte M, Guzzo J, Hartke A, Hawkins T, Hols P, Hutkins R,
Kleerebezem M, Kok J, Kuipers O, Lubbers M, Maguin E, McKay L, Mills D, Nauta A,
Overbeek R, Pel H, Pridmore D, Saier M, van Sinderen D, Sorokin A, Steele J, O'Sullivan D, de
Vos W, Weimer B, Zagorec M and Siezen R. Discovering lactic acid bacteria by genomics.
Antonie Van Leeuwenhoek 2002; 82: 29-58.
Klaenhammer TR, Azcarate-Peril MA, Altermann E and Barrangou R. Influence of the dairy
environment on gene expression and substrate utilization in lactic acid bacteria. J Nutr 2007;
137: 748S-50S.
Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers OP, Leer R, Tarchini R,
Peters SA, Sandbrink HM, Fiers MWEJ, Stiekema W, Lankhorst RMK, Bron PA, Hoffer SM,
Groot MNN, Kerkhoven R, de Vries M, Ursing B, de Vos WM and Siezen RJ. Complete
genome sequence of Lactobacillus plantarum WCFS1. Proc Natl Acad Sci U S A 2003; 100:
1990-1995.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
56
References
Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues.
A novel approach to testing for induced point mutations in mammals. Humangenetik 1975; 26:
231-243.
Koistinen KM, Hassinen VH, Gynther PAM, Lehesranta SJ, Keinänen SI, Kokko HI, Oksanen
EJ, Tervahauta AI, Auriola S and Kärenlampi SO. Birch PR-10c is induced by factors causing
oxidative stress but appears not to confer tolerance to these agents. New Phytol 2002; 155: 381391.
Krakowka S, Eaton KA, Rings DM and Argenzio RA. Production of gastroesophageal erosions
and ulcers (GEU) in gnotobiotic swine monoinfected with fermentative commensal bacteria and
fed high-carbohydrate diet. Vet Pathol 1998; 35: 274-282.
Larsen N, Boye M, Siegumfeldt H and Jakobsen M. Differential expression of proteins and
genes in the lag phase of Lactococcus lactis subsp. lactis grown in synthetic medium and
reconstituted skim milk. Appl Environ Microbiol 2006; 72: 1173-1179.
Lawlor PG, Lynch PB, Gardiner GE, Caffrey PJ and O’Doherty JV. Effect of liquid feeding
weaned pigs on growth performance to harvest. J Anim Sci 2002; 80: 1725-1735.
Lehesranta SJ, Davies HV, Shepherd LVT, Nunan N, McNicol JW, Auriola S, Koistinen KM,
Suomalainen S, Kokko HI and Kärenlampi SO. Comparison of tuber proteomes of potato
varieties, landraces, and genetically modified lines. Plant Physiol 2005; 138: 1690-1699.
Loubiere P, Salou P, Leroy MJ, Lindley ND and Pareilleux A. Electrogenic Malate Uptake and
Improved Growth Energetics of the Malolactic Bacterium Leuconostoc-Oenos Grown on
Glucose-Malate Mixtures. J Bacteriol 1992; 174: 5302-5308.
Luesink EJ, van Herpen RE, Grossiord BP, Kuipers OP and de Vos WM. Transcriptional
activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus
lactis are mediated by the catabolite control protein CcpA. Mol Microbiol 1998; 30: 789-798.
Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, Pavlov A, Pavlova N,
Karamychev V, Polouchine N, Shakhova V, Grigoriev I, Lou Y, Rohksar D, Lucas S, Huang K,
Goodstein DM, Hawkins T, Plengvidhya V, Welker D, Hughes J, Goh Y, Benson A, Baldwin K,
Lee JH, Díaz-Muñiz I, Dosti B, Smeianov V, Wechter W, Barabote R, Lorca G, Altermann E,
Barrangou R, Ganesan B, Xie Y, Rawsthorne H, Tamir D, Parker C, Breidt F, Broadbent J,
Hutkins R, O'Sullivan D, Steele J, Unlu G, Saier M, Klaenhammer T, Richardson P, Kozyavkin
S, Weimer B and Mills D. Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci
U S A 2006; 103: 15611-15616.
Mäki M. Lactic Acid Bacteria in Vegetable Fermentations. In S. Salminen, A. von Wright, and
A. Ouwehand (ed.), Lactic Acid Bacteria: Microbial and Functional Aspects, 3rd ed. Marcel
Dekker, New York, NY, 2004, p. 419-430.
Manso MA, Léonil J, Jan G and Gagnaire V. Application of proteomics to the characterisation of
milk and dairy products. Int Dairy J 2005; 15: 845-855.
Manso MA, Léonil J, Piot M and Gagnaire V. Isolation and characterisation of a Lactobacillus
helveticus ITG LH1 peptidase-rich sub-proteome. Int J Food Microbiol 2005; 105: 119-129.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
57
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
McFeeters RF. Fermentation microorganisms and flavor changes in fermented foods. J Food Sci
2004; 69: M35-M37.
Mikkelsen LL and Jensen BB. Performance and microbial activity in the gastrointestinal tract of
piglets fed fermented liquid feed at weaning. J Anim Feed Sci 1998; 7: 211-215.
MLC (Meat and Livestock Commission). General Guidelines on Liquid Feeding for Pigs, UK,
2003.
Molenaar D, Bringel F, Schuren FH, de Vos WM, Siezen RJ and Kleerebezem M. Exploring
Lactobacillus plantarum genome diversity by using microarrays. J Bacteriol 2005; 187: 61196127.
Monteoliva L and Albar JP. Differential proteomics: an overview of gel and non-gel based
approaches. Brief Funct Genomic Proteomic 2004; 3: 220-239.
Morelli L, Vogensen FK, von Wright A. Genetics of Lactic Acid Bacteria. In S. Salminen, A.
von Wright, and A. Ouwehand (ed.), Lactic Acid Bacteria: Microbial and Functional Aspects,
3rd ed. Marcel Dekker, New York, NY, 2004, p. 249-293.
Mullen AM, Stapleton PC, Corcoran D, Hamill RM and White A. Understanding meat quality
through the application of genomic and proteomic approaches. Meat Sci 2006; 74: 3-16.
Muscariello L, Marasco R, De Felice M and Sacco M. The functional ccpA gene is required for
carbon catabolite repression in Lactobacillus plantarum. Appl Environ Microbiol 2001; 67:
2903-2907.
Niven SJ, Beal JD and Brooks PH. The effect of controlled fermentation on the fate of synthetic
lysine in liquid diets for pigs. Anim Feed Sci Technol 2006; 129: 304-315.
NRC (National Research Council). Nutrient Requirements of Swine. 10th Revised Edition. A
report of the Committee on Animal Nutrition. Washington, D.C. National Academy Press, 1998.
O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975;
250: 4007-4021.
Palmfeldt J, Levander F, Hahn-Hägerdal B and James P. Acidic proteome of growing and resting
Lactococcus lactis metabolizing maltose. Proteomics 2004; 4: 3881-3898.
Passos FV, Fleming HP, Hassan HM and McFeeters RF. Effect of malic acid on the growth
kinetics of Lactobacillus plantarum. Appl Microbiol Biotechnol 2003; 63: 207-211.
Pavan S, Hols P, Delcour J, Geoffroy MC, Grangette C, Kleerebezem M and Mercenier A.
Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study
in vivo biological effects. Appl Environ Microbiol 2000; 66: 4427-4432.
Pieterse B, Leer RJ, Schuren FH and van der Werf MJ. Unravelling the multiple effects of lactic
acid stress on Lactobacillus plantarum by transcription profiling. Microbiology-Sgm 2005; 151:
3881-3894.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
58
References
Pietrogrande MC, Marchetti N, Dondi F and Righetti PG. Spot overlapping in two-dimensional
polyacrylamide gel electrophoresis maps: relevance to proteomics. Electrophoresis 2003; 24:
217-224.
Robertson ID, Accioly JM, Moore KM, Driesen SJ, Pethick DW and Hampson DJ. Risk factors
for gastric ulcers in Australian pigs at slaughter. Prev Vet Med 2002; 53: 293-303.
Russell PJ, Geary TM, Brooks PH and Campbell A. Performance, water use and effluent output
of weaner pigs fed ad libitum with either dry pellets or liquid feed and the role of microbial
activity in the liquid feed. J Sci Food Agric 1996; 72: 8-16.
Salovaara H. Lactic Acid Bacteria in Cereal-Based Products. In S. Salminen, A. von Wright, and
A. Ouwehand (ed.), Lactic Acid Bacteria: Microbial and Functional Aspects, 3rd ed. Marcel
Dekker, New York, NY, 2004, p. 431-452.
Savard T, Beaulieu C, Gardner NJ and Champagne CP. Characterization of spoilage yeasts
isolated from fermented vegetables and Inhibition by lactic, acetic and propionic acids. Food
Microbiol 2002; 19: 363-373.
Scheele GA. Two-dimensional gel analysis of soluble proteins. Charaterization of guinea pig
exocrine pancreatic proteins. J Biol Chem 1975; 250: 5375-5385.
Scholten RH, van der Peet-Schwering CM, Verstegen MW, den Hartog LA, Schrama JW and
Vesseur PC. Fermented co-products and fermented compound diets for pigs: a review. Anim
Feed Sci Technol 1999; 82: 1-19.
Scholten RH, van der Peet-Schwering CM, den Hartog LA, Balk M, Schrama JW and Verstegen
MW. Fermented wheat in liquid diets: Effects on gastrointestinal characteristics in weanling
piglets. J Anim Sci 2002; 80: 1179-1186.
Shevchenko A, Wilm M, Vorm O and Mann M. Mass spectrometric sequencing of proteins from
silver stained polyacrylamide gels. Anal Chem 1996; 68: 850-858.
Siezen RJ, van Enckevort FH, Kleerebezem M and Teusink B. Genome data mining of lactic
acid bacteria: the impact of bioinformatics. Curr Opin Biotechnol 2004; 15: 105-115.
Titgemeyer F and Hillen W. Global control of sugar metabolism: a Gram-positive solution.
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology 2002;
82: 59-71.
van de Guchte M, Serror P, Chervaux C, Smokvina T, Ehrlich SD and Maguin E. Stress
responses in lactic acid bacteria. Antonie Van Leeuwenhoek 2002; 82: 187-216.
van Winsen RL, Urlings BA, Lipman LJ, Snijders JM, Keuzenkamp D, Verheijden JH and van
Knapen F. Effect of fermented feed on the microbial population of the gastrointestinal tracts of
pigs. Appl Environ Microbiol 2001; 67: 3071-3076.
van Winsen RL, Lipman LJ, Biesterveld S, Urlings BA, Snijders JM and van Knapen F.
Mechanism of Salmonella reduction in fermented pig feed. J Sci Food Agric 2001; 81: 342-346.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
59
Carme Plumed-Ferrer: Lactobacillus plantarum: From application to protein expression
Weinberg ZG, Muck RE and Weimer PJ. The survival of silage inoculant lactic acid bacteria in
rumen fluid. J Appl Microbiol 2003; 94: 1066-1071.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 220: 1-60 (2007)
60
Kuopio University Publications C. Natural and Environmental Sciences
C 199. Tarvainen, Tanja. Computational Methods for Light Transport in Optical Tomography.
2006. 123 p. Acad. Diss.
C 200. Heikkinen, Päivi. Studies on Cancer-related Effects of Radiofrequency Electromagnetic
Fields. 2006. 165 p. Acad. Diss.
C 201. Laatikainen, Tarja. Pesticide induced responses in ectomycorrhizal fungi and symbiont Scots
pine seedlings.
2006. 180 p. Acad. Diss.
C 202. Tiitta, Markku. Non-destructive methods for characterisation of wood material.
2006. 70 p. Acad. Diss.
C 203. Lehesranta, Satu. Proteomics in the Detection of Unintended Effects in Genetically
Modified Crop Plants.
2006. 71 p. Acad. Diss.
C 204. Boman, Eeva. Radiotherapy forward and inverse problem applying Boltzmann transport
equation.
2007. 138 p. Acad. Diss.
C 205. Saarakkala, Simo. Pre-Clinical Ultrasound Diagnostics of Articular Cartilage and
Subchondral Bone.
2007. 96 p. Acad. Diss.
C 206. Korhonen, Samuli-Petrus. FLUFF-BALL, a Fuzzy Superposition and QSAR Technique Towards an Automated Computational Detection of Biologically Active Compounds Using
Multivariate Methods.
2007. 154 p. Acad. Diss.
C 207. Matilainen, Merja. Identification and characterization of target genes of the nuclear
reseptors VDR and PPARs: implementing in silico methods into the analysis of nuclear receptor
regulomes.
2007. 112 p. Acad. Diss.
C 208. Anttonen, Mikko J. Evaluation of Means to Increase the Content of Bioactive Phenolic
Compounds in Soft Fruits.
2007. 93 p. Acad. Diss.
C 209. Pirkanniemi, Kari. Complexing agents: a study of short term toxicity, catalytic oxidative
degradation and concentrations in industrial waste waters.
2007. 83 p. Acad. Diss.
C 210. Leppänen, Teemu. Effect of fiber orientation on cockling of paper.
2007. 96 p. Acad. Diss.
C 211. Nieminen, Heikki. Acoustic Properties of Articular Cartilage: Effect of Structure,
Composition and Mechanical Loading.
2007. 80 p. Acad. Diss.
C 212. Tossavainen, Olli-Pekka. Shape estimation in electrical impedance tomography.
2007. 64 p. Acad. Diss.
C 213. Georgiadis, Stefanos. State-Space Modeling and Bayesian Methods for Evoked
Potential Estimation.
2007. 179 p. Acad. Diss.