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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. 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