Biotechnology of Lactic Acid Bacteria E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 1116

Veröffentlichungsjahr: 2015

Beschreibung

Lactic acid bacteria (LAB) have historically been used as starter cultures for the production of fermented foods, especially dairy products. Over recent years, new areas have had a strong impact on LAB studies: the application of �omics� tools; the study of complex microbial ecosystems, the discovery of new LAB species, and the use of LAB as powerhouses in the food and medical industries.

This second edition of Biotechnology of Lactic Acid Bacteria: Novel Applications addresses the major advances in the fields over the last five years. Thoroughly revised and updated, the book includes new chapters. Among them:

• The current status of LAB systematics;
• The role of LAB in the human intestinal microbiome and the intestinal tract of animals and its impact on the health and disease state of the host;
• The involvement of LAB in fruit and vegetable fermentations;
• The production of nutraceuticals and aroma compounds by LAB; and
• The formation of biofilms by LAB.

This book is an essential reference for established researchers and scientists, clinical and advanced students, university professors and instructors, nutritionists and food technologists working on food microbiology, physiology and biotechnology of lactic acid bacteria.

Leseprobe

Table of Contents

Cover

Title Page

List of Contributors

Editors

Contributors

Preface

Chapter 1: Updates on Metabolism in Lactic Acid Bacteria in Light of “Omic” Technologies

1.1. Sugar Metabolism

1.2. Citrate Metabolism and Formation of Aroma Compounds

1.3. The Proteolytic System of Lactic Acid Bacteria

1.4. LAB Metabolism in Light of Genomics, Comparative Genomics, and Metagenomics

1.5. Novel Aspects of Metabolism Regulation in the Post-genomic Age

1.6. Functional Genomics and Metabolism

1.7. Systems Biology of LAB

Acknowledgments

References

Chapter 2: Systematics of Lactic Acid Bacteria: Current Status

2.1. Families and Genera of Lactic Acid Bacteria

2.2. A Focus on the Family Lactobacillaceae

2.3. Taxonomic Tools in the Genomic Era

References

Chapter 3: Genomic Evolution of Lactic Acid Bacteria: From Single Gene Function to the Pan-genome

3.1. The Genomics Revolution

3.2. Genomic Adaptations of LAB to the Environment

3.3. “Probiotic Islands”?

3.4. Stress Resistance and Quorum Sensing Mechanisms

3.5. The Impact of Genome Sequencing on Characterization, Taxonomy, and Pan-genome Development of Lactic Acid Bacteria

3.6. Functional Genomic Studies to Unveil Novel LAB Utilities

3.7. Conclusions

References

Chapter 4: Lactic Acid Bacteria: Comparative Genomic Analyses of Transport Systems

4.1. Introduction

4.2. Channel-forming Proteins

4.3. The Major Facilitator Superfamily

4.4. Other Large Superfamilies of Secondary Carriers

4.5. ABC Transporters

4.6. Heavy Metal Transporters

4.7. P-type ATPases in Prokaryotes

4.8. The Prokaryote-specific Phosphotransferase System (PTS)

4.9. Multidrug Resistance Pumps

4.10. Nutrient Transport in LAB

4.11. Conclusions and Perspectives

Acknowledgments

References

Chapter 5: Novel Developments in Bacteriocins from Lactic Acid Bacteria

5.1. Introduction

5.2. Characteristics and Classification of Bacteriocins

5.3. Mode of Action

5.4. Bacteriocin Resistance

5.5. Applications

5.6. Future Perspectives

References

Chapter 6: Bacteriophages of Lactic Acid Bacteria and Biotechnological Tools

6.1. Introduction

6.2. Bacteriophages of Lactic Acid Bacteria

6.3. Antiphage Strategies

6.4. Phage-Based Molecular Tools

6.5. LAB Phages as Biocontrol Tools

6.6. Conclusions

References

Chapter 7: Lactic Acid Bacteria and the Human Intestinal Microbiome

7.1. Introduction

7.2. Ecology of the Human Intestinal Tract

7.3. A Case Study: The Lactobacillus rhamnosus Species

7.4. Concluding Perspectives and Future Directions

Acknowledgments

References

Chapter 8: Probiotics and Functional Foods in Immunosupressed Hosts

8.1. Introduction

8.2. Probiotic Fermented Milk in a Malnutrition Model

8.3. Probiotic Administration in Stress Process

8.4. Conclusions

Acknowledgments

References

Chapter 9: Lactic Acid Bacteria in Animal Production and Health

9.1. Introduction

9.2. Lactic Acid Bacteria and Probiotics

9.3. Classifications and Regulatory Criteria of Probiotics in Animal Health

9.4. Probiotic LAB and Animal Production Sectors

9.5. Conclusions

References

Chapter 10: Proteomics for Studying Probiotic Traits

10.1. Introduction

10.2. Mass Spectrometric Methodologies in Proteomics

10.3. Proteomics for Studying Molecular Mechanisms of Probiotic Action

10.4. Concluding Remarks and Future Directions

References

Chapter 11: Engineering Lactic Acid Bacteria and Bifidobacteria for Mucosal Delivery of Health Molecules

11.1. Introduction

11.2.

Lactococcus lactis

: A Pioneer Bacterium

11.3.

Lactobacillus

spp. as a Delivery Vector

11.4. Bifidobacteria as a New Live Delivery Vehicle

11.5. Engineering Genetic Tools for Protein and DNA Delivery

11.6. Therapeutic Applications

11.7. Allergy

11.8. Autoimmune Diseases

11.9. Infectious Diseases

References

Chapter 12: Lactic Acid Bacteria for Dairy Fermentations: Specialized Starter Cultures to Improve Dairy Products

12.1. Introduction

12.2. Adjunct Cultures

12.3. Phage-Resistant Starters

12.4. New Sources of Starter Strains

12.5. Conclusions

References

Chapter 13:

Lactobacillus sakei

in Meat Fermentation

13.1. Introduction

13.2. Genomics and Diversity of the Species

Lactobacillus sakei

13.3. Post-genomic Vision of Meat Fitness Traits of

Lactobacillus sakei

13.4. Conclusions

References

Chapter 14: Vegetable and Fruit Fermentation by Lactic Acid Bacteria

14.1. Introduction

14.2. Lactic Acid Bacteria Microbiota of Raw Vegetables and Fruits

14.3. Fermentation of Vegetable Products

14.4. Main Fermented Vegetable Products

14.5. Physiology and Biochemistry of LAB during Vegetable and Fruit Fermentation

14.6. Food Phenolic Compounds: Antimicrobial Activity and Microbial Responses

14.7. Health-promoting Properties of Fermented Vegetables and Fruits

14.8. Alternative Sources of Novel Probiotics Candidates

14.9. Vehicles for Delivering Probiotics

14.10. Conclusions

References

Chapter 15: Lactic Acid Bacteria and Malolactic Fermentation in Wine

15.1. Introduction

15.2. The Lactic Acid Bacteria of Wine

15.3. The

Oenococcus Oeni

Species

15.4. Evolution of Lactic Acid Bacteria during Winemaking

15.5. Lactic Acid Bacteria Metabolism and its Impact on Wine Quality (Table 15.2)

15.6. Controlling the Malolactic Fermentation

15.7. Conclusions

References

Chapter 16: The Functional Role of Lactic Acid Bacteria in Cocoa Bean Fermentation

16.1. Introduction

16.2. Cocoa Crop Cultivation and Harvest

16.3. The Cocoa Pulp or Fermentation Substrate

16.4. Fresh, Unfermented Cocoa Beans

16.5. Cocoa Bean Fermentation

16.6. Succession of Microorganisms during Cocoa Bean Fermentation

16.7. Biochemical Changes in the Cocoa Beans during Fermentation and Drying

16.8. Optimal Fermentation Course and End of Fermentation

16.9. Further Processing of Fermented Cocoa Beans

16.10. Use of Starter Cultures for Cocoa Bean Fermentation

16.11. Concluding Remarks

References

Chapter 17: B-Group Vitamins Production by Probiotic Lactic Acid Bacteria

17.1. Introduction

17.2. B-Group Vitamins

17.3. Probiotics

In Situ

17.4. Conclusions

Acknowledgments

References

Chapter 18: Nutraceutics and High Value Metabolites Produced by Lactic Acid Bacteria

18.1. Introduction

18.2. Nutraceutics

18.3. Exopolysaccharides

18.4. Commodity Chemicals

18.5. Conclusions

References

Chapter 19: Production of Flavor Compounds by Lactic Acid Bacteria in Fermented Foods

19.1. Introduction

19.2. Flavor and Aroma Compounds

19.3. LAB of Fermented Foods and their Role in Flavor Formation

19.4. Biotic and Abiotic Factors Modulating the Contribution of LAB to Flavor Formation

19.5. Conclusions and Research Perspectives

References

Chapter 20: Lactic Acid Bacteria Biofilms

20.1. Lactic Acid Bacteria Biofilms are Ubiquitous in a Wide Variety of Environments from Nature to Domesticated Settings

20.2. Biofilm Life Cycle and Bacterial Factors Involved in LAB Biofilm Lifestyle

20.3. Health and Biotechnological Potential of LAB Biofilms and Underlying Mechanisms

20.4. Conclusions

Acknowledgments

References

Index

End User License Agreement

List of Tables

Chapter 03

Table 3.1. Lactic acid bacteria genomes, number of complete or partially complete sequences per genus. Summarized from http://www.ncbi.nlm.nih.gov/genome/browse/.

Table 3.2. Components of the

Lactobacillus helveticus

CNRZ32 proteolytic enzyme system: a look before and after genome sequence determination (Cogan et al., 2007). Reproduced with permission.

Table 3.3. Pan- and core genomes, based on gene families, for specific strains from five genera of LAB bacteria. Reproduced with permission (Lukjancenko et al., 2012).

Chapter 04

Table 4.1. Summary of functionally characterized aquaporins and glycerol facilitators in LAB.

Table 4.2. Summary of functionally characterized Pore-Forming Toxins in LAB.

Table 4.3. Functional types of Major Facilitator Superfamily (MFS) carriers and their relative occurrences in five different prokaryotic groups of organisms.

Table 4.4. Summary of functionally characterized secondary carriers in LAB.

Table 4.5. Distribution of RND superfamily members in five groups of prokaryotes.

Table 4.6. Distribution of DMT superfamily members in five groups of prokaryotes.

Table 4.7. Distribution of MOP superfamily members in five groups of prokaryotes.

Table 4.8. Organism-type distribution of the ABC1, ABC2, and ABC3-type exporters of the ABC functional superfamily.

Table 4.9. Summary of functionally characterized ABC transporters in LAB.

Table 4.10. Functional types of ABC exporter, expressed in percent, identified in different types of prokaryotes. The percentages are in bold print, while the relative proportions of ABC1, ABC2 and ABC3 types are provided below the percentages. CHO, complex carbohydrates; AAs, amino acids.

Table 4.11. Distribution of eight families of secondary carriers specific for inorganic divalent cations in five groups of organisms. All values are expressed as the average numbers of transporters in each family divided by the number of genomes examined.

Table 4.12. P-type ATPase superfamily representation (%) in five types of organisms. The values are for the individual families found in LAB.

Table 4.13. Summary of functionally characterized PTS systems in LAB.

Chapter 06

Table 6.1. Strategies applied in phage control

Chapter 07

Table 7.1. List of LAB and bifidobacteria detected in the human intestinal tract as previously described (Molin et al. 1993; Ahrne et al. 1998; Heilig et al. 2002; Vaughan et al. 2002; Bello et al. 2003; Walter 2008).

Table 7.2. Host–microbe interaction factors in

Lact. rhamnosus

strain GG promoting ecological fitness in the human gastrointestinal tract. Representative and non-exhaustive list of reference(s) are shown for each factor.

Chapter 08

Table 8.1. Immune cells in the small intestine of malnourished and re-nourished mice.

Table 8.2. Secretory IgA in the intestinal fluid and immune cells in the small intestine of stressed mice.

Chapter 09

Table 9.1. Types of microorganisms used as probiotics in livestock and animal productions.

Chapter 14

Table 14.1. Main criteria and corresponding metabolic traits to select starters for vegetable and fruit fermentation.

Table 14.2. Metabolism of phenolic compounds by LAB.

Chapter 15

Table 15.1. List of lactic acid bacteria species in the wine environment.

Table 15.2. Main metabolisms known in wine

Chapter 16

Table 16.1. Diversity of lactic acid bacteria associated with cocoa bean fermentations in different countries (original species names are used).

Ent

.,

Enterococcus

;

L

.,

Lactococcus

;

Lact

.,

Lactobacillus

;

Leuc

.,

Leuconostoc

;

O

.,

Oenococcus

;

Ped

.,

Pediococcus

;

W

.,

Weissella

.

Table 16.2. Functional role of lactic acid bacteria associated with cocoa bean fermentation.

Chapter 17

Table 17.1. Vitamin enrichment by lactic acid bacteria in fermented foods.

Chapter 19

Table 19.1. Origin and flavor notes of some important aroma compounds in fermented dairy products.

Table 19.2. Main LAB species in some examples of fermented foods and their role in the formation of flavor.

Table 19.3. Examples of strain and species-dependent traits related to the formation of aroma compounds in fermented foods.

Chapter 20

Table 20.1. Some examples of LAB biofilms identified along the food chain and in domesticated environments.

Table 20.2. Bacterial factors involved in different steps of biofilm formation in LAB.

List of Illustrations

Chapter 01

Figure 1.1. Pathways of glucose metabolism. (a) Homofermentative pathway. (b) Mixed-acid metabolism. (c) Heterofermentative pathway. (d) Leloir pathway. Reactions are catalyzed by the following enzymes: 1, glucokinase (GLK); 2, glucose-phosphate isomerase (GPI); 3, phosphofructokinase (PFK); 4, fructose-bisphosphate aldolase (FBPA); 5, triose-phosphate isomerase (TPI); 6, glyceraldehyde-phosphate dehydrogenase (GAPDH); 7, phosphoglycerate kinase (PGK); 8, phosphoglycerate mutase (PMG); 9, enolase (ENO); 10, pyruvate kinase (PK); 11, lactate dehydrogenase (LDH); 12, pyruvate dehydrogenase (PDH); 13, pyruvate formate lyase (PFL); 14, acetaldehyde dehydrogenase (ACDH); 15, alcohol dehydrogenase (ADHE); 16, phosphotransacetylase (PTA); 17, acetate kinase (ACK); 18, α-acetolactate synthase (ALS); 19, α-acetolactate decarboxylase (ALD); 20, 2,3-butanediol dehydrogenase (BDH); 21, diacetyl reductase (DR); 22, glucose-6-P dehydrogenase (G6PDH); 23, 6-P-gluconate dehydrogenase (6PGDH); 24, ribulose-5-P-3-epimerase (RPPE); 25, D-xylulose-5P phosphoketolase (XPK); 26, galactokinase (GK); 27, galactose-1-P-uridylyltransferase (GPUDLT); 28, UDP-galactose-1-epimerase (UDPE); 29, phosphoglucomutase (PGM).

Figure 1.2. Citrate metabolism in

Lactococcus

,

Leuconostoc

, and

Weissella

species. Key for the enzymes: CL, citrate lyase; OAD, oxaloacetate decarboxylase; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase; ALS, α-acetolactate synthase; ADC, α-acetolactate decarboxylase; DAR, diacetyl acetoin reductase; BDH, 2,3-butanediol dehydrogenase; Tppi, thiamine pyrophosphate.

Figure 1.3. Diagram of the proteolytic systems of lactic acid bacteria. (a) Extracellular components: PrtP, cell-envelope proteinase; PrtM, proteinase maduration protein; Opp, oligopetide permease; DtpT, the ion linked trasnsporter for di-and tripeptides; and Opt, the ABC transporter for peptides. (b) Intracelullar components: pool of about 20–25 peptidases, including general (PepN, PepC) and specific (PepX, PepQ) peptidases, and amino acid catabolic enzymes (carboxylases, aminotransferases, etc.).

Figure 1.4. Distribution of predicted transcription factors (TFs) in selected Lactobacillaceae (a) and Streptococcaceae (b) genomes. The least-represented TFs families (AbrB, AsnC, BirA, CcpN, CodY, ComK, CtsR, DtxR, Fis, Fur, GlnL, GutM, HrcA, HTH_11, IclR, LexA, NiaR, NrdR, NrtR, PF04394, PurR, Rex, ROK, SdaR, SfsA, SorC, YobV) are not presented on the ideograms. Row data derive from (Ravcheev et al. 2013). Graphical presentation of these data has been developed with Circos (v0.64) (Krzywinski et al. 2009). Strains of presented

Lactobacillales

species:

L. lactis cremoris

(

Lactococcus lactis

subsp.

cremoris

SK11),

L. lactis lactis

(

Lactococcus lactis

subsp.

lactis

IL1403),

S. thermophilus

(

Streptococcus thermophilus

CNRZ1066),

S. agalactiae

(

Streptococcus agalactiae

2603V/R)

S. uberis

(

Streptococcus uberis

0140J),

S. equi

(

Streptococcus equi

MGCS10565),

S

.

dysgalactiae

(

Streptococcus dysgalactiae

GGS_124),

S. pyogenes

(

Streptococcus pyogenes

M1 GAS),

S

.

gallolyticus

(

Streptococcus gallolyticus

UCN34),

S

.

mutans

(

Streptococcus mutans

UA159),

S. suis

(

Streptococcus suis

05ZYH33),

S. mitis

(

Streptococcus mitis

B6),

S. pneumoniae

(

Streptococcus pneumoniae

TIGR4),

S. gordonii

(

Streptococcus gordonii

CH1),

S. sanguinis

(

Streptococcus sanguinis

SK36),

L. sakei

(

Lactobacillus sakei

23K),

L. casei

(

Lactobacillus casei

ATCC 334),

L. rhamnosus

(

Lactobacillus rhamnosus

GG),

L. delbrueckii

(

Lactobacillus delbrueckii

ATCC BAA-365),

L. acidophilus

(

Lactobacillus acidophilus

NCFM),

L. helveticus

(

Lactobacillus helveticus

DPC 4571),

L. johnsonii

(

Lactobacillus johnsonii

NCC 533),

P. pentosaceus

(

Pediococcus pentosaceus

ATCC 25745),

L. brevis

(

Lactobacillus brevis

ATCC 367),

L. plantarum

(

Lactobacillus plantarum

WCFS1),

L. fermentum

(

Lactobacillus fermentum

IFO 3956),

L. reuteri

(

Lactobacillus reuteri

JCM 1112),

O. oeni

(

Oenococcus oeni

PSU-1),

L. mesenteroides

(

Leuconostoc mesenteroides

ATCC 8293),

L. salivarius

(

Lactobacillus salivarius

UCC118).

Chapter 02

Figure 2.1. Phylogenetic tree depicting the relationship between families of order Lactobacillales based on 16S rRNA gene sequence. The tree was calculated using Neighbor-Joining and the number of differences as a method. Bootstrap values (1000 replicates) are reported in percentages at nodes. The scale bar represents the number of substitutions per site.

Chapter 03

Figure 3.1. COG statistics for the pan- and core genomes of nonpathogenic and pathogenic isolates from the genera

Lactobacillus

,

Lactococcus

,

Leuconostoc

,

Enterococcus

,

Streptococcus

, and

Bifidobacterium.

Figure 3.2 Organization of the genome region of selected LAB containing the autoinducer-2 (AI-2) producer gene

lux

S. The comparison was performed using the Multi-Genome Region Comparison tool in the Comprehensive Microbial Resource website at JCVI (http://www.jcvi.org/). The cutoff selection for protein matches was 40% minimum similarity. The two chromosomal regions of

Lact. delbrueckii

subsp.

bulgaricus

ATCC BAA-365 containing

lux

S are indicated.

Figure 3.3. Whole-genome alignment of

Strep. thermophilus

CNRZ1066 versus

Strep. thermophilus

LMG 18311 using MUMmer (Maximal Unique Match) (Kurtz et al. 2004). Minimum Match Length was set at 150. Unique regions in each chromosome are depicted as green (unique to CNRZ1066) and red dots (unique to LMG18311).

Chapter 04

Figure 4.1. Schematic depiction of the structures of the three families of integral membrane ABC exporters that catalyze substrate efflux using ATP hydrolysis for energy coupling. (a) ABC1 proteins are 6-TMS permeases that arose by intragenic triplication of a 2-TMS hairpin-encoding gene. (b) ABC2 proteins are 6-TMS permeases that arose by intragenic duplication of a gene encoding a 3-TMS primordial structure giving a 6-TMS protein with the two repeat units having opposite orientation in the membrane. (c) ABC3 proteins are 4-, 8-, and 10-TMS permeases where duplication of the primordial 4-TMS element gave rise to the 8- and 10-TMS proteins. In the 10-TMS proteins, the extra two TMSs separate the two 4-TMS repeat units. (Wang et al., 2009; Zheng et al., 2013).

Figure 4.2. Four independently evolving families within the PTS functional superfamily. This schematic figure illustrates independent evolutionary origins for the four currently recognized families. (a, left) The Glc/Fru/Lac superfamily, (b, center) the Man family, (c, right) the Asc/Gat superfamily, and (d, bottom center) the Dha family.

Chapter 05

Figure 5.1. Classification scheme of the bacteriocins of Gram (+) bacteria, revised from Cotter et al. (2005b). The main distinction is made between the Class I lantibiotics, which undergo extensive post-translational modifications, and the Class II non-modified bacteriocins. The amino acid sequence and structural features of one example bacteriocin of each different class are shown.

Chapter 06

Figure 6.1. Electron micrograph of negatively stained bacteriophage A2 that infects strains of

Lact. casei

and

Lact. paracasei.

Figure 6.2. Natural phage resistance mechanisms described in lactic acid bacteria. R/M: restriction/modification systems; CRISPR-

cas

: clustered regularly interspaced short palindromic repeats (CRISPR) loci and

cas

(CRISPR-associated) genes.

Chapter 07

Figure 7.1. Hypervariable regions identified in Lact. rhamnosus strain GG chromosome when compared to other

Lact. rhamnosus

isolates. These regions, also termed lifestyle islands, mostly relate to carbohydrate metabolism, host interaction and signaling, DNA restriction/modification systems, CRISPR-

cas

locus and prophages.

Figure 7.2. Architecture and composition of

Lact. rhamnosus

GG cell envelope associated with different cell-wall components. The cell membrane is typically covered with peptidoglycans that are decorated with mucus-binding pili, LPXTG proteins, glycoproteins, lipoteichoic acid (LTA), wall teichoic acids (WTA). A layer of exopolysaccharides (EPS) is juxtaposed onto the peptidoglycans.

Chapter 08

Figure 8.1. Improvement of the intestinal mucosal and the immune system by a PFM as dietary supplement after a malnutrition period. (a) Impairment of the intestinal mucosa by malnutrition. Malnutrition induces histological changes in the intestinal mucosa, with length diminution of the villi. The number of IgA producing cells diminished in the small intestine of malnourished mice. Also a decrease in the number of immune cells, such as MQ, DC, B and T lymphocytes (CD4 and CD8), was observed, as in cytokines producing cells (IL-10, IL-4, IL-6, IL-12, and TNFα). The intestinal microbiota showed a significant increase in the enterobacteria population. (b) Improvement of the intestinal mucosa of malnourished animals with PFM. The re-nutrition with PFM as a dietary supplement restores the length of the intestinal villi Significant increases in the number of IgA producing cells in the lamina propria of the intestinal villi were observed. The number of MQ, DC, BL, and T lymphocytes (CD4 and CD8) also improved. The cytokine producing cells (IL-10, IL-4, IL-6, IL-12 and TNF-α) increased mainly IFN-γ. The PFM induced increases in the bifidobacteria population accompanied by a diminution of enterobacteria. IEC: Intestinal epithelial cell; GC: goblet cell; PP: Peyer’s patch; LP lamina propria; MQ: macrophages; DC: dendritic cells; BL: B lymphocytes; S-IgA: secretory IgA.

Chapter 10

Figure 10.1. Workflow generally applied in proteomic studies.

Figure 10.2. Response mechanisms triggered by both acid and bile exposure in lactobacilli and bifidobacteria.

Figure 10.3. Response mechanisms specifically triggered by bile exposure in lactobacilli and bifidobacteria.

Chapter 11

Figure 11.1. Schematic representation of broad choice of bacteria, expression systems, and product localizations mainly used in mucosal delivery.

Chapter 12

Figure 12.1. Simplified representation of CRISPR-

cas

system locus. CRISPR repeats are represented as black diamonds and spacers as numbered white boxes. The asterisk indicates the terminal repeat.

Chapter 13

Figure 13.1. Accessory genome or flexible gene pool of

Lact. sakei

.

Figure 13.2. Main functional categories associated with the flexible gene pool of

Lact. sakei

.

Chapter 14

Figure 14.1. Example of biotechnology protocol to ferment raw vegetables and fruits.

Figure 14.2. Example of some metabolic pathways adopted by

Lactobacillus plantarum

during fermentation of vegetable and fruit juices. Ile, isoleucine; Leu, leucine; Val, valine; His, histidine; Glu, glutamic acid; BcAT, branched-chain aminotransferase; KDC, α-keto acid decarboxylase; ADH, alcohol dehydrogenase; MLE, malolactic enzyme; HDC, histidine decarboxylase

Chapter 15

Figure 15.1. Example of the LAB species found before and after alcoholic fermentation as identified by rpoB/PCR-DGGE.

Chapter 16

Figure 16.1. Opened cocoa pod with beans embedded in a mucilaginous pulp.

Figure 16.2. (a) Cocoa bean. (b) Cocoa bean storage cells.

Figure 16.3. (a–f) The cocoa bean heap fermentation is the most simple and most commonly used method of fermentation on small farms. It requires the simplest equipment at practically no cost, so that it can be run by a family. In a heap fermentation process the cocoa pulp–bean mass is piled on banana or plantain leaves, which are spread out in a circle on the ground, sometimes around a central hole in the ground or raised above soil level to allow easy pulp drainage. When the heap is complete, it is covered with more leaves and these are often held in place by small logs. The cover protects the fermenting cocoa pulp–bean mass against surface mold growth and keeps the heat inside. In general, sweatings are allowed to flow away and penetrate into the ground. The size of the heaps varies widely; heaps from about 25 to 2000 kg are common. This cocoa bean fermentation method is used throughout West-Africa and almost exclusively in Ghana, where farmers are fermenting their beans in heaps of 200 to 500 kg for six days. The heap fermentation method has been used to produce some of the world’s most qualitative standard cocoa available, especially in Ghana.

Figure 16.4. (a) Community dynamics and (b) metabolite kinetics during cocoa pulp–bean mass fermentation.

Figure 16.5. Biochemical changes and diffusion processes in pulp and beans during the cocoa bean fermentation process (after Lopez, 1986).

Chapter 17

Figure 17.1. Main intermediates of the cobalamin biological synthesis. Arrow with incomplete lines indicate the existence of more than three enzymatic steps involved in obtaining the intermediate.

Chapter 18

Figure 18.1. Chemical structures of

D

-mannitol,

D

-sorbitol,

D

-erythritol,

D

-galactitol,

D

-xylitol,

D

-ribitol,

D

-lactitol, and

D

-maltitol.

Figure 18.2. Discovering of bioactive peptides.

Figure 18.3. Schematic representation of GABA production. Glutamate is taken up by the electrogenic

L

-glutamate/GABA antiporter, the decarboxylation of

L

-glutamate via glutamate decarboxylase (GAD) consumes an intracellular H

+

and contributes to the generation of proton motive force by GABA. PLP: pyridoxal-5’-phosphate.

Chapter 19

Figure 19.1. Numbers of papers published in relation to the formation of flavor compounds by lactic acid bacteria in different fermented foods. Interrogation of the ISI Web of Science database in June 2014, over the period 1994-2014.

Figure 19.2. Schematic overview of conversion pathways leading to the formation of the main flavor compounds by lactic acid bacteria in fermented foods. Enzymes: extracellular enzymes or intracellular enzymes released from lysed cells; broken arrows: chemical (non enzymatic) reactions; red, bold: flavor compounds; blue, italics: main enzymes involved.

Figure 19.3. General scheme illustrating the dynamic of flavor development in fermented foods and the interactions within this ecosystem that influence flavor formation.

Chapter 20

Figure 20.1. The ubiquity of LAB biofilms in nature including plants, silage, cheese manufacturing environment, and mucosae of man and animals. Images embedded in the arrow are from Jean Weber ©INRA, Alain Fraval ©INRA, Jean-Marie Bossennec ©INRA, Christian Slagmulder ©INRA, Serge Carre ©INRA, and Sophie Normant ©INRA and were obtained through the INRA media library (http://mediatheque.inra.fr/). The central image showing a lactobacilli biofilm is from Thierry Meylheuc and Alexis Canette (MIMA2 platform, http://www6.jouy.inra.fr/mima2_eng/).

Figure 20.2. Biofilm lifecycle schemed as three main steps: (I) bacterial adhesion, (II) biofilm maturation through matrix biosynthesis and cell proliferation, and (III) biofilm decline and cell dispersal. Arrows within the biofilm matrix represent water channels.

Figure 20.3. The pili of the lactic acid bacterium

Lactococcus lactis

affect the architecture of biofilms. (a) Non-piliated

L. lactis

biofilm and (b) piliated

L. lactis

biofilm obtained from confocal image series. (c)

L. lactis

pili observed using negative staining and transmission electron microscopy. Images (a) and (b) are from Julien Deschamps and image (c) from Alexis Cannette (http://www6.jouy.inra.fr/mima2_eng/).

Guide

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Biotechnology of Lactic Acid Bacteria

Novel Applications

Second Edition

Edited by

Fernanda Mozzi, Raúl R. Raya, and Graciela M. Vignolo

This edition first published 2016© 2016 by John Wiley & Sons, Ltd.

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List of Contributors

Editors

Fernanda Mozzi. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Raúl R. Raya. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Graciela M. Vignolo. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Contributors

Tamara Aleksandrzak-Piekarczyk. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland

Thibault Allain. INRA, Commensal and Probiotics-Host Interactions Laboratory, UMR 1319 Micalis, FranceAgroParisTech, UMR1319, France

Camille Aubry. INRA, Commensal and Probiotics-Host Interactions Laboratory, UMR 1319 Micalis, FranceAgroParisTech, UMR1319, France

M. Andrea Azcarate-Peril. Department of Cell Biology and Physiology, and Microbiome Core Facility, University of North Carolina at Chapel Hill, Chapel Hill, USA

Luis G. Bermúdez-Humarán. INRA, Commensal and Probiotics-Host Interactions Laboratory, UMR 1319 Micalis, FranceAgroParisTech, UMR1319, France

Ana Binetti. Instituto de Lactología Industrial (INLAIN), Universidad Nacional del Litoral-CONICET, Argentina

Damien Bouchard. INRA, AGROCAMPUS OUEST UMR1253 Science et Technologie du Lait et de l'Œuf, France

Dag A. Brede. Department of Chemistry, Biotechnology and Life Science, Norwegian University of Life Sciences, Norway

Romain Briandet. INRA and AgroParisTech, UMR1319 Micalis, France

Mariángeles Briggiler Marcó. Instituto de Lactología Industrial (INLAIN), Universidad Nacional del Litoral-CONICET, Argentina

Domenico Carminati. Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie (CRA-FLC), Italy

Marie-Christine Champomier-Vergès. INRA, UMR 1319 Micalis, FranceAgroParisTech, UMR Micalis, France

Jean-Marc Chatel. INRA, Commensal and Probiotics-Host Interactions Laboratory, UMR 1319 Micalis, FranceAgroParisTech, UMR1319, France

Alejandra de Moreno de LeBlanc. Laboratorio de Inmunología, Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Willem M. de Vos. Department of Veterinary Biosciences, University of Helsinki, Finland Laboratory of Microbiology, Wageningen University, The Netherlands

Luc De Vuyst. Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Department of Bioengineering Sciences, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Belgium

Raffaella Di Cagno. Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Italy

Dzung B. Diep. Department of Chemistry, Biotechnology and Life Science, Norwegian University of Life Sciences, Norway

Grace L. Douglas. Human Health & Performance Directorate, NASA Johnson Space Center, Houston, USA

François P. Douillard. Department of Veterinary Biosciences, University of Helsinki, Finland

Sergine Even. INRA, AGROCAMPUS OUEST UMR1253 Science et Technologie du Lait et de l’Œuf, France

Giovanna E. Felis. Department of Biotechnology, University of Verona, Italy

María Fernández. Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Spain

Pasquale Filannino. Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Italy

Christina Gabrielsen. Department of Chemistry, Biotechnology and Life Science, Norwegian University of Life Sciences, Norway

Pilar García. DairySafe Group, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Spain

Giorgio Giraffa. Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie (CRA-FLC), Italy

Marco Gobbetti. Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Italy

Daniela Guglielmotti. Instituto de Lactología Industrial (INLAIN), Universidad Nacional del Litoral-CONICET, Argentina

Elvira M. Hebert. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Marianela Juárez del Valle. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Todd R. Klaenhammer. Department of Food, Bioprocessing, and Nutrition Sciences, and Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, USA

Magdalena Kowalczyk. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland

Jonathan Emiliano Laiño. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Philippe Langella. INRA, Commensal and Probiotics-Host Interactions Laboratory, UMR 1319 Micalis, FranceAgroParisTech, UMR1319, France

Yves Le Loir. INRA, AGROCAMPUS OUEST UMR1253 Science et Technologie du Lait et de l'Œuf, France

Jean Guy LeBlanc. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Aline Lonvaud-Funel. University of Bordeaux, ISVV, France

Graciela L. Lorca. Department of Microbiology and Cell Science, Genetics Institute and Institute of Food and Agricultural Sciences, University of Florida, Gainesville, USA

Sylvie Lortal. INRA, AGROCAMPUS OUEST UMR1253 Science et Technologie du Lait et de l’OEuf, France

Carolina Maldonado Galdeano. Laboratorio de Inmunología, Centro de Referencia para Lactobacilos (CERELA)-CONICET, ArgentinaCatedra de Inmunologia, Instituto de Microbiologia, Facultad de Bioquimica, Quimica y Farmacia, Universidad Nacional de Tucuman, Argentina

Beatriz Martínez. DairySafe Group, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Spain

Baltasar Mayo. Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Spain

Maria Fiorella Mazzeo. Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell'Alimentazione, CNR, Italy

Fernanda Mozzi. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Jane M. Natividad. INRA, Commensal and Probiotics-Host Interactions Laboratory, UMR 1319 Micalis, FranceAgroParisTech, UMR1319, France

Ingolf F. Nes. Department of Chemistry, Biotechnology and Life Science, Norwegian University of Life Sciences, Norway

Ivanna Novotny Nuñez. Laboratorio de Inmunología, Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Martin Manuel Palomar. Laboratorio de Inmunología, Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Gabriela Perdigón. Laboratorio de Inmunología, Centro de Referencia para Lactobacilos (CERELA)-CONICET, ArgentinaCatedra de Inmunologia, Instituto de Microbiologia, Facultad de Bioquimica, Quimica y Farmacia, Universidad Nacional de Tucuman, Argentina

Jean-Christophe Piard. INRA and AgroParisTech, UMR1319 Micalis, France

Mariana Piuri. Departamento de Química Biológica, FCEyN, Universidad de Buenos Aires, Argentina

Tomislav Pogačić. Department of Dairy Science, Faculty of Agriculture University of Zagreb, Croatia

Raúl R. Raya. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Jorge Reinheimer. Instituto de Lactología Industrial (INLAIN), Universidad Nacional del Litoral-CONICET, Argentina

Ana Rodríguez González. DairySafe Group, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Spain

Milton H. Saier, Jr. Division of Biological Sciences, University of California at San Diego, San Diego, USA

Elisa Salvetti. Department of Biotechnology, University of Verona, Italy

Graciela Savoy de Giori. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Fernando Sesma. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Rosa Anna Siciliano. Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell'Alimentazione, CNR, Italy

María Pía Taranto. Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Anne Thierry. INRA, AGROCAMPUS OUEST UMR1253 Science et Technologie du Lait et de l’OEuf, France

Sandra Torriani. Department of Biotechnology, University of Verona, Italy

Taylor A. Twiddy. Department of Microbiology and Cell Science, Genetics Institute and Institute of Food and Agricultural Sciences, University of Florida, Gainesville, USA

Magalie Weber. INRA, AGROCAMPUS OUES, UMR1253 Science et Technologie du Lait et de l’OEuf, France

Stefan Weckx. Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Department of Bioengineering Sciences, Faculty of Sciences and Bioengineering Sciences, Belgium

Miriam Zago. Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie (CRA-FLC), Italy

Monique Zagorec. INRA, UMR 1014 Secalim, FranceLUNAM Université, Oniris, France

Preface

We have witnessed an explosion of new findings in the field of Lactic Acid Bacteria (LAB) over the last five years that have passed since the previous edition of our book. Rapid advances in the “omics” (genomics, proteomics, pangenomics, metagenomics) sciences and next generation sequencing technologies have revolutionized the characterization of LAB; the discovery of new LAB species has been achieved and complete genomes of all major groups are now available. The insights arisen from these basic studies have been translated into innovation leading to the use of LAB in applications beyond classic food fermentations. Thus, LAB have been engineered or manipulated for their use as live vaccines or as microbial factories for the production of food ingredients, nutraceuticals, commodity chemicals, and other high-value metabolites. LAB microbiota involved in the fermentation of vegetables, fruits, cocoa, wine, meat, and dairy products, as well as their contribution to sensory and safety, have been updated and/or included as new fields. Results from comparative and functional genomics have been used to understand the response of LAB to their environment, leading to better understanding of their adaption and safety in traditional/industrial foods and their interactions with the human host. Thus, chapters in this new edition of our book have been updated or present for the first time a wide range of topics including basic issues (metabolism, biodiversity, biofilms and transport systems), comprehensive information on new advanced approaches (comparative and functional genomics), human-health LAB-related aspects, LAB safety, as well as traditional and novel biotechnological applications. We hope this book finds its audience among microbiologists, food scientists, nutritionists, clinical and advanced students. The editors greatly appreciate the hard work and the time dedicated of many well-known leading scientists who contributed to this book. We also thank to CERELA, CONICET, and FONCyT from Argentina.

This book is devoted to the memory of our very good friend and colleague, Fernando Sesma, who passed away in July 2014.

Chapter 1Updates on Metabolism in Lactic Acid Bacteria in Light of “Omic” Technologies

Magdalena Kowalczyk1, Baltasar Mayo2, María Fernández2, and Tamara Aleksandrzak-Piekarczyk1,*

1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland

2Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Spain

1.1. Sugar Metabolism

Sugars are the primary carbon and energy source for LAB that are grown for fermented food and feed production as well as in laboratory media. Many different transport systems are involved in LAB carbohydrate uptake, including phosphotransferase systems (PTS), ATP-binding cassettes (ABC), and glycoside–pentoside–hexuronide transporters. In Lactococcus lactis, glucose is imported by either a mannose or cellobiose PTS or one or more non-PTS permease(s) (Castro et al. 2009). LAB prefer glucose but can also metabolize several common hexoses; however, the ability to ferment other sugars is strain dependent. Dairy LAB can use milk’s most abundant sugar, lactose, as a carbon source, whereas plant-associated bacteria utilize a large variety of other carbohydrates, including β-glucosides (Aleksandrzak-Piekarczyk 2013). Furthermore, phenotypic and genotypic analyses of twenty L. lactis subsp. lactis and cremoris genotypes showed strain-to-strain variations (Fernandez et al. 2011). These two groups had distinctive carbohydrate fermentation and enzyme activity profiles with cremoris genotypes exhibiting broader profiles.

Monosaccharides incorporated by the cell or liberated in the cytoplasm by disaccharide hydrolysis enter glycolysis at the glucose-6P (G6P) level or are processed by the Leloir pathway (Figure 1.1). In L. lactis, lactose that is transported by the PTS system is hydrolyzed to galactose-6P, transformed by the tagatose pathway (Tag6P), and then enters glycolysis at the triose phosphate level. In some LAB, only the glucose moiety of lactose is fermented, while the galactose moiety is excreted; this leads to galactose accumulation in the medium, which yields poor-quality dairy products (Neves et al. 2010; Aleksandrzak-Piekarczyk, 2013). Galactose can be imported by the non-PTS permease GalP and metabolized via the Leloir pathway (galMKTE). Alternatively, galactose can be imported by PTSLac (lacFE) and further metabolized to triose phosphates by the Tag6P pathway (lacABCD). Recently, an alternative uptake route was discovered that consists of galactose translocation via the galactose PTS, followed by Gal6P dephosphorylation to galactose, which is further metabolized via the Leloir pathway (Neves et al. 2010). This knowledge has been used to genetically engineer strains that enhance galactose consumption rates by up to 50% (Neves et al. 2010).

Figure 1.1. Pathways of glucose metabolism. (a) Homofermentative pathway. (b) Mixed-acid metabolism. (c) Heterofermentative pathway. (d) Leloir pathway. Reactions are catalyzed by the following enzymes: 1, glucokinase (GLK); 2, glucose-phosphate isomerase (GPI); 3, phosphofructokinase (PFK); 4, fructose-bisphosphate aldolase (FBPA); 5, triose-phosphate isomerase (TPI); 6, glyceraldehyde-phosphate dehydrogenase (GAPDH); 7, phosphoglycerate kinase (PGK); 8, phosphoglycerate mutase (PMG); 9, enolase (ENO); 10, pyruvate kinase (PK); 11, lactate dehydrogenase (LDH); 12, pyruvate dehydrogenase (PDH); 13, pyruvate formate lyase (PFL); 14, acetaldehyde dehydrogenase (ACDH); 15, alcohol dehydrogenase (ADHE); 16, phosphotransacetylase (PTA); 17, acetate kinase (ACK); 18, α-acetolactate synthase (ALS); 19, α-acetolactate decarboxylase (ALD); 20, 2,3-butanediol dehydrogenase (BDH); 21, diacetyl reductase (DR); 22, glucose-6-P dehydrogenase (G6PDH); 23, 6-P-gluconate dehydrogenase (6PGDH); 24, ribulose-5-P-3-epimerase (RPPE); 25, D-xylulose-5P phosphoketolase (XPK); 26, galactokinase (GK); 27, galactose-1-P-uridylyltransferase (GPUDLT); 28, UDP-galactose-1-epimerase (UDPE); 29, phosphoglucomutase (PGM).

Sugar fermentation leads to the formation of lactic acid alone or in combination with other organic acids and ethanol. Variations in the metabolic products of LAB have yielded three categories of fermentation: homofermentation, mixed-acid metabolism, and heterofermentation (Figure 1.1). These three types of fermentation and their regulatory mechanisms have been comprehensively reviewed in the first edition of this book (Mayo et al. 2010). Currently, the control and regulation of glycolytic flux in LAB are not fully understood (Martinussen et al. 2013). The control of glycolytic flux in L. lactis is not due to the actions of a single enzyme, sugar transport, or ATP-dependent mechanism. However, a combination of these mechanisms cannot be ruled out as a possible explanation (Martinussen et al. 2013).

In recent years, studies have shifted from digestible disaccharides to indigestible higher oligosaccharides as interest in intestinal microbial ecology and the commercial use of prebiotic oligosaccharides has emerged. Mono- and disaccharide metabolism is well understood; however, few data are available on the metabolism of higher oligosaccharides, which are abundant in cereals, milk, fruits, and the upper intestinal tract of animals. The metabolism of four major oligosaccharide groups have been examined in detail: (i) starch, maltodextrins, and isomalto-oligosaccharides (IMO); (ii) fructo-oligosaccharides (FOS); (iii) β-galacto-oligosaccharides (βGOS); and (iv) raffinose-family oligosaccharides and α-galacto-oligosaccharides (ROF and αGOS, respectively) (Gänzle and Follador 2012).

In addition to metabolizing sugar, LAB are able to direct sugar towards exopolysaccharide (EPS) biosynthesis. These long-chain saccharides are loosely attached to the cell surface to form capsule-like structures or are secreted into the environment. EPS production by lactobacilli has been previously discussed in a comprehensive review (Badel et al. 2011). Several studies of biopolymer diversity in LAB from cereal (Bounaix et al. 2009, 2010; Palomba et al. 2012) and in intestinal LAB (Salazar et al. 2009; Górska-Frączek et al. 2011, 2013; Sims et al. 2011) have also been published since the first edition of this book.

EPSs come in many different structures, sizes, and sugar compositions and are classified into two groups: homopolysaccharides (HoPS), which consist of one type of monosacharide (α-D-glucan, β-D-glucan, fructan, or a polygalactan); and heteropolysaccharides (HePS), which consist of different types of monosaccharides (D-glucose, D-galactose, L-rhamnose, and their derivatives). HoPS are synthesized extracellularly by highly specific glycosyltransferase enzymes as well as glucan- or fructan-sucrases. HoPS synthesis specifically requires sucrose as a substrate and the energy generated by its hydrolysis. The crystal structures, reaction and product specificities of glucansucrases as well as structural analyses of α-glucan polymers have been recently reviewed (Leemhuis et al. 2013).

HePS are synthesized from glucose, galactose, or other monosaccharides by the combined actions of several types of glycosyltransferases. HePS biosynthesis involves four major consecutive steps: (i) sugar transport into the cytoplasm, (ii) sugar-1P synthesis, (iii) polymerization of repeating unit precursors, and (iv) EPS export outside the cell. The synthesis of two EPSs in Lactobacillus johnsonii FI9785 is dependent on the 14-kb eps gene cluster; however, the precise regulation of EPS biosynthesis has yet to be identified (Dertli et al. 2013). It is thought that EPS production can be regulated at each of these four steps. Genome sequencing of EPS-related genes and their organization (Koryszewska-Baginska et al. 2014) may provide additional insight into whether this is the case.

1.1.1. Practical Aspects of Sugar Catabolism

The ability of LAB to ferment sugars has been widely utilized in various foods’ production. However, LAB also have the potential for increasing the production value of biofuels and biochemical products due to their robustness and tolerance for ethanol, low pH, and high temperatures (Martinussen et al. 2013). Focus has increased on optimizing lactate production from natural substrates such as starchy or lignocellulosic materials from agricultural, agro-industrial, and forestry sources due to their abundance, low price, high polysaccharide content, and renewability (Okano et al. 2009; Abdel-Rahman et al. 2011; Castillo Martinez et al. 2013). In addition, EPS from LAB can play an important role in the food industry as an emulsifier, thickener, viscosifier, and stabilizer. EPS has been used in the rheology and texture of fermented milks (Ramchandran and Shah 2009) and other fermented products, such as sourdough (Katina et al. 2009; Galle et al. 2010) and cereal-based beverages (Zannini et al. 2013). It may also improve the quality, safety, and acceptability of gluten-free bread (Moroni et al. 2009) and replace hydrocolloids in sorghum sourdough (Galle et al. 2011). EPSs from LAB are also of great interest to agro-food industries since their vast structural diversity may lead to innovative applications. However, the majority of LAB only produce low levels of polysaccharides; therefore, optimized methodologies for increased EPS production and recovery are still required (Notararigo et al. 2013).

EPS from LAB also has beneficial physiological properties for humans (Patel et al. 2011). Cell-bound EPSs from Lactobacillus acidophilus 606 (Kim et al. 2010) and Lactobacillus plantarum 70810 (Wang et al. 2014) have been shown to have antitumor properties. EPSs have also been shown to exhibit immunomodulatory activity for macrophages (Liu et al. 2011; Ciszek-Lenda et al. 2011) as well as intestinal epithelial cells (Patten et al. 2014). In addition, EPSs produced by Lactobacillus reuteri can inhibit enterotoxigenic Escherichia coli-induced hemagglutination of porcine erythrocytes, which further indicates that EPS has therapeutic potential (Wang et al. 2010). EPS from Lact. plantarum 70810 has a metal binding capacity and could be used as a potential biosorbent for lead removal from the environment (Feng et al. 2012). LAB can also produce a variety of functional oligosaccharides that can be used as prebiotics (Pepe et al. 2013), nutraceuticals, sweeteners, humectants, drugs against colon cancer, and immune stimulators (Patel et al. 2011). Some probiotic LAB can also utilize prebiotic compounds, including non-digestible FOSs, inulin-type fructans, or β-glucans (Russo et al. 2012), which stimulate the growth of beneficial commensals in the gastrointestinal tract.

1.2. Citrate Metabolism and Formation of Aroma Compounds

In addition to sugars, several LAB species can metabolize citrate. Citrate fermentation in LAB leads to the production of volatile compounds. In fermented dairy products, these compounds are C4 compounds, such as diacetyl, acetoin, and butanediol, which are responsible for the typical aroma of many fermented dairy products. Therefore, citrate metabolizing LAB, such as L. lactis subsp. lactis biovar. diacetylactis (L. diacetylactis) and some Leuconostoc and Weissella species, are currently used as starter and adjunct cultures for the production of these C4 compounds. However, in other fermented products, such as wine, beer, and sausages, the volatile compounds produced from the fermentation of citrate by LAB are considered off-flavors, and their presence should be avoided. Citrate utilization by LAB has been previously described in detail (Quintans et al. 2008) and summarized in the first edition of this book (Mayo et al. 2010). Therefore, in this chapter, we are presenting only citrate metabolism in LAB in the context of recent achievements.

1.2.1. Citrate Transport

Citrate transport is a limiting step for citrate utilization and is performed by a variety of membrane-associated permeases. In contrast, volatile compounds formed in the cytoplasm are secreted without requiring specific transporters. Most LAB species internalize citrate using a 2-hydroxycarboxylate (2-HCT) transporter, which can transport dicarboxylic and tricarboxylic acids. The 2-HCT family of transporters includes CitP from Lactococcus, Leuconostoc, and Weissella (Pudlik and Lolkema 2010). CitP is an antiport transporter that exchanges H-citrate2− and lactate1− to generate a membrane potential (Figure 1.2). In L. diacetylactis, CitP is encoded by the citQRP operon located on the “citrate plasmid” (Drider et al. 2004; Kelly et al. 2010). In L. diacetylactis, transcription of the promoters that control cit operons are specifically activated by low pH environments as an adaptive response to acid stress. This has been confirmed by transcriptomics analysis of L. diacetylactis in milk (Raynaud et al. 2005) and cheese (Cretenet et al. 2010). In Weissella paramesenteroides and Leuconostoc mesenteroides subsp. cremoris, the citP genes (citMCDEFGRP) are located in a plasmidic or chromosomal citI cluster, respectively (Martı&c.acute;n et al. 1999, 2000; Bekal et al. 1998). Enlarged citrate plasmids (15 to 23 kbp) have also been observed in wild (non-starter) strains of L. diacetylactis (Drici et al. 2010; Kelly et al. 2010).

Figure 1.2. Citrate metabolism in Lactococcus, Leuconostoc, and Weissella species. Key for the enzymes: CL, citrate lyase; OAD, oxaloacetate decarboxylase; LDH, lactate dehydrogenase; PDC, pyruvate decarboxylase; ALS, α-acetolactate synthase; ADC, α-acetolactate decarboxylase; DAR, diacetyl acetoin reductase; BDH, 2,3-butanediol dehydrogenase; Tppi, thiamine pyrophosphate.

In Enterococcus faecalis and Lactobacillus casei, citrate is transported by CitM and CitH transporters, respectively, both belonging to the family of citrate-metal symporters (CitMHS) (for a review see Lensbouer and Doyle 2010). CitMHS transporters transport citrate in cation (Ca2+, Mn2+, or Fe3+) complexes. Recently, CitH in Lact. casei has been shown to be a proton symporter that transports a Ca2+-citrate complex and uses Ca2+ as a substrate (Mortera et al. 2013). Other types of citrate transporters have also been identified in Lact. plantarum, Oenococcus oeni (for a review see Mayo et al. 2010), and the atypical citrate-fermenting wild L. diacetylactis strain (Passerini et al. 2013b).

1.2.2. Conversion of Citrate into Pyruvate and Production of Aroma Compounds

Once inside the cell, citrate is converted into acetate and oxaloacetate in a reaction catalyzed by the citrate lyase (CL) enzyme complex (Figure 1.2). Next, oxaloacetate is decarboxylated by oxaloacetate decarboxylase (OAD), which generates pyruvate and CO2 (Figure 1.2). Analysis of various LAB genomes have identified genes encoding the α-, β-, and δ-subunits of OAD (Makarova et al. 2006). However, the physiological role of OAD remains poorly understood in LAB species; only enzymes from E. faecalis (Repizo et al. 2013) and Lact. casei (Mortera et al. 2013) have been recently investigated. In other LAB species, including L. diacetylactis, W. paramesenteroides, Leuc. mesenteroides, Lact. plantarum, and O. oeni, oxaloacetate is decarboxylated by the soluble and cytoplasmic malic enzyme (ME) (Sender et al. 2004). Surprisingly, the cit locus of E. faecalis has been recently shown to contain genes that encode both OAD and ME (Espariz et al. 2011).

Pyruvate metabolism in LAB can yield different end products, including lactate, formate, acetate, and ethanol as well as the important aroma compounds diacetyl, acetoin, and butanediol (Neves et al. 2005).

1.2.3. Conversion of Citrate into Succinate

Some LAB species cannot truly convert citrate into pyruvate. Instead, the CitT transporter generates succinate via malate and fumarate. Furthermore, the complete tricarboxylic acids (TCA) pathway has recently been identified in the Lact. casei genome using in silico analysis (Díaz-Muñiz et al. 2006). In this LAB species, the dominant end-products of citrate metabolism were acetic acid and L-lactic acid at both excess and limiting amounts of carbohydrates. Trace amounts of D-lactic acid, acetoin, formic acid, ethanol, and diacetyl confirm OAD activity; however, succinic acid, malic acid, and butanendiol were not observed (Díaz-Muñiz et al. 2006; Mortera el al. 2013).

1.2.4. Bioenergetics of Citrate Metabolism

The co-metabolism of glucose and citrate produces different physiological effects in homofermentative and heterofermentative LAB. In homofermentative LAB, citrate utilization has a protective effect against acid stress. In heterofermentative LAB, “citrolactic” fermentation generates one extra mol of ATP per mol of citrate. In milk, L. diacetylactis metabolizes lactose and produces lactic acid, which is exchanged by the antiporter CitP for citrate during excretion. L. diacetylactis is homofermentative and converts glucose into lactate, generating two moles of ATP per mol of glucose. The NAD+ consumed in the first steps of this pathway is regenerated during the transformation of pyruvate into lactate, thereby maintaining the redox potential. In the presence of glucose and citrate, each mol of citrate produces one mol of pyruvate without generating NADH. This excess of pyruvate is diverted to α-acetolactate synthesis and the subsequent production of aroma compounds. Similar to L. diacetylactis, the higher biomass of Lact. casei during Ca2+-citrate and carbohydrate co-metabolism has been attributed to counteracting the growth inhibition of carbohydrate metabolism that is induced by acidification in its final stages (Mortera et al. 2013).

1.3. The Proteolytic System of Lactic Acid Bacteria

L. lactis strains isolated from dairy products are characterized by a high number of amino acid auxotrophies and the ability to utilize milk proteins as an amino acid source. These properties are common to dairy strains even from distant geographic locations in Asia, Europe, North America, and New Zealand (Rademaker et al. 2007; Kelly et al. 2010). The use of environmental proteins as an amino acid source in LAB explains the importance of their proteolytic system. LAB depend on this system to obtain the essential amino acids that are then used as precursors for not only peptides and proteins but also for many other biomolecules. Amino acids are the precursors of aromatic compounds, which are important to the final flavor of food products. Proteolytic activity also generates other molecules, such as bioactive peptides, that have functions related to the probiotic properties of LAB.

The proteolytic system in LAB can be divided into several steps: protein degradation, peptide transport, peptide degradation, and amino acid catabolism (Figure 1.3).

Figure 1.3. Diagram of the proteolytic systems of lactic acid bacteria. (a) Extracellular components: PrtP, cell-envelope proteinase; PrtM, proteinase maduration protein; Opp, oligopetide permease; DtpT, the ion linked trasnsporter for di-and tripeptides; and Opt, the ABC transporter for peptides. (b) Intracelullar components: pool of about 20–25 peptidases, including general (PepN, PepC) and specific (PepX, PepQ) peptidases, and amino acid catabolic enzymes (carboxylases, aminotransferases, etc.).

1.3.1. Protein Degradation

Studies of protein degradation in LAB initially focused on casein degradation using L. lactis as a model organism. Casein hydrolysis in LAB is initiated by a cell-envelope proteinase (CEP), which degrades proteins into oligopeptides, and gene deletion studies have shown that LAB are unable to grow in milk in the absence of a functional CEP. However, since CEP is extracellular, peptides produced by CEP can also be consumed by protease-negative variants, allowing them to survive in culture (Bachmann et al. 2012).

Lactocepins are a diverse group of CEPs that belong to the subtilisin protein family of serine proteases. CEPs are anchored to the cell wall via sortase A (SrtA) (Dandoy et al. 2011). LAB lactocepins are encoded by prtP, prtB, prtS, and/or prtH, which differ in their number of functional domains. CEP distribution varies widely among strains. Overall, the most abundant paralog among LAB is prtH3, which is carried by over 80% of strains tested, followed by paralogs of prtH and prtH4 (Broadbent et al. 2011). Most LAB possess only one CEP. However, four CEP genes (prtH, prtH2, prtH3, and prtH4) have been identified in Lactobacillus helveticus CNRZ32 (Jensen et al. 2009). The presence of several protease genes in Lact. helveticus could explain its high proteolytic efficiency. Only prtH2 is common to all characterized Lact. helveticus strains (Genay et al. 2009). However, analysis of the proteinase in BGRA43 showed that the only active gene was prtH. CEP activation requires the maturation proteinase PrtM. Two PrtMs (PrtM1 and PrtM2) have been identified in Lact. helveticus CNRZ32 (Savijoki et al. 2006). Additional studies (Genay et al. 2009; Broadbent et al. 2011) have reported that PrtM1 is required for PrtH activation, while PrtM2 plays a role in the activation of other CEP paralogs.

As mentioned above, CEP activity was initially evaluated using casein as a substrate. However, LAB strains isolated from non-milk or non-fermentation environments have also exhibited casein hydrolysis. For instance, Lact. helveticus BGRA43, which is isolated from human feces, has strong proteolytic activity and is able to completely hydrolyze αs1-, β-, and κ-caseins (Strahinic et al. 2013). In addition, the lactocepin secreted by Lactobacillus paracasei VSL#3 can selectively degrade cell-associated and tissue-distributed IP-10 and other proinflammatory chemokines in vitro (von Schillde et al. 2012). These findings indicate that lactocepin, which is encoded by prtP, is highly selective despite targeting a broad spectrum of cleavage sites. Therefore, additional protein characteristics, such as surface charge and/or three-dimensional structure, determine whether a protein is cleaved or not. The selective degradation of pro-inflammatory chemokines could be also related to differences in the regulation of prtP expression.

In Streptococcus thermophilus, the cell wall associated proteinase PrtS is highly conserved (95% identity) with the PrtS of Streptococcus suis. Although recent studies have suggested that PrtS contributes to the virulence of Strep. suis (Bonifait el al. 2010), the primary role of PrtS in Strep. thermophilus strains is cleaving casein to oligopeptides. This function is clearly related to the adaptation of Strep. thermophilus to dairy environments; analyses of prtS distribution in Strep. thermophilus found that it occurs infrequently in historical specimens but frequently in more recent industrial ones. Furthermore, this “ecological” island confers an important metabolic trait for milk adaptation and appears to be laterally transferred in Strep. thermophilus. Together, these data suggest that Strep. thermophilus evolved via gene acquisition and selection as the result of the environmental pressures of food production (Delorme et al. 2010).

The second stage of protein degradation is the transport of di-, tri-, and oligo-peptides into the cell. Three oligo-, di-, and tri-peptide transport systems (Opp, Dpp and DtpT, respectively) have been described in LAB. Lact. acidophilus, Lactobacillus brevis, Lact. casei, Lactobacillus rhamnosus, and L. lactis, possess all three of these peptide transport systems. Some Lact. helveticus strains, such as DPC4571, also have three peptide transport systems, while others, such as H10, only have two (Opp and DtpT). These results indicate that the proteolytic systems differ between different strains of even the same species. Finally, Lact. reuteri only has one functional peptide transport system (DtpT) (Liu et al. 2010).

1.3.2. Peptidases