**3.3. Instability of EPS production**

The instability of hetero-EPS production has been reviewed by de Vuyst et al. [8]. Briefly, a loss in the ability to produce slime may be caused by repeated subculture of bacterial strains or incubation at high temperatures. The loss of plasmids from ropy mesophilic LAB strains is generally the reason for loss of slime production. On the other hand, thermophilic LAB, namely, *Lb. delbrueckii* subsp. *bulgaricus* and *Str. thermophilus*, have been shown to lack plasmids encoding components required for slime production. These species can usually recover the ability to produce slime following loss due to culture conditions. Thus, genetic instability could be a consequence of the actions of mobile genetic elements such as insertion sequences. Recently, the EPS gene cluster in *Lb. fermentum* TDS030603 was reported to be located in chromosomal DNA [67].

Priming glycosyl transferases are thought to be crucial for EPS biosynthesis and disruption of the priming glycosyl transferase gene generates non-EPS-producing mutants. Tsuda et al. generated the EPS-producing mutant strain 301102S from the non-EPS-producing *Lb. plantarum* 301102 following exposure to the mutagens acridine orange and novobiocin [4]. The activities of α-phosphoglucomutase (PGM), UDP-glucose pyrophosphorylase (UGP), and UDP-galactose 4-epimerase (UGE) were measured in parental and mutant strains by using the method of Mozzi [68], and were found to be almost the same for both [Tsuda & Miyamoto, unpublished data]. Next, priming glycosyl transferase genes in parental and mutant strains were amplified with the thermal cycler. Primers were designed to amplify a priming glycosyl transferase gene referring to complete *Lb. plantarum* WCFS1 genome sequenced [69]. PCR products were subjected to restriction digestion, which allowed identification of putative priming glycosyl transferase gene. PCR products were also applied to single strand conformation polymorphism (SSCP) analysis for detecting point mutations. However, both parental and mutant strains had the same priming glycosyl transferase gene sequence, and similar levels of activities of the PGM, UGP, and UGE enzymes. Thus, although priming glycosyl transferases are essential, other factors may also be necessary for EPS production, and a mutation affecting EPS production may occure in another gene. Morona et al. reported that an autophosphorylating protein-tyrosine kinase is essential for encapsulation in *Streptococcus pneumoniae* [70]. A point mutation in the gene encoding the autophosphorylating protein-tyrosine kinase affecting the ATP-binding domain resulted in loss of EPS production.

### **4. Polysaccharides and oligosaccharides for colon health**

524 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

polymerized to become a hetero-EPS (Fig. 3).

**Figure 3.** Outline of biosynthesis of hetero EPS.

**3.3. Instability of EPS production** 

located in chromosomal DNA [67].

PGM: α-phosphoglucomutase, UGP: UDP-glucose pyrophospholyrase UGE: UDP-galactose 4-epimerase, TGP: dTDP-glucose pyrophospholyrase TRS: dTDP-rhamnose synthetic enzyme system, PMI: phosphomannoisomerase PMM: phosphomannomutase, GMP: GDP-mannose pyrophospholyrase

substrate. The repeating unit is exported through the bacterial membrane, and is

The instability of hetero-EPS production has been reviewed by de Vuyst et al. [8]. Briefly, a loss in the ability to produce slime may be caused by repeated subculture of bacterial strains or incubation at high temperatures. The loss of plasmids from ropy mesophilic LAB strains is generally the reason for loss of slime production. On the other hand, thermophilic LAB, namely, *Lb. delbrueckii* subsp. *bulgaricus* and *Str. thermophilus*, have been shown to lack plasmids encoding components required for slime production. These species can usually recover the ability to produce slime following loss due to culture conditions. Thus, genetic instability could be a consequence of the actions of mobile genetic elements such as insertion sequences. Recently, the EPS gene cluster in *Lb. fermentum* TDS030603 was reported to be

Priming glycosyl transferases are thought to be crucial for EPS biosynthesis and disruption of the priming glycosyl transferase gene generates non-EPS-producing EPS produced by LAB have various functional roles in human or animal health including immunomodulatory properties, antiviral activity, antioxidant activity, and antihypertensive activity [1, 55, 71, 72], and have also been used as food additives for texture improvement. These properties have been extensively reviewed [8, 9, 56, 73, 74]. Besides these properties, prebiotics based on LAB and oligosaccharides have other health benefits. Prebiotics are usually non-digestible oligosaccharides that selectively stimulate the growth and activity of a limited number of bacterial species in the colon, such as bifidobacteria and lactobacilli, and therefore, improve host health. Detrimental bacteria may form substances such as ammonia, hydrogen sulfide, indles, and amines that are noxious to the host. However, beneficial bacteria such as bifidobacteria and lactobacilli inhibit the proliferation of detrimental bacteria, and their cell components stimulate the host immune system [75]. Gastrointestinal microflora consist of approximately 1014 colony forming units (cfu)/g of various types of both detrimental and beneficial bacteria, and the numbers and composition vary greatly along the gastrointestinal tract. The balance of the gastrointestinal micro flora influences different aspects of host health such as bowel movement, tympanites flatulence, and the absorption of nutrients. Many factors may upset this balance, including stress, consumption of antibiotics, infection, food poisoning, and the natural ageing process. To redress this balance, the growth and activities of beneficial bacteria may be enhanced by specific ingredients in foods.


Exopolysaccharides of Lactic Acid Bacteria for Food and Colon Health Applications 527

Various oligosaccharides have been identified as prebiotics, that can increase the number of *Bifidobacterium* in the host colon. Galacto-oligosaccharides (GOS) and fructooligosaccharides (FOS) are considered important prebiotics. Other carbohydrates including gluco-oligosaccharides, isomalto-oligosaccharides, lactulose, mannan-oligosaccharides, and nigero-oligosaccharides are also considered prebiotics. Increased numbers of bifidobacteria and/or lactobacilli in the colon have been shown to have beneficial effects, although the specific mixtures of populations of these genera necessary to provide health-promoting effects has not yet been determined. This is because the beneficial effects are likely to be due to improvement in the balance of coloni micro flora. However, difference do exist in the micro flora among individuals. To function most effectively, prebiotics must be resistant to digestive processes in the stomach and small bowel, so that they can come into contact with

The food for specified health use (FOSHU) system was introduced in Japan in 1991. FOSHU refers to foods containing ingredients that provide health benefits and have officially approved physiological effects on the human body. FOSHU is intended to be consumed for the maintenance or promotion of health or for special health uses, for example, to control conditions such as blood pressure or blood cholesterol. To be defined as FOSHU, it is important to assess the safety of the food as well as the effectiveness of health promotion, and this assessment must be approved by the Ministry of Health, Labour and Welfare in Japan. At present (2012), 990 foods are recognized as FOSHU, and of these, 86 provide gastrointestinal health benefit. Foods for balancing gastrointestinal micro flora contain galactosylsucrose, soy oligosaccharides, lactulose, GOS, FOS, isomalto-oligosaccharides, raffinose, xylo-oligosaccharides, mannobiose, and brewer's yeast cell wall as functional

GOS are well-known type of prebiotic oligosaccharides found in human milk. The concentration of oligosaccharides is 100 times higher in human breast milk than in bovine milk [76]. Many studies have shown that breast-fed infants have intestinal microflora dominated by bifidobacteria. The reason for this phenomenon is thought to be that the oligosaccharides in breast milk, including GOS, can reach the upper gut without being digested where the bifidobacteria can utilize them. At present, GOS is produced by the enzymatic treatment of lactose by β-galactosidase. GOS produced in this manner usually have degrees of polymerization (DP) between 2 and 10. Furthermore, the type of glycosidic linkage is determined by the reaction conditions: final products usually possess β-1,2, β-1,3, or β-1,4 linkages. GOS is given a caloric value of 2 kcal/g in Japan and Europe for food-

The effect of GOS on defecation has been studied in healthy volunteers. Defecation frequency was significantly increased, and faeces became significantly softer after the subjects drank a beverage containing 5.0 g of GOS, on a daily basis. Therefore,

the bacteria growing in the large intestine.

ingredients.

**4.1. GOS** 

labelling purpose.

**Table 2.** Monosaccharide ratio in hetero EPS

Glc: glucose, Gal: galactose, Rha: rhamnose, Fuc: fucose, NAc Glu: N-acetyl glucosamine, NAc Gal: Nacetyl galactosamine, GlcA: glucuronic acid.

Various oligosaccharides have been identified as prebiotics, that can increase the number of *Bifidobacterium* in the host colon. Galacto-oligosaccharides (GOS) and fructooligosaccharides (FOS) are considered important prebiotics. Other carbohydrates including gluco-oligosaccharides, isomalto-oligosaccharides, lactulose, mannan-oligosaccharides, and nigero-oligosaccharides are also considered prebiotics. Increased numbers of bifidobacteria and/or lactobacilli in the colon have been shown to have beneficial effects, although the specific mixtures of populations of these genera necessary to provide health-promoting effects has not yet been determined. This is because the beneficial effects are likely to be due to improvement in the balance of coloni micro flora. However, difference do exist in the micro flora among individuals. To function most effectively, prebiotics must be resistant to digestive processes in the stomach and small bowel, so that they can come into contact with the bacteria growing in the large intestine.

The food for specified health use (FOSHU) system was introduced in Japan in 1991. FOSHU refers to foods containing ingredients that provide health benefits and have officially approved physiological effects on the human body. FOSHU is intended to be consumed for the maintenance or promotion of health or for special health uses, for example, to control conditions such as blood pressure or blood cholesterol. To be defined as FOSHU, it is important to assess the safety of the food as well as the effectiveness of health promotion, and this assessment must be approved by the Ministry of Health, Labour and Welfare in Japan. At present (2012), 990 foods are recognized as FOSHU, and of these, 86 provide gastrointestinal health benefit. Foods for balancing gastrointestinal micro flora contain galactosylsucrose, soy oligosaccharides, lactulose, GOS, FOS, isomalto-oligosaccharides, raffinose, xylo-oligosaccharides, mannobiose, and brewer's yeast cell wall as functional ingredients.

### **4.1. GOS**

526 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

OLL 1073R-

*Streprococcus thermophilus* 

*Lactococcus lactis* subsp. *cremoris*

*Lactobacillus delbrueckii* subsp. *bulgaricus*

*Lb.* 

*kefiranofaciens* 

K1

**Table 2.** Monosaccharide ratio in hetero EPS

acetyl galactosamine, GlcA: glucuronic acid.

Speceis Strain Glc Gal Rha Fuc NAc

CNCMI 733 1 2 1 [25] SFi39 1 1 [26] SFi12 1 3 2 [26] LY03 1 4 [27] OR901 5 2 [28] MR-1C 5 2 1 [29]

NIZO B891 3 2 [30] Ropy352 2 3 [31] NIZO B39 2 3 2 [32] SBT 0495 2 2 1 [33]

<sup>1</sup>1 1.6 [34]

NCFB 2772 1 2.4 [35] Lb18 1 1 [36]

rr 1 5 1 [38] NCFB 2772 1 7 0.8 [35]

766 2 1 [40] 2091 1 2 [41] Lb161 5 2 [42]

GG 1 4 1 [44]

EP56 3 1 1 [45]

1 1 [48]

EU23 1 1 [37]

*Lb. helveticus* TN-4 1 1 [39]

*Lb. rhamnosus* RW-9595M 2 1 4 [43]

*Lb. plantarum* EP56 3 1 1 [45]

*Lb. pentosus* LPS26 1 2 2 [46] *Lb. paracasei* 34-1 3 1 1 [47]

*Lb. sake* 0-1 3 2 [49]

Glc: glucose, Gal: galactose, Rha: rhamnose, Fuc: fucose, NAc Glu: N-acetyl glucosamine, NAc Gal: N-

Gal GlcA Gly Reference

GOS are well-known type of prebiotic oligosaccharides found in human milk. The concentration of oligosaccharides is 100 times higher in human breast milk than in bovine milk [76]. Many studies have shown that breast-fed infants have intestinal microflora dominated by bifidobacteria. The reason for this phenomenon is thought to be that the oligosaccharides in breast milk, including GOS, can reach the upper gut without being digested where the bifidobacteria can utilize them. At present, GOS is produced by the enzymatic treatment of lactose by β-galactosidase. GOS produced in this manner usually have degrees of polymerization (DP) between 2 and 10. Furthermore, the type of glycosidic linkage is determined by the reaction conditions: final products usually possess β-1,2, β-1,3, or β-1,4 linkages. GOS is given a caloric value of 2 kcal/g in Japan and Europe for foodlabelling purpose.

The effect of GOS on defecation has been studied in healthy volunteers. Defecation frequency was significantly increased, and faeces became significantly softer after the subjects drank a beverage containing 5.0 g of GOS, on a daily basis. Therefore,

consumption of a beverage containing 5.0 g of GOS can improve defecation in individuals with a tendency for constipation [77]. Ishikawa et al. reported that the number of faecal bifidobacteria increased significantly after subjects consumed 2.5 g of GOS/day for 3 weeks [78]. GOS utilization by enterobacteria was further investigated in vitro. The trisaccharide forms of GOS were utilized by *Bifidobacterium*, *Lactobacillus acidophilus, Lb. reuteri*, *Bacteroides, Clostridium perfringens, Klebsiella pnumoniae, Enterococcus faecium*, and the tetra-saccharide forms were utilized by *Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis,* and *Ent. faecium*. These results suggest that a higher DP of GOS enhanced selectivity, and that the tetrasaccharide forms of GOS are specifically utilized by bifidobacteria. Similarly, *Bifidobacterium lactis* DR10 utilizes trisaccharide and tetra-saccharide forms of GOS, whereas *Lb. rhamnosus* DR20 prefers disaccharides and monosaccharides [79]. Barboza reported that *Bif. breve* and *Bif. longum* subsp. *infantis* can consume GOS with a DP ranging from 3 to 8 [80]. Furthermore, *Bif. longum* subsp. *infantis* preferentially consume GOS with a DP of 4, and *Bif. adolescentis* utilizes GOS with DP of 3. In addition, the structure of GOS influences its utilization by lactobacilli and bifidobacteria [81]. Trisaccharides of 4'-GOS (β-1,4 linkage) and 6'-GOS (β-1,6 linkage) can be used as the sole carbon source. Almost all lactobacilli and bifidobacteria tested preferred to utilize 4'-GOS, while *Lb. acidophilus, Lb. reuteri*, and *Lb. casei* could utilize both 4'- and 6'-GOS. GOS are used to stimulate beneficial bacteria, but can also be utilized by bacteroides and clostridia [82]. GOS selectivity may be enhanced by altering the structure and increasing the DP.

Exopolysaccharides of Lactic Acid Bacteria for Food and Colon Health Applications 529

time PCR [86]. The composition of bifidobacteria in the gut microflora was studied by clone library analysis in ten volunteers. All ten volunteers carried *Bif. longum,* and nine of these also carried *Bif. adolescentis*. The consumption of inulin (10 g/d) increased the number of bifidobacteria in faeces with *Bif. adolescentis* showing the highest increase response among *Bifidobacterium* species. Rossi et al. reported that only 8 of 55 *Bifidobacterium* strains fermented inulin in pure cultures, although inulin increased the number of bifidobacteria in faecal culture [87]. They, therefore, suggested that most bifidobacteria were not able to utilize long fructans in the absence of other intestinal bacteria that can hydrolyze fructans, and that fermentation of oligosaccharides in the colon is the result of a complex metabolic

It is not clear which oligosaccharides are the most suitable substrates for the selective growth of specific beneficial species or strains. Several research group have suggested useful methods to investigate the potential prebiotic activity of oligosaccharides [88-92]. Potential prebiotic activities were determined on the basis of the changes in the growth of beneficial and undesirable bacteria, such as bifidobacteria, lactobacilli, clostridia, and bacteroides. Such methods can evaluate the ability of specific strains to utilize a particular prebiotic, and a comparison of the prebiotic activities of oligosaccharides by using these methods could help in the choice of prebiotics for improving the gastrointestinal microflora on an individual basis. However, it is important to understand that only a limited group of bacteria can be chosen from the gastrointestinal microflora by using these methods, and that polysaccharides and oligo-saccharides are fermented by numerous species in the

Oligosaccharides produced by beneficial bacteria or their enzymes may enhance the growth of beneficial bacteria. A novel GOS mixture produced using *Bif. longum* NCIBM 41171 galactosidases increased the proportion of bifidobacteria in faeces relative to commercial GOS [93]. In the above-described study, oligosaccharides synthesized by the enzymes from *Bifidobacterium* strains were favored by the producer strains [83]. These studies suggest that the oligosaccharides produced by beneficial bacteria are selectively utilized by the producer strain, because the enzymes required for their degradation are already available. In addition, glycosyltransferases may possess both hydrolytic and transglycosylation activities [94], and glycosidases and glycosyltransferases may coexist in the same strains. Schwab et al. reported the production of novel oligosaccharides [95]. Hetero-oligosaccharides were produced from lactose, mannose, fucose, and N-acetylglucosamine by using crude cell extracts and whole cells of LAB and bifidobacteria. These hetero-oligosaccharides contained mannose, fucose, and N-acetylglucosamine, and could be digested by LAB strains. The prebiotic activities of these oligosaccharides were not investigated; however, a similar approach using probiotic and intestinal beneficial bacteria may lead to the production of

sequence carried out by numerous species.

gastrointestinal tract.

highly selective prebiotics.

**4.3. Selection of high-efficiency prebiotics** 

The use of beneficial bacteria or their enzymes in the synthesis of prebiotics may be a good way to produce prebiotics with high specificity. Rabiu reported that five different GOS were produced using β-galactosidase extracted from five different *Bifidobacterium* species, and that each GOS showed an increased growth rate in producer strains, except for *Bif. adolescentis* [83]. The utilization of these GOS by faecal bacteria was investigated using commercial GOS as control. The number of *Bacteroides* was decreased with GOS from bifidobacteria, whereas both GOS extracts and commercial GOS increased the number of bifidobacteria, lactobacilli, and clostridia.
