**4.2. FOS**

FOS is used as a generic term for all β-2,1 linear fructans with a variable DP. Inulin and oligofructose are common forms of FOS that are widely found in nature. Chicory inulin has a DP of 2-60, and the product of its partial enzymatic hydrolysis is oligofructose or FOS with a DP of 2-10.

The effect of FOS intake on intestinal microflora was studied in humans. The number of bifidobacteria in faeces was significantly increased during the FOS intake (1 g/d) period, and a significant increase in stool frequency and a softening effect on stool were observed [84]. FOS increased the level of bifidobacteria in faeces, whereas that of bacteroides, clostridia, and fusobacteria decreased in subjects that were fed FOS (15 g/d) for 15 days [85]. Another study measured the increase in number of *Bifidobacterium* species in faeces by using realtime 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 sequence carried out by numerous species.

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

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

and increasing the DP.

**4.2. FOS** 

a DP of 2-10.

bifidobacteria, lactobacilli, and clostridia.

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

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

FOS is used as a generic term for all β-2,1 linear fructans with a variable DP. Inulin and oligofructose are common forms of FOS that are widely found in nature. Chicory inulin has a DP of 2-60, and the product of its partial enzymatic hydrolysis is oligofructose or FOS with

The effect of FOS intake on intestinal microflora was studied in humans. The number of bifidobacteria in faeces was significantly increased during the FOS intake (1 g/d) period, and a significant increase in stool frequency and a softening effect on stool were observed [84]. FOS increased the level of bifidobacteria in faeces, whereas that of bacteroides, clostridia, and fusobacteria decreased in subjects that were fed FOS (15 g/d) for 15 days [85]. Another study measured the increase in number of *Bifidobacterium* species in faeces by using realIt 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 gastrointestinal tract.

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 highly selective prebiotics.

The dietary fiber, arabinoxylan is the predominant hemicellulose from cereals and exhibits prebiotic activity [96]. The addition of water-unextractable arabinoxylans increased the population of bifidobacteria and bacteroides in a medium inoculated with faecal slurry. Polysaccharides are not usually utilized by microorganisms. Remarkably, however, *Bifidobacterium bifidum* DSM20456 can utilize the EPS produced by *Pediococcus pentosaceus*, *Lb. plantarum*, *Weissella cibaria*, and *Weissella confusa,* and some growth is observed in cas of *Bif. longum*, *Bif. adolescentis*, and *Lb. acidophilus* [97]. For EPS production by LAB, reduced yields were frequently observed after the maximal level had been reached, which might be caused by the enzymes produced by the bacteria [98]. Tsuda and Miyamoto investigated the prebiotic activity of EPS produced by *Lb. plantarum* 301102S [52], a mutant strain derived from *Lb. plantarum* 301102. Oral administration of the parental strain 301102 showed the survivability and proliferation in porcine gastrointestinal tract [99]. The potential prebiotic activities of EPS, GOS, and inulin were measured in 37 LAB strains, and the activity scores of EPS in the strains 301102 and 301102S were highest. This suggests that the EPS produced by the mutant strain is utilized by the same strain 301102S and the parental strain, and that the parental strain has enzymes that can degrade the EPS.

Exopolysaccharides of Lactic Acid Bacteria for Food and Colon Health Applications 531

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