Prebiotic effects of non-digestible oligo- and polysaccharides

This thesis is the result of work carried out within a four-year multi-disciplined program, entitled ' Non-digestible oligosaccharides in foods and feed'. Within the project, four Ph.D. students were employed at the Food Chemistry, Food Microbiology, Human Nutrition and Animal Nutrition groups of the Wageningen Agricultural University. This thesis describes the studies carried out at the Food Microbiology group.Oligosaccharides are carbohydrates, with an average chain length of 2-10 sugar residues. Most oligosaccharides cannot be digested by the enzymes in the upper gut, nor can these compounds be absorbed. These oligosaccharides are considered non-digestible, and reach the large intestine unaltered. Non-digestible oligosaccharides (NDOs) are mainly of vegetable origin and are a normal part of the human diet. Some of the natural NDOs are now produced commercially using enzymatic methods.Most NDOs are completely or partially degraded and fermented by the bacterial populations in the large intestine. Some of the NDOs are considered to have a beneficial effect on the health of the host, due to the specific fermentation by two groups of intestinal bacteria, the bifidobacteria and lactobacilli. Commercial NDOs are marketed as a healthy ingredient, due to this selective fermentation, in several Western countries.Chapter 1 describes the gastrointestinal tract and the bacterial composition in each part thereof. The same chapter gives an overview of the current knowledge of the fermentation of NDOs by intestinal bacteria and the effects on the host health, as far as known.Chapters 2 and 3 describe the effect of the two types of NDOs, currently available on the Dutch market, on the etiology of dental caries. When consumed, residues of NDOs in foods may remain in the oral cavity. In the oral cavity many different bacteria are capable of degrading and fermenting carbohydrates, which results in the formation of acid and, possibly, dental lesions and caries. NDOs, being carbohydrates, may thus be fermented and are, in theory, a risk factor for dental caries.In Chapter 2 the degradation and fermentation of fructooligosaccharides (FOS) by the oral microflora is described. It can be concluded that this class of NDOs can be fermented by the most common bacterial species. These NDOs can be considered cariogenic, in vitro , but in vivo studies have to be carried out to determine the actual risk for dental caries.In Chapter 3, the degradation and fermentation of transgalactosyl-oligosaccharides (TOS) by the oral microflora is described. It was concluded that this class of NDOs is not, or very slowly, degraded and fermented. These NDOs are not considered a risk factor for dental caries.Within the framework of the project, the Food Chemistry group synthesized and purified a large number of oligosaccharide mixtures from plant cell walls. As these purifications are laborious and the total quantities of pure oligosaccharides are very small, it was decided to determine the fermentation of plant cell wall compounds by intestinal bacteria. These plant cell wall compounds are available in large quantities and thus could be used for screening studies.Chapter 4 describes the degradation and fermentation of such a plant cell wall polysaccharide, xyloglucan. Xyloglucan is present in many edible plants, but it is commercially prepared from tamarind seeds. The more (chemically) complex the compound the more enzymes are necessary for degradation, and the less bacteria are capable of fermenting the compound. Xyloglucan has a relatively simple chemical structure, but, surprisingly, only very few intestinal bacteria were capable of degrading this compound. The second remarkable conclusion was that most of the bacteria capable of degrading xyloglucan, belonged to the genus Clostridium . Previously, this genus has not been considered of major importance for polysaccharide degradation in the intestine.Chapter 5 describes the degradation of a second plant cell wall polysaccharide, guar gum, a galactomannan. Like xyloglucan, galactomannans are part of the cell wall of many plants. Guar is commercially produced from the seeds of the Cyamopsis tetragonoloba tree and used as a thickening agent in many foods. Guar also has a relatively simple structure. Nevertheless, only three different bacterial species, capable of degrading guar, could be isolated from human and animal faeces. One of these, Bifidobacterium dentium was considered to be mainly an oral species, but, using guar, could also be isolated from faeces. The same species could also be isolated from samples of saliva from 19 out of 20 volunteers. A second species, Streptococcus bovis could only be isolated from animal faeces, whereas the third species, Clostridium butyricum was present in human and animal faeces. The latter species produced large amounts of gas, and can thus be considered responsible for the increased flatulence observed after the ingestion of guar.Chapter 6 describes the differences in the fermentation of different oligosaccharides by human faecal inocula. In addition three polysaccharides were used in these studies. All donors had received the same diet and four samples were taken from each volunteer. The results show large differences between test compounds within the same volunteer, and large differences between volunteers on the same test compound. It can be concluded that the fermentation is largely dependent on host (genetic) factors, and not on dietary factors. It was also concluded that formation of gas is correlated with the formation of butyric acid. Butyric acid is considered to be important for the health of the intestinal wall. Gas production can thus be used as a simple screening method for butyrate production.Within the project the variations in the bacterial composition of human and pig faeces have been studied. It was concluded in the early stages of the project that no methods existed for the reliable quantification of two major intestinal bacterial groups, bifidobacteria and lactobacilli. Two new methods had to be developed for the quantification of these bacterial groups.Chapter 7 describes the development of a new medium for bifidobacteria, the RB medium. Selectivity is based on raffinose, propionate and lithiumchloride. The medium is not yet an ideal medium for the isolation and quantification for bifidobacteria but, compared with media currently used, it is more selective.Chapter 8 describes the development of a new medium for lactobacilli, the LAMVAB medium. Selectivity is based on vancomycin and a low pH (5.0). The combination of vancomycin and low pH inhibits practically all other intestinal bacteria. LAMVAB has successfully been employed to isolate lactobacilli from faeces from a large number of animals.The two newly developed media were compared with two other media, that are used regularly. The results of this comparison is described in Chapter 9 . The media were used to isolate bifidobacteria and lactobacilli in human and cat faeces and pig small intestinal contents. The three media for bifidobacteria performed equally well for human faeces, but for the other two kinds of samples, the RB medium performed better. For lactobacilli, LAMVAB performed better for all three types of samples tested.Chapter 10 discusses the results of this thesis and some recommendations for further research are given.As conclusion it can be stated that very few NDOs are degraded and fermented selectively by bifidobacteria. This was confirmed in Chapters 2 and 6, in which degradation and fermentation of FOS by other bacterial groups is described.Although FOS and TOS are found to be possibly cariogenic, it is not likely that either oligosaccharide will cause caries under normal conditions.Xyloglucan and guar are degraded only by a limited number of bacteria. Unexpectedly, clostridia played a major role in the degradation of both substrates. Both substrates may be a good substrate for the production of new NDOs, but considering the results it is unlikely that these oligosaccharides are a good substrate for lactobacilli or bifidobacteria.RB and LAMVAB are new media, which are suitable for the quantitative isolation of bifidobacteria and lactobacilli from human faeces. LAMVAB is also suitable for animal faeces. Both media are more selective than the media used at present.