Fermentation of fructooligosaccharides and inulin by bifidobacteria: a comparative study of pure and fecal cultures

The utilization of fructooligosaccharides (FOS) and inulin by 55 Bifidobacterium strains was investigated. Whereas FOS were fermented by most strains, only eight grew when inulin was used as the carbon source. Residual carbohydrates were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection after batch fermentation. A strain-dependent capability to degrade fructans of different lengths was observed. During batch fermentation on inulin, the short fructans disappeared first, and then the longer ones were gradually consumed. However, growth occurred through a single uninterrupted exponential phase without exhibiting polyauxic behavior in relation to the chain length. Cellular beta-fructofuranosidases were found in all of the 21 Bifidobacterium strains tested. Four strains were tested for extracellular hydrolytic activity against fructans, and only the two strains which ferment inulin showed this activity. Batch cultures inoculated with human fecal slurries confirmed the bifidogenic effect of both FOS and inulin and indicated that other intestinal microbial groups also grow on these carbon sources. We observed that bifidobacteria grew by cross-feeding on mono- and oligosaccharides produced by primary inulin intestinal degraders, as evidenced by the high hydrolytic activity of fecal supernatants. FOS and inulin greatly affected the production of short-chain fatty acids in fecal cultures; butyrate was the major fermentation product on inulin, whereas mostly acetate and lactate were produced on FOS.     

In vitro study of Prebiotic Properties of Levan-type Exopolysaccharides from Lactobacilli and Non-digestible Carbohydrates Using Denaturing Gradient Gel Electrophoresis

Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.     

Exopolysaccharides Produced by Intestinal Bifidobacterium Strains Act as Fermentable Substrates for Human Intestinal Bacteria

Eleven exopolysaccharides (EPS) isolated from different human intestinal Bifidobacterium strains were tested in fecal slurry batch cultures and compared with glucose and the prebiotic inulin for their abilities to act as fermentable substrates for intestinal bacteria. During incubation, the increases in levels of short-chain fatty acids (SCFA) were considerably more pronounced in cultures with EPS, glucose, and inulin than in controls without carbohydrates added, indicating that the substrates assayed were fermented by intestinal bacteria. Shifts in molar proportions of SCFA during incubation with EPS and inulin caused a decrease in the acetic acid-to-propionic acid ratio, a possible indicator of the hypolipidemic effect of prebiotics, with the lowest values for this parameter being obtained for EPS from the species Bifidobacterium longum and from Bifidobacterium pseudocatenulatum strain C52. This behavior was contrary to that found with glucose, a carbohydrate not considered to be a prebiotic and for which a clear increase of this ratio was obtained during incubation. Quantitative real-time PCR showed that EPS exerted a moderate bifidogenic effect, which was comparable to that of inulin for some polymers but which was lower than that found for glucose. PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments using universal primers was employed to analyze microbial groups other than bifidobacteria. Changes in banding patterns during incubation with EPS indicated microbial rearrangements of Bacteroides and Escherichia coli relatives. Moreover, the use of EPS from B. pseudocatenulatum in fecal cultures from some individuals accounted for the prevalence of Desulfovibrio and Faecalibacterium prausnitzii, whereas incubation with EPS from B. longum supported populations close to Anaerostipes, Prevotella, and/or Oscillospira. Thus, EPS synthesized by intestinal bifidobacteria could act as fermentable substrates for microorganisms in the human gut environment, modifying interactions among intestinal populations.     

Development of Bifidobacterium lactis Bb 12 on β‐(2,6)‐Linked Fructan‐Containing Substrate

β‐(2,1)‐linked fructan of plant origin (inulin) and the related oligosaccharides (FOS) as non‐digestible carbohydrates, i.e., potent prebiotics, can stimulate the growth of various probiotic lactic acid bacteria, including a number of bifidobacteria strains. The related β‐(2,6)‐linked fructans of microbial origin (levan and FOS), however, have scarcely been investigated in this respect. Therefore, the bifidogenic properties of various fructans, i.e., inulin, levan, fructooligosaccharides (FOS) and fructan syrup (FS), were tested as glucose substitutes in MRS media and were compared concerning their effect on the commercial strain Bifidobacterium lactis Bb 12. Although glucose was the preferred substrate for growth and biomass formation, FS exhibited a comparable cell growth (8.4 × 10⁷ counts/mL and 1.0 × 10⁷ counts/mL, respectively) and acidification power (84 °T and 74 °T, respectively) during 48 h of fermentation, as well as an increase in lactic acid and decrease in acetic acid formation. Bifidobacterium lactis Bb 12 did not utilize inulin as a sole carbon source as judged from the 60 % decrease in cell count and the insignificant (0.1 pH unit) acidification of the growth medium, whereas levan provided a noticeable increase in cell count and acidification (0.4 pH units) during 48 h of fermentation. FOS preparation appeared to be a satisfactory carbon source for this strain, but lower acidification power (56 °T) and cell counts were observed as compared to glucose‐ or FOS‐containing media (2.6 % and 22 %, respectively). The products obtained under conditions of mild lactic acid hydrolysis of levan (37 °C, pH 3.3, 24 h) enhanced the cell count (7–10 %) and acidification power (by a factor of 2.7) of Bifidobacterium lactis Bb 12.     

Comparison of the prebiotic properties of native chicory and synthetic inulins using swine fecal cultures

ABSTRACT

Inulin-type fructans are known to exert different effects on the fermentation profile depending on the average and range of the degree of polymerization (DP). Here, swine fecal cultures were used to investigate the prebiotic properties of native chicory inulin (NIN), extracted from the chicory root, and synthetic inulin (SIN), which has a narrower DP distribution than NIN. Both NIN and SIN showed prebiotic effects, but NIN exhibited a significant decrease in pH and increase in the production of propionate and butyrate compared to SIN. There were also differences in the production of succinate and lactate, the precursors of propionate and butyrate, and the relative abundance of associated genes. Furthermore, NIN induced the growth of certain species of Bifidobacterium and Lactobacillus more strongly than SIN. These results suggest that NIN and SIN exhibit different prebiotic properties due to differences in DP, and that NIN might be more beneficial to host health.     

Metabolism of fructo-oligosaccharides by Bifidobacterium spp.

Summary Bifidobacterium adolescentis ATCC 15703, B. longum ATCC 15707, and B. thermophilum ATCC 25525 were examined for the ability to grow with fructo-oligosaccharides (FOS) as carbohydrate sources. The three species produced cell-associated -fructosidases (inulinases) capable of hydrolysing FOS. Maximum activity was obtained with short-chain FOS with degrees of polymerization (DP) of between three and five (neosugars). The B. thermophilum inulinase was induced by inulin, a long-chain FOS with DP=35, while the enzymes from the other two strains were constitutive. Production of inulinase by all three strains was regulated by catabolite repression. Inulinase activity of the three Bifidobacterium spp. was similar when grown with 0.5% inulin as the carbohydrate source; however, B. thermophilum grew much more rapidly. All three strains utilized crude Jerusalem artichoke flour (JAF) as a carbohydrate source, suggesting that JAF might have commercial application as a food or feed additive to stimulate bifidobacteria in the gut.Contribution no. 802 from the Food Research Centre     

Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10⁷) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.     

Interactions between Bifidobacterium and Bacteroides Species in Cofermentations Are Affected by Carbon Sources,Including Exopolysaccharides Produced by Bifidobacteria

Cocultures of strains from two Bifidobacterium and two Bacteroides species were performed with exopolysaccharides (EPS) previously purified from bifidobacteria, with inulin, or with glucose as the carbon source. Bifidobacterium longum NB667 and Bifidobacterium breve IPLA20004 grew in glucose but showed poor or no growth in complex carbohydrates (inulin, EPS E44, and EPS R1), whereas Bacteroides grew well in the four carbon sources tested. In the presence of glucose, the growth of Bacteroides thetaiotaomicron DSM-2079 was inhibited by B. breve, whereas it remained unaffected in the presence of B. longum. Ba. fragilis DSM-2151 contributed to a greater survival of B. longum, promoting changes in the synthesis of short-chain fatty acids (SCFA) and organic acids in coculture with respect to monocultures. In complex carbohydrates, cocultures of bifidobacterium strains with Ba. thetaiotaomicron did not modify the behavior of Bacteroides nor improve the poor growth of bifidobacteria. The metabolic activity of Ba. fragilis in coculture with bifidobacteria was not affected by EPS, but greater survival of bifidobacteria at late stages of incubation occurred in cocultures than in monocultures, leading to a higher production of acetic acid than in monocultures. Therefore, cocultures of Bifidobacterium and Bacteroides can behave differently against fermentable carbohydrates as a function of the specific characteristics of the strains from each species. These results stress the importance of considering specific species and strain interactions and not simply higher taxonomic divisions in the relationship among intestinal microbial populations and their different responses to probiotics and prebiotics.     

Characterization of a novel fructosyltransferase from Lactobacillus reuteri that synthesizes high-molecular-weight inulin and inulin oligosaccharides

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with beta-(2-->1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10(7)) with beta-(2-->1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.     

Lactobacillus paracasei subsp. paracasei 8700:2 Degrades Inulin-Type Fructans Exhibiting Different Degrees of Polymerization

Ten strains of lactobacilli were assessed for their capacity to degrade inulin-type fructans, which are well-known prebiotics. Both oligofructose and inulin were tested. The dairy isolate Lactobacillus acidophilus IBB 801 degraded only oligofructose. The human isolate Lactobacillus paracasei subsp. paracasei 8700:2 degraded oligofructose and long-chain inulin and grew rapidly on both energy sources. In both cases, fractions of different degrees of polymerization were fermented. Moreover, large and short fractions of oligofructose were degraded simultaneously. When L. paracasei subsp. paracasei 8700:2 grew on oligofructose-enriched inulin, oligofructose was preferentially metabolized. In all cases, lactic acid was the main metabolic end product. Significant amounts of acetic acid, formic acid, and ethanol were produced when long-chain inulin or oligofructose-enriched inulin was used as the sole energy source.     

Fermentation of Fructooligosaccharides by Lactic Acid Bacteria and Bifidobacteria

Lactic acid bacteria and bifidobacteria were screened of their ability to ferment fructooligosaccharides (FOS) on MRS agar. Of 28 strains of lactic acid bacteria and bifidobacteria examined, 12 of 16 Lactobacillus strains and 7 of 8 Bifidobacterium strains fermented FOS. Only strains that gave a positive reaction by the agar method reached high cell densities in broth containing FOS.     

In vitro Effects of Prebiotics and Synbiotics on Apis cerana Gut Microbiota

This study aimed to investigate in vitro effects of the selected prebiotics alone, and in combination with two potential probiotic Lactobacillus strains on the microbial composition of Apis cerana gut microbiota and acid production. Four prebiotics, inulin, fructo-oligosaccharides, xylo-oligosaccharides, and isomalto-oligosaccharides were chosen, and glucose served as the carbon source. Supplementation of this four prebiotics increased numbers of Bifidobacterium and lactic acid bacteria while decreasing the pH value of in vitro fermentation broth inoculated with A. cerana gut microbiota compared to glucose. Then, two potential probiotics derived from A. cerana gut at different dosages, Lactobacillus helveticus KM7 and Limosilactobacillus reuteri LP4 were added with isomalto-oligosaccharides in fermentation broth inoculated with A. cerana gut microbiota, respectively. The most pronounced impact was observed with isomalto-oligosaccharides. Compared to isomalto-oligosaccharides alone, the combination of isomalto-oligosaccharides with both lactobacilli strains induced the growth of Bifidobacterium, LAB, and total bacteria and reduced the proliferation of Enterococcus and fungi. Consistent with these results, the altered metabolic activity was observed as lowered pH in in vitro culture of gut microbiota supplemented with isomalto-oligosaccharides and lactobacilli strains. The symbiotic impact varied with the types and concentration of Lactobacillus strains and fermentation time. The more effective ability was observed with IMO combined with L. helveticus KM7. These results suggested that isomalto-oligosaccharides could be a potential prebiotic and symbiotic with certain lactobacilli strains on A. cerana gut microbiota.     

猪源乳酸杆菌与益生元组合筛选及其体外发酵特性

[目的]对3株乳酸杆菌和4种寡糖类益生元进行组合筛选,并探究其对猪结肠微生物体外发酵特性的影响.[方法]将3株乳酸杆菌(罗伊氏乳杆菌L45、植物乳杆菌L47和罗伊氏乳杆菌L63)分别添加至以菊粉(inulin)、低聚果糖(FOS)、低聚半乳糖(GOS)或乳果糖(lactulose)为唯一碳源的培养基中,结合菌株24 h...     

In vitro fermentation of linear and alpha-1,2-branched dextrans by the human fecal microbiota

The role of structure and molecular weight in fermentation selectivity in linear α-1,6 dextrans and dextrans with α-1,2 branching was investigated. Fermentation by gut bacteria was determined in anaerobic, pH-controlled fecal batch cultures after 36 h. Inulin (1%, wt/vol), which is a known prebiotic, was used as a control. Samples were obtained at 0, 10, 24, and 36 h of fermentation for bacterial enumeration by fluorescent in situ hybridization and short-chain fatty acid analyses. The gas production of the substrate fermentation was investigated in non-pH-controlled, fecal batch culture tubes after 36 h. Linear and branched 1-kDa dextrans produced significant increases in Bifidobacterium populations. The degree of α-1,2 branching did not influence the Bifidobacterium populations; however, α-1,2 branching increased the dietary fiber content, implying a decrease in digestibility. Other measured bacteria were unaffected by the test substrates except for the Bacteroides-Prevotella group, the growth levels of which were increased on inulin and 6- and 70-kDa dextrans, and the Faecalibacterium prausnitzii group, the growth levels of which were decreased on inulin and 1-kDa dextrans. A considerable increase in short-chain fatty acid concentration was measured following the fermentation of all dextrans and inulin. Gas production rates were similar among all dextrans tested but were significantly slower than that for inulin. The linear 1-kDa dextran produced lower total gas and shorter time to attain maximal gas production compared to those of the 70-kDa dextran (branched) and inulin. These findings indicate that dextrans induce a selective effect on the gut flora, short-chain fatty acids, and gas production depending on their length.     

In Vitro Kinetic Analysis of Fermentation of Prebiotic Inulin-Type Fructans by Bifidobacterium Species Reveals Four Different Phenotypes

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of 18 Bifidobacterium strains grown on fructose, oligofructose, or inulin were performed. A principal component analysis of the data sets, expanded with the results of a genetic screen concerning the presence of a β-fructofuranosidase gene previously encountered in Bifidobacterium animalis subsp. lactis DSM 10140^T, revealed the existence of four clusters among the bifidobacteria tested. Strains belonging to a first cluster could not degrade oligofructose or inulin. Strains in a second cluster could degrade oligofructose, displaying a preferential breakdown mechanism, but did not grow on inulin. Fructose consumption was faster than oligofructose degradation. A third cluster was composed of strains that degraded all oligofructose fractions simultaneously and could partially break down inulin. Oligofructose degradation was substantially faster than fructose consumption. A fourth, smaller cluster consisted of strains that shared high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin. For all strains, a metabolic shift toward more acetate, formate, and ethanol production, at the expense of lactate production, was observed during growth on less readily fermentable energy sources. No correlation between breakdown patterns and the presence of the β-fructofuranosidase gene could be detected. These variations indicate niche-specific adaptation of bifidobacteria and could have in vivo implications on the strain specificity of the stimulatory effect of inulin-type fructans on bifidobacteria.     

In vitro study of prebiotic properties of levan-type exopolysaccharides from Lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis

Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.     

Cloning,expression and functional validation of a β-fructofuranosidase from Lactobacillus plantarum

Fructooligosaccharides (FOS) are prebiotics that selectively stimulate the growth and activity of lactobacilli and bifidobacteria. These strains metabolize FOS with endogenous β-fructofuranosidase. In this study, a β-fructofuranosidase gene from Lactobacillus plantarum ST-III designated sacA was cloned into Escherichia coli, and the properties of the recombinant protein (SacA) were examined. The sacA gene encodes a peptide of 501 amino acids with a predicted molecular weight of 56.7 kDa. Sequence alignment revealed the presence of three highly conserved motifs, NDPNG, RDP and EC, indicating that the enzyme belongs to glycoside hydrolase family 32. The predicted three-dimensional structure of the SacA enzyme was similar to β-fructofuranosidases of bifidobacteria, such that it contained a five-blade β-propeller module and a β-sandwich domain with one additional N-terminal α-helix. The optimal reaction temperature and pH of the enzyme were 37 °C and 6.0, respectively. Substrate hydrolysis and kinetic parameters demonstrated that β-fructofuranosidase from L. plantarum ST-III liberated fructosyl residues from the non-reducing terminus of fructans, such as sucrose, FOS, levan or inulin, and FOS was the preferred substrate. The expression of the sacA gene in a non-FOS-fermenting strain, Lactobacillus rhamnosus GG, enabled the recombinant strain to metabolize FOS and sucrose.     

Different effects of difructose anhydride III and inulin-type fructans on caecal microbiota in rats

The effects of different kinds of inulin-type fructans on caecal microbiota were evaluated in rats. Four groups of male Wistar rats were fed either a control diet, or diets containing 5% inulin, 5% fructooligosaccharides (FOS), or 5% difructose anhydride III (DFAIII) for two weeks. In the DFAIII group, caecal propionate, butyrate, counts of bifidobacteria, and total anaerobes were lower than in the inulin group, while caecal propionate, succinate, counts of bifidobacteria, and total anaerobes were lower than in the FOS group. Compared to controls, in the DFAIII group the counts of clostridia in caecum were increased by 3 log units. However, this change was statistically not significant. There were no differences between inulin and FOS groups for the pool of short chain fatty acids in caecum and bacterial counts. Results indicate that DFAIII has different effects on caecal microbiota compared to inulin and FOS and that these differences are most likely due to the alpha(3-->2) bonds in DFAIII.     

Degradation of Pectins with Different Degrees of Esterification by Bacteroides thetaiotaomicron Isolated from Human Gut Flora

A complete human fecal flora and cultures of defined species obtained from fecal flora were investigated in vitro to determine their ability to ferment the dietary fiber pectin. Bacteroides thetaiotaomicron was tested as a pectin-degrading microorganism alone and in coculture with Escherichia coli. Macromolecular pectins with different degrees of esterification were used as substrates in microbial degradation studies. The levels of oligogalacturonic acids formed in batch cultures were estimated during a 24- or 48-h incubation period by using high-performance thin-layer chromatography and high-performance anion-exchange chromatography. The spectrum and the amount of unsaturated oligogalacturonic acids formed as intermediate products of pectin fermentation changed permanently in the culture media during incubation with the complete fecal flora. After 24 h, no oligogalacturonic acids were detected. The pectin-degrading activities of pure cultures of B. thetaiotaomicron were lower than the pectin-degrading activity of a complete fecal flora. Cocultures of B. thetaiotaomicron and E. coli exhibited intermediate levels of degradation activity. In pure cultures of E. coli no pectin-degrading activity was found. Additionally, the rate of pectin degradation was affected by the degree of esterification of the substrate. Saturated oligogalacturonic acids were not found during pectin fermentation. The disappearance of oligogalacturonic acids in the later stages of fermentation with both the complete fecal flora and B. thetaiotaomicron was accompanied by increased formation of short-chain fatty acids.     

Propionibacterium acidipropionici CRL1198 influences the production of acids and the growth of bacterial genera stimulated by inulin in a murine model of cecal slurries

Different attempts have been made to improve the health status of humans and animals by increasing the intestinal production of short-chain fatty acids (SCFA) derived from non-digestible carbohydrates fermentation. In this paper we investigate the in vitro production of short-chain fatty acids (SCFA) after addition of inulin, propionibacteria or a combination of both in an experimental model of mice cecal slurries. The development of bacterial genera which are usually stimulated by inulin addition was also investigated. According to our experimental data, acetic acid and butyric acids concentrations increased after incubation in slurries that had no supplements. By contrast, butyric acid concentrations remained in the basal value when supplements were used. Fermentation of only inulin did not increase the concentration of total SCFA. Propionibacterium acidipropionici CRL1198 improved the production of propionic acid in cecal slurries when it was added alone, but the effect was more noticeable in the combination with inulin. A modulation of the global fermentative activity of the cecal microbiota was evidenced by the increase on the ratio propionic acid/SCFA in supplementations with propionibacteria. Statistical analysis of data demonstrated that samples from homogenates with propionibacteria alone or combined with inulin belong to the same cluster. The presence of propionibacteria limited the growth of Bacteroides fragilis and Clostridium hystoliticum groups in slurries with and without inulin. The growth of Bifidobacterium was not modified and the stimulating effect of inulin on lactobacilli disappeared in the presence of propionibacteria. In conclusion, dairy propionibacteria are potential candidates to develop new functional foods helpful to ensure the intestinal production of SCFA during inulin supplementation and to control the overgrowth of bacteria belonging to Bacteroides and Clostridium genera.