Summer Meeting 2013: growth and physiology of bifidobacteria

Bifidobacteria are a minor fraction of the human colon microbiota with interesting properties for carbohydrate degradation. Monosaccharides such as glucose and fructose are degraded through the bifid shunt, a dedicated pathway involving phosphoketolase activity. Its stoechiometry learns that three moles of acetate and two moles of lactate are produced per two moles of glucose or fructose that are degraded. However, deviations from this 3 : 2 ratio occur, depending on the rate of substrate consumption. Slower growth rates favour the production of acetate and pyruvate catabolites (such as formate) at the cost of lactate. Interestingly, bifidobacteria are capable to degrade inulin‐type fructans (ITF) (oligofructose and inulin) and arabinoxylan‐oligosaccharides (AXOS). Beta‐fructofuranosidase activity enables bifidobacteria to degrade ITF. However, this property is strain‐dependent. Some strains consume both fructose and oligofructose, with different preferences and degradation rates. Small oligosaccharides (degree of polymerization or DP of 2–7) are taken up, in a sequential order, indicating intracellular degradation and as such giving these bacteria a competitive advantage towards other inulin‐type fructan degraders such as lactobacilli, bacteroides and roseburias. Other strains consume long fractions of oligofructose and inulin. Exceptionally, oligosaccharides with a DP of up to 20 (long‐chain inulin) are consumed by specific strains. Also, the degradation of AXOS by α‐arabinofuranosidase and β‐xylosidase is strain‐dependent. Particular strains consume the arabinose substituents, whether or not together with a consumption of the xylose backbones of AXOS, either up to xylotetraose or higher and either extra‐ or intracellularly. The production of high amounts of acetate that accompanies inulin‐type fructan degradation by bifidobacteria cross‐feeds other colon bacteria involved in the production of butyrate. However, bifidobacterial strain‐dependent differences in prebiotic degradation indicate the existence of niche‐specific adaptations and hence mechanisms to avoid competition among each other and to favour coexistence with other colon bacteria.     

Chemopreventive effects from prebiotic inulin towards microbial 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine bioactivation

Aims:  Using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME), we investigated the chemopreventive potential of prebiotic chicory inulin towards the in vitro bioactivation of 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) by human intestinal microbiota.
Methods and Results:  HPLC data revealed that inulin significantly decreased the formation of the genotoxic PhIP-M1 metabolite, with the highest inhibitory activity in the colon ascendens (87% decrease). Interestingly, this chemopreventive effect correlated with alterations of bacterial community composition and metabolism in the different colon compartments. Conventional culture-based techniques and PCR-DGGE analysis on the SHIME colon suspension revealed significant bifidogenic effects during inulin treatment, whereas the overall microbial community kept relatively unchanged. Additionally, the production of short-chain fatty acids increased with 12%, 3% and 7%, while ammonia concentrations decreased with 3%, 4% and 3% in the ascending, transverse and descending colon compartments, respectively.
Conclusions:  These results indicate that the prebiotic effects from inulin may also purport protective effects towards microbial PhIP bioactivation.
Significance and Impact of the Study:  As the colonic microbiota may contribute significantly to the carcinogenic potential of PhIP, the search for dietary constituents that decrease the formation of this harmful metabolite, may help in preventing its risk towards human health.     

Arabinoxylan‐oligosaccharides (AXOS) affect the protein/carbohydrate fermentation balance and microbial population dynamics of the Simulator of Human Intestinal Microbial Ecosystem

Arabinoxylan‐oligosaccharides (AXOS) are a recently newly discovered class of candidate prebiotics as – depending on their structure – they are fermented in different regions of gastrointestinal tract. This can have an impact on the protein/carbohydrate fermentation balance in the large intestine and, thus, affect the generation of potentially toxic metabolites in the colon originating from proteolytic activity. In this study, we screened different AXOS preparations for their impact on the in vitro intestinal fermentation activity and microbial community structure. Short‐term fermentation experiments with AXOS with an average degree of polymerization (avDP) of 29 allowed part of the oligosaccharides to reach the distal colon, and decreased the concentration of proteolytic markers, whereas AXOS with lower avDP were primarily fermented in the proximal colon. Additionally, prolonged supplementation of AXOS with avDP 29 to the Simulator of Human Intestinal Microbial Ecosystem (SHIME) reactor decreased levels of the toxic proteolytic markers phenol and p‐cresol in the two distal colon compartments and increased concentrations of beneficial short‐chain fatty acids (SCFA) in all colon vessels (25–48%). Denaturant gradient gel electrophoresis (DGGE) analysis indicated that AXOS supplementation only slightly modified the total microbial community, implying that the observed effects on fermentation markers are mainly caused by changes in fermentation activity. Finally, specific quantitative PCR (qPCR) analysis showed that AXOS supplementation significantly increased the amount of health‐promoting lactobacilli as well as of Bacteroides–Prevotella and Clostridium coccoides–Eubacterium rectale groups. These data allow concluding that AXOS are promising candidates to modulate the microbial metabolism in the distal colon.     

Inulin-type fructans of longer degree of polymerization exert more pronounced in vitro prebiotic effects

AIMS: We assessed to what extent fructans of different degrees of polymerization (DP) differ in their prebiotic effectiveness towards in vitro microbial communities from the proximal and distal colon. METHODS AND RESULTS: Two short chain fructans - oligofructose (DP 2-20) and inulin (DP 3-60) - were administered to the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) at 2.5 g day(-1). The influence of fructan addition towards fermentation activity and microbial community composition from the different SHIME colon compartments were evaluated. Both fructans exerted prebiotic effects with significantly higher butyrate and propionate production and stimulation of lactic acid-producing bacteria. Compared with oligofructose, it was noted that it took more time before significant effects from inulin addition were observed. Yet, the higher short-chain fatty acid production and lower proteolytic activity showed that the prebiotic effects from inulin were more pronounced than oligofructose. Also, the bifidogenic effects from inulin vs oligofructose were higher in the distal colon compartments and this effect was prolonged in the distal colon once the addition was stopped. CONCLUSIONS: Inulin has more pronounced prebiotic effects than oligofructose towards both fermentation activity and bacterial community composition in the SHIME model. SIGNIFICANCE AND IMPACT OF THE STUDY: Its slower fermentation rate and higher prebiotic potency makes inulin a more interesting compound than oligofructose to beneficially influence the microbial community from both the proximal and distal colon regions.     

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.     

In vitro fermentation of broiler cecal content: the role of lactobacilli and pH value on the composition of microbiota and end products fermentation

Aim:  To assess the probiotic effects of Lactobacillus agilis JCM 1048 and L. salivarius ssp . salicinius JCM 1230 and the pH on the cecal microflora of chicken and metabolic end products.
Methods and Results:  An in vitro system, operated with batch bioreactor, was used for this assessment. Selected bacterial species were monitored at two pH values, over 24 h of batch culture incubation. The concentration of short chain fatty acids (SCFA) and lactate in the fermented material was also determined. The addition of L. agilis JCM 1048 and L. salivarius ssp . salicinius JCM 1230 into vessel 2 (Cc + P) increased the total anaerobes, lactobacilli and bifidobacteria after 24 h incubation. Moreover, lactobacilli supplementation decreased the total aerobes and streptococci, but it did not have any effects on coliforms. The supplementation of lactobacilli in vessel 2 (Cc + P) was found to significantly increase the production of lactate, propionate and butyrate. Furthermore, pH did not alter the formation of butyrate, whereas the production of acetate and propionate was significantly decreased at pH = 5·8.
Conclusions:  L. agilis JCM 1048 and L. salivarius ssp . salicinius JCM 1230, as probiotic bacteria, have the ability to re-establish proper microbial balance by the formation of lactate as well as propionate, and stimulate butyrate-producing bacteria to produce butyrate in the chicken cecum.
Significance and Impact of the Study:  This study was the first to report this under in vitro conditions, highlighting the probiotic roles of the two Lactobacillus strains in broiler cecal fermentation at different initial pH. These useful data can be helpful in improving the fermentation process in chicken cecum.     

Prebiotic effects of chicory inulin in the simulator of the human intestinal microbial ecosystem

The prebiotic potential of native chicory inulin was assessed in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) by monitoring microbial community from the colon compartments, its metabolic activity and community structure. Inulin addition selected for a higher short chain fatty acid production with shifts towards propionic and butyric acid. Conventional culture-based techniques and PCR-denaturing gradient gel electrophoresis analysis showed no remarkable changes in the overall microbial community from the colon compartments of the SHIME, whereas selective effects were seen for lactic acid bacteria. Quantitative PCR with bifidobacteria-specific primers revealed a significant increase with more than 1 log CFU ml(-1) from the proximal to distal colon, in contrast to culture-based techniques, which only showed a minor bifidogenic effect in the ascending colon. Our results indicate that inulin purports prebiotic effects from the proximal to distal colon and that real-time PCR is a more precise technique to detect differences in bifidobacterial populations whereas conventional culturing techniques are much more variable.     

Arabinoxylans and inulin differentially modulate the mucosal and luminal gut microbiota and mucin-degradation in humanized rats

The endogenous gut microbiota affects the host in many ways. Prebiotics should favour beneficial intestinal microbes and thus improve host health. In this study, we investigated how a novel class of potential prebiotic long-chain arabinoxylans (LC-AX) and the well-established prebiotic inulin (IN) modulate the gut microbiota of humanized rats. Six weeks after axenic rats were inoculated with a human faecal microbiota, their colonic microbiota was similar to this inoculum (～ 70%), whereas their caecal microbiota was enriched with Verrucomicrobia and Firmicutes concomitant with lower abundance of Bacteroidetes. Moreover, different Bifidobacterium species colonized the lumen (B. adolescentis) and mucus (B. longum and B. bifidum). Both LC-AX and IN increased SCFA levels and induced a shift from acetate towards health-promoting propionate and butyrate respectively. By applying a high-resolution phylogenetic micro-array (HITChip) at the site of fermentation (caecum), IN and LC-AX were shown to stimulate bacterial groups with known butyrate-producers (Roseburia intestinalis, Eubacterium rectale, Anaerostipes caccae) and bifidobacteria (B. longum) respectively. Prebiotic administration also resulted in lower caecal abundances of the mucin-degrading Akkermansia muciniphila and potentially more mucin production by the host. Both factors might explain the increased caecal mucin levels for LC-AX (threefold) and IN (sixfold). These mucins were degraded along the colon, resulting in high faecal abundances of Akkermansia muciniphila for LC-AX and especially IN-treated rats. Finally, the microbial changes caused an adaptation period for the host with less weight gain, after which the host fine-tuned the interaction with this altered microbiota. Our results demonstrate that next to IN, LC-AX are promising prebiotic compounds by stimulating production of health-promoting metabolites by specific microbes in the proximal regions. Further, prebiotic supplementation shifted mucin degradation to distal regions, where mucin-degraders may produce beneficial metabolites (e.g. propionate by Akkermansia muciniphila), so that prebiotics may potentially improve gut health along the entire length of the intestine.     

An in vitro study of the probiotic potential of a bile-salt-hydrolyzing Lactobacillus fermentum strain,and determination of its cholesterol-lowering properties

This study evaluated the use of a bile-salt-hydrolyzing Lactobacillus fermentum strain as a probiotic with potential hypocholesterolemic properties. The effect of L. fermentum on representative microbial populations and overall metabolic activity of the human intestinal microbiota was investigated using a three-stage continuous culture system. Also, the use of galactooligosaccharides as a prebiotic to enhance growth and/or activity of the Lactobacillus strain was evaluated. Administration of L. fermentum resulted in a decrease in the overall bifidobacterial population (ca. 1 log unit). In the in vitro system, no significant changes were observed in the total bacterial, Lactobacillus, Bacteroides, and clostridial populations through L. fermentum supplementation. Acetate production decreased by 9 to 27%, while the propionate and butyrate concentrations increased considerably (50 to 90% and 52 to 157%, respectively). A general, although lesser, increase in the production of lactate was observed with the administration of the L. fermentum strain. Supplementation of the prebiotic to the culture medium did not cause statistically significant changes in either the numbers or the activity of the microbiota, although an increase in the butyrate production was seen (29 to 39%). Results from this in vitro study suggest that L. fermentum KC5b is a candidate probiotic which may affect cholesterol metabolism. The short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation, are probably the major mechanism involved in the purported cholesterol-lowering properties of this strain.     

Mutual Cross-Feeding Interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 Explain the Bifidogenic and Butyrogenic Effects of Arabinoxylan Oligosaccharides

Arabinoxylan oligosaccharides (AXOS) are a promising class of prebiotics that have the potential to stimulate the growth of bifidobacteria and the production of butyrate in the human colon, known as the bifidogenic and butyrogenic effects, respectively. Although these dual effects of AXOS are considered beneficial for human health, their underlying mechanisms are still far from being understood. Therefore, this study investigated the metabolic interactions between Bifidobacterium longum subsp. longum NCC2705 (B. longum NCC2705), an acetate producer and arabinose substituent degrader of AXOS, and Eubacterium rectale ATCC 33656, an acetate-converting butyrate producer. Both strains belong to prevalent species of the human colon microbiota. The strains were grown on AXOS during mono- and coculture fermentations, and their growth, AXOS consumption, metabolite production, and expression of key genes were monitored. The results showed that the growth of both strains and gene expression in both strains were affected by cocultivation and that these effects could be linked to changes in carbohydrate consumption and concomitant metabolite production. The consumption of the arabinose substituents of AXOS by B. longum NCC2705 with the concomitant production of acetate allowed E. rectale ATCC 33656 to produce butyrate (by means of a butyryl coenzyme A [CoA]:acetate CoA-transferase), explaining the butyrogenic effect of AXOS. Eubacterium rectale ATCC 33656 released xylose from the AXOS substrate, which favored the B. longum NCC2705 production of acetate, explaining the bifidogenic effect of AXOS. Hence, those interactions represent mutual cross-feeding mechanisms that favor the coexistence of bifidobacterial strains and butyrate producers in the same ecological niche. In conclusion, this study provides new insights into the bifidogenic and butyrogenic effects of AXOS.     

An In Vitro Study of the Probiotic Potential of a Bile-Salt-Hydrolyzing Lactobacillus fermentum Strain, and Determination of Its Cholesterol-Lowering Properties

This study evaluated the use of a bile-salt-hydrolyzing Lactobacillus fermentum strain as a probiotic with potential hypocholesterolemic properties. The effect of L. fermentum on representative microbial populations and overall metabolic activity of the human intestinal microbiota was investigated using a three-stage continuous culture system. Also, the use of galactooligosaccharides as a prebiotic to enhance growth and/or activity of the Lactobacillus strain was evaluated. Administration of L. fermentum resulted in a decrease in the overall bifidobacterial population (ca. 1 log unit). In the in vitro system, no significant changes were observed in the total bacterial, Lactobacillus, Bacteroides, and clostridial populations through L. fermentum supplementation. Acetate production decreased by 9 to 27%, while the propionate and butyrate concentrations increased considerably (50 to 90% and 52 to 157%, respectively). A general, although lesser, increase in the production of lactate was observed with the administration of the L. fermentum strain. Supplementation of the prebiotic to the culture medium did not cause statistically significant changes in either the numbers or the activity of the microbiota, although an increase in the butyrate production was seen (29 to 39%). Results from this in vitro study suggest that L. fermentum KC5b is a candidate probiotic which may affect cholesterol metabolism. The short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation, are probably the major mechanism involved in the purported cholesterol-lowering properties of this strain.     

Fermentation RS3 derived from sago and rice starch with Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629

Resistant starch type 3 (RS3) is retrograded starch which is not digested by human starch degrading enzyme, and will thus undergo bacterial degradation in the colon. The main fermentation products are the Short Chain Fatty Acid (SCFA): acetate, propionate and butyrate. SCFA has significant benefit impact on the metabolism of the host. The objectives of this research were to study the SCFA profile produced by colonic butyrate producing bacteria grown in medium containing RS3. RS3 was made from sago or rice starch treated with amylase, pullulanase and the combination of amylase and pullulanase. Fermentation study was performed by using Clostridium butyricum BCC B2571 or Eubacterium rectale DSM 17629, which has been identified as capable of degradation of starch residue and also regarded as beneficial bacteria. Experimental result revealed that enzyme hydrolysis of retrograded sago or rice starch was beneficial to RS formation. RS3 derived from sago contained higher RS (31-38%) than those derived from rice starch (21-26%). This study indicated that C. butyricum BCC B2571 produced acetate, propionate and butyrate at molar ratio of 1.8 : 1 : 1, when the medium was supplemented with RSSA at concentration 1%. In the medium containing similar substrate, E. rectale DSM 17629 produced acetate, propionate and butyrate at molar ratio of 1.7 : 1 : 1.2. High levels of acetate, propionate and butyrate at molar ratio of 1.8 : 1 : 1.1 was also produced by E. rectale DSM 17629 in medium supplemented with RSSP at concentration 1%. The results showed that both bacteria responded differently to the RS3 supplementation. Such result provided insight into the possibility of designing RS3 as prebiotic with featured regarding SCFA released in the human colon with potential health implication.     

Propionate and butyrate metabolism in rat or sheep hepatocytes

The capacities of isolated hepatocytes to metabolize volatile fatty acids have been compared in rat and sheep hepatocytes. In both species, acetate utilization in vitro was quite limited. Significant species differences for propionate and butyrate consumption were found: propionate utilization by rat hepatocytes was relatively limited and plateaued at about 0.8-1.0 mM, whereas butyrate utilization was approx. 2-times higher. In contrast, ruminant hepatocytes exhibited a lower rate of butyrate utilization, but propionate metabolism was much more active than in rat liver cells. With relatively low concentrations of substrates (max. 2 mM), only propionate, compared to lactate or alanine, had a significant glucogenicity with hepatocytes from fed sheep. In both species, butyrate inhibited propionate consumption, although to a larger extent in sheep. The conversion of [2-14C]propionate to glucose by sheep hepatocytes was inhibited by 2 mM butyrate (60%) or ammonia (30%); 1 mM oleate or 10 mM glucose were ineffective. The basal rate of ammonia utilization by sheep hepatocytes was much lower than in rat and was unaffected upon addition of ornithine. Ammonia metabolism was markedly enhanced by butyrate and, in contrast to rat liver cells, also by propionate.     

The Ability of Bifidobacteria To Degrade Arabinoxylan Oligosaccharide Constituents and Derived Oligosaccharides Is Strain Dependent

Arabinoxylan oligosaccharides (AXOS) are prebiotic carbohydrates with promising health-promoting properties that stimulate the activity of specific colon bacteria, in particular bifidobacteria. However, the mechanisms by which bifidobacterial strains break down these compounds in the colon is still unknown. This study investigates AXOS consumption of a large number of bifidobacterial strains (36), belonging to 11 different species, systematically. To determine their degradation mechanisms, all strains were grown on a mixture of arabinose and xylose, xylo-oligosaccharides, and complex AXOS molecules as the sole added energy sources. Based on principal component and cluster analyses of their different arabinose substituent and/or xylose backbone consumption patterns, five clusters that were species independent could be distinguished among the bifidobacterial strains tested. In parallel, the strains were screened for the presence of genes encoding several putative AXOS-degrading enzymes, but no clear-cut correlation could be made with the different degradation mechanisms. The intra- and interspecies differences in the consumption patterns of AXOS indicate that bifidobacterial strains could avoid competition among each other or even could cooperate jointly to degrade these complex prebiotics. The knowledge gained on the AXOS degradation mechanisms in bifidobacteria can be of importance in the rational design of prebiotics with tailor-made composition and thus increased specificity in the colon.     

Non Digestible Oligosaccharides Modulate the Gut Microbiota to Control the Development of Leukemia and Associated Cachexia in Mice

We tested the hypothesis that changing the gut microbiota using pectic oligosaccharides (POS) or inulin (INU) differently modulates the progression of leukemia and related metabolic disorders. Mice were transplanted with Bcr-Abl-transfected proB lymphocytes mimicking leukemia and received either POS or INU in their diet (5%) for 2 weeks. Combination of pyrosequencing, PCR-DGGE and qPCR analyses of the 16S rRNA gene revealed that POS decreased microbial diversity and richness of caecal microbiota whereas it increased Bifidobacterium spp., Roseburia spp. and Bacteroides spp. (affecting specifically B. dorei) to a higher extent than INU. INU supplementation increased the portal SCFA propionate and butyrate, and decreased cancer cell invasion in the liver. POS treatment did not affect hepatic cancer cell invasion, but was more efficient than INU to decrease the metabolic alterations. Indeed, POS better than INU delayed anorexia linked to cancer progression. In addition, POS treatment increased acetate in the caecal content, changed the fatty acid profile inside adipose tissue and counteracted the induction of markers controlling β-oxidation, thereby hampering fat mass loss. Non digestible carbohydrates with prebiotic properties may constitute a new nutritional strategy to modulate gut microbiota with positive consequences on cancer progression and associated cachexia.     

Thermodynamics of microbial growth coupled to metabolism of glucose, ethanol, short-chain organic acids, and hydrogen

A literature compilation demonstrated a linear relationship between microbial growth yield and the free energy of aerobic and anaerobic (respiratory and/or fermentative) metabolism of glucose, ethanol, formate, acetate, lactate, propionate, butyrate, and H(2). This relationship provides a means to estimate growth yields for modeling microbial redox metabolism in soil and sedimentary environments.     

Short chain fatty acids but not lactate or succinate stimulate mucus release in the rat colon

Background: Short chain fatty acids (SCFAs) affect various intestinal functions. Mucus is an important physiological component of the intestinal mucosal barrier. However, the effect of SCFAs or other organic acids on the intestinal mucus release is poorly understood. The aim of this study was to investigate whether lumen SCFA stimulates mucus release into the rat colon. Methods: A solution of SCFA, lactate or succinate was infused into the colon of anesthetized rats, and we then measured the hexose content of the effluent. We also examined the influence of cholinergic antagonists on the effects of SCFA. Results: A SCFA mixture (75 mM acetate, 35 mM propionate and 20 mM butyrate) or individual SCFAs (130 mM) increased the mucus release into the colon in a similar manner. The individual SCFAs, but not lactate or succinate, stimulated colonic mucus secretion in similar concentration-dependent manners. Butyrate stimulated colonic mucus secretion at 20 mM, but acetate, propionate, lactate and succinate at this concentration did not. Pretreatment with an anti-cholinergic agent diminished the stimulatory effects of SCFAs on mucus secretion. Conclusions: Lumen SCFAs, but not lactate or succinate, stimulate mucus release from the rat colon via a cholinergic nerve mechanism.     

In vitro comparison of the prebiotic effects of two inulin-type fructans

Faecal cultures were used to compare the prebiotic effects of a new fructan containing high solubility inulin (HSI) and of a well-established prebiotic containing oligofructose (OF) with a negative control (CT). Changes in the intestinal microbiota, pH, ammonia, volatile organic acids and lactic acid were monitored during incubation. Molecular techniques for microbial enumeration indicated that both HSI and OF led to a significant increase in bifidobacteria (P< or =0.05) and lactobacilli (P< or =0.05) compared to the control. Significant changes in the pH and levels of ammonia with both inulin-type fructans were observed, as well as higher levels of acetic, lactic and formic acids (P< or =0.05). The fermentative metabolism appeared to be faster on OF than on HSI. Both OF and HSI showed clear prebiotic effects, but had differences in fermentation kinetics because of to the different degree of polymerization (DP). This study provides proof for the prebiotic effectiveness of HSI, and shows that inulin-type fructans with higher DP might have a prolonged bifidogenic effect, thus could extend the saccharolytic metabolism and low pH to the distal parts of the colon.     

Inhibition of histone-deacetylase activity by short-chain fatty acids and some polyphenol metabolites formed in the colon

Colorectal cancer is the most abundant cause of cancer mortality in the Western world. Nutrition and the microbial flora are considered to have a marked influence on the risk of colorectal cancer, the formation of butyrate and other short-chain fatty acids (SCFAs) possibly playing a major role as chemopreventive products of microbial fermentation in the colon. In this study, we investigated the effects of butyrate, other SCFAs, and of a number of phenolic SCFA and trans-cinnamic acid derivatives formed during the intestinal degradation of polyphenolic constituents of fruits and vegetables on global histone deacetylase (HDAC) activity in nuclear extracts from colon carcinoma cell cultures using tert-butoxycarbonyl-lysine (acetylated)-4-amino-7-methylcoumarin (Boc-Lys(Ac)-AMC) as substrate. Inhibition of HDAC activity, e.g., by butyrate, is related to a suppression of malignant transformation and a stimulation of apoptosis of precancerous colonic cells. In nuclear extracts from HT-29 human colon carcinoma cells, butyrate was found to be the most potent HDAC inhibitor (IC(50)=0.09 mM), while other SCFAs such as propionate were less potent. In the same assay, p-coumaric acid (IC(50)=0.19 mM), 3-(4-OH-phenyl)-propionate (IC(50)=0.62 mM) and caffeic acid (IC(50)=0.85 mM) were the most potent HDAC inhibitors among the polyphenol metabolites tested. Interestingly, butyrate was also the most potent HDAC inhibitor in a whole-cell HeLa Mad 38-based reporter gene assay, while all polyphenol metabolites and all other SCFAs tested were much less potent; some were completely inactive. The findings suggest that butyrate plays an outstanding role as endogenous HDAC inhibitor in the colon, and that other SCFAs and HDAC-inhibitory polyphenol metabolites present in the colon seem to play a much smaller role, probably because of their limited levels, their marked cytotoxicity and/or their limited intracellular availability.     

In vitro determination of prebiotic properties of oligosaccharides derived from an orange juice manufacturing by-product stream

Fermentation properties of oligosaccharides derived from orange peel pectin were assessed in mixed fecal bacterial culture. The orange peel oligosaccharide fraction contained glucose in addition to rhamnogalacturonan and xylogalacturonan pectic oligosaccharides. Twenty-four-hour, temperature- and pH-controlled, stirred anaerobic fecal batch cultures were used to determine the effects that oligosaccharides derived from orange products had on the composition of the fecal microbiota. The effects were measured through fluorescent in situ hybridization to determine changes in bacterial populations, fermentation end products were analyzed by high-performance liquid chromatography to assess short-chain fatty acid concentrations, and subsequently, a prebiotic index (PI) was determined. Pectic oligosaccharides (POS) were able to increase the bifidobacterial and Eubacterium rectale numbers, albeit resulting in a lower prebiotic index than that from fructo-oligosaccharide metabolism. Orange albedo maintained the growth of most bacterial populations and gave a PI similar to that of soluble starch. Fermentation of POS resulted in an increase in the Eubacterium rectale numbers and concomitantly increased butyrate production. In conclusion, this study has shown that POS can have a beneficial effect on the fecal microflora; however, a classical prebiotic effect was not found. An increase in the Eubacterium rectale population was found, and butyrate levels increased, which is of potential benefit to the host.