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.     

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.     

Comparison of prebiotic effects of arabinoxylan oligosaccharides and inulin in a simulator of the human intestinal microbial ecosystem

In this study, the prebiotic potential of arabinoxylan oligosaccharides (AXOS) was compared with inulin in two simulators of the human intestinal microbial ecosystem. Microbial breakdown of both oligosaccharides and short-chain fatty acid production was colon compartment specific, with ascending and transverse colon being the predominant site of inulin and AXOS degradation, respectively. Lactate levels (+5.5 mM) increased in the ascending colon during AXOS supplementation, while propionate levels (+5.1 mM) increased in the transverse colon. The concomitant decrease in lactate in the transverse colon suggests that propionate was partially formed over the acrylate pathway. Furthermore, AXOS supplementation strongly decreased butyrate in the ascending colon, this in parallel with a decrease in Roseburia spp. and Bacteroides / Prevotella / Porphyromonas (−1.4 and −2.0 log CFU) levels. Inulin treatment had moderate effects on lactate, propionate and butyrate levels. Denaturing gradient gel electrophoresis analysis revealed that inulin changed microbial metabolism by modulating the microbial community composition. In contrast, AXOS primarily affected microbial metabolism by 'switching on' AXOS-degrading enzymes (xylanase, arabinofuranosidase and xylosidase), without significantly affecting microbial community composition. Our results demonstrate that AXOS has a higher potency than inulin to shift part of the sugar fermentation toward the distal colon parts. Furthermore, due to its stronger propionate-stimulating effect, AXOS is a candidate prebiotic capable of lowering cholesterol and beneficially affecting fat metabolism of the host.     

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

Abstract

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 α(3→2) bonds in DFAIII.     

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.     

Cloning and functional analysis of a high DP fructan:fructan 1-fructosyl transferase from Echinops ritro (Asteraceae): comparison of the native and recombinant enzymes

Inulin-type fructans are the simplest and most studied fructans and have become increasingly popular as prebiotic health-improving compounds. A natural variation in the degree of polymerization (DP) of inulins is observed within the family of the Asteraceae. Globe thistle (Echinops ritro), artichoke (Cynara scolymus), and Viguiera discolor biosynthesize fructans with a considerably higher DP than Cichorium intybus (chicory), Helianthus tuberosus (Jerusalem artichoke), and Dahlia variabilis. The higher DP in some species can be explained by the presence of special fructan:fructan 1-fructosyl transferases (high DP 1-FFTs), different from the classical low DP 1-FFTs. Here, the RT-PCR-based cloning of a high DP 1-FFT cDNA from Echinops ritro is described, starting from peptide sequence information derived from the purified native high DP 1-FFT enzyme. The cDNA was successfully expressed in Pichia pastoris. A comparison is made between the mass fingerprints of the native, heterodimeric enzyme and its recombinant, monomeric counterpart (mass fingerprints and kinetical analysis) showing that they have very similar properties. The recombinant enzyme is a functional 1-FFT lacking invertase and 1-SST activities, but shows a small intrinsic 1-FEH activity. The enzyme is capable of producing a high DP inulin pattern in vitro, similar to the one observed in vivo. Depending on conditions, the enzyme is able to produce fructo-oligosaccharides (FOS) as well. Therefore, the enzyme might be suitable for both FOS and high DP inulin production in bioreactors. Alternatively, introduction of the high DP 1-FFT gene in chicory, a crop widely used for inulin extraction, could lead to an increase in DP which is useful for a number of specific industrial applications. 1-FFT expression analysis correlates well with high DP fructan accumulation in vivo, suggesting that the enzyme is responsible for high DP fructan formation in planta.     

典型煤层水微生物产甲烷潜力及其群落结构研究

内蒙古自治区二连盆地、海拉尔盆地是我国重要的煤层气产区,其中生物成因煤层气是煤层气的重要来源,但复杂物质转化产甲烷相关微生物群落结构及功能尚不清楚。【目的】研究煤层水中的微生物代谢挥发性脂肪酸产甲烷的生理特征及群落特征。【方法】以内蒙古自治区二连盆地和海拉尔盆地的四口煤层气井水作为接种物,分别添加乙酸钠、丙酸钠和丁酸钠厌氧培养;定期监测挥发性脂肪酸降解过程中甲烷和底物的变化趋势,应用高通量测序技术,分析原始煤层气井水及稳定期产甲烷菌液的微生物群落结构。【结果】除海拉尔盆地H303煤层气井微生物不能代谢丙酸外,其他样品均具备代谢乙酸、丙酸和丁酸产生甲烷的能力,其生理生态参数存在显著差异,产甲烷延滞期依次是乙酸<丁酸<丙酸;最大比产甲烷速率和底物转化效率依次是丙酸<乙酸<丁酸。富集培养后,古菌群落结构与煤层气井水的来源显著相关,二连盆地优势古菌为氢营养型产甲烷古菌Methanocalculus (相对丰度13.5%–63.4%)和复合营养型产甲烷古菌Methanosarcina (7.9%–51.3%),海拉尔盆地的优势古菌为氢营养型产甲烷古菌Methanobact...     

Molecular properties and prebiotic effect of inulin obtained from artichoke (Cynara scolymus L.)

A high molecular weight inulin has been prepared from artichoke (Cynara scolymus L.) agroindustrial wastes using environmentally benign aqueous extraction procedures. Physico-chemical analysis of the properties of artichoke inulin was carried out. Its average degree of polymerization was 46, which is higher than for Jerusalem artichoke, chicory, and dahlia inulins. GC-MS confirmed that the main constituent monosaccharide in artichoke inulin was fructose and its degradation by inulinase indicated that it contained the expected beta-2,1-fructan bonds. The FT-IR spectrum was identical to that of chicory inulin. These data indicate that artichoke inulin will be suitable for use in a wide range of food applications. The health-promoting prebiotic effects of artichoke inulin were demonstrated in an extensive microbiological study showing a long lasting bifidogenic effect on Bifidobacterium bifidum ATCC 29521 cultures and also in mixed cultures of colonic bacteria.     

Prebiotic effectiveness of inulin extracted from edible burdock

To investigate the prebiotic potential of burdock inulin (B-INU), the in vitro and in vivo effects of B-INU on bacterial growth were studied. B-INU significantly stimulated the growth of bifidobacteria in Man-Rogosa-Sharp (MRS) medium, anaerobically. Compared with chicory inulin (C-INU), long-chain inulin (L-INU) and fructooligosaccharides (FOS), 1% (w/v) B-INU promoted the specific growth rate of beneficial bacteria. The decreases of media pH with B-INU were almost the same as that with C-INU and FOS. In vivo, B-INU significantly increased the number of lactobacilli and bifidobacteria (P<0.05) in cecal content. Mice fed with B-INU, C-INU and FOS for 14 days had greater number of cecal beneficial bacteria population than those fed with L-INU for 14 days. In addition, all fructans did not cause any side effects, such as eructation and bloating. Results indicated that inulin extracted from edible burdock showed prebiotic properties that could promote health.     

Production of inulooligosaccharides from chicory extract by endoinulinase from Xanthomonas oryzae No. 5

Inulooligosaccharides (IOS) production from chicory extract was carried out using endoinulinase obtained from a new isolate, Xanthomonas oryzae No. 5. The IOS production from chicory extract was maximum when 50 g/liter of chicory extract was utilized as the substrate. As the substrate concentration increased, the IOS production accordingly decreased probably due to substrate inhibition. For a comparative study, enzyme reactions were carried out from pure inulin as substrate. Though total IOS contents indicated higher IOS yield with pure inulin compared to that of chicory extract, the distribution of inulooligosaccharide components between pure inulin and chicory extract was not significantly different; i.e. DP5 and higher oligosaccharides are major products in case of both chicory extract and pure inulin as substrate. A considerable amount of oligofructose (about 30%, w/w), which were originally present in chicory extract, resulted in the change of the enzyme kinetics. A reaction pH 7 was found to be most suitable for enzyme reaction. The initial reaction rates increased with increasing enzyme dosage, although the relative composition of the IOS produced remain unchanged.     

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.     

Simultaneous saccharification of inulin and ethanol fermentation by recombinantSaccharomyces cerevisiae secreting inulinase

VariousSaccharomyces cerevisiae strains were transformed with a 2 μ-based multicopy expression plasmid, pYIGP, carryingKluyveromyces marxianus inulinase gene under the control ofGAPDH promoter. Among them two strains, SEY2102 and 2805, showed high levels of cell growth and inulinase expression, and were selected to study their fermentation properties on inulin. Jerusalem artichoke inulin was more effective for cell growth (10∼11 g-dry wt./L at 48 hr) and inulinase expression (1.0 units/mL with SEY2102/pYIGP and 2.5 units/mL with 2805/pYIGP) than other inulin sources such as dahlia and chicory. It was also found that maximal ethanol production of 9 g/L was obtained from Jerusalem artichoke inulin at the early stationary phase (around 30 hr), indicating that recombinantS. cerevisiae cells secreting exoinulinase could be used for the simultaneous saccharification of inulin and ethanol fermentation.     

Metabolism of chicory fructooligosaccharides by bifidobacteria

Two types of chicory fructooligosaccharides (Fibruline Instant and Fibrulose F97) were metabolised by Bifidobacterium longum, B. infantis and B. angulatum. Chromatographic analysis of the medium after 120 h revealed a consumption of all the fructose oligomers present in the commercial chicory fructooligosaccharide mixtures for all the strains. Maximum measurable degree of polymerisation of the substrates before fermentation was 41. The higher biomass production was reached with B. infantis (1.4 and 1.7 g dry wt l^–1) for its cultivation on medium complemented, respectively, with Fibruline Instant and Fibrulose F97 as substrate. These results give the opportunity to use chicory fructooligosaccharides as a prebiotic.     

Production of flavour esters by Rhizopus oryzae

Microbial production of different alipathic esters with flavour characteristic has been studied. Lyophilized whole cells of Rhizopus oryzae CBS 112-07 were found to be particularly suitable to catalyse the synthesis of different flavour esters (hexyl acetate, propionate, butyrate, caprylate; geranyl acetate, propionate, butyrate and 2- and 3-methylbutyl acetate, butyrate) in n-heptane. This strain was therefore utilized for the semipreparative production of geranyl butyrate by semicontinous and continous addition of the substrates with satisfactory yields (144 g l^–1 in 264 h and 142 g l^–1 in 48 h respectively).     

Metabolic properties and kinetics of methanogenic granules

Two types of mesophilic methanogenic granules (R- and F-granules) were developed on different synthetic feeds containing acetate, propionate and butyrate as major carbon sources and their metabolic properties were characterized. The metabolic activities of granules on acetate, formate and H₂-CO₂ were related to the feed composition used for their development. These granules performed a reversible reaction between H₂ production from formate and formate synthesis from H₂ plus bicarbonate. Both types of granules exhibited high activity on normal and branched volatile fatty acids with three to five carbons and low activity on ethanol and glucose. The granules performed a reversible isomerization between isobutyrate and butyrate during butyrate or isobutyrate degradation. Valerate and 2-methylbutyrate were produced and consumed during propionate-butyrate degradation. The respective apparent K _m (mm) for various substrates in disrupted R- and F-granules was: acetate, 0.43 and 0.41; propionate, 0.056 and 0.038; butyrate, 0.15 and 0.19; isobutyrate, 0.12 and 0.19; valerate, 0.15 and 0.098. Both granules had an optimum temperature range from 40 to 50° C for H₂-CO₂ and formate utilization and 40° C for acetate, propionate and butyrate utilization and a similar optimum pH. Correspondence to: J. G. Zeikus     

Enzymatic production of inulo-oligosaccharides from chicory juice

Batchwise production of inulo-oligosaccharide from chicory juice was carried out by an endoinulinase from Pseudo-monas sp. The maximum yield of oligosaccharides (OS) was about 80% in total sugar basis with substrate at 30–100 g/l. Compared with pure inulin of the same origin as a substrate, the same OS yield was obtained but it showed quite a different product distribution in degree of polymerization (DP) and sugar composition, where DP2, DP3 and DP4 were major components. © Rapid Science Ltd. 1998     

猪小肠微生物对不同蛋白源的体外发酵特性比较

【目的】采用体外发酵技术比较小肠微生物对不同蛋白源的发酵规律。【方法】以成年猪的十二指肠、空肠和回肠内容物为接种物,以豆粕、菜粕或鱼粉水解物上清液为氮源底物,于发酵的0、4、8、12 h分别测定发酵液p H、微生物蛋白、氨氮和挥发性脂肪酸含量,同时提取细菌DNA并进行定量分析。【结果】添加含氮底物的空肠组和回肠组氨氮浓度和菌体蛋白浓度均相对增加,尤其是酶解菜粕组菌体蛋白合成量较高;十二指肠组菌体蛋白浓度以及氨氮含量不断减少。各发酵组乳酸和挥发酸快速积累,4–8 h积累量最大;8 h后空肠组和回肠组乳酸和挥发酸含量相对稳定,而十二指肠组后期丙酸含量增加约2 mmol/L,并伴随着乳酸含量的相对减少。同时,各组中总细菌、拟杆菌门、厚壁菌门和乳酸杆菌数量相对增加,且略高于无氮组,但不同蛋白源组间无显著差异。【结论】在体外培养条件下,空肠和回肠微生物具有相似的发酵规律,均能快速利用培养液中的含氮物质合成菌体蛋白;十二指肠微生物具有较强的产挥发酸能力,能够转化乳酸并大量产生丁酸和丙酸,这有利于宿主营养功能和肠道健康。     

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.     

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.