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Background Fructo-oligosaccharides (FOS), inulin, and galacto-oligosaccharides (GOS) are widely recognized prebiotics that profoundly affect the intestinal microbiota, including stimulation of bifidobacteria and lactobacilli, and are reported to elicit several health benefits. The combination of dietary FOS and inulin with calcium phosphate was reported to stimulate commensal Lactobacillus populations and protect the host against pathogenic Enterobacteriaceae, but little is known about the effects of GOS in diets with a different level of calcium phosphate. Methods We investigated the microbiome changes elicited by dietary supplementation with GOS or inulin using diets with high (100 mmol/kg) and low (30 mmol/kg) calcium phosphate levels in adult Wistar rats. Rats were acclimatized to the respective experimental diets for 14 days, after which fecal material was collected, DNA was extracted from fecal material, and the V3‑V4 region of the bacterial 16S rRNA gene was amplified with PCR, followed by microbial composition analysis. In tandem, the organic acid profiles of the fecal material were analyzed. Results Feeding rats non-supplemented (no prebiotic-added) diets revealed that diets rich in calcium phosphate favored members of the Firmicutes and increased fecal lactic, succinic, acetic, propionic, and butyric acid levels. In contrast, relatively low dietary calcium phosphate levels promoted the abundance of mucin degrading genera like Akkermansia and Bacteroides, and resulted in increased fecal propionic acid levels and modest increases in lactic and butyric acid levels. Irrespective of the calcium phosphate levels, supplementation with GOS or inulin strongly stimulated Bifidobacterium, while only high calcium phosphate diets increased the endogenous Faecalibaculum populations. Conclusions Despite the prebiotic’s substantial difference in chemical structure, sugar composition, oligomer size, and the microbial degradation pathway involved in their utilization, inulin and GOS modulated the gut microbiota very similarly, in a manner that strongly depended on the dietary calcium phosphate level. Therefore, our study implies that the collection of detailed diet information including micronutrient balance is necessary to correctly assess diet-driven microbiota analysis.

Anti-cancer T-cell responses are often halted due to the immune-suppressive micro-environment, in part related to tumor-associated macrophages. In the current study, we assessed indigestible β-glucans (oatβG, curdlan, grifolan, schizophyllan, lentinan, yeast whole glucan particles (yWGP), zymosan and two additional yeast-derived β-glucans a and b) for their physicochemical properties as well as their effects on the plasticity of human monocyte-derived macrophages that were polarized with IL-4 to immune-suppressive macrophages. Beta-glucans were LPS/LTA free, and tested for solubility, molecular masses, protein and monosaccharide contents. Curdlan, yeast-b and zymosan re-polarized M(IL-4) macrophages towards an M1-like phenotype, in particular showing enhanced gene expression of CCR7, ICAM1 and CD80, and secretion of TNF-α and IL-6. Notably, differential gene expression, pathway analysis as well as protein expressions demonstrated that M(IL-4) macrophages treated with curdlan, yeast-b or zymosan demonstrated enhanced production of chemo-attractants, such as CCL3, CCL4, and CXCL8, which contribute to recruitment of monocytes and neutrophils. The secretion of chemo-attractants was confirmed when using patient-derived melanoma-infiltrating immune cells. Taken together, the bacterial-derived curdlan as well as the yeast-derived β-glucans yeast-b and zymosan have the unique ability to preferentially skew macrophages towards a chemo-attractant-producing phenotype that may aid in anti-cancer immune responses. Electronic supplementary material The online version of this article (10.1007/s00262-020-02707-4) contains supplementary material, which is available to authorized users.

SCOPE: Non-digestible carbohydrates (NDCs) such as native chicory inulin and 2'-fucosyllactose (2'-FL) are added to infant formula to mimic some of the human milk oligosaccharide (HMO) functions. It is unknown whether combining inulin and 2'-FL influences their fermentation kinetics and whether the immune-modulatory effects of these NDCs are different under normal and inflammatory-prone Th2-polarizing conditions.METHODS AND RESULTS: We investigated the in vitro fermentation of 2'-FL and native chicory inulin, fermented individually and combined, using fecal inocula of 8-week-old infants. Native inulin was fermented in a size-dependent fashion and expedited the fermentation of 2'-FL. Fermentation of both native inulin and 2'FL increased the relative abundance of Bifidobacterium, which coincided with the production of acetate and lactate. The fermentation digesta of all fermentations differentially influenced both dendritic cell and T-cell cytokine responses under normal culture conditions or in presence of the Th2-polarizing cytokines IL-33 and TSLP, with the most pronounced effect for IL-1β in the presence of TSLP.CONCLUSIONS: Our findings show that native inulin can expedite the fermentation of 2'-FL by infant fecal microbiota and that these NDC fermentation digesta have different effects under normal and Th2-polarizing conditions, indicating that infants with different immune backgrounds might benefit from tailored NDC formulations.

The aim of the current study was to investigate whether degradation of rapeseed meal (RSM) by a swine gut microbiota consortium was improved by modifying RSM by treatment with cellulase (CELL), two pectinases (PECT), or alkaline (ALK) compared to untreated RSM and to assess whether microbiota composition and activity changed. The predicted relative abundances of carbohydrate digestion and absorption, glycolysis, pentose phosphate pathway, and pyruvate metabolism were significantly increased upon CELL and ALK feeding, and CELL and ALK also exhibited increased total short-chain fatty acid (SCFA) production compared to CON. Megasphaera, Prevotella, and Desulfovibrio were significantly positively correlated with SCFA production. Findings were validated in ileal cannulated pigs, which showed that CELL and ALK increased fiber degradation of RSM. In conclusion, CELL and ALK rather than PECT1 or PECT2 increased fiber degradation in RSM, and this information could guide feed additive strategies to improve efficiency and productivity in the swine industry.

To study the Chinese human milk N-glycome over lactation, N-glycans were released and separated from serum proteins, purified by solid-phase extraction, and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 66 different putative N-glycans were found in the colostrum (week 1) and mature milk (week 4) of seven Chinese mothers. A clear difference was observed between milk of five secretor and two nonsecretor mothers, based on the type and relative amounts of the individual N-glycans. The relative levels of the total neutral nonfucosylated and the fucosylated N-glycans in milk of five secretor mothers increased and decreased over lactation, respectively. This pattern could not be observed for the milk from the two nonsecretor mothers. Overall, this was the first study that provided detailed information on individual N-glycans in milk among mothers and over time as well as that fucosylation of N-glycans in milk was associated with the mother's secretor status.

Citrus pectins were studied by enzymatic fingerprinting using a simultaneous enzyme treatment with endo-polygalacturonase (endo-PG) from Kluyveromyces fragilis and pectin lyase (PL) from Aspergillus niger to reveal the methyl-ester distribution patterns over the pectin backbone. Using HILIC-MS combined with HPAEC enabled the separation and identification of the diagnostic oligomers released. Structural information on the pectins was provided by using novel descriptive parameters such as degree of blockiness of methyl-esterified oligomers by PG (DBPGme) and degree of blockiness of methyl-esterified oligomers by PL (DBPLme). This approach enabled us to clearly differentiate citrus pectins with various methyl-esterification patterns. The simultaneous use of PG and PL showed additional information, which is not revealed in digests using PG or PL alone. This approach can be valuable to differentiate pectins having the same DM and to get specific structural information on pectins and therefore to be able to better predict their physical and biochemical functionalities.

SCOPE: Pectins have anti-inflammatory properties on intestinal immunity through direct interactions on Toll-like receptors (TLRs) in the small intestine or via stimulating microbiota-dependent effects in the large intestine. Both the degree of methyl-esterification (DM) and the distribution of methyl-esters (degree of blockiness; DB) of pectins contribute to this influence on immunity but whether and how the DB impacts immunity through microbiota-dependent effects in the large intestine is unknown. Therefore, we tested pectins that structurally differ in DB in a mouse model with Citrobacter rodentium-induced colitis and studied the impact on the intestinal microbiota composition and associated attenuation of inflammation.METHODS AND RESULTS: Both low and high DB pectins induced a more rich and diverse microbiota composition. These pectins also lowered the bacterial load of C. rodentium in caecal digesta. Through these effects, both low and high DB pectins attenuated C. rodentium-induced colitis resulting in reduced intestinal damage, reduced numbers of Th1-cells which are increased in case of C. rodentium-induced colitis, and reduced levels of GATA3+ Tregs which are related to tissue inflammation.CONCLUSION: Pectins prevent C. rodentium-induced colonic inflammation by lowering the C. rodentium load in the caecum independently of the DB. This article is protected by copyright. All rights reserved.

Human milk is widely acknowledged as the best food for infants, and that is not just because of nutritional features. Human milk also contains a plethora of bioactive molecules, including a large set of human milk oligosaccharides (hMOs). Especially fucosylated hMOs have received attention for their anti-adhesive effects on pathogens, preventing attachment to the intestine and infection. Because hMOs are generally challenging to produce in sufficient quantities to study and ultimately apply in (medical) infant formula, novel compounds that are inspired by hMO structures (so-called “mimics”) are interesting compounds to produce and evaluate for their biological effects. Here we present our thorough study into the digestion, fermentation and anti-adhesive capacity of the novel compound di-fucosyl-β-cyclodextrin (DFβCD), which was inspired by the molecular structures of hMOs. We establish that DFβCD is not digested by α-amylase and also resistant to fermentation by microbial enzymes from a 9 month-old infant inoculum. In addition, we reveal that DFβCD blocks adhesion of enterotoxigenic E. coli (ETEC) to Caco-2 cells, especially when DFβCD is pre-incubated with ETEC prior to addition to the Caco-2 cells. This suggests that DFβCD functions through a decoy effect. We expect that our results inspire the generation and biological evaluation of other fucosylated hMOs and mimics, to obtain a comprehensive overview of the anti-adhesive power of fucosylated glycans., The novel hMO-mimic DFβCD shows promising characteristics to function as a decoy substrate against ETEC adhesion.

Scope Next to galacto‐oligosaccharides (GOS), starch‐derived isomalto‐oligosaccharide preparation (IMO) and isomalto/malto‐polysaccharides (IMMP) could potentially be used as prebiotics in infant formulas. However, it remains largely unknown how the specific molecular structures of these non‐digestible carbohydrates (NDCs) impact fermentability and immune responses in infants. Methods and Results In vitro fermentation of GOS, IMO and IMMP using infant fecal inoculum of 2‐ and 8‐week‐old infants shows that only GOS and IMO are fermented by infant fecal microbiota. The degradation of GOS and IMO coincides with an increase in Bifidobacterium and production of acetate and lactate, which is more pronounced with GOS. Individual isomers with an (1↔1)‐linkage or di‐substituted reducing terminal glucose residue are more resistant to fermentation. GOS, IMO, and IMMP fermentation digesta attenuates cytokine profiles in immature dendritic cells (DCs), but the extent is dependent on the infants age and NDC structure. Conclusion The IMO preparation, containing reducing and non‐reducing isomers, shows similar fermentation patterns as GOS in fecal microbiota of 2‐week‐old infants. Knowledge obtained on the substrate specificities of infant fecal microbiota and the subsequent regulatory effects of GOS, IMO and IMMP on DC responses might contribute to the design of tailored NDC mixtures for infants of different age groups., The in vitro fermentation of galacto‐oligosaccharide (GOS), isomalto‐oligosaccharide preparation (IMO) and isomalto/malto‐polysaccharides (IMMP) by pooled fecal inocula of 2‐ and 8‐week‐old infants shows that next to the size, the highly variable structure of oligosaccharides shows a huge impact on the fermentability of non‐digestible carbohydrates (NDCs) by infant fecal microbiota. GOS, IMO as well as IMMP fermentation digesta attenuated cytokine profiles in immature dendritic cells of which the extent is dependent on the infants age and NDC structure.

Globally increased life expectancy strongly triggered interest to delay the onset of frailty, which has been associated with alterations in compositional and functional characteristics of intestinal microbiota. In the current study, we used an in vitro batch incubation model to compare the metabolic capacity of the faecal microbiota of adults (n = 6) versus pre-frail elderly (n = 6) to degrade various glycosidic carbohydrates, including galacto-oligosaccharides, 2′-fucosyllactose, chicory fructo-oligosaccharides and inulin, and isomalto/malto-polysaccharides. The in vitro metabolic capacity was also compared with an in vivo GOS intervention study based on the same subjects. Analysis of 16S rRNA gene sequences and metabolites revealed distinct portions of variation in overall microbiota and metabolite composition during incubation being explained by individuality of the subjects and carbon source. In addition, the age group of the subjects also had significant impact on microbiota variation, carbohydrate degradation and metabolite production. This was accompanied by elevated increase in the relative abundance of Bifidobacterium in the microbiota of adults compared to that of pre-frail elderly and significantly decreased effectiveness to degrade galacto-oligosaccharides by the latter group. Altogether, the carbohydrate degradation in elderly was different compared to adults, with some carbohydrates showing decreased degradation rates. Longer interventions periods may be required to enhance bifidobacterial abundance in the microbiota of pre-frail elderly and thereby to obtain associated prebiotic health benefits.

Contains fulltext : 233384.pdf (Publisher’s version ) (Open Access) To follow human milk oligosaccharide (HMO) biosynthesis and in vivometabolization, mother milk and infant feces from 68 mother–infant dyads at 2, 6, and 12 weeks postpartum were analyzed, with 18 major HMOs quantitated. Fucosylated and neutral core HMO levels in milk were dependent on mothers' Lewis/Secretor status, whereas most sialylated HMO levels were independent. Infant fecal excretion of HMOs gradually declined with age, especially for neutral core structures. Although decreasing in absolute concentrations in milk during lactation, the relative abundance of total fucosylated HMOs increased in both milk and feces. Mono-fucosylated HMOs were more consumed than those decorated with two fucose moieties. More (α2-3)-sialylated HMOs were degraded than (α2-6)-sialylated HMOs. The transition speed of HMO metabolization from nonspecific or structure-specific consumption stage to the complete consumption stage was individual-dependent. Variation was associated with mode and place of delivery, where caesarean section or early exposure to hospital environment delayed the transition. 14 p.

Human milk oligosaccharides (hMOs) are unique bioactive components in human milk. 3-Fucosyllactose (3-FL) is an abundantly present hMO that can be produced in sufficient amounts to allow application in infant formula. Lacto-N-triaose II (LNT2) can be obtained by acid hydrolysis of lacto-N-neotetraose (LNnT). Both 3-FL and LNT2 have been shown to have health benefits, but their impact on infant microbiota composition and microbial metabolic products such as short-chain fatty acids (SCFAs) is unknown. To gain more insight in fermentability, we performed in vitro fermentation studies of 3-FL and LNT2 using pooled fecal microbiota from 12-week-old infants. The commonly investigated galacto-oligosaccharides (GOS)/inulin (9 : 1) served as control. Compared to GOS/inulin, we observed a delayed utilization of 3-FL, which was utilized at 60.3% after 36 h of fermentation, and induced the gradual production of acetic acid and lactic acid. 3-FL specifically enriched bacteria of Bacteroides and Enterococcus genus. LNT2 was fermented much faster. After 14 h of fermentation, 90.1% was already utilized, and production of acetic acid, succinic acid, lactic acid and butyric acid was observed. LNT2 specifically increased the abundance of Collinsella, as well as Bifidobacterium. The GOS present in the GOS/inulin mixture was completely fermented after 14 h, while for inulin, only low DP was rapidly utilized after 14 h. To determine whether the fermentation might lead to enhanced colonization of commensal bacteria to gut epithelial cells, we investigated adhesion of the commensal Lactobacillus plantarum WCFS1 to Caco-2 cells. The fermentation digesta of LNT2 collected after 14 h, 24 h, and 36 h, and GOS/inulin after 24 h of fermentation significantly increased the adhesion of L. plantarum WCFS1 to Caco-2 cells, while 3-FL had no such effect. Our findings illustrate that fermentation of hMOs is very structure-dependent and different from the commonly applied GOS/inulin, which might lead to differential potencies to stimulate adhesion of commensal cells to gut epithelium and consequent microbial colonization. This knowledge might contribute to the design of tailored infant formulas containing specific hMO molecules to meet the need of infants during the transition from breastfeeding to formula.

Human milk oligosaccharides (hMOs) are important bioactive components in mother's milk contributing to infant health by supporting colonization and growth of gut microbes. In particular, Bifidobacterium genus is considered to be supported by hMOs. Approximately 200 different hMOs have been discovered and characterized, but only a few abundant hMOs can be produced in sufficient amounts to be applied in infant formula. These hMOs are usually supplied in infant formula as single molecule, and it is unknown which and how individual hMOs support growth of individual gut bacteria. To investigate how individual hMOs influence growth of several relevant intestinal bacteria species, we studied the effects of three hMOs (2 '-fucosyllactose, 3-fucosyllactose, and 6 '-sialyllactose) and an hMO acid hydrolysate (lacto-N-triose) on three Bifidobacteria and one Faecalibacterium and introduced a co-culture system of two bacterial strains to study possible cross-feeding in presence and absence of hMOs. We observed that in monoculture, Bifidobacterium longum subsp. infantis could grow well on all hMOs but in a structure-dependent way. Faecalibacterium prausnitzii reached a lower cell density on the hMOs in stationary phase compared to glucose, while B. longum subsp. longum and Bifidobacterium adolescentis were not able to grow on the tested hMOs. In a co-culture of B. longum subsp. infantis with F. prausnitzii, different effects were observed with the different hMOs; 6 '-sialyllactose, rather than 2 '-fucosyllactose, 3-fucosyllactose, and lacto-N-triose, was able to promote the growth of B. longum subsp. infantis. Our observations demonstrate that effects of hMOs on the tested gut microbiota are hMO-specific and provide new means to support growth of these specific beneficial microorganisms in the intestine.

This study provided insights into the degradation of human milk proteins in an in vitro infant digestion model by comparing colostrum (week 1) and mature milk (week 4) of 7 Chinese mothers individually. In this study, we adapted the exiting INFOGEST in vitro model, to conditions representative to infants (0 to 3 month-old). The level of undigested proteins was analyzed by LC-MS/MS after gel-electrophoretic separation and in-gel digestion. The BCA protein assay showed that the total undigested milk protein content decreased from the start to the end of digestion with variations between mothers, especially in the gastric phase (25–80%). Undigested proteins could also still be found after the intestinal phase, ranging from 0.5 to 4.2% of initial protein content. Based on LC-MS/MS analysis, milk protein digestion varied between the mothers individually, especially during the gastric phase. No differences could be observed between protein digestion from colostrum and mature milk after the intestinal phase. The highest levels of proteins remaining after intestinal digestion can be linked to the group immune-active proteins, for all mothers. The level of protease inhibitors and total protein content in the milk did not correlate with the overall proteolysis during digestion. The results also showed that milk serum proteins partly remained after the gastric phase, with 33% remaining from colostrum and 37% remaining from mature milk. More than 40 milk serum proteins were detected after the intestinal phase. Some of the highly abundant milk serum proteins (lactoferrin, serum albumin, bile salt-activated lipase, immunoglobulins, α1-antichymotrypsin) were still partially present intact after the intestinal phase, for all mothers. Caseins were also not completely digested in the gastric phase, with 35% remaining from colostrum and 13% remaining from mature milk. Caseins, on the other hand, were almost completely digested after the intestinal phase. The complete degradation of caseins into peptides might be related to their structural features. Overall, this study showed that digestion differed for the various human milk proteins by adapting an in vitro digestion model to infant physiological conditions, with the main differences between digestion of the milk from individual mothers being observed after gastric digestion.

Scope Inulin‐type fructans are commonly applied in infant formula to support development of gut microbiota and immunity. These inulin‐type fructans are considered to be fermented by gut microbiota, but it is unknown how fermentation impacts immune modulating capacity and whether the process of fermentation is dependent on the infant's age. Methods and results The in vitro fermentation of chicory fructo‐oligosaccharides (FOS) and native inulin are investigated using pooled fecal inocula of two‐ and eight‐week‐old infants. Both inocula primarily utilize the trisaccharides in FOS, while they almost completely utilize native inulin with degree of polymerization (DP) 3–8. Fecal microbiota of eight‐week‐old infants degrades longer chains of native inulin up to DP 16. This correlates with a higher abundance of Bifidobacterium and higher production of acetate and lactate after 26 h of fermentation. Fermented FOS and native inulin attenuate pro‐inflammatory cytokines produced by immature dendritic cells (DCs), but profiles and magnitude of attenuation are stronger with native inulin than with FOS. Conclusion The findings demonstrate that fermentation of FOS and native inulin is dependent on the infant's age and fructan structure. Fermentation enhances attenuating effects of pro‐inflammatory responses in DCs, which depend mainly on microbial metabolites formed during fermentation., This in vitro fermentation setup using pooled fecal inocula of two‐ and eight‐week‐old infants showed that chicory fructo‐oligosaccharides and native inulin attenuate pro‐inflammatory responses in infants. The extent of attenuation depends on the microbial metabolites formed during fermentation of fructans, which is affected by the infant's age and corresponding microbial functionality, as well as the size and structure of fructans.

Synbiotics are food supplements that combine probiotics and prebiotics to synergistically elicit a health effect in humans. Lactobacillus plantarum exhibits remarkable genetic and phenotypic diversity, in particular in strain-specific carbohydrate utilization capacities, and several strains are marketed as probiotics. We have screened 77 L. plantarum strains for their abilities to utilize specific prebiotic fibers, revealing variable and strain-specific growth efficiencies on isomalto- and galactooligosaccharides. We identified a single strain within the screening panel that was able to effectively utilize inulin and fructooligosaccharides (FOS), which did not support efficient growth of the rest of the strains. In the panel we tested, we did not find strains that could utilize arabinoxylooligosaccharides or sulfated fucoidan. The strain-specific growth phenotype on isomaltooligosaccharides was further analyzed using high-performance anion-exchange chromatography, which revealed distinct substrate utilization phenotypes within the strain panel. The strain-specific phenotypes could be linked to the strains’ genotypes by identifying gene clusters coding for carbohydrate membrane transport systems that are predicted to be involved in the utilization of isomaltose and other (unidentified) oligosaccharides in the isomaltooligosaccharide substrate. IMPORTANCE Synbiotics combine prebiotics and probiotics to synergistically enhance the health benefits associated with these ingredients. Lactobacillus plantarum is encountered as a natural inhabitant of the gastrointestinal tract, and specific strains are marketed as probiotics based on their strain-specific health-promoting activities. Strain-specific stimulation of growth through prebiotic substrates could enhance the persistence and/or activity of L. plantarumin situ. Our study establishes a high-throughput screening model for prebiotic substrate utilization by individual strains of bacteria, which can be readily employed for synbiotic matchmaking approaches that aim to enhance the intestinal delivery of probiotics through strain-specific, selective growth stimulation.

Several factors affect gut microbiota development in early life, among which breastfeeding plays a key role. We followed 24 mother-infant pairs to investigate the associations between concentrations of selected human milk oligosaccharides (HMOs) in breastmilk, infant faeces, and the faecal microbiota composition in healthy, breastfed infants at two, six and 12 weeks of age. Lactation duration had a significant effect on breastmilk HMO content, which decreased with time, except for 3-fucosyllactose (3FL) and Lacto-N-fucopentaose III (LNFP III). We confirmed that microbiota composition was strongly influenced by infant age and was associated with mode of delivery and breastmilk LNFP III concentration at two weeks, with infant sex, delivery mode, and concentrations of 3′sialyllactose (3′SL) in milk at six weeks, and infant sex and Lacto-N-hexaose (LNH) in milk at 12 weeks of age. Correlations between levels of individual breastmilk HMOs and relative abundance of OTUs found in infant faeces, including the most predominant Bifidobacterium OTUs, were weak and varied with age. The faecal concentration of HMOs decreased with age and were strongly and negatively correlated with relative abundance of OTUs within genera Bifidobacterium, Parabacteroides, Escherichia-Shigella, Bacteroides, Actinomyces, Veillonella, Lachnospiraceae Incertae Sedis, and Erysipelotrichaceae Incertae Sedis, indicating the likely importance of these taxa for HMO metabolism in vivo.

Starch digestion in the proximal small intestine of pigs exceeds in vitro predictions, suggesting a currently underestimated role for the stomach in starch digestion kinetics. This study aimed to investigate the role of amylase action in the porcine stomach on starch digestion kinetics, including hydrolysis by porcine saliva and degradation by bacterial enzymes present in the stomach. We studied the hydrolysis of starch in pigs fed barley based diets, in which starch was included as isolated powder, ground barley, or extruded barley. We identified soluble maltodextrins originating from starch breakdown in stomach digesta, especially in pigs fed extruded barley. Furthermore, we observed bacterial cells embedded in granular starch with electron microscopy, for pigs fed diets containing isolated barley starch. These observations lead us to measure starch hydrolysis in a dynamic stomach model over the course of a 225 min incubation, in which the pH was step-wise decreased from 6.5 to 2.0. Using this method, feed was either exposed to an enzyme extract obtained from stomach digesta or to porcine saliva. Up to 30 % of starch was hydrolysed into maltodextrins with a degree of polymerisation (DP)

Several Lactobacillus plantarum strains are marketed as probiotics for their potential health benefits. Prebiotics, e.g., galacto-oligosaccharides (GOS), have the potential to selectively stimulate the growth of L. plantarum probiotic strains based on their phenotypic diversity in carbohydrate utilization, and thereby enhance their health promoting effects in the host in a strain-specific manner. Previously, we have shown that GOS variably promotes the strain-specific growth of L. plantarum. In this study we investigated this variation by molecular analysis of GOS utilization by L. plantarum. HPAEC-PAD analysis revealed two distinct GOS utilization phenotypes in L. plantarum. Linking these phenotypes to the strain-specific genotypes led to the identification of a lac operon encoding a β-galactosidase (lacA), a permease (lacS), and a divergently oriented regulator (lacR), that are predicted to be involved in the utilization of higher degree of polymerization (DP) constituents present in GOS (specifically DP of 3–4). Mutation of lacA and lacS in L. plantarum NC8 resulted in reduced growth on GOS, and HPAEC analysis confirmed the role of these genes in the import and utilization of higher-DP GOS constituents. Overall, the results enable the design of highly-selective synbiotic combinations of L. plantarum strain-specific probiotics and specific GOS-prebiotic fractions.

The human intestinal tract harbors diverse and complex microbial communities that have a vast metabolic capacity including the breakdown of complex carbohydrates into short chain fatty acids, acetate, propionate, and butyrate. As butyrate is beneficial for gut health there is much attention on butyrogenic bacteria and their role in the colonic anaerobic food chain. However, our understanding how production of butyrate by gut microorganisms is controlled by interactions between different species and environmental nutrient availability is very limited. To address this, we set up experimental in vitro co-culture systems to study the metabolic interactions of Anaerostipes rhamnosivorans, a butyrate producer with each of its partners; Blautia hydrogenotrophica, an acetogen; Methanobrevibacter smithii, a methanogen and Bacteroides thetaiotaomicron, a versatile degrader of plant cell wall pectins; through corresponding specific cross-feeding. In all co-cultures, A. rhamnosivorans was able to benefit from its partner for enhanced butyrate formation compared to monocultures. Interspecies transfer of hydrogen or formate from A. rhamnosivorans to the acetogen B. hydrogenotrophica and in turn of acetate from the acetogen to the butyrogen were essential for butyrate formation. A. rhamnosivorans grown on glucose supported growth of M. smithii via interspecies formate/hydrogen transfer enhancing butyrate formation. In the co-culture with pectin, lactate was released by B. thetaiotaomicron which was concomitantly used by A. rhamnosivorans for the production of butyrate. Our findings indicate enhanced butyrate formation through microbe-microbe interactions between A. rhamnosivorans and an acetogen, a methanogen or a pectin-degrader. Such microbial interactions enhancing butyrate formation may be beneficial for colonic health.

Aging is accompanied with increased frailty and comorbidities, which is potentially associated with microbiome perturbations. Dietary fibers could contribute to healthy aging by beneficially impacting gut microbiota and metabolite profiles. We aimed to compare young adults with elderly and investigate the effect of pectin supplementation on fecal microbiota composition, short chain fatty acids (SCFAs), and exhaled volatile organic compounds (VOCs) while using a randomized, double-blind, placebo-controlled parallel design. Fifty-two young adults and 48 elderly consumed 15 g/day sugar beet pectin or maltodextrin for four weeks. Fecal and exhaled breath samples were collected before and after the intervention period. Fecal samples were used for microbiota profiling by 16S rRNA gene amplicon sequencing, and for analysis of SCFAs by gas chromatography (GC). Breath was used for VOC analysis by GC-tof-MS. Young adults and elderly showed similar fecal SCFA and exhaled VOC profiles. Additionally, fecal microbiota profiles were similar, with five genera significantly different in relative abundance. Pectin supplementation did not significantly alter fecal microbiota, SCFA or exhaled VOC profiles in elderly or young adults. In conclusion, aside from some minor differences in microbial composition, healthy elderly and young adults showed comparable fecal microbiota composition and activity, which were not altered by pectin supplementation.

Scope Understanding biological functions of different free human milk oligosaccharides (HMOs) in shaping gastrointestinal tract microbiota during infancy is of great interest. We examined a link between HMOs in maternal milk and infant faecal microbiota composition and investigated the role of microbiota in degrading HMOs within the GI tract of healthy, breastfed, one-month old infants. Methods and results Maternal breast milk and corresponding infant faeces originated from the KOALA Birth Cohort. HMOs were quantified in milk and infant faecal samples using PGC-UPLC-MS and HPAEC-PAD. Faecal microbiota composition was characterised using Illumina HiSeq amplicon 16S rRNA sequencing. The composition associated with gender, mode of delivery, and milk HMOs: Lacto-N-fucopentaose I and 2'-Fucosyllactose. Overall, Bifidobacterium, Bacteroides, Escherichia-Shigella and Parabacteroides were predominating genera. We detected three different patterns in the infant faecal microbiota structure. Gastrointestinal degradation of HMOs was strongly associated with faecal microbiota composition, and there was a link between utilisation of specific HMOs and abundance of various phylotypes (OTUs). Conclusions HMOs in maternal milk are among important factors shaping GI tract microbiota composition in one-month old breastfed infants. Infant's ability to metabolise different HMOs strongly correlate with faecal microbiota composition, and with phylotypes within genera Bifidobacterium, Bacteroides and Lactobacillus. This article is protected by copyright. All rights reserved.

To better understand the variability of the type and level of serum proteins in human milk, the milk serum proteome of Chinese mothers during lactation was investigated using proteomic techniques and compared to the milk serum proteome of Dutch mothers. This showed that total milk serum protein concentrations in Chinese human milk decreased over a 20-week lactation period, although with variation between mothers in the rate of decrease. Variation was also found in the composition of serum proteins in both colostrum and mature milk, although immune-active proteins, enzymes, and transport proteins were the most abundant for all mothers. These three protein groups account for many of the 15 most abundant proteins, with these 15 proteins covering more than 95% of the total protein concentrations, in both the Chinese and Dutch milk serum proteome. The Dutch and Chinese milk serum proteome were also compared based on 166 common milk serum proteins, which showed that 22% of the 166 serum proteins differed in level. These differences were observed mainly in colostrum and concern several highly abundant proteins. This study also showed that protease inhibitors, which are highly correlated to immune-active proteins, are present in variable amounts in human milk and could be relevant during digestion.

The potential prebiotic effect of fibres (alcohol insoluble solids fractions) from fruit by-products - orange bagasses and passion fruit peels and their degradation by human gut microbiota was tested in an in vitro colon system. Standard medium and inulin were used as controls. Orange bagasses (A-OB1 and A-OB2) had similar chemical composition but differed regarding fermentation profile. A-OB2 resulted in a more diverse bacterial community than A-OB1 and produced more SCFA, with increased Ruminococcus and Lachnospira. Carbohydrate utilization was higher on A-OB2 probably due to higher ratio soluble to insoluble fibres. Isolated fibres from passion fruit peels presented similar chemical composition and fermentation profiling. Bacteroides and Ruminococcus were the main genera stimulated. Negligible lactate and succinate production represent slow fermentation, a protective feature against colon cancer. This study provided evidence that the tested fruit by-products have the potential to be used for selective modulation of the gut microbiota.

The aim of this study was to test the compatibility of two selected Lactobacillus strains with different prebiotic carbohydrates in order to develop a synbiotic cheese. Initially, the growth of the probiotic lactobacilli with the prebiotic formulations was monitored in optimized growth medium and in milk. Afterwards, semi-hard cheeses were manufactured with the selected combination of Lb. paracasei INIA P272 and fructo-oligosaccharides (FOS). Results showed that the addition of FOS did not affect Lb. paracasei viability in the cheese after 28 days of ripening, but a higher viability of this strain was achieved in the presence of FOS when cheese was tested in a colonic model. Thus, the inclusion of FOS in the cheese could have a beneficial effect by increasing the probiotic strain viability in the host.

Consumption of non-starch polysaccharides (NSP) is associated with reduced risk of obesity. This study aimed to compare the effects of cereals (oats) and legumes (soybean), rich in different classes of NSP, on appetite regulation and fat accumulation in rats. Soy pectin fermented more efficient than cereal arabinoxylan in rats. Soy pectin and oat β-glucan were utilized mainly in the caecum of rats. Only small amount of maltodextrin, cello-oligosaccharides and xylo-oligosaccharides were detected in the digesta. Caecal fermentation of soy pectin produced significantly higher concentration of short chain fatty acids (SCFAs) compared to the control. Retroperitoneal (RP) fat-pad weight was significantly lower for rats fed with soybean meal enriched diet than for controls. An inverse correlation between rat RP fat-pad weight and concentration (and proportion) of butyrate was observed. Consumption of soy pectin and oat β-glucan enriched foods to produce targeted SCFAs in vivo could be a potential strategy to lower fat mass accumulation and a potential tool to manage obesity.

The prevalence of acute respiratory infections and their impact on quality of life underlies the need for efficacious solutions that are safe, sustainable and economically viable. Polysaccharides in several (traditional) plant extracts have been shown to be immunostimulatory, and some studies suggest beneficial effects against respiratory infections. The aim of this study was to (i) identify the active polysaccharide constituents from affordable and renewable crops (bell pepper and carrot) using activity-guided fractionation, (ii) evaluate in vitro effects on innate immune responses (phagocytosis and cytokine secretion), microbiota modulation and production of short chain fatty acids, followed by (iii) the evaluation of effects of a bell pepper extract enriched for the active component in a human proof of concept study. We identified rhamnogalacturonan-I (RG-I) as the nutricophore responsible for the immunostimulatory activity with substantial structural and functional equivalence between bell pepper (bp) and carrot (c). The in vitro studies showed that bpRG-I and cRG-I comprise similar immune- and microbiota modulatory potential and the human study demonstrated that bpRG-I was well tolerated and enhanced innate immune responsiveness in vivo. This is an important step towards testing the efficacy of RG-I from bpRG-I or cRG-I in an infection trial in humans.

Scope: We aimed to investigate and compare the effects of four types of pectins on dietary fiber (DF) fermentation, microbiota composition, and short chain fatty acid (SCFA) production throughout the large intestine in rats. Methods and results: Male Wistar rats were given diets supplemented with or without 3% structurally different pectins for 7 weeks. Different fermentation patterns of pectins and different location of fermentation of pectin and diet arabinoxylans (AXs) in the large intestine were observed. During cecal fermentation, sugar beet pectin significantly stimulated Lactobacillus (p

Oat beta-Glucans were studied for their immunological impact before and after enzymatic digestion in order to enhance the efficacy of oat beta-Glucans for application in functional foods. Oat beta-Glucan is reported to have minimal impact compared to its fungal counterpart in vitro. Digestion with endo-glucanase enhanced its efficacy towards stimulating MCP-1, RANTES, IL-8, and IL-4 production in human dendritic cells as compared to the nondigested beta-Glucan. This effect resulted from an enhanced activation of the Dectin-1 receptor. Our data suggest that the immune-stimulation was dependent on the beta-(1-3) linkages and the reduced particle size of digested beta-Glucans. Thus, we show that enzymatic pre-digestion of dietary fibres such as oat beta-Glucan enhances its impact on specific immune receptors. We also demonstrate that particle size and/or molecular weight of oat beta-Glucans and exposure of specific binding sites for the receptors might be important tools for designing efficacious functional feed and food additives. (C) 2015 Elsevier Ltd. All rights reserved.

A pectic polysaccharide from rose hip (RH) fruits has been obtained by extraction with 1% aqueous citric acid. It was found that the polysaccharide fraction mainly consisted of galacturonic acid (45.5%) next to galactose (5.5%) and arabinose (4.7%). RH pectin is having a relatively high degree of methylesterification (62%) and acetylation (10%) and consists of different molecular weight populations in the range of 10-100 kDa. Enzymatic fingerprinting was performed using a combination of pectin lyase (PL) and endo-polygalacturonase. Detailed information about the structure and level of galacturonic acid oligomers released was obtained using LC-HILIC-MS/ELSD and HPAEC. Predominantly, unsaturated and methyl-esterified oligomers (DP 3-5) were released indicating that high proportions of methylesterified 'PL degradable' areas were present within the pectin. The data revealed that homogalacturonan is the main building block of the extracted pectin and consists of long methylesterified/acetylated GalA sequences interspersed with small blocks of non-methyl-esterified GalA units.

Sugar beet pulp (SBP) and lemon peel wastes (LPW) were used to obtain two mixtures of pectic oligosaccharides (denoted as SBPOS and LPOS, respectively). Oligogalacturonides in LPOS showed a larger molecular weight, higher degree of methylation and lower degree of acetylation than the ones in SBPOS. The suitability of pectic oligosaccharides, pectins from SBP and LPW and commercial FOS for causing prebiotic effects were compared by in vitro fermentation and fluorescence in situ hybridization using human faecal inocula and eight different probes. The joint populations of bifidobacteria and lactobacilli increased from 19% up to 29%, 34% and 32% in cultures with LPOS, SBPOS and FOS, respectively. Faecalibacterium and Roseburia also increased their counts with all the substrates (especially with LPOS). The highest concentrations of organic acids were observed in media containing oligosaccharides. This work confirms that pectic oligosaccharides present better prebiotic properties than pectins, and similar or better than FOS.

Replacing dairy components from milk replacer (MR) with vegetable products has been previously associated with decreased protein and fat digestibility in milk-fed calves resulting in lower live weight gain. In this experiment, the major carbohydrate source in MR, lactose, was partly replaced with gelatinized corn starch (GCS) to determine the effect on protein and fat digestibility in milk-fed calves. In total, 16 male Holstein-Friesian calves received either MR with lactose as the carbohydrate source (control) or 18% GCS at the expense of lactose. In the adaptation period, calves were exposed to an increasing dose of GCS for 14 weeks. The indigestible marker cobalt ethylenediaminetetraacetic acid was incorporated into the MR for calculating apparent nutrient digestibility, whereas a pulse dose of chromium (Cr) chloride was fed with the last MR meal 4 h before slaughter as an indicator of passage rates. The calves were anesthetized and exsanguinated at 30 weeks of age. The small intestine was divided in three; small intestine 1 and 2 (SI1 and SI2, respectively) and the terminal ileum (last ~100 cm of small intestine) and samples of digesta were collected. Small intestinal digesta was analysed for α-amylase, lipase and trypsin activity. Digestibility of protein was determined for SI1, SI2, ileum and total tract, whereas digestibility of fat was determined for SI1, SI2 and total tract. Apparent protein digestibility in the small intestine did not differ between treatments but was higher in control calves at total tract level. Apparent crude fat digestibility tended to be increased in SI1 and SI2 for GCS calves, but no difference was found at total tract level. Activity of α-amylase in SI2 and lipase in both SI1 and SI2 was higher in GCS calves. Activity of trypsin tended to be higher in control calves and was higher in SI1 compared with SI2. A lower recovery of Cr in SI2 and a higher recovery of Cr in the large intestine suggest an increased rate of passage for GCS calves. Including 18% of GCS in a milk replacer at the expense of lactose increased passage rate and decreased apparent total tract protein digestibility. In the small intestine, protein digestion did not decrease when feeding GCS and fat digestion even tended to increase. Overall, effects on digestion might be levelled when partially replacing lactose with GCS, because starch digestion is lower than that of lactose but fat digestion may be slightly increased when feeding GCS.

Genes encoding pectic enzymes were introduced to wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing β-galactosidase (β-Gal-14 mutant) or rhamnogalacturonan lyase (RGL-18 mutant). After sequential extraction, β-Gal-14 hot buffer-soluble solids (HBSS) of pectin contained 54% less galactose than Karnico HBSS, representing shorter galactan side chains. The individual pectin populations of β-Gal-14 HBSS showed different modifications extended to the two sub-populations as obtained by ion-exchange chromatography. Compared to wild-type, RGL-18 HBSS contained 27% more galacturonic acid and 55% less Gal on fresh potato weight basis, which was due to the removal of galactan-rich rhamnogalacturonan I (RG-I) segments. All pectin populations of RGL-18 showed consistently low levels of RG-I segments. Transgenic modification showed side effects on the methyl-esterification and acetyl substitution of RGL-18 HBSS (DM = 53, DA = 21), but not of the β-Gal-14 HBSS in comparison to wild-type (DM = 29, DA = 54).

The aim of the current study was to investigate whether degradation of rapeseed meal (RSM) by a swine gut microbiota consortium was improved by modifying RSM by treatment with cellulase (CELL), two pectinases (PECT), or alkaline (ALK) compared to untreated RSM and to assess whether microbiota composition and activity changed. The predicted relative abundances of carbohydrate digestion and absorption, glycolysis, pentose phosphate pathway, and pyruvate metabolism were significantly increased upon CELL and ALK feeding, and CELL and ALK also exhibited increased total short-chain fatty acid (SCFA) production compared to CON.

Infants are known for their high inter-individual variability in gut microbiota composition in the first months of life. This poses difficulties when predicting the fate of non-digestible carbohydrates (NDCs) in the infant gut using in vitro models, since numerous experiments with individual faecal inocula of different infants are required. In this study an in vitro fermentation experiment was performed with galacto-oligosaccharides (GOS) using both individual and pooled faecal inocula of five two-week-old infants. It was shown that pooled faecal inoculum can be used to judge the fermentability of GOS, as a similar trend in total organic acid production and relative increase in Bifidobacterium was observed for the pooled faecal inoculum and four out of five individual faecal inocula. An additional fermentation using pooled faecal inoculum of five other infants of the same age confirmed these findings. Additionally, we provided evidence for both size- and isomer-specific fermentation of GOS by infant microbiota, which reveals the potential for the production of tailored NDC mixtures to meet the needs of specific subgroups of infants. Hence, the use of pooled faecal inocula contributes to increasing our knowledge on structure-specific effects of NDCs in infants efficiently.

Scope: The prevalence of metabolic-syndrome-related disease has strongly increased. Nutritional intervention strategies appear attractive, particularly with novel prebiotics. Isomalto/malto-polysaccharides (IMMPs) represent promising novel prebiotics that promote proliferation of beneficial bacteria in vitro. The present study investigates for the first time the in vivo effects of IMMP in mice. Methods and results: C57BL/6 wild-type mice received control or IMMP-containing (10%, w/w) diets for 3 weeks. IMMP leads to significantly more fecal bulk (+26%, p

Scope: This study characterize intestinal fermentation of isomalto/malto-polysaccharides (IMMPs), by monitoring degradation of IMMPs, production of short chain fatty acids (SCFAs), lactic acid, and succinic acid as well as enzyme activity and microbiota composition. Methods and results: IMMP-94 (94% α-(1→6) glycosidic linkages), IMMP-96, IMMP-27, and IMMP-dig27 (IMMP-27 after removal of digestible starch segments) are fermented batchwise in vitro using human fecal inoculum. Fermentation digesta samples are taken for analysis in time up till 48 h. The fermentation of α-(1→6) glycosidic linkages in IMMP-94, IMMP-96, and IMMP-dig27 starts after 12 h and finishes within 48 h. IMMP-27 fermentation starts directly after inoculation utilizing α-(1→4) linked glucosyl residues; however, the utilization of α-(1→6) linked glucoses is delayed and start only after the depletion of α-(1→4) linked glucose moieties. SCFAs are produced in high amounts with acetic acid and succinic acid being the major products next to propionic acid and butyric acid. The polysaccharide fraction is degraded into isomalto-oligosaccharides (IMOs) mainly by extracellular enzymes. The smaller IMOs are further degraded by cell-associated enzymes. Overall microbial diversity and the relative abundance of Bifidobacterium and Lactobacillus, significantly increase during the fermentation of IMMPs. Conclusion: IMMP containing segments of α-(1→6) linked glucose units are slowly fermentable fibers with prebiotic potential.

To study the variability in human milk oligosaccharide (HMO) composition of Chinese human milk over a 20-wk lactation period, HMO profiles of 30 mothers were analyzed using CE-LIF. This study showed that total HMO concentrations in Chinese human milk decreased significantly over a 20-wk lactation period, independent of the mother's SeLe status, although with individual variations. In addition, total acidic and neutral HMO concentrations in Chinese human milk decreased over lactation, and levels are driven by their mother's SeLe status. Analysis showed that total neutral fucosylated HMO concentrations in Chinese human milk were higher in the two secretor groups as compared to the nonsecretor group. On the basis of the total neutral fucosylated HMO concentrations in Chinese human milk, HMO profiles within the Se+Le+ group can be divided into two subgroups. HMOs that differed in level between Se+Le+ subgroups were 2′FL, DF-L, LNFP I, and F-LNO. HMO profiles in Dutch human milk also showed Se+Le+ subgroup division, with 2′FL, LNT, and F-LNO as the driving force.

Background: The proportion of starch disappearing from the small intestinal lumen is generally lower in ruminants than in monogastric animals, and there are indications that the starch digestion capacity in ruminants is limited.Objectives: Milk-fed calves were used to study the rate-limiting enzyme in starch hydrolysis and to quantify starch fermentation in ruminants.Methods: Forty male Holstein-Friesian calves were fed milk replacer containing either lactose (control) or 1 of 4 corn starch products. The following starch products differed in the enzyme ratios required for their complete hydrolysis to glucose: gelatinized starch [alpha-amylase and (iso)maltase], maltodextrin [(iso)maltase and alpha-amylase], maltodextrin with alpha-1,6-branching (isomaltase, maltase, and alpha-amylase), and maltose (maltase). In the adaptation period, calves were stepwise exposed to an increasing dose of the starch product for 14 wk to allow maximal adaptation of all enzyme systems involved. In the experimental period, apparent total tract and ileal starch product disappearance, total tract starch product fermentation, and alpha-amylase, maltase, and isomaltase activities were determined at 18% inclusion of the starch product.Results: Maltase and isomaltase activities in the brush border did not increase for any of the starch product treatments. Luminal alpha-amylase activity was lower in the proximal (3 9 +/- 3.2 and 2.7 +/- 1.7 U/mg Co for control and starch product calves, respectively) but greater in the distal small intestine of starch-fed calves than in control calves (0.0 +/- 0.0 and 6.4 +/- 1.5 U/mg Co for control and starch product calves, respectively; means +/- SEs for control and means +/- pooled SEMs for starch product treatments). Apparent deal (61.6% +/- 6.3%) and total tract (99.1% +/- 0.4%) starch product disappearance did not differ between starch product treatments, suggesting that maltase activity limits starch digestion in ruminants. Total tract starch product fermentation averaged 414 +/- 43 g/d, corresponding to 89% of intake, of which Half was fermented before the terminal ileum, regardless of starch product treatment.Conclusion: Fermentation, rather than enzymatic digestion, is the main reason for small intestinal starch disappearance in milk-fed calves.

A commercially available resistant maltodextrin (RMD), which is made out of corn starch by chemically and enzymatically modification, consists of atypical starch linkages and a low average molecular mass of 2 kDa. These characteristics of RMD make it rather complicated to identify with any chromatographic or masspectrometric method at this moment. A batch fermentation with human inocula over 48 h under anaerobic conditions showed that the degradation of the indigestible RMD is slow in comparison to dietary fibres like FOS. The RMD is only fermentable for ca. 60% and no specific oligosaccharide within the RMD population seems to be preferentially utilised by the microbiota present. Short chain fatty acid (SCFA) production, with acetic acid as the main SCFA, started after ca. 5 h of fermentation, while the increase of SCFA at 11 h was concurrent with the major degradation of the oligosaccharides of RMD. The microbiota composition as analysed by HITchip, revealed that RMD slightly stimulated the growth of bifidobacteria in comparison to the blank. The activity of carbohydrate degrading enzymes, produced by the microbiota during the in vitro fermentation, revealed that potential capacity to degrade typical starch linkages was high, but the RMD was only slowly and partly degradable during the incubation of 20 h.

A gene encoding a putative pectinesterase from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli. The resulting recombinant enzyme ( Bli PME) was purified and characterized as a pectin methylesterase. The enzyme showed maximum activity at pH 8.0 and 50 °C. Bli PME is able to release up to 100% of the methylesters from lime pectin (DM 34–76 → DM 0) and up to 73% of all methylesters from SBPs (DM 30–73 → DM 14). Bli PME efficiently de-methylesterifies lemon pectins and SBPs in a blockwise manner and is quite tolerant towards the acetyl groups present within the SBPs. Detailed analysis of the Bli PME-modified pectins using HILIC–MSn and the classical calcium reactivity measurement showed that the enzyme generates pectins with low methylesterification (lime and SBP) and high acetyl content (SBP) while creating blocks of nonmethylesterified galacturonic acid residues. The high activity of Bli PME towards highly methylesterified and acetylated pectins makes this novel esterase more efficient in removing methylesters from highly esterified beet pectin compared to other PMEs, e.g. Aspergillus niger PME.

Scope: We aimed to investigate the effects of three different soluble pectins on the digestion of other consumed carbohydrates, and the consequent alterations of microbiota composition and SCFA levels in the intestine of pigs.Methods and results: Piglets were fed a low-methyl esterified pectin enriched diet (LMP), a high-methyl esterified pectin enriched diet (HMP), a hydrothermal treated soybean meal enriched diet (aSBM) or a control diet (CONT). LMP significantly decreased the ileal digestibility of starch resulting in more starch fermentation in the proximal colon. In the ileum, low-methyl esterified pectin present was more efficiently fermented by the microbiota than high-methyl esterified pectin present which was mainly fermented by the microbiota in the proximal colon. Treated soybean meal was mainly fermented in the proximal colon and shifted the fermentation of cereal dietary fiber to more distal parts, resulting in high SCFA levels in the mid colon. LMP, HMP, and aSBM decreased the relative abundance of the genus Lactobacillus and increased that of Prevotella in the colon.Conclusion: The LMP, HMP, and aSBM, differently affected the digestion processes compared to the control diet and shaped the colonic microbiota from a Lactobacillus-dominating flora to a Prevotella-dominating community, with potential health-promoting effects.

Contains fulltext : 170614.pdf (Publisher’s version ) (Open Access) It has been shown in vitro that only specific dietary fibers contribute to immunity, but studies in vivo are not conclusive. Here, we investigated degree of polymerization (DP) dependent effects of beta2-->1-fructans on immunity via microbiota-dependent and -independent effects. To this end, conventional or germ-free mice received short- or long-chain beta2-->1-fructan for 5 days. Immune cell populations in the spleen, mesenteric lymph nodes (MLNs), and Peyer's patches (PPs) were analyzed with flow cytometry, genome-wide gene expression in the ileum was measured with microarray, and gut microbiota composition was analyzed with 16S rRNA sequencing of fecal samples. We found that beta2-->1-fructans modulated immunity by both microbiota and microbiota-independent effects. Moreover, effects were dependent on the chain-length of the beta2-->1-fructans type polymer. Both short- and long-chain beta2-->1-fructans enhanced T-helper 1 cells in PPs, whereas only short-chain beta2-->1-fructans increased regulatory T cells and CD11b-CD103- dendritic cells (DCs) in the MLN. A common feature after short- and long-chain beta2-->1-fructan treatment was enhanced 2-alpha-l-fucosyltransferase 2 expression and other IL-22-dependent genes in the ileum of conventional mice. These effects were not associated with shifts in gut microbiota composition, or altered production of short-chain fatty acids. Both short- and long-chain beta2-->1-fructans also induced immune effects in germ-free animals, demonstrating direct effect independent from the gut microbiota. Also, these effects were dependent on the chain-length of the beta2-->1-fructans. Short-chain beta2-->1-fructan induced lower CD80 expression by CD11b-CD103- DCs in PPs, whereas long-chain beta2-->1-fructan specifically modulated B cell responses in germ-free mice. In conclusion, support of immunity is determined by the chemical structure of beta2-->1-fructans and is partially microbiota independent.

This study simulates the fermentation process of barley ?-glucan and sugar beet pectin in the human colon and monitors the degradation products formed. Additionally, immune effects of the degradation products were investigated.Immunostimulatory activity of fermentation digesta was investigated using bone marrow derived dendritic cells (BMDCs) from toll-like receptor 2/4 (TLR2/4) knockout mice, which were unresponsive to microbe-associated molecular patterns. Cytokine responses were elicited to dietary fibers and not to the SCFA and microbiota. The fermentation digesta were analyzed for their SCFA profiles and glycan metabolites over time. During fermentation the amount of insoluble precipitating fibers increased and induced as well as soluble molecules of lower molecular mass greater amounts of cytokines in BMDCs than the parental fiber. Additionally, high amounts of cytokines can be attributed to soluble galactose-rich beet pectin molecules.The fermentation of the two fibers led to fiber-specific amounts of SCFA, glycosidic metabolites, and different immunomodulatory properties. BMDC from TLR2/4 knockout mice did not respond to the digest microbiota and SCFA, making it a useful approach to study temporal effects of fermentation on the immunomodulatory effects of fibers.? 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Screening of eight carbohydrate acetyl esterases for their activity towards xanthan resulted in the recognition of one active esterase. AXE3, a CAZy family CE1 acetyl xylan esterase originating from Myceliophthora thermophila C1, removed 31% of all acetyl groups present in xanthan after a 48 h incubation. AXE3 activity towards xanthan was only observed when xanthan molecules were in the disordered conformation. Optimal performance towards xanthan was observed at 53 °C in the complete absence of salt, a condition favouring the disordered conformation. AXE3-deacetylated xanthan was hydrolyzed using cellulases and analyzed for its repeating units using UPLC–HILIC–ELSD/ESI–MS. This showed that AXE3 specifically removes the acetyl groups positioned on the inner mannose and that acetyl groups positioned on the outer mannose are not removed at all. After a prolonged incubation at optimal conditions, 57% of all acetyl groups, representing 70% of all acetyl groups on the inner mannose units, were hydrolyzed.

The aim of this research was to study the in vitro fermentation of chicory root pulp (CRP) and ensiled CRP (ECRP) using human fecal inoculum. Analysis of carbohydrate levels in fermentation digests showed that 51% of all CRP carbohydrates were utilized after 24 h of fermentation. For ECRP, having the same cell wall polysaccharide composition as CRP, but with solubilization of 4 times more of CRP pectin due to ensiling, the fermentation was quicker than with CRP as 11% more carbohydrates were utilized within the first 12 h. The level of fiber utilization for ECRP after 24 h was increased by 8% compared to CRP. This effect on fiber utilization from ECRP seemed to arise from (i) increased levels of soluble pectin fibers (arabinan, homogalacturonan, and galactan) and (ii) ahypothesized more open structure of the remaining cell walls in ECRP, which was more accessible to degradation than the CRP cell wall network. KEYWORDS: chicory root pulp, ensiled chicory root pulp, insoluble

Enzymatic fingerprinting was applied to sugar beet pectins (SBPs) modified by either plant or fungal pectin methyl esterases and alkali catalyzed de-esterification to reveal the ester distributions over the pectin backbone. A simultaneous pectin lyase (PL) treatment to the commonly used endo-polygalacturonase (endo-PG) degradation showed to be effective in degrading both high and low methylesterified and/or acetylated homogalaturonan regions of SBP simultaneously. Using LC-HILIC–MS/ELSD, we studied in detail all the diagnostic oligomers present, enabling us to discriminate between differently prepared sugar beet pectins having various levels of methylesterification and acetylation. Furthermore, distinction between commercially extracted and de-esterified sugar beet pectin having different patterns of substitution was achieved by using novel descriptive pectin parameters. In addition to DBabs approach for nonmethylesterified sequences degradable by endo-PG, the “degree of hydrolysis” (DHPG) representing all partially saturated methylesterified and/or acetylated galacturonic acid (GalA) moieties was introduced as a new parameter. Consequently, the description DHPL has been introduced to quantify all esterified unsaturated GalA oligomers.

Supplementation of a Trichoderma longibrachiatum preparation to an industrial Aspergillus niger/Talaromyces emersonii enzyme mixture demonstrated synergy for the saccharification of corn silage water-unextractable solids (WUS). Sub-fractions of the crude T. longibrachiatum preparation obtained after chromatography were analyzed regarding their hydrolytic activity. An acetyl xylan esterase 1 [Axe1, carbohydrate esterase (CE) family 5]-enriched sub-fraction closely mimicked the hydrolytic gain as obtained by supplementation of the complete, crude enzyme mixture (increase of 50%, 62% and 29% for Xyl, Ara and Glc, respectively). The acetic acid released from model polysaccharides (WUS) and oligosaccharides [neutral (AcXOS) and acidic (AcUXOS) xylo-oligosaccharides] by Axe1 was two and up to six times higher compared to the acetic acid released by acetyl xylan esterase A (AxeA, CE 1). Characterization of Axe1 treated AcXOS and AcUXOS revealed deacetylation of oligosaccharides that were not deacetylated by AxeA or the A. niger/T. emersonii preparation.

Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear.We hypothesized that β2→1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether β2→1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 β2→1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by β2→1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain β2→1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain β2→1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with β2→1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that β2→1-fructans are specific ligands for TLR2. To conclude, β2→1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of β2→1-fructan-mediated health effects. © 2014 American Society for Nutrition.Chemicals/CAS: inulinase, 9025-67-6; phorbol 13 acetate 12 myristate, 16561-29-8; toll like receptor 2, 203811-81-8

Prebiotics are considered to have potential to reduce disturbances in the gut microbiota induced by antibiotics. Results in literature are, however, not consistent. The current in vitro study conducted in a fermentation screening platform allowed to unambiguously compare the impact of galacto-oligosaccharides (GOS) on adult gut microbiota composition and activity upon treatment with four antibiotics at two doses. The changes in relative abundance of bacteria upon antibiotic treatment and the growth of Bifidobacterium and Lactobacillus upon GOS addition were antibiotic and dose dependant. This conclusion explains discrepancies in literature and indicates that particular combinations of GOS antibiotic should be studied. The combination GOS–Amoxicillin was especially of interest as, after decrease in Bifidobacterium levels, a recovery of mainly Bifidobacterium longum was observed and could be correlated with specific degradation patterns of GOS. Next to different degradation profiles of individual GOS, an accumulation of monosaccharides and intermediate organic acids was observed in antibiotic-treated microbiota as compared to nontreated microbiota. This showed that although GOS were utilized and beneficial bacteria could grow in 3 of 4 antibiotics tested, the metabolic activity of an antibiotic-treated microbiota was still disturbed as compared to the nontreated microbiota.