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Insoluble fiber degradation by supplemented enzymes was previously shown to improve fermentation in poultry, and has been further postulated to disrupt the cereal cell wall matrix, thus improving nutrient digestion. Here, we characterized insoluble feed-derived polysaccharides and lignin in digesta from broilers fed wheat-soybean and maize-soybean diets without or with xylanase/glucanase supplementation. Enzyme supplementation in wheat-soybean diet increased the yield of water-extractable arabinoxylan (AX) in the ileum. Still, most AX (> 73 %) remained insoluble across wheat-soybean and maize-soybean diets. Analysis of so-far largely ignored lignin demonstrated that a lignin-rich fiber fraction accumulated in the gizzard, while both insoluble AX and lignin reaching the ileum appeared to be excreted unfermented. More than 20 % of water-insoluble AX was extracted by 1 M NaOH and 11–20 % was sequentially extracted by 4 M NaOH, alongside other hemicelluloses, from ileal digesta and excreta across all diets. These findings showed that enzyme-supplementation did not impact AX extractability by alkali, under the current experimental conditions. It is, therefore, suggested that the degradation of insoluble AX by dietary xylanase in vivo mainly results in arabinoxylo-oligosaccharide release, which is not accompanied by a more loose cell wall architecture.

Citrus pectins have demonstrated health benefits through direct interaction with Toll-like receptor 2. Methyl-ester distribution patterns over the homogalacturonan were found to contribute to such immunomodulatory activity, therefore molecular interactions with TLR2 were studied. Molecular-docking analysis was performed using four GalA-heptamers, GalA7Me0, GalA7Me1,6, GalA7Me1,7 and GalA7Me2,5. The molecular relations were measured in various possible conformations. Furthermore, commercial citrus pectins were characterized by enzymatic fingerprinting using polygalacturonase and pectin-lyase to determine their methyl-ester distribution patterns. The response of 12 structurally different pectic polymers on TLR2 binding and the molecular docking with four pectic oligomers clearly demonstrated interactions with human-TLR2 in a structure-dependent way, where blocks of (non)methyl-esterified GalA were shown to inhibit TLR2/1 dimerization. Our results may be used to understand the immunomodulatory effects of certain pectins via TLR2. Knowledge of how pectins with certain methyl-ester distribution patterns bind to TLRs may lead to tailored pectins to prevent inflammation.

Contains fulltext : 290903.pdf (Publisher’s version ) (Open Access) Human milk oligosaccharides (HMOs) are one of the most abundant solid components in a mother's milk. Animal studies have confirmed a link between early life exposure to HMOs and better cognitive outcomes in the offspring. Human studies on HMOs and associations with later child cognition are scarce. In this preregistered longitudinal study, we investigated whether human milk 2'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, grouped fucosylated HMOs, and grouped sialylated HMOs, assessed during the first twelve postnatal weeks, are associated with better child executive functions at age three years. At infant age two, six, and twelve weeks, a sample of human milk was collected by mothers who were exclusively (n = 45) or partially breastfeeding (n = 18). HMO composition was analysed by use of porous graphitized carbon-ultra high-performance liquid chromatography-mass spectrometry. Executive functions were assessed at age three years with two executive function questionnaires independently filled in by mothers and their partners, and four behavioural tasks. Multiple regression analyses were performed in R. Results indicated that concentrations of 2'-fucosyllactose and grouped fucosylated HMOs were associated with better executive functions, while concentrations of grouped sialylated HMOs were associated with worse executive functions at age three years. Future studies on HMOs that sample frequently during the first months of life and experimental HMO administration studies in exclusively formula-fed infants can further reveal associations with child cognitive development and uncover potential causality and sensitive periods. 23 p.

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.

Pomelo peel, a by-product of fruit processing, is an excellent source of pectin. We aimed to study the effects of thermal treatments on the extraction and in vitro fermentation patterns of pectins from pomelo peel. We used subcritical water extraction and hot water extraction, with or without chelator (citric acid or EDTA-2Na) assistance to obtain the pectins. The pectin fractions were characterized by their constituent monosaccharide composition and molecular weight (Mw) distribution. The degree of methyl esterification of pomelo pectins was determined using FT-IR spectroscopy. We also compared the fermentation patterns of the pectin using an in vitro simulated human gut fermentation model, monitored Mw changes during fermentation by high performance size exclusion chromatography (HPSEC), and analyzed short-chain fatty acid (SCFA) using gas chromatograph. Our results showed that both chelator assistance and subcritical water treatment increased the pectin yield. Chelator-assisted extraction increased the pectin Mw and decreased the degree of methyl esterification (DM) whereas, subcritical water treatment had the opposite effect. Pectin with low rhamnogalacturonan-I (RG-I) and high homogalacturonan (HG) domain can be obtained from the used pomelo peels by citric acid-assisted extraction. Although gut microbiota intensively utilized uronic acid (UA) of pomelo during early fermentation stages, constituent neutral monosaccharides were consumed even faster than UA. Pomelo pectins enhanced the relative abundance of Bacteroides and Prevotella 9, and stimulated the production of SCFA, particularly acetic acid. Generally, pomelo pectin could be considered a new source for modulating gut microbiota towards a healthy pattern in functional foods.

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.

Dietary fibers such as fructans can protect the intestinal epithelial barrier integrity, but the mechanisms underlying this protection are not completely understood. We aimed to study the protective effect of β(2→1)-β(2→6) branched graminan-type fructans (GTFs) on gut epithelial barrier function that was disrupted by three different agents which impact the barrier function

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.

Galacto-oligosaccharides (GOS) are used in infant formula to replace the health effects of human milk oligosaccharides, which appear to be dependent upon the structure of the individual oligosaccharides present. However, a comprehensive overview of the structure-specific effects is still limited as a result of the high structural complexity of GOS. In this study, porous graphitic carbon (PGC) was used as the stationary phase during ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). This approach resulted in the recognition of more than 100 different GOS structures in one single run, including reducing and non-reducing GOS isomers. Using nuclear magnetic resonance-validated structures of GOS trisaccharides, we discovered MS fragmentation rules to distinguish reducing isomers with a mono- and disubstituted terminal glucose by UHPLC-PGC-MS. UHPLC-PGC-MS enabled effective recognition of structural features of individual GOS components in complex GOS preparations and during, e.g., biological conversion reactions. Hence, this study lays the groundwork for future research into structure-specific health effects of GOS.

The performance of pigs is affected by the rate of nutrient absorption in the gastrointestinal tract, which depends in turn strongly on the rate of stomach emptying. The 13C breath test provides a non-invasive diagnostic tool to measure gastric emptying patterns. Despite the wide acceptance of this method in human intervention studies, it has not found its way to the domain of animal sciences. In this study, we used the breath test to measure gastric emptying in young growing pigs using [1-13C] octanoic acid to trace digesta solids and [1-13C] glycine to study liquids. Pigs were fed a starch-rich diet, varying in starch source (isolated starch from barley, maize or high-amylose maize) or form (isolated barley starch, ground barley or extruded barley), after which 13CO2 enrichment was frequently measured during 11 h. Outliers in 13CO2 enrichment in the response curve of each pig were identified with a Cook 1/4s distance outlier test in combination with a leave-one-out analysis. Effects of experimental treatments on breath test parameters were tested using a GLM. In general, pigs were easy to train and the tailor-made mask allowed effortless sampling. Gastric emptying of all pigs followed a biphasic pattern, with a higher 13C recovery during the first peak. The first peak in gastric emptying of solids reached its maximum enrichment within 2 h after feeding in all cases. For digesta liquids, this peak was reached earlier for pigs fed ground barley (2.2 h after feeding), compared to pigs fed diets containing isolated starch (2.8 h after feeding). The second peak in gastric emptying of solids was reached later for pigs fed ground barley (5.9 h after feeding), compared with pigs fed extruded barley (4.5 h after feeding) and pigs fed diets containing isolated barley starch (4.8 h after feeding). In conclusion, the 13C breath test is a convenient, non-invasive tool to gain more insights into the gastric emptying pattern of pigs.

In this study, we systematically investigated the structural characterization and

Previously, arabinoxylan (AX) depolymerization by dietary endo-xylanase was observed in the broiler ileum, but released arabinoxylo-oligosaccharides (AXOS) were not characterized in detail. This study aimed at extracting and identifying AXOS released in vivo in broilers, in order to delineate the influence of endo-xylanase on AX utilization. Hereto, digesta from the gizzard, ileum, ceca and excreta of broilers fed a wheat-soybean diet without (Con) or with endo-xylanase supplementation (Enz) were assessed. Soluble AX content in the ileum was higher for Enz diet (26.9%) than for Con diet (18.8%), indicating a different type and amount of AX entering the ceca. Removal of maltodextrins and fructans enabled monitoring of AX depolymerization to AXOS (Enz diet) using HPSEC-RI and HPAEC-PAD. A recently developed HILIC-MSn methodology allowed AXOS (DP 4–10) identification in ileal digesta and excreta. Xylanase-induced AXOS formation coincided with decreased total tract AX recovery, which indicated improved AX hindgut utilization.

Arabinoxylans (AXs) display biological activities that depend on their chemical structures. To structurally characterize and distinguish AXs using a non-enzymatic approach, various TEMPO-oxidized AXs were partially acid-hydrolysed to obtain diagnostic oligosaccharides (OS). Arabinurono-xylo-oligomer alditols (AUXOS-A) with degree of polymerization 2–5, comprising one and two arabinuronic acid (AraA) substituents were identified in the UHPLC-PGC–MS profiles of three TEMPO-oxidized AXs, namely wheat (ox-WAX), partially-debranched WAX (ox-pD-WAX), and rye (ox-RAX). Characterization of these AUXOS-A highlighted that single-substitution of the Xyl unit preferably occurs at position O-3 for these samples, and that ox-WAX has both more single substituted and more double-substituted xylose residues in its backbone than the other AXs. Characteristic UHPLC-PGC–MS OS profiles, differing in OS abundance and composition, were obtained for each AX. Thus, partial acid-hydrolysis of TEMPO-oxidized AXs with analysis of the released OS by UHPLC-PGC-MS is a promising novel non-enzymatic approach to distinguish AXs and obtain insights into their structures.

Purpose of the study: A major challenge in cardiology remains in finding a therapy for cardiac fibrosis. Inhibition of galectin-3 with pectins attenuates fibrosis in animal models of heart failure. The purpose of this study is to identify pectins with the strongest galectin-3 inhibitory capacity. We evaluated the in vitro inhibitory capacity, identified potent pectins, and tested if this potency could be validated in a mouse model of myocardial fibrosis. Methods: Various pectin fractions were screened in vitro. Modified rhubarb pectin (EMRP) was identified as the most potent inhibitor of galectin-3 and compared to the well-known modified citrus pectin (MCP). Our findings were validated in a mouse model of myocardial fibrosis, which was induced by angiotensin II (Ang II) infusion. Results: Ang II infusion was associated with a 4–5-fold increase in fibrosis signal in the tissue of the left ventricle, compared to the control group (0•22±0•10 to 1•08±0•53%; P < 0•001). After treatment with rhubarb pectin, fibrosis was reduced by 57% vs. Ang II alone while this reduction was 30% with the well-known MCP (P = NS, P < 0•05). Treatment was associated with a reduced cardiac inflammatory response and preserved cardiac function. Conclusion: The galectin-3 inhibitor natural rhubarb pectin has a superior inhibitory capacity over established pectins, substantially attenuates cardiac fibrosis, and preserves cardiac function in vivo. Bioactive pectins are natural sources of galectin-3 inhibitors and may be helpful in the prevention of heart failure or other diseases characterized by fibrosis. Funding: Dr. Meijers is supported by the Mandema-Stipendium of the Junior Scientific Masterclass 2020-10, University Medical Center Groningen and by the Netherlands Heart Foundation (Dekkerbeurs 2021)Dr. de Boer is supported by the Netherlands Heart Foundation (CVON SHE-PREDICTS-HF, grant 2017-21; CVON RED-CVD, grant 2017-11; CVON PREDICT2, grant 2018-30; and CVON DOUBLE DOSE, grant 2020B005), by a grant from the leDucq Foundation (Cure PhosphoLambaN induced Cardiomyopathy (Cure-PLaN), and by a grant from the European Research Council (ERC CoG 818715, SECRETE-HF).

In this study, we systematically investigated the structural characterization and in vitro fermentation patterns of crude black mulberry fruit polysaccharides (BMPs), either extracted by water (BMP) or by enzymatic treatment. Different enzymatic treatments were pectinase-extracted (PE)-BMP, pectin lyase-extracted (PL)-BMP, cellulase-extracted (CE)-BMP, and compound enzymes-extracted (M)-BMP (pectinase:pectin lyase:cellulase = 1:1:1). Our results show that enzymatic treatment improved the polysaccharide yield and led to a different chemical composition and structure for the polysaccharides. Change dynamics during the in vitro fermentation indicated that BMPs could indeed be degraded and consumed by human fecal microbiota and that different BMPs showed different degrees of fermentability. In addition, BMPs stimulated the growth of Bacteroidetes and Firmicutes, inhibited the growth of Fusobacteria and Proteobacteria (except for CE-BMP), and induced the production of short-chain fatty acids (SCFAs). Furthermore, we found that BMP and PL-BMP exhibited better fermentability and prebiotic potential than the other polysaccharides.

The potential of travelling wave ion mobility spectroscopy in combination with collision induced dissociation tandem mass spectrometry (CID-TWIMS-MS/MS) to separate cereal-derived isomeric arabinoxylanoligosaccharides (A)XOS was investigated. Three trisaccharide, four tetrasaccharide, and four pentasaccharide (A)XOS isomers were analyzed by positive and negative ionization TWIMS-MS and CID-TWIMS-MS/MS. The triand pentasaccharide isomers were distinguishable by the ATDs of the precursor ions. The CID-TWIMS-MS/MS could separate most of the isomeric fragment ions produced from tetra- and pentasaccharide (A)XOS. Finally, the base peak mobility spectrum is introduced as a practical tool for (A)XOS fingerprinting.

Identification of arabinoxylo-oligosaccharides (AXOS) within complex mixtures is an ongoing analytical challenge. Here, we established a strategy based on hydrophilic interaction chromatography coupled to collision induced dissociation-mass spectrometry (HILIC-MSn) to identify a variety of enzyme-derived AXOS structures. Oligosaccharide reduction with sodium borohydride remarkably improved chromatographic separation of isomers, and improved the recognition of oligosaccharide ends in MS-fragmentation patterns. Localization of arabinosyl substituents was facilitated by decreased intensity of Z ions relative to corresponding Y ions, when fragmentation occurred in the vicinity of substituents. Interestingly, the same B fragment ions (MS2) from HILIC-separated AXOS isomers showed distinct MS3 spectral fingerprints, being diagnostic for the linkage type of arabinosyl substituents. HILIC-MSn identification of AXOS was strengthened by using specific and well-characterized arabinofuranosidases. The detailed characterization of AXOS isomers currently achieved can be applied for studying AXOS functionality in complex (biological) matrices. Overall, the present strategy contributes to the comprehensive carbohydrate sequencing.

An autoclave treatment at 121 °C on periodate-oxidized plant polysaccharides and mixes thereof was investigated for the release of oligosaccharides to obtain a generic polysaccharide depolymerization method for polysaccharides fingerprinting. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) analysis of the oligosaccharides released showed that each polysaccharide had a unique oligosaccharides profile, even the same type of polysaccharide derived from different sources and/or having different fine structures (e.g. class of (arabino)xylans, galactomannans, glucans, or pectic materials). Various polysaccharide classes present in a polysaccharide mix could be identified based on significantly different (p < 0.05) marker m/z values present in the mass spectrum. Principal component analysis and hierarchical cluster analysis of the obtained MALDI-TOF MS data highlighted the structural heterogeneity of the polysaccharides studied, and clustered polysaccharide samples with resembling oligosaccharide profiles. Our approach represents a step further towards a generic and accessible identification of plant polysaccharides individually or in a mixture.

Dietary fibers such as fructans can protect the intestinal epithelial barrier integrity, but the mechanisms underlying this protection are not completely understood. We aimed to study the protective effect of beta(2 -> 1)-beta(2 -> 6) branched graminan-type fructans (GTFs) on gut epithelial barrier function that was disrupted by three different agents which impact the barrier function via different cellular mechanisms. The effects of GTFs were compared with those of linear beta(2 -> 1) inulin-type fructans (ITFs). T84 intestinal epithelial monolayers were incubated with GTFs and ITFs. Afterwards, the monolayers were challenged with the barrier disruptors calcium ionophore A23187, 12-myristate 13-acetate (PMA) and deoxynivalenol (DON). Transepithelial resistance was measured with an electric cell-substrate impedance sensing system. All fructans studied prevented the barrier disruption induced by A23187. ITF II protected from the disruptive effects of PMA. However, none of the studied fructans influenced the disruption induced by DON. As a measure of disruption-induced inflammation, interleukin-8 (IL-8) production by the intestinal epithelium was determined by ELISA. The production of IL-8 induced by A23187 was decreased by all fructans, whereas IL-8 production induced by DON decreased only upon pre-treatment with ITF II. None of the studied fructans prevented PMA induced IL-8 production. GTFs just like ITFs can influence the barrier function and inflammatory processes in gut epithelial cells in a structure-dependent fashion. These distinct protective effects are dependent on the different signaling pathways that lead to gut barrier disruption.

Graminan-type fructans (GTFs) have demonstrated immune benefits. However, mechanisms underlying these benefits are unknown. We studied GTFs interaction with Toll-like receptors (TLRs), performed molecular docking and determined their impact on dendritic cells (DCs). Effects of GTFs were compared with those of inulin-type fructans (ITFs). Whereas ITFs only contained β(2→1)-linked fructans, GTFs showed higher complexity as it contains additional β(2→6)-linkages. GTFs activated NF-κB/AP-1 through MyD88 and TRIF pathways. GTFs stimulated TLR3, 7 and 9 while ITFs activated TLR2 and TLR4. GTFs strongly inhibited TLR2 and TLR4, while ITFs did not inhibit any TLR. Molecular docking demonstrated interactions of fructans with TLR2, 3, and 4 in a structure dependent fashion. Moreover, ITFs and GTFs attenuated pro-inflammatory cytokine production of stimulated DCs. These findings demonstrate immunomodulatory effects of GTFs via TLRs and attenuation of cytokine production in dendritic cells by GTFs and long-chain ITF.

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.

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

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.

In this study, we present an enzymatic fingerprinting method for the characterization of isomalto/malto-polysaccharides (IMMPs). IMMPs are produced by the modification of starch with the 4,6-α-glucanotransferase (GTFB) enzyme and consist of α-(1→4), α-(1→6) and α-(1→4,6) linked glucoses. Enzymes were used separately, simultaneously or in successive order to specifically degrade and/or reveal IMMP substructures. The enzymatic digests were subsequently analysed with HPSEC and HPAEC to reveal the chain length distribution (CLD) of different IMMP substructures. The presence of amylose in the substrate resulted in the formation of linear α-(1→6) linked glycosidic chains (13.5 kDa) in the former amylopectin fraction. The length of these chains indicates that GTFB transferase activity on amylopectin is more likely to elongate single amylopectin chains than to provide an even distribution. Enzymatic fingerprinting also revealed that the GTFB enzyme is capable of introducing large (20 kDa) linear α-(1→6) linked glycosidic chains in the α-glucan substrate.

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.

Detailed knowledge on the fate of dietary components inside the human intestinal tract is lacking. Access to this inner world of digestion is now possible through novel human gastrointestinal sampling capsules. Due to the novelty of such devices, no methodology has been published to stabilise and analyse the resulting samples. A complicating factor is that excretion of such capsules in faeces may take days, while degradation of the dietary components continues. Therefore a stabilising reagent should be pre-loaded in the capsule to ensure the measurement of a representative sample. Considering the small volume of recovered samples, analytical methods must be optimized to collect as many data as possible from little material. We present a complete workflow for stabilising and analysing the fermentation status of dietary fibres in such samples, including microbiota, fibre degradation, and short chain fatty acids. The final quenching reagent was designed based on safety and effectiveness to inhibit fructo- and galacto-oligosaccharides degradation and short chain fatty acids production by human ileostomy microbiota, and subsequently validated in faecal samples. The final composition of the stock quenching reagent is 175 mM Tris, 525 mM NaCl, 35 mM EDTA, 12% SDS, and 8 M urea at pH 8.5.