Revealing Glycosylation Patterns in In Vitro-Produced Mucus Exposed to Pasteurized Mucus-Associated Intestinal Microbes by MALDI-TOF-MS and PGC-LC-MS/MS.

The human intestinal mucus layer protects against pathogenic microorganisms and harmful substances, whereas it also provides an important colonization niche for mutualistic microbes. The main functional components of mucus are heavily glycosylated proteins, called mucins. Mucins can be cleaved and utilized by intestinal microbes. The mechanisms between intestinal microbes and the regulation of mucin glycosylation are still poorly understood. In this study, in vitro mucus was produced by HT29-MTX-E12 cells under Semi-Wet interface with Mechanical Stimulation. Cells were exposed to pasteurized nonpathogenic bacteria Akkermansia muciniphila, Ruminococcus gnavus, and Bacteroides fragilis to evaluate influence on glycosylation patterns. Following an optimized protocol, O- and N-glycans were efficiently and reproducibly released, identified, and semiquantified using MALDI-TOF-MS and PGC-LC-MS/MS. Exposure of cells to bacteria demonstrated increased diversity of sialylated O-glycans and increased abundance of high mannose N-glycans in in vitro produced mucus. Furthermore, changes in glycan ratios were observed. It is speculated that bacterial components interact with the enzymatic processes in glycan production and that pasteurized bacteria influence glycosyltransferases or genes involved. These results highlight the influence of pasteurized bacteria on glycosylation patterns, stress the intrinsic relationship between glycosylation and microbiota, and show the potential of using in vitro produced mucus to study glycosylation behavior.

A novel "microbiota-host interaction model" to study the real-time effects of fermentation of non-digestible carbohydrate (NDCs) on gut barrier function.

In this study, an in vitro co-culture model using an electric cell-substrate impedance sensing system (ECIS) for testing the impact of real-time fermentation of non-digestible carbohydrates (NDCs) by the intestinal microbiota on gut barrier function was established. We applied Lactobacillus plantarum WCFS1 as a model intestinal bacterium and alginate-pectin as immobilization polymers as well as a source of NDCs to determine the impact of pectin fermentation on the barrier function of T84 gut epithelial cells. In the first design, L. plantarum WCFS1 was encapsulated in an alginate capsule followed by embedding in an agar layer to mimic a firm mucus layer that might be present in the colon. In this experimental design, the presence of the agar layer interfered with the transepithelial electrical resistance (TEER) measurement of T84 cells. Subsequently, we removed the agar layer and used encapsulated bacteria in an alginate gel and found that the TEER measurement was adequate. The encapsulation of the L. plantarum WCFS1 does avoid direct contact with cells. Also, the encapsulation system allows higher amounts of packing densities of L. plantarum WCFS1 in a limited space which can limit the oxygen concentration within the capsule and therefore create anaerobic conditions. To test this design, T84 cells were co-incubated with L. plantarum alginate-capsules supplemented with graded loads of fermentable pectin (0, 4, and 8 mg/ml per capsule) to investigate the effect of pectin fermentation on gut barrier function. We observed that as the pectin content in the L. plantarum capsules increased, pectin showed a gradually stronger protective effect on the TEER of the gut epithelium. This could partly be explained by enhanced SCFA production as both lactate and acetate were enhanced in L. plantarum containing alginate capsules with 8 mg/ml pectin. Overall, this newly designed in vitro co-culture model allows for studying the impact of bacteria-derived fermentation products but also for studying the direct effects of NDCs on gut barrier function in a relatively high-throughput way.

Structural Characterization of Disaccharides Using Cyclic Ion Mobility Spectrometry and Monosaccharide Standards.

To understand the mode of action of bioactive oligosaccharides, such as prebiotics, in-depth knowledge about all structural features, including monosaccharide composition, linkage type, and anomeric configuration, is necessary. Current analytical techniques provide limited information about structural features within complex mixtures unless preceded by extensive purification. In this study, we propose an approach employing cyclic ion mobility spectrometry (cIMS) for the in-depth characterization of oligosaccharides, here demonstrated for disaccharides. We were able to separate galactose and glucose anomers by exploiting the high ion mobility resolution of cIMS. Using the obtained monosaccharide mobilograms as references, we determined the composition and anomeric configuration of 4ß-galactobiose by studying the monosaccharide fragments generated by collision-induced dissociation (CID) before the ion mobility separation. Drift times and individual MS2 spectra of partially resolved reducing-end anomers of 4ß-galactobiose, 4ß-galactosylglucose (lactose), and 4ß-glucosylglucose (cellobiose) were obtained by deconvolution using CID fragmentation induced in the transfer region between the cIMS cell and TOF analyzer. The composition and anomeric configuration of the reducing end anomers of these disaccharides were identified using cIMS2 approaches, where first each anomer was isolated using cIMS and individually fragmented, and the monosaccharide fragments were again separated by cIMS for comparison with monosaccharide standards. With these results we demonstrate the promising application of cIMS for the structural characterization of isomeric oligosaccharides.

Prebiotic utilisation provides Lactiplantibacillus plantarum a competitive advantage in vitro, but is not reflected by an increased intestinal fitness.

Synbiotics combine the concepts of probiotics and prebiotics to synergistically enhance the health-associated effects of both components. Previously, we have shown that the intestinal persistence of inulin-utilizing L. plantarum Lp900 is significantly increased in rats fed an inulin-supplemented, high-calcium diet. Here we employed a competitive population dynamics approach to demonstrate that inulin and GOS can selectively enrich L. plantarum strains that utilize these substrates for growth during in vitro cultivation, but that such enrichment did not occur during intestinal transit in rats fed a GOS or inulin-supplemented diet. The intestinal persistence of all L. plantarum strains increased irrespective of their prebiotic utilization phenotype, which was dependent on the calcium level of the diet. Analysis of fecal microbiota and intestinal persistence decline rates indicated that prebiotic utilization capacity did not selectively stimulate intestinal persistence in prebiotic supplemented diets. Moreover, microbiota and organic acid profile analyses indicate that the prebiotic utilizing probiotic strains are vastly outcompeted by the endogenous prebiotic-utilizing microbiota, and that the collective enhanced persistence of all L. plantarum strains is most likely explained by their well-established tolerance to organic acids.

Assessing high-temperature and pressure extraction of bioactive water-soluble polysaccharides from passion fruit mesocarp.

The mesocarp (albedo) of passion fruit is considered a waste product but rich in soluble fibers, especially pectins. Biological activity and health benefits of pectins have recently emerged, especially in colorectal cancer and attenuating inflammation. Pectin conventional extraction often uses mineral acids, which can be hazardous to the environment, and alternatives can be costly. Here, we assessed a high-temperature and pressure method to extract pectin from the passion fruit albedo and evaluated the differences from the water-soluble fractions extracted. HPSEC, HPAEC, FTIR-ATR, and HSQC-NMR were performed to identify and confirm the highly methylated homogalacturonan structures. The heat-modified samples showed a decreased molecular size compared to the untreated sample. Colorectal cancer cell lines showed reduced viability after being treated with different doses of modified samples, with two of them, LW-MP3 and 4, showing the most potent effects. All samples were detected inside cells by immunofluorescence assay. It was observed that LW-MP3 and 4 upregulated the p53 protein, indicating cell-cycle arrest and the cleaved caspase-9 in one of the cell lines, with LW-MP4 enhancing cell death by apoptosis. Since the modified samples were composed of hydrolyzed homogalacturonans, those probably were the responsible structures for these anti-cancer effects.

Intraintestinal fermentation of fructo- and galacto-oligosaccharides and the fate of short-chain fatty acids in humans.

Consumption of fructo- (FOS) and galacto-oligosaccharides (GOS) has health benefits which have been linked in part to short-chain fatty acids (SCFA) production by the gut microbiota. However, detailed knowledge of this process in the human intestine is lacking. We aimed to determine the acute fermentation kinetics of a FOS:GOS mixture in healthy males using a naso-intestinal catheter for sampling directly in the ileum or colon. We studied the fate of SCFA as substrates for glucose and lipid metabolism by the host after infusion of 13C-SCFA. In the human distal ileum, no fermentation of FOS:GOS, nor SCFA production, or bacterial cross-feeding was observed. The relative composition of intestinal microbiota changed rapidly during the test day, which demonstrates the relevance of postprandial intestinal sampling to track acute responses of the microbial community toward interventions. SCFA were vividly taken up and metabolized by the host as shown by incorporation of 13C in various host metabolites.

The action of endo-xylanase and endo-glucanase on cereal cell wall polysaccharides and its implications for starch digestion kinetics in an in vitro poultry model.

Endo-xylanase and endo-glucanase are supplemented to poultry diets in order to improve nutrient digestion and non-starch polysaccharide (NSP) fermentation. Here, the action of these enzymes on alcohol insoluble solids (AIS) from wheat and maize grains as well as its implications for starch digestion in milled grains were evaluated in vitro, under conditions mimicking the poultry digestive tract. For wheat AIS, GH11 endo-xylanase depolymerized soluble arabinoxylan (AX) during the gizzard phase, and proceeded to release insoluble AX under small intestine conditions. At the end of the in vitro digestion (480 min), the endo-xylanase, combined with a GH7 endo-ß-1,4-glucanase, released 30.5 % of total AX and 18.1 % of total glucan in the form of arabinoxylo- and gluco-oligosaccharides, as detected by HPAEC-PAD and MALDI-TOF-MS. For maize AIS, the combined enzyme action released 2.2 % and 7.0 % of total AX and glucan, respectively. Analogous in vitro digestion experiments of whole grains demonstrated that the enzymatic release of oligomers coincided with altered grain microstructure, as examined by SEM. In the present study, cell wall hydrolysis did not affect in vitro starch digestion kinetics for cereal grains. This study contributes to understanding the action of feed enzymes on cereal NSP under conditions mimicking the poultry digestive tract.

Toxicological evaluation of a pumpkin-derived pectin preparation: in vitro genotoxicity studies and a 13-week oral toxicity study in Sprague-Dawley rats.

The safety of a rhamnogalacturonan-I-enriched pectin extract (G3P-01) from pumpkin (Cucurbita moschata var. Dickinson) was evaluated for use as an ingredient in food and dietary supplements. G3P-01 was tested in a battery of genetic toxicity studies including reverse mutagenicity and in vitro micronucleus assay. In addition, Sprague-Dawley rats were randomized and orally dosed with G3P-01 incorporated in animal diet at concentrations of 0, 9000, 18,000, and 36,000 ppm daily for 13-weeks (n=10/sex/group) in line with OECD guidelines (TG 408). The results of the in vitro bacterial reverse mutation assay and micronucleus assay in TK6 cells demonstrated a lack of genotoxicity. The 13-week oral toxicity study in Sprague-Dawley rats demonstrated that the test article, G3P-01 was well tolerated; there were no mortalities and no adverse effects on clinical, gross pathology, hematology, blood chemistry, and histological evaluation of the essential organs of the animals. The present study demonstrates that G3P-01 is non-genotoxic and is safe when ingested in diet at concentrations up to 36, 000 ppm. The subchronic no-observed-adverse-effect level (NOAEL) for G3P-01 was concluded to be 36,000 ppm, equivalent to 1,899 and 2,361 mg/kg/day for male and female rats respectively.

Dextran and levan exopolysaccharides from tempeh-associated lactic acid bacteria with bioactivity against enterotoxigenic Escherichia coli (ETEC).

Soybean tempeh contains bioactive carbohydrate that can reduce the severity of diarrhea by inhibiting enterotoxigenic Escherichia coli (ETEC) adhesion to mammalian epithelial cells. Lactic acid bacteria (LAB) are known to be present abundantly in soybean tempeh. Some LAB species can produce exopolysaccharides (EPS) with anti-adhesion bioactivity against ETEC but there has been no report of anti-adhesion bioactive EPS from tempeh-associated LAB. We isolated EPS-producing LAB from tempeh-related sources, identified them, unambiguously elucidated their EPS structure and assessed the bioactivity of their EPS against ETEC. Pediococcus pentosaceus TL, Leuconostoc mesenteroides WA and L. mesenteroides WN produced both dextran (α-1,6 linked glucan; >1000 kDa) and levan (ß-2,6 linked fructan; 650-760 kDa) in varying amounts and Leuconostoc citreum TR produced gel-forming α-1,6-mixed linkage dextran (829 kDa). All four isolates produced EPS that could adhere to ETEC cells and inhibit auto-aggregation of ETEC. EPS-PpTL, EPS-LmWA and EPS-LmWN were more bioactive towards pig-associated ETEC K88 while EPS-LcTR was more bioactive against human-associated ETEC H10407. Our finding is the first to report on the bioactivity of dextran against ETEC. Tempeh is a promising source of LAB isolates that can produce bioactive EPS against ETEC adhesion and aggregation.

Rice-derived arabinoxylan fibers are particle size-dependent inducers of trained immunity in a human macrophage-intestinal epithelial cell co-culture model.

Arabinoxylans have been identified for a wide range of purported health-promoting applications, primarily attributed to its immunomodulatory effects. Previously, we have reported the ability of arabinoxylans to induce non-specific memory in innate immune cells, commonly referred to as "trained innate immunity". In the present study, we investigated the effect of particle size on innate immune training and resilience in primary human macrophages as well as in a more physiologically relevant macrophage-intestinal epithelial cell co-culture model. We demonstrated that smaller (>45 & < 90 µm) compared to larger (>90 µm) particle size fractions of rice bran-derived arabinoxylan preparations have a higher enhancing effect on training and resilience in both models. Smaller particle size fractions elevated TNF-α production in primary macrophages and enhanced Dectin-1 receptor activation in reporter cell lines compared to larger particles. Responses were arabinoxylan source specific as only the rice-derived arabinoxylans showed these immune-supportive effects. This particle size-dependent induction of trained immunity was confirmed in the established co-culture model. These findings demonstrate the influence of particle size on the immunomodulatory potential of arabinoxylans, provide further insight into the structure-activity relationship, and offer new opportunities to optimize the immune-enhancing effects of these dietary fibers.

Presence of digestible starch impacts in vitro fermentation of resistant starch.

Starch is an important energy source for humans. Starch escaping digestion in the small intestine will transit to the colon to be fermented by gut microbes. Many gut microbes express α-amylases that can degrade soluble starch, but only a few are able to degrade intrinsic resistant starch (RS), which is insoluble and highly resistant to digestion (≥80% RS). We studied the in vitro fermentability of eight retrograded starches (RS-3 preparations) differing in rapidly digestible starch content (≥70%, 35-50%, ≤15%) by a pooled adult faecal inoculum and found that fermentability depends on the digestible starch fraction. Digestible starch was readily fermented yielding acetate and lactate, whereas resistant starch was fermented much slower generating acetate and butyrate. Primarily Bifidobacterium increased in relative abundance upon digestible starch fermentation, whereas resistant starch fermentation also increased relative abundance of Ruminococcus and Lachnospiraceae. The presence of small fractions of total digestible starch (±25%) within RS-3 preparations influenced the fermentation rate and microbiota composition, after which the resistant starch fraction was hardly fermented. By short-chain fatty acid quantification, we observed that six individual faecal inocula obtained from infants and adults were able to ferment digestible starch, whereas only one adult faecal inoculum was fermenting intrinsic RS-3. This suggests that, in contrast to digestible starch, intrinsic RS-3 is only fermentable when specific microbes are present. Our data illustrates that awareness is required for the presence of digestible starch during in vitro fermentation of resistant starch, since such digestible fraction might influence and overrule the evalution of the prebiotic potential of resistant starches.

Physicochemical, structural, and functional characterization of pectin extracted from quince and pomegranate peel: A comparative study.

Pectin's physicochemical, structural, and functional characteristics vary widely depending on the source of extraction. In this study, pectins were extracted from seedless quince and pomegranate peel, and their physicochemical, structural, and functional properties were investigated. A Box-Behnken Design with three factors and three levels was applied to optimize the pectin extraction yield from each matrix. As a result, the best extraction yields for quince pectin (QP) and pomegranate peel pectin (PPP) were 11.44 and 12.08 % (w/w), respectively. Both extracted pectins exhibit a linear structure, with the homogalacturonan domain dominating the rhamnogalacturonan I. Both pectins are highly methyl-esterified (DM > 69 %) with a higher degree of acetylation for PPP than QP, with 12 and 8 %, respectively. Unlike QP, PPP has a narrow, homogenous distribution and greater molecular weight (120 kDa). Regarding functionality, 1 g of QP could retain 4.92 g of water, and both pectin emulsions were more stable at room temperature than at 4 °C. When the concentration of QP is increased, rheological measurements demonstrate that it exhibits pseudoplastic behavior. Finally, QP can be used as a thickener, whereas PPP can be utilized as starting material for chemical changes to create multifunctional pectins.

The European Polysaccharide Network of Excellence (EPNOE) research roadmap 2040: Advanced strategies for exploiting the vast potential of polysaccharides as renewable bioresources.

Polysaccharides are among the most abundant bioresources on earth and consequently need to play a pivotal role when addressing existential scientific challenges like climate change and the shift from fossil-based to sustainable biobased materials. The Research Roadmap 2040 of the European Polysaccharide Network of Excellence (EPNOE) provides an expert's view on how future research and development strategies need to evolve to fully exploit the vast potential of polysaccharides as renewable bioresources. It is addressed to academic researchers, companies, as well as policymakers and covers five strategic areas that are of great importance in the context of polysaccharide related research: (I) Materials & Engineering, (II) Food & Nutrition, (III) Biomedical Applications, (IV) Chemistry, Biology & Physics, and (V) Skills & Education. Each section summarizes the state of research, identifies challenges that are currently faced, project achievements and developments that are expected in the upcoming 20 years, and finally provides outlines on how future research activities need to evolve.

Microbiota-dependent influence of prebiotics on the resilience of infant gut microbiota to amoxicillin/clavulanate perturbation in an in vitro colon model.

Antibiotic exposure disturbs the developing infant gut microbiota. The capacity of the gut microbiota to recover from this disturbance (resilience) depends on the type of antibiotic. In this study, infant gut microbiota was exposed to a combination of amoxicillin and clavulanate (amoxicillin/clavulanate) in an in vitro colon model (TIM-2) with fecal-derived microbiota from 1-month-old (1-M; a mixed-taxa community type) as well as 3-month-old (3-M; Bifidobacterium dominated community type) breastfed infants. We investigated the effect of two common infant prebiotics, 2'-fucosyllactose (2'-FL) or galacto-oligosaccharides (GOS), on the resilience of infant gut microbiota to amoxicillin/clavulanate-induced changes in microbiota composition and activity. Amoxicillin/clavulanate treatment decreased alpha diversity and induced a temporary shift of microbiota to a community dominated by enterobacteria. Moreover, antibiotic treatment increased succinate and lactate in both 1- and 3-M colon models, while decreasing the production of short-chain (SCFA) and branched-chain fatty acids (BFCA). The prebiotic effect on the microbiota recovery depended on the fermenting capacity of antibiotic-exposed microbiota. In the 1-M colon model, the supplementation of 2'-FL supported the recovery of microbiota and restored the production of propionate and butyrate. In the 3-M colon model, GOS supplementation supported the recovery of microbiota and increased the production of acetate and butyrate.

A wide diversity exists in pectin structure from thirteen apple cultivars.

To emphasize that differences in pectin structure among cultivars play a crucial role in the texture and quality of fruits and vegetables, the sugar content and methyl-esterification of pectin fractions from 13 apple cultivars was studied. Cell wall polysaccharides were isolated as alcohol-insoluble solids (AIS) and subsequently extracted to yield water-soluble solids (WSS) and chelating-soluble solids (ChSS). All fractions contained significant amounts of galacturonic acid, while sugar compositions varied between cultivars. AIS and WSS pectins showed a degree of methyl-esterification (DM) > 50 %, while ChSS pectins had either a medium (∼50 %) or low (<30 %) DM. Homogalacturonan as major structure was studied using enzymatic fingerprinting. Methyl-ester distribution of pectin was described by degrees of blockiness and -hydrolysis. Novel descriptive parameters were obtained by measuring the levels of methyl-esterified oligomers released by endo-PG (DBPGme) and PL (DBPLme). Pectin fractions differed in relative amounts of non-, moderately-, and highly methyl-esterified segments. WSS pectins were mostly lacking non-esterified GalA sequences, while ChSS pectins had medium DM and many non-methyl-esterified blocks or a low DM with many intermediate methyl-esterified GalA blocks. These findings will be of help to better understand physicochemical properties of apple and its products.

Agaricus subrufescens fermented rye affects the development of intestinal microbiota, local intestinal and innate immunity in suckling-to-nursery pigs.

BACKGROUND: Agaricus subrufescens is considered as one of the most important culinary-medicinal mushrooms around the world. It has been widely suggested to be used for the development of functional food ingredients to promote human health ascribed to the various properties (e.g., anti-inflammatory, antioxidant, and immunomodulatory activities). In this context, the interest in A. subrufescens based feed ingredients as alternatives for antibiotics has also been fuelled during an era of reduced/banned antibiotics use. This study aimed to investigate the effects of a fermented feed additive -rye overgrown with mycelium (ROM) of A. subrufescens-on pig intestinal microbiota, mucosal gene expression and local and systemic immunity during early life. Piglets received ROM or a tap water placebo (Ctrl) perorally every other day from day 2 after birth until 2 weeks post-weaning. Eight animals per treatment were euthanized and dissected on days 27, 44 and 70. RESULTS: The results showed ROM piglets had a lower inter-individual variation of faecal microbiota composition before weaning and a lower relative abundance of proteobacterial genera in jejunum (Undibacterium and Solobacterium) and caecum (Intestinibacter and Succinivibrionaceae_UCG_001) on day 70, as compared to Ctrl piglets. ROM supplementation also influenced gut mucosal gene expression in both ileum and caecum on day 44. In ileum, ROM pigs showed increased expression of TJP1/ZO1 but decreased expression of CLDN3, CLDN5 and MUC2 than Ctrl pigs. Genes involved in TLR signalling (e.g., TICAM2, IRAK4 and LY96) were more expressed but MYD88 and TOLLIP were less expressed in ROM pigs than Ctrl animals. NOS2 and HIF1A involved in redox signalling were either decreased or increased in ROM pigs, respectively. In caecum, differentially expressed genes between two groups were mainly shown as increased expression (e.g., MUC2, PDGFRB, TOLLIP, TNFAIP3 and MYD88) in ROM pigs. Moreover, ROM animals showed higher NK cell activation in blood and enhanced IL-10 production in ex vivo stimulated MLN cells before weaning. CONCLUSIONS: Collectively, these results suggest that ROM supplementation in early life modulates gut microbiota and (local) immune system development. Consequently, ROM supplementation may contribute to improving health of pigs during the weaning transition period and reducing antibiotics use.

Fucosylated Human Milk Oligosaccharides during the First 12 Postnatal Weeks Are Associated with Better Executive Functions in Toddlers.

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.

TLR 2/1 interaction of pectin depends on its chemical structure and conformation.

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.

The fate of insoluble arabinoxylan and lignin in broilers: Influence of cereal type and dietary enzymes.

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.

Combining galacto-oligosaccharides and 2'-fucosyllactose alters their fermentation kinetics by infant fecal microbiota and influences AhR-receptor dependent cytokine responses in immature dendritic cells.

Galacto-oligosaccharides (GOS) and 2'-fucosyllactose (2'-FL) are non-digestible carbohydrates (NDCs) that are often added to infant formula to replace the functionalities of human milk oligosaccharides (HMOs). It is not known if combining GOS and 2'-FL will affect their fermentation kinetics and subsequent immune-modulatory effects such as AhR-receptor stimulation. Here, we used an in vitro set-up for the fermentation of 2'-FL and GOS, either individually or combined, by fecal microbiota of 8-week-old infants. We found that GOS was fermented two times faster by the infant fecal microbiota when combined with 2'-FL, while the combination of GOS and 2'-FL did not result in a complete degradation of 2'-FL. Fermentation of both GOS and 2'-FL increased the relative abundance of Bifidobacterium, which coincided with the production of acetate and lactate. Digesta of the fermentations influenced dendritic cell cytokine secretion differently under normal conditions and in the presence of the AhR-receptor blocker CH223191. We show that, combining GOS and 2'-FL accelerates GOS fermentation by the infant fecal microbiota of 8-week-old infants. In addition, we show that the fermentation digesta of GOS and 2'-FL, either fermented individually or combined, can attenuate DC cytokine responses in a similar and in an AhR-receptor dependent way.