Characterizing microbiota-independent effects of oligosaccharides on intestinal epithelial cells: insight into the role of structure and size : Structure-activity relationships of non-digestible oligosaccharides.

PURPOSE: The direct effects of galacto-oligosaccharides (GOS), including Vivinal® GOS syrup (VGOS) and purified Vivinal® GOS (PGOS), on the epithelial integrity and corresponding interleukin-8 (IL-8/CXCL8) release were examined in a Caco-2 cell model for intestinal barrier dysfunction. To investigate structure-activity relationships, the effects of individual DP fractions of VGOS were evaluated. Moreover, the obtained results with GOS were compared with Caco-2 monolayers incubated with fructo-oligosaccharides (FOS) and inulin. METHODS: Caco-2 monolayers were pretreated (24 h) with or without specific oligosaccharides or DP fractions of VGOS (DP2 to DP6) before being exposed for 12 or 24 h to the fungal toxin deoxynivalenol (DON). Transepithelial electrical resistance and lucifer yellow permeability were measured to investigate barrier integrity. A calcium switch assay was used to study the reassembly of tight junction proteins. Release of CXCL8, a typical marker for inflammation, was quantified by ELISA. RESULTS: In comparison with PGOS, FOS and inulin, VGOS showed the most pronounced protective effect on the DON-induced impairment of the monolayer integrity, acceleration of the tight junction reassembly and the subsequent CXCL8 release. DP2 and DP3 in concentrations occurring in VGOS prevented the DON-induced epithelial barrier disruption, which could be related to their high prevalence in VGOS. However, no effects of the separate DP GOS fractions were observed on CXCL8 release. CONCLUSIONS: This comparative study demonstrates the direct, microbiota-independent effects of oligosaccharides on the intestinal barrier function and shows the differences between individual galacto- and fructo-oligosaccharides. This microbiota-independent effect of oligosaccharides depends on the oligosaccharide structure, DP length and concentration.

In Vitro Fermentation of Porcine Milk Oligosaccharides and Galacto-oligosaccharides Using Piglet Fecal Inoculum.

In this study, the in vitro fermentation by piglet fecal inoculum of galacto-oligosaccharides (GOS) and porcine milk oligosaccharides (PMOs) was investigated to identify possible preferences for individual oligosaccharide structures by piglet microbiota. First, acidic PMOs and GOS with degrees of polymerization 4-7 were depleted within 12 h of fermentation, whereas fucosylated and phosphorylated PMOs were partially resistant to fermentation. GOS structures containing ß1-3 and ß1-2 linkages were preferably fermented over GOS containing ß1-4 and ß1-6 linkages. Upon in vitro fermentation, acetate and butyrate were produced as the main organic acids. GOS fermentation by piglet inoculum showed a unique fermentation pattern with respect to preference of GOS size and organic acids production.

Milk Oligosaccharide Variation in Sow Milk and Milk Oligosaccharide Fermentation in Piglet Intestine.

Porcine milk oligosaccharides (PMOs) were analyzed in six colostrum and two mature milk samples from Dutch Landrace sows. In total, 35 PMOs were recognized of which 13 were new for the PMO literature: neutral HexNAc-Hex, ß4'-galactosyllactose, putative GalNAc(α/ß1-3)Gal(ß1-4)Glc, lacto-N-fucopentaose-II, lacto-N-tetraose, galactose substituted lacto-N-neohexaose, lacto-N-hexaose and difucosyl-lacto-N-hexaose, and acidic Neu5Ac(α2-6)GlcNAc(ß1-3)Gal(ß1-4)Glc, sialyllacto-N-tetraose-a and -b, Neu5Ac2-Hex3, and sialyllacto-N-fucopentaose-II. PMOs were analyzed using capillary electrophoresis with laser-induced florescence detection or mass spectrometry and using liquid chromatography with mass spectrometry. Interindividual variation regarding PMO presence and concentration was observed between porcine milks. Within a limited sample set, a 43% decrease of the major PMOs was found during a 1 w lactation period. Interestingly, while some PMOs decreased, some other PMOs increased in concentration. PMOs were also monitored in fecal samples of suckling piglets. In feces of 1-2 d old piglets, few intact PMOs were found, indicating considerable PMO fermentation at early stage of life.

Oligosaccharides in Urine, Blood, and Feces of Piglets Fed Milk Replacer Containing Galacto-oligosaccharides.

Human milk oligosaccharides (HMOs) are absorbed into the blood (about 1% of the HMO intake) and subsequently excreted in urine, where they may protect the infant from pathogen infection. As dietary galacto-oligosaccharides (GOS) have partial structural similarities with HMOs, this study investigated the presence of GOS and oligosaccharides originating from milk replacer in blood serum, urine, and cecal and fecal samples of piglets, as a model for human infants. Using liquid chromatography-mass spectrometry and capillary electrophoresis with fluorescence detection, oligosaccharides originating from piglet diet including 3'-sialyllactose and specific GOS ranging from degree of polymerization 3 to 6 were detected in blood serum and in urine of piglets. In blood serum, GOS levels ranged from 16 to 23 µg/mL, representing about 0.1% of the GOS daily intake. In urine, approximately 0.85 g of GOS/g of creatinine was found. Cecum digesta and feces contained low amounts of oligosaccharides, suggesting an extensive GOS intestinal fermentation in piglets.

Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic ß-N-acetylhexosaminidases.

This paper describes the discovery and characterization of two novel ß-N-acetylhexosaminidases HEX1 and HEX2, capable of catalyzing the synthesis of human milk oligosaccharides (HMO) backbone structures with fair yields using chitin oligomers as ß-N-acetylglucosamine (GlcNAc) donor. The enzyme-encoding genes were identified by functional screening of a soil-derived metagenomic library. The ß-N-acetylhexosaminidases were expressed in Escherichia coli with an N-terminal His6-tag and were purified by nickel affinity chromatography. The sequence similarities of the enzymes with their respective closest homologues are 59 % for HEX1 and 51 % for HEX2 on the protein level. Both ß-N-acetylhexosaminidases are classified into glycosyl hydrolase family 20 (GH 20) are able to hydrolyze para-nitrophenyl-ß-N-acetylglucosamine (pNP-GlcNAc) as well as para-nitrophenyl-ß-N-acetylgalactosamine (pNP-GalNAc) and exhibit pH optima of 8 and 6 for HEX1 and HEX2, respectively. The enzymes are able to hydrolyze N-acetylchitooligosaccharides with a degree of polymerization of two, three, and four. The major findings were, that HEX1 and HEX2 catalyze trans-glycosylation reactions with lactose as acceptor, giving rise to the human milk oligosaccharide precursor lacto-N-triose II (LNT2) with yields of 2 and 8 % based on the donor substrate. In total, trans-glycosylation reactions were tested with the disaccharide acceptors ß-lactose, sucrose, and maltose, as well as with the monosaccharides galactose and glucose resulting in the successful attachment of GlcNAc to the acceptor in all cases.