Preparative scale purification of fucosyl-N-acetylglucosamine disaccharides and their evaluation as potential prebiotics and antiadhesins

Abstract

Fucosyl-N-acetylglucosamine disaccharides are important core structures that form part of human mucosal and milk glyco-complexes. We have previously shown that AlfB and AlfC α-L-fucosidases from Lactobacillus casei are able to synthesize fucosyl-α-1,3--N-acetylglucosamine (Fuc-α1,3-GlcNAc) and fucosyl-α-1,6-N-acetylglucosamine (Fuc-α1,6-GlcNAc), respectively, in transglycosylation reactions. Here, these reactions were performed in a semipreparative scale, and the produced disaccharides were purified. The maximum yields obtained of Fuc-α1,3-GlcNAc and Fuc-α1,6-GlcNAc were 4.2 and 9.3 g/l, respectively. The purified fucosyl-disaccharides were then analyzed for their prebiotic effect in vitro using strains from the Lactobacillus casei/paracasei/rhamnosus group and from Bifidobacterium species. The results revealed that 6 out of 11 L. casei strains and 2 out of 6 L. rhamnosus strains tested were able to ferment Fuc-α1,3-GlcNAc, and L. casei BL87 and L. rhamnosus BL327 strains were also able to ferment Fuc-α1,6-GlcNAc. DNA hybridization experiments suggested that the metabolism of Fuc-α1,3-GlcNAc in those strains relies in an α-L-fucosidase homologous to AlfB. Bifidobacterium breve and Bibidobacterium pseudocatenolatum species also metabolized Fuc-α1,3-GlcNAc. Notably, L-fucose was excreted from all the Lactobacillus and Bifidobacterium strains fermenting fucosyl-disaccharides, except from strains L. rhamnosus BL358 and BL377, indicating that in these latest strains, L-fucose was catabolized. The fucosyl-disaccharides were also tested for their inhibitory potential of pathogen adhesion to human colon adenocarcinoma epithelial (HT29) cell line. Enteropathogenic Escherichia coli (EPEC) strains isolated from infantile gastroenteritis were used, and the results showed that both fucosyl-disaccharides inhibited adhesion to different extents of certain EPEC strains to HT29 cells in tissue culture.

Unique Microbial Catabolic Pathway for the Human Core N -Glycan Constituent Fucosyl-α-1,6- N -Acetylglucosamine-Asparagine

“…Analytical and semipreparative HPLC analyses. Transfucosylation reaction products were purified by high-performance liquid chromatography (HPLC) using a preparative Rezex RCM-monosaccharide column (Phenomenex) as previously described (65). Appropriate fractions were pooled, concentrated, and analyzed by using an analytical Rezex RSO-oligosaccharide column (Phenomenex) in the case of 6=FN-Asn synthesis and an analytical Rezex RCM-monosaccharide column (Phenomenex) in the case of N2F N-glycan, 6=FucGal, and 6=FucGlc synthesis.…”

Human milk and mucosal lacto- and galacto-N-biose synthesis by transgalactosylation and their prebiotic potential in Lactobacillus species

“…Positive probiotic effects of Lactobacillus species have been shown on immune system stimulation, atopic disease, and several types of diarrhea in children among others (Ashraf and Shah 2014;Kalliomaki et al 2003;Pedone et al 2000;Rodriguez-Diaz and Monedero 2013;Szajewska et al 2013). We have previously demonstrated that fucosyl-Nacetylglucosamine disaccharides, that form part of HMOs and also part of glyco-complexes from the mucosa, were fermented by strains of L. casei, L. rhamnosus, B. breve, and B. pseudocatenulatum (Becerra et al 2015), suggesting a potential prebiotic effect for those carbohydrates. Additionally, we have characterized in L. casei strain BL23 the gnb operon (gnbREFGBCDA) that is involved in the transport and catabolism of LNB and GNB (Bidart et al 2014).…”

“…The transgalactosylation reaction products were analyzed and purified by HPLC (Jasco PU2080Plus system) using analytical and preparative Rezex RCM-Monosaccharide columns (Phenomenex, Torrance, CA, USA), respectively, as described previously (Becerra et al 2015). Di-and monosaccharides were confirmed by comparison of their retention times with those of standards (LNB, GNB, galactose, GlcNAc, and GalNAc).…”

“…The Lactobacillus strains were grown at 37°C under static conditions in MRS basal medium as previously described (Becerra et al 2015). Lacto-N-biose (LNB) and galacto-Nbiose (GNB) utilization was determined by measuring growth in 96-well microtiter plates in a POLARstar Omega microplate reader (BMG Labtech, Offenburg, Germany) at 37°C under static conditions.…”

Metabolismo y síntesis de oligosacáridos de la leche humana mediante la utilización de enzimas glicosil hidrolasas de Lactobacillus casei

“…los distintos disacáridos que forman parte de oligosacáridos más complejos. Se ha analizado el potencial inmunomodulador de la LNB y GNB sintetizadas aquí, y de los fucosil-N-acetilglucosamina disacáridos, Fuc1-3 y Fuc1-6, sintetizados previamente en nuestro laboratorio (Becerra et al, 2015;Rodriguez-Diaz et al, 2013). Para los ensayos de análisis del potencial inmunomodulador se utilizaron dos modelos descritos en la sección de materiales y métodos, el de incubación de los disacáridos con la línea celular de epitelio intestinal HT-29 en co-cultivo con células mononucleares de sangre periférica (PBMC) en placas transwell, y el de incubación directa de los disacáridos con PBMC.…”