Structure determination of the exopolysaccharide produced by Lactobacillus rhamnosus strains RW-9595M and R

Abstract

Exopolysaccharides (EPSs) were isolated and purified from Lactobacillusrhamnosus strains RW-9595M, which has been shown to possess cytokine-stimulating activity, and R grown under various fermentation conditions (carbon source, incubation temperature and duration). Identical 1H NMR spectra were obtained in all cases. Molecular masses were determined by gel permeation chromatography. The primary structure was elucidated using chemical and spectroscopic techniques. Organic acid, monosaccharide and absolute configuration analyses gave the following composition: pyruvate, 1; d-glucose, 2; d-galactose, 1; and l-rhamnose, 4. Methylation analysis indicated the presence of three residues of 3-linked rhamnose, and one residue each of 2,3-linked rhamnose, 2-linked glucose, 3-linked glucose and 4,6-linked galactose. The EPS was submitted to periodate oxidation followed by borohydride reduction. Monosaccharide analysis of the resulting polysaccharide gave the new composition: rhamnose, 4; and glucose, 1. Methylation analysis confirmed the loss of the 2-linked glucose and 4,6-linked galactose residues. On the basis of one- and two-dimensional 1H and 13C NMR data, the structure of the native EPS was consistent with the following heptasaccharide repeating unit: {3Rhaα-3Glcβ-3[Gal4,6(R)Pyα-2]Rhaα-3Rhaα-3Rhaα-2Glcα-}n where Rha corresponds to rhamnose (6-deoxymannose) and Py corresponds to pyruvate acetal. Complete 1H and 13C assignments are reported for the native and the corresponding pyruvate-hydrolysed polysaccharide. Electrospray MS and MS/MS data are given for the oligosaccharide produced by Smith degradation.