Structure characterization of an exopolysaccharide produced by Bifidobacterium animalis RH

Abstract

An exopolysaccharide fraction (EPSb) produced by Bifidobacterium animalis RH isolated from the feces of centenarians was purified to illustrate its structure and conformational characterization. Results from Fourier-transform infrared spectrometry, gel permeation chromatography, high-pH anion-exchange chromatography with pulsed amperometric detection, periodate oxidation and Smith degradation, methylation and gas chromatography–mass spectrometry, and nuclear magnetic response analysis indicated that EPSb (M_w = 21.3 kDa) was composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), and mannose (Man) in a molar ratio of 0.4:0.3:1.6:0.8:1.2. This compound had a backbone of (1 → 3,4)-linked Man, (1 → 4)-linked Rha, (1 → 4)-linked Gal, and (1 → 4)-linked Glc. It was branched with Gal and terminated with Gal and Glc residues. The molecular structure of EPSb was observed via atomic force microscopy. EPSb showed spherical lumps and a ring-like network. Conformational analysis revealed the non-triple helical conformation of EPSb.

Introduction

The human large intestine contains a complex microflora. Bifidobacteria are often the predominant species in the gut microbiota of healthy humans. Aside from their wide range of therapeutic values in humans, these microorganisms have long been recognized as bacteria with probiotic, nutritive, and therapeutic properties. The health and nutritional benefits ascribed to bifidobacteria include maintenance of healthy intestinal microflora and integrity, improvement of lactose digestibility and tolerance, antitumorigenic activity, reduction of serum cholesterol levels, synthesis of B-complex vitamins, and absorption of calcium (Hoover, 1993).

Several lactic acid bacteria (LAB) produce exopolysaccharides (EPSs) that are secreted into the growth media. Since 1990, several structural studies and functionality of EPSs produced by different strains of LAB have been reported (Frengova, Simova, & Beshkova, 1997). The production of EPS is a relatively novel characteristic in the genus Bifidobacterium. Given that bifidobacteria are obligated anaerobes, certain precautions are required to prevent the toxic effects of oxygen when they are cultivated for industrial applications. Thus, compared with the genera Lactobacillus, Streptococcus, and Lactococcus, little is known on the structure and bioactivities of EPS produced by the genus Bifidobacterium. Structures of the cell wall polysaccharides from Bifidobacterium longum YIT4016 and Bifidobacterium catemulatum YIT4028 (Nagaoka et al., 1995, Nagaoka et al., 1996), as well as EPSs from Bifidobacterium longum have been reported (Robert, 1995, Andaloussi, 1995). They were all heteropolysaccharides composed of galactose (Gal), glucose (Glc), and rhamnose (Rha), among others.

Several lactobacilli and bifidobacteria used as probiotic bacteria produce EPSs. The health-promoting effect of EPS-producing strains is suggested to be related to the biological activities of these biopolymers. EPSs are beneficial to human health as probiotics because of their anti-tumor, anti-ulcer, immunomodulating, and cholesterol-lowering activities (Ruas-Madiedo, Hugenholtz, & Zoon, 2002). The molecular structure and conformation of polysaccharides are important in determining the bioactivities of these compounds (Ye et al., 2008, Tao et al., 2006, Qi et al., 2005).

New technologies, such as atomic force microscopy (AFM) and laser light scattering, have emerged as powerful tools for single-molecule force spectroscopy experiments and micro-cosmos observation. These techniques can be used to characterize the surface structures and molecular weight of aqueous samples (Chen et al., 2009). AFM has recently been used to study the molecular structure of some polysaccharides (Zhang et al., 2007, Iijima et al., 2007, Ikeda and Shishido, 2005). However, studies on EPSs produced by LAB, especially bifidobacterium, are lacking.

We isolated an EPS-producing strain B. animalis RH from the feces of centenarians in Bama (Guangxi, China), which was listed as one of the five officially certified villages of longevity by the International Society of Natural Medicine in 1991. To the best of our knowledge, only a few studies have focused on the structure, especially conformational analysis, of EPS from B. animalis. Accordingly, in the present study, we reported the chemical structure and conformational characterization of EPSb, one of the fractions from B. animalis RH.