Immunomodulatory activity of non starch polysaccharides isolated from green gram (Vigna radiata).

Green gram, rich in dietary fiber is known to enhance the function of immune system. However information pertaining to the immunomodulatory potential of its non starch polysaccharides (NSPs) is scanty. Hence, five different NSPs were extracted successively using water (WSP), hot water (55 o C, HWSP), EDTA (0.5%, Pectins) and alkali (10%, Hemicellulose A and B) which varied in their arabinose to galactose ratio, sugar, protein, uronic acid contents, molecular weight distribution and immunomodulatory activity. Hemicellulose B was relatively rich in carbohydrate content (~95%) and also possessed potent immunomodulatory activity among the various NSPs. Hemicellulose B was further fractionated on DEAE-cellulose column into six different fractions by eluting step-wise with water, ammonium carbonate (0.1, 0.2, 0.3 M AC) and sodium hydroxide (0.1 and 0.2 M NaOH). 0.1 M AC eluted fraction was found to be the major one amounting to ~ 50% yield and showed relatively significant (p < 0.001) activity towards splenocyte proliferation and macrophage activation as compared with rest of the DEAE eluted fractions.

Purification and characterization of a novel α-D-glucosidase from Lactobacillus fermentum with unique substrate specificity towards resistant starch.

Resistant starch is not digestible in the small intestine and is fermented by lactic acid bacteria in the large intestine into short chain fatty acids, such as acetate, propionate and butyrate, which result in several health benefits in analogy with dietary fibre components. The mode and mechanism of resistant starch degradation by lactic acid bacteria is still not understood. In the present study, we have purified α-D-glucosidase from Lactobacillus fermentum NCDC 156 by employing three sequential steps i.e. ultra filtration, DEAE-cellulose and Sephadex G-100 chromatographies. It was found to be a monomeric protein (~50 kDa). The optimum pH and temperature of this enzyme were found to be 5.5 and 37°C, respectively. Under optimised conditions with p-nitrophenyl-D-glucopyranoside as the substrate, the enzyme exhibited a Km of 0.97 mM. Its activity was inhibited by Hg2+ and oxalic acid. N-terminal blocked purified enzyme was subjected to lysyl endopeptidase digestion and the resultant peptides were subjected to BLAST analysis to understand their homology with other α-D-glucosidases from lactobacillus species.

Antioxidant property of synbiotic combination of Lactobacillus sp. and wheat bran xylo-oligosaccharides.

Wheat bran water unextractable portion (WB-WUP) was subjected to xylanase treatment to obtain a mixture of xylo-oligosaccharides (XOS). XOS mixture was purified on charcoal-celite column and the individual oligosaccharides were separated on a Bio-Gel P-2 column. The sugar composition of the purified oligosaccharides was determined by GLC and their structure was deduced by ESI-MS and (1)H and (13)C NMR. The major oligosaccharides identified were xylobiose and xylotriose (consisting of arabinose). Five strains of lactobacilli (probiotics), XOS (prebiotics) and a combination of both (synbiotics) in milk (as medium) were monitored for antioxidant activity. DPPH radical scavenging activity (~70 %) as well as ferric reducing power (~80 mg/100 ml FeSO4eq) were significantly higher (p < 0.05) in all the synbiotic preparations compared to that of control. The present study indicated that the synbiotic preparations consisting of XOS and lactobacilli can be effectively used as dietary supplement.

Purification and characterization of ß-D-xylosidase from Lactobacillus brevis grown on xylo-oligosaccharides.

In the recent years there has been a growing interest in the use of oligosaccharides as prebiotics to modulate gut microbiota with an aim to improve the gut health. Though xylo-oligosaccharides (XOS) have been increasingly used as prebiotics, information pertaining to the enzymes used by lactobacilli to degrade these substrates is scanty. Present investigation reports the purification and characterization of ß-D-xylosidase from Lactobacillus brevis NCDC01 grown on XOS. Three sequential steps consisting of ultra-filtration, DEAE cellulose ion-exchange and Sephacryl S-100 gel filtration chromatographies were employed to purify the enzyme to apparent homogeneity and it was found to be monomeric on SDS-PAGE with an apparent molecular mass of ~58.0 kDa. The pH and temperature optima were 6.0 and 40 °C respectively. The enzyme remained stable over a pH range of 5.5-7.5 and up to 50 °C for 30 min. Under optimum pH and temperature with p-nitrophenyl ß-D-xylopyranoside as a substrate, the enzyme exhibited a K(m) of 0.87 mM. The enzyme does not require any metal ion for activity or stability but is completely inhibited by Hg(2+), Pb(2+), p-chloromercuribenzoate (PCMB), oxalic acid and citric acid. This is perhaps the first report on the purification and characterization of ß-D-xylosidase from Lactobacillus brevis NCDC01.

Prebiotic Activity of Purified Xylobiose Obtained from Ragi (Eleusine coracana, Indaf-15) Bran.

The role of prebiotics in improving human health has attracted global attention and the research is mostly focused on the strains belonging to the genera Bifidobacterium and Lactobacillus. Non-digestible oligosaccharides hold significant role in recent research due to their prebiotic nature. Soluble polysaccharides (SP, 14.4%), isolated from ragi bran consisted mainly of arabinose and xylose with minor quantities of rhamnose, mannose, galactose and glucose. Ragi bran SP subjected to purified endoxylanase (from 96 h ragi malt) treatment to obtain xylo-oligosaccharides which were further purified on Biogel P-2 followed by HPLC. The purified oligosaccharide yielded (RO-1; 17.9%) was identified as xylobiose by electrospray ionization mass spectrometry (282 + 23 = 305) and (1)HNMR. In vitro studies carried out using Bifidobacterium and Lactobacillus sp. proved the prebiotic nature of the crude xylo-oligosaccharides (XOs) and RO-1. Acetate was found to be the chief short chain fatty acid released during fermentation of both crude XOs and purified xylobiose and 24 h bacterial culture showed high xylanase activity (1020-1690 µU min(-1)).