Cellulolytic potential of probiotic Bacillus Subtilis AMS6 isolated from traditional fermented soybean (Churpi): An in-vitro study with regards to application as an animal feed additive.

The aim of the present study is to evaluate the probiotic attributes of Bacillus subtilis AMS6 isolated from fermented soybean (Churpi). This isolate exhibited tolerance to low pH (pH 2.0) and bile salt (0.3%), capability to autoaggregate and coaggregate. AMS6 also showed highest antibacterial activity against the pathogenic indicator strain Salmonella enterica typhimurium (MTCC 1252) and susceptibility towards different antibiotics tested. The isolate was effective in inhibiting the adherence of food borne pathogens to Caco-2 epithelial cell lines, and was also found to be non-hemolytic which further strengthen the candidature of the isolate as a potential probiotic. Further studies revealed B. subtilis AMS6 showed cellulolytic activity (0.54±0.05 filter paper units mL(-1)) at 37°C. The isolate was found to hydrolyze carboxymethyl cellulose, filter paper and maize (Zea mays) straw. The maize straw digestion was confirmed by scanning electron microscopy studies. The isolate was able to degrade filter paper within 96h of incubation. A full length cellulase gene of AMS6 was amplified using degenerate primers consisting of 1499 nucleotides. The ORF encoded for a protein of 499 amino acids residues with a predicted molecular mass of 55.04kDa. The amino acids sequence consisted of a glycosyl hydrolase family 5 domain at N-terminal; Glycosyl hydrolase catalytic core and a CBM-3 cellulose binding domain at its C terminal. The study suggests potential probiotic B. subtilis AMS6 as a promising candidate envisaging its application as an animal feed additive for enhanced fiber digestion and gut health of animal.

Cloning and extracellular expression of a raw starch digesting α-amylase (Blamy-I) and its application in bioethanol production from a non-conventional source of starch.

The aim of this study was to clone and efficiently express a raw starch-digesting α-amylase enzyme in the culture media and also to investigate the potential application of this recombinant enzyme in the digestion of non-conventional raw starch for bioethanol production. A raw starch digesting α-amylase gene isolated from Bacillus licheniformis strain AS08E was cloned and extracellularly expressed in E. coli cells using the native signal peptide. The mature recombinant α-amylase (Blamy-I) consisting of 483 amino acid residues was found to be homogenous with a mass of 55.3 kDa (by SDS-PAGE analysis) and a predicted pI of 6.05. Structural and functional analysis of Blamy-I revealed the presence of an extra Ca(2+) -binding region between the A and C domains responsible for higher thermostability of this enzyme. The statistical optimization of E. coli culture conditions resulted in an approximately eightfold increase in extracellular expression of Blamy-I as compared to its production under non-optimized conditions. Blamy-I demonstrated optimum enzyme activity at 80 °C and pH 10.0, and efficiently hydrolyzed raw starch isolated from a non-conventional, underutilized jack fruit seeds. Further utilization of this starch for bioethanol production using Blamy-I and Saccharomyces cerevisiae also proved to be highly promising.

In vitro evaluation of celluloytic Bacillus amyloliquefaciens AMS1 isolated from traditional fermented soybean (Churpi) as an animal probiotic.

A microorganism showing probiotic attributes and hydrolyzing carboxymethylcellulose was isolated from traditional fermented soybean (Churpi) and identified as Bacillus amyloliquefaciens by analysis of 16S rRNA gene sequence and named as B. amyloliquefaciens AMS1. The potentiality of this isolate as probiotic was investigated in vitro and it showed gastrointestinal transit tolerance, cell surface hydrophobicity, cell aggregation and antimicrobial activity. The isolate was found to be non-hemolytic which further strengthens its candidature as a potential probiotic. The maize straw digestion was confirmed by scanning electron microscopy studies. The isolate was able to degrade filter paper within 96 hours of incubation. This study explores the possibility of combining the cellulase degrading ability of a microbe with its probiotic attributes to enhance gut health of animal and digestibility of the feed.