Characterization of bacteriocin produced by Brevibacillus laterosporus Tk3 isolated from ‘tatwakhar’ – a flour prepared from seeds of Aesculus indica in remote areas of Himachal Pradesh India

Abstract

Bacteriocin producing strain Brevibacillus laterosporus TK3, was isolated from ‘Tatwakhar’- a flour prepared from seeds of Indian Horse Chestnut (Aesculus indica). Bacterial strain Brevibacillus laterosporus TK3 identified by morphological, biochemical techniques followed by 16S rRNA gene sequencing. The 16S rRNA sequence of bacteriocin producer was deposited in NCBI GenBank under accession no. KP861913.1. Bacteriocin of Brevibacillus laterosporus TK3 showed strong antagonistic activity against food spoiling/pathogenic bacteria viz. Listeria monocytogenes, Staphylococcus aureus and Clostridium perfringens. Bacteriocin production by Brevibacillus laterosporus TK3 was enhanced by optimizing production time, pH of the medium, inoculum size and incubation temperature. Maximum bacteriocin activity (6000 AU/ml) was recorded/obtained in basal salt medium of pH 5.5 with an inoculum size of 1.5 OD at 10% and incubation period of 24h at 35 °C. The bacteriocin was purified by single step gel exclusion chromatography. Molecular weight of active bacteriocin from Brevibacillus laterosporous TK3 was found to be 6 kDa according to SDS PAGE. The molecular mass of purified bacteriocin was confirmed as 5953.89 Da by MALDI TOF analysis. The purified bacteriocin was found desirable/suitable for food preservation as it showed wide spectrum of antimicrobial activity, resistance to high temperature, wide pH range and sensitivity to proteolytic enzymes thus, making it safe for human consumption.