STUDIES ON SAFETY AND SHELF STABILITY OF BACILLUS PROBIOTICS

Abstract

Safety of probiotics has always been an area of concern. The present study evaluates the safety aspects of novel probiotic
Bacillus species as per the guidelines recommended jointly by Indian Council of Medical Research (ICMR) and
Department of Biotechnology (DBT). It also includes assessment of viability during storage since the number of viable
probiotic bacteria per gram of product or per serving should be mentioned on its label. Fifteen probiotic Bacillus isolates
were subjected to the study of haemolytic activity, testing for enterotoxin genes by PCR technique, antibiotic sensitivity
tests by Kirby Bauer method, and identification of the isolates by 16S rRNA and MALDI-TOF-MS studies. Five of these
cultures were found to be non-haemolytic which is a desirable probiotic characteristic. Three of these acid and bile
tolerant isolates were tested for antibiotic sensitivity. They were found to be sensitive to most antibiotics used, thus safe
for consumption, in terms of antibiotic resistance transfer to normal flora and pathogens. The three Bacillus isolates
tested negative for frequently encountered enterotoxin genes in Bacillus species-- nheA, nheB, bal, hblA, entFM and
bceT. As per the WHO/FAO joint working committee recommendations, minimum viable numbers of the probiotic
strains after six months storage at different temperatures was evaluated using the spread plate method. One of these
cultures, identified as Brevibacillus borstelensis was found to be the most stable. Its GenBank accession number is
MK156349. The culture has been identified to be a non-pathogenic strain. Thus, the novel prospective probiotic
bacterium proved to be safe for consumption.