216 CHARACTERIZATION OF BACTERIOCIN PRODUCER “LACTOBACILLUS BREVIS UN” AS POTENTIALPROBIOTIC STRAIN Neha Gautam* 1 and Nivedita Sharma 2 Address(es): Neha Gautam, 1 Department of Biotechnology and Microbiology, St. Bede’s College, Navbahar, Shimla (H.P.)-171002, India. 2 Microbiology Research Laboratory, Department of Basic Sciences, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan (H.P.) – 173 230, India. *Corresponding author: neha_mbg@yahoo.com ABSTRACT Keywords: Autoaggregation, Bacteriocin, Bile salt, Hydrophobicity, MRS, Probiotic INTRODUCTION Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (FAO, 2002).They are also called “friendly bacteria” or “good bacteria”can be used as complementary/alternative medicine and are not presently considered to be part of conventional medicine (Saikhon and Jairath 2010). In today’s world overuse of antibiotics and lesser consumption of traditional fermented food products by human population have disrupted the beneficial microflora of gastrointestinal tract. Due to lack of beneficial bacteria, GI track is prone to the attack of pathogenic organisms which may cause immune dysfunctions such as allergies, asthma, eczema, depression, bipolar disease, ADHD etc. Therefore, probiotics are recommended by doctors and, more frequently by nutritionists, after a course of antibiotics to re-stimulate immune functions (Green, 2010). Ingestion of probiotics improves human immune function. Most commonly used probiotics are species of Lactobacilli and Bifidobacteria. Most probiotic products consist of one or more species of bacteria from one or both of these types.L. brevis is widely seen in nature and has been found in fermented foods of both plant and animal origin as well as in human intestinal flora (Yakabeet al., 2009) and can therefore, be used as a probiotic. L. brevis has been patented several times. Though, several workers have claimed the effectiveness of L. brevisas a probiotic but many strains of L. brevis are still unrecognized, as different strains of a species exhibit differences in acid and bile resistance, ability to colonize the gastrointestinal (GI) tract, clinical efficacy, and the health benefits they confer. Therefore, in present investigation an effort has been made to place bacteriocin producing new isolate of “Dhulliachar” - L. brevis UN into a class of probiotics that could be utilized in dietary supplements. MATERIAL AND METHODS Isolation, screening and identification of lactic acid bacteria Lactic acid bacterial strain was isolated from “Dhulliachar”which is being consumed by the people of North East region of India. Isolation was carried out on De Man Ragaosa Sharpe (MRS) agar (Hi-media make) under anaerobic conditions by standard spread plate method (Aneja et al., 2003). Screening of isolates was done on the basis of morphological, physiological, biochemical characteristics and antagonistic potential (Gautam et al., 2014; Barefoot and Klaenhammer 1983). Colour, form, margin, elevation and texture of each isolated strain were noted down. Gram staining, catalase test, oxidase test, citrate utilization test, gas production from glucose, casein hydrolysis, H2S production and sugar fermentation were performed with isolated strains by standard microbiological techniques. Serious food borne pathogens/food spoilage bacteria i.e. Listeria monocytogenes MTCC 839 and Leuconostoc mesenteroides MTCC 107, Enterococcus faecalis MTCC 2729, Lactobacillus plantarum CRI, Bacillus cereus CRI, Clostridium perfringens MTCC 1739, Pectobacterium caratovorum MTCC 1428, Escherichia coli IGMC and Staphylococcus aureus IGMC, Aeromonas hydrophila IGMC were used to study antimicrobial potential of isolated strains. The test strains were procured from Institute of Microbial Technology (IMTECH), Chandigarh, India, Central Research Institute, Kasuali, India and Indira Gandhi Medical College (IGMC), Shimla, India. All these test strains were revived twice for 24 h at 37 0 C before performing the experiments, as all these indicators were preserved in 40 % glycerol at -20 o C.The bit disk method (Gautam and Sharma 2009; Kimura, 1998; Sharma et al., 2009) and well diffusion method (Del et al., 2000) were used to study antimicrobial potential of isolated strains. The gram positive, catalase-negative rods having maximum antagonistic activity were selected for further studies. Out of 12 isolates UN was selected. On the basis of 16S rRNA gene technique UN identified as Lactobacillus brevis. The sequences so obtained were submitted in National Centre for Biotechnology Information (NCBI) to get an accession numbers. L. brevis UN registered under the accession no. JX046150 (Gautam et al., 2014). Probiotic attributes Probiotic potential of L. brevis UN was studied by evaluating various factors viz., bacteriocin producing potential, lactic acid production, autoaggregation capacity, adhesion to solvents, bile salt tolerance and antibiotic sensitivity. Bacteriocin production L. brevis UN (10 %) was inoculated in MRS broth and incubated at 35 0 C, at 120 rpm for 30 h in orbital shaker. The isolate with 1.99 OD was centrifuged at 18,000 rpm for 20 min at 4 o C. The supernatant was filtered and collected in a sterilized test tube. This collected supernatant was neutralized to pH 7.0 (with sterilized 1N NaOH) and catalase was added (2 mg in 20 ml).Well diffusion method was repeated with this preparation against respective indicators. Further bacteriocin activity in cell free supernatants was determined by activity unit per milliliter (AU/ml). Activity unit per ml was determined as the inverse of the last Present study was carried out to classify a bacteriocin producer bacterial isolate, Lactobacillus brevis UN under the category of probiotics. L. brevisUN isolated from “Dhulliachar” which is a powdered mixture of seeds of Cucurbita pepo and Sesamum indicum consumed by the people of North East region of India as a condiment of food.Isolate was identified by conventional and molecular techniques. Isolated strain was tested for different probiotic attributes. Lactic acid production and auto aggregation capacity of L. brevis UN were 1.44 % and 40.54 % respectively. Isolate showed maximum adhesion of 42 % for xylene, survived at pH 1.0 after 180 minutes, exhibited some degree of bile salt tolerance, showed bacteriocin production potential and found sensitive to most of the antibiotics. Overall cumulative probiotic potential for L. brevis UN was 95.83 %. L. brevisUN fulfill the criteria of potential probiotic therefore, it can be used by food industries in probiotic preparation. ARTICLE INFO Received 23. 6. 2014 Revised 14. 5. 2015 Accepted 2. 9. 2015 Published 1. 12. 2015 Regular article doi: 10.15414/jmbfs.2015/16.5.3.216-220