Detection of Heat Stable Bacteriocin from Lactobacillus acidophilus NCIM5426 by Liquid Chromatography/mass Spectrometry

Abstract

Bacteriocins from lactic acid bacteria (LAB) are natural antimicrobial peptides or proteins with interesting potential applications in food preservation and health care. The present study was aimed to isolate bacteriocinogenic LAB from dairy products, fermented food and environmental waste samples. One thirty five colonies of LAB were isolated and screened for bacteriocin production by agar overlay method. Among them, Lactobacillus acidophilus NCIM5426 isolated from home made paneer showed maximum zone of inhibition i.e 22-24mm in diameter against food borne ( Listeria monocytogenes , Staphylococcus aureus ) and human pathogens ( Escherichia coli , Salmonella typhi ). The antibacterial compound from the culture supernatant was found to be proteinaceous in nature and therefore, identified as bacteriocin. The bacteriocin of L. acidophilus NCIM5426 was found to be heat-stable (121°C for 15 min) and active over a wide pH range of 4.0-10.0. It showed stability (60%) for 30 days at room temperature (30-32°C). Addition of surfactants (EDTA, SDS, Hexadecyl trimethylamonium bromide) up to 1% to crude bacteriocin showed increase in antibacterial activity where as metal ions (Calcium Chloride, Zinc Sulphate and Mercuric Chloride) in low concentration (0.5-1mgL -1 ) decreased the activity. The bacteriocin was purified to its homogeneity by ammonium sulfate precipitation followed by gel filtration chromatography and HPLC. Molecular weight of bacteriocin was found to be 5.6 and 5.8 KDa by SDS page and LC/MS respectively. Production and activity of bacteriocin was significant (50%) even at higher salt (NaCl) concentration i.e 3%. Our present study demonstrates the possibility of using L. acidophilus NCIM5426 or its bacteriocin as a biopreservative in dairy industry.