BIOCHEMICAL AND GENETIC CHARACTERIZATION OF BACTERIOCIN PRODUCED BY Brevibacillus laterosporus TK3 AND TO EVALUATE ITS APPLICATION AS BIOPRESERVATIVE

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Date
2018-10
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UHF,NAUNI
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ABSTRACT The present investigation was carried out to identify the bacterial isolate TK3 (already isolated from Tatwakhar fermented flour) through biochemical and genetic tools, potential for bacteriocin production, optimization of growth parameters to enhance the maximum bacteriocin production, purification, characterization of purified bacteriocin on biochemical level and application of purified bacteriocin as food biopreservative. Isolate TK3 was identified as Brevibacillus laterosporus TK3 by 16S rRNA gene technique and registered in NCBI under accession no. KP861913.1. Maximum bacteriocin production was achieved by growing Brevibacillus laterosporus TK3 on basal salt media at pH 5.5, temperature 35oC with an inoculum size of 1.5 OD @ 10% at 24th h of incubation i.e. early stationary phase. Crude bacteriocin from Brevibacillus laterosporus TK3 was partially purified by 30% ammonium sulfate precipitation. Gel exclusion chromatography on sephadex G-75 was used for further purification of bacteriocin and its molecular weight was found to be 5.953 kDa by MALDI-TOF mass spectrometry. Activity units increased from 6×103 AU/ml to 1×104 AU/ml. Purified bacteriocin titers of Brevibacillus laterosporus TK3 increased by 36.7% against L. monocytogenes and 31.3% against S. aureus. The PMF searches with MASCOT search engine using swissProt database of MALDI TOF MS analysis showed matching of two values (1106.44 and 1154.21) with sakacin from Lactobacillus sakei GN having a score of 34 with protein sequence coverage of 31 % with some unmatched peptides though which confers to the novel nature of this bacteriocin. Bacteriocin was found to have very high thermostability with only slight decrease in activity even after heating up to 121oC, was most active within pH range of 5.0 to 8.0 with slight decrease in activity when the pH shifted to acidic and alkaline range and was sensitive to proteolytic enzymes trypsin and proteinase k. Bacteriocin has shown very high storage stability as it retained its 80% and 75% residual activity against L. monocytogenes and S. aureus respectively even after 12 months storage period. The use of bacteriocin produced by Brevibacillus laterosporus TK3 as food biopreservative to enhance the shelf life storage of chicken cubes, minced chicken and cheese has been found almost as effective as chemical preservatives in controlling the total bacterial count as well as containing the growth of deadly food borne pathogens viz. L. monocytogenes and S. aureus thus making this bacteriocin as a good alternative to replace synthetic food additives- sodium nitrite and sorbic acid for safer preservation of food products in food industry.
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