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Subject: Microbiology × Author: GAUTAM, NEHA × End date: 2013 × Start date: 2010 ×
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    ThesisItemOpen Access
    DETECTION AND CHARACTERIZATION OF GENE ENCODING BACTERIOCIN IN LACTIC ACID BACTERIA AND TO STUDY PRESERVATIVE POTENTIAL OF PURIFIED BACTERIOCIN
    (2013) GAUTAM, NEHA; SHARMA, NIVEDITA
    ABSTRACT The present investigation was carried out to isolate most efficient bacteriocin producing potential probiotic lactic acid bacteria from rare and unexplored food sources, their screening, identification, optimization to enhance the maximum bacteriocin producing potential, purification, characterization of purified bacteriocin on biochemical as well as on molecular level and application of bacteriocin as food biopreservatives. In addition, probiotic attributes of bacteriocin producing lactic acid bacteria and its role in cell mediated preservation was studied. In total 53 bacterial isolates were isolated from fermented/ non fermented food sources. out of all, 41 bacterial isolates which were catalase –ve were screened against ten spoilage causing food borne pathogens by bit/disc method. Among all, 8 isolates were further secreened to test bacteriocin production potential on the basis of their wide inhibitory spectrum against tested pathogens. Finally two isolates UN and G2 were selected for further studies being hyperbacteriocin producers, which were isolated from Dhulliachar and Gundruck respectively which are traditional fermented food products of North east India. Isolate UN was identified as Lactobacillus brevis while G2 identified as Lactobacillus spicheri by 16S r RNA gene technique and registered in NCBI under accession no. JX46150 and JX48191 respectively. Bacteriocin production was optimized through classical one variable at a time method. Both the isolates showed maximum bacteriocin production at early stationary phase, at pH4.0, temperature 350C with an inoculum size of 1.5 OD @ 10 %. Bacteriocins from both the isolates were purified by single step gel exclusion chromatography and their molecular weights were found to be 14 kDa and 43 kDa respectively. Activity units increased from 2×103 to 8×103 AU/ml in both cases. Purified bacteriocin titers of L. brevis UN increased by 87.5 % against L. monocytogenes, 66.6 % against S.aureus and 75 % against C. perfringens. In case of L. spicheri G2 bacteriocin titers increased by 75 % , against L. monocytogenes, S. aureus and 15 % against C. perfringens respectively. Purified bacteriocins of both the isolates were characterized by studying the effect of temperature, pH , proteolytic enzymes and storage stability on them. Both purified bacteriocins were maximum active against all the tested pathogens at neutral pH, both were found to have moderate thermostability and were sensitive to proteolytic enzymes trypsin and proteinase k. Molecular determinants for bacteriocin production in L. brevis UN showed that gene for bacteriocin production was plasmid bound. 1H-NMR revealed the unique combinations of different amino acids in biochemical structure of purified bacteriocin which has been reported for the first time in present study. Both isolates were tested for their probiotic attributes and were found to have sound probiotic potential.The use of both the strains in bacteriocin mediated preservation and cell mediated preservation have been found quite satisfactory. The purified bacteriocins produced by L. brevis UN and L.spicheri G2 showed resistance to the spoilage causing microorganisms in milk and apple juice. L. brevis UN and L. spicheri G2 were used successfully to prepare healthy and refreshing probiotics drinks viz. bioyogurt I, II, III and nutritionally rich cereal based probiotic product.