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Molecular genetic studies of pediocin-like bacteriocin in Pediococcus, Enterococcus and Lactobacillus sp.

Manjulata Devi, S. (2012) Molecular genetic studies of pediocin-like bacteriocin in Pediococcus, Enterococcus and Lactobacillus sp. PhD thesis, University of Mysore.

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Abstract

The aim of the study was to isolate intergeneric or interspecific lactic acid bacteria (LAB) producing the pediocin PA-1 like bacteriocin from different sources. The isolates obtained from vegetable sources displayed strong anti-listerial activity and were considered for further characterization. The pediocin specific gene PCR and dot-blot hybridization helped in the rapid detection of the pediocin PA-1 like bacteriocin. Further, the culture filtrate (CF) of the selected isolates was found to have class IIa bacteriocin properties, which include, stability at wide temperatures and pH, proteinaceous in nature and the anti-microbial peptide was found to be 4.6 KDa. The physiological, biochemical and molecular typing techniques like RAPD and RFLP, followed by 16S rDNA sequencing confirmed the taxonomical status of the LAB producing the putative pediocin PA-1 like bacteriocin. The isolates were identified as Streptococcus equinus Ac1 NCIM 5418, Pediococcus acidilactici NCIM 5419, Ped. pentosaceus NCIM 5420, Lactobacillus plantarum Acr2 and, three isolates of Enterococcus faecium NCIM 5421, NCIM 5422 and NCIM 5423, respectively. The bacteriocin production at different temperatures, pH and salt concentrations was studied to investigate the optimal condition for application of these isolates as a starter culture or as a biopreservative in either acidic or non-acidic foods. The isolates Pediococcus acidilactici NCIM 5419 and NCIM 5424, Ped. pentosaceus NCIM 5420, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 were selected to determine the genetic determinants of the bacteriocin encoded. The use of class IIa bacteriocin specific primers, plasmid curing, immunity assay and Southern hybridization with pedB as probe, revealed that all the isolates produced plasmid encoded pediocin PA-1 like bacteriocin. It was observed by PCR analysis that the selected isolates had a conserved operon arranged in pedABCD order. The amplification of the flanking regions was performed by inverse PCR and obtained a variation in the amplicon sizes ranging from 6 to 12 Kb. A variation in the amplicon size was observed in the downstream region in Lactobacillus plantarum Acr2, indicating a deletion or recombination event. The PCR assays, Southern hybridization, sequencing of amplified products, phylogenetic tree revealed the presence of mobile genetic elements (MGEs) like repB, ISLpl1, relaxases (mobA) and integrases (tyrosine recombinase) in the flanking regions of the operon. Hence, such MGEs are found to be responsible for the transfer of pediocin operon among LAB.The in situ production of the pediocin PA-1 like bacteriocin and the fermentation ability by Pediococcus acidilactici NCIM 5424, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 in soymilk was investigated. The isolates Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 showed ability of fermentation within 4 and 6 h respectively, and production of bacteriocin around 25600 AU/ml, unlike NCIM 5424. No significant difference was observed (P>0.05) in the viable count during storage at 4 oC for 15 days, when co-cultivated with Listeria monocytogenes ScottA. The pH, titratable acidity, pediocin activity, anti-oxidant property and sensory attributes for Enterococcus faecium NCIM 5423 were studied. An acceptable score for sensory features was achieved for the soymilk fermented by Ent. faecium NCIM 5423. The transfer of plasmid encoded pediocin PA-1 like bacteriocin from Pediococcus acidilactici NCIM 5424, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 to Ent. faecalis JH2-2 was determined. In the present study, the conjugal transfer under in vitro (filter mating method) and in situ (soymilk model) conditions were used. The in situ method gave more transfer frequency, ranging from 10-7 to 10-4 transconjugants per recipient cell. The isolate Enterococcus faecium NCIM 5423 was able to transfer the bacteriocin only under in situ conditions, whereas the isolate Enterococcus faecium 5423 transferred the bacteriocin under both the methods. No transconjugants were obtained between the mating pair Lact. plantarum Acr2 to Ent. faecalis JH2-2. Several molecular techniques were used to characterize the obtained transconjugants. The physiological conditions like pH and temperature were found to influence the production of bacteriocin in the transconjugants. The results of these experiments suggest the horizontal gene transfer and the natural spread of pediocin PA-1 like bacteriocin among LAB present in their close vicinity by means of conjugation. The dissemination of pediocin PA-1 like bacteriocin under in situ conditions is noteworthy, and such bacteriocin producers can be useful in the fermentation of dairy products and gives a scope for the development of starter cultures, and as a bio-preservative and bio-protective culture in the fermentation of dairy and meat products.

Item Type: Thesis (PhD)
Uncontrolled Keywords: lactic acid bacteria, pediocin, bacteriocin production
Subjects: 600 Technology > 08 Food technology > 09 Food Microbiology
Divisions: Food Microbiology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 03 Mar 2014 07:43
Last Modified: 03 Mar 2014 07:43
URI: http://ir.cftri.com/id/eprint/11354

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