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Antibacterial peptides of Bacillus species active against food-borne pathogens.

Nithya, V. (2012) Antibacterial peptides of Bacillus species active against food-borne pathogens. PhD thesis, University of Mysore.

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Abstract

Bacillus species are usually found in a number of food products, in which they cooperate with other microorganisms during fermentation and releases valuable compounds including enzymes and antimicrobial substances. Antibacterial substances produced by food-grade Bacillus spp. can find potential biopreservatives in food systems. In this study, Bacillus spp. were selectively isolated from various food sources based on their antibacterial activity against Micrococcus luteus ATCC 9341. Among 78 such isolates, 25 potent isolates were selected for further studies, and were characterized and differentiated by phenological and molecular approaches. From these 25 isolates, seven isolates were taken which showed considerable diversity in their characteristic features and were identified as B. subtilis Ec1, B. thuringiensis Ik15, B. licheniformis Me1, B. cereus Ik11, Bacillus flexus Hk1, B. megaterium Pk12 and B. amyloliquefaciens Bk1. These cultures have been deposited in Microbial Culture Collection, National Centre for Cell Science, India. Most of the isolates displayed wide range of inhibitory activity against both Gram-positive and Gramnegative food-borne pathogenic bacteria. The antibacterial substances produced by these isolates were partially characterised. The culture, B. licheniformis Me1 produces an antibacterial substance which is proteinaceous in nature, stable over a wide temperature and pH, and exhibits wide range of inhibitory spectrum. Based on these results, B. licheniformis Me1 was selected for further studies. The antibacterial peptide (ABP) produced by B. licheniformis Me1 was purified from culture supernatant using ammonium sulphate, butanol extraction and HPLC. A single active peak was obtained after RP-HPLC which showed an m/z value of 279.28, determined by mass-spectroscopy. The tricine SDS-PAGE analysis of the purified ABP revealed that it is low molecular weight (MW) peptide of 3 to 3.4 kDa. The effect of ABP on food-borne pathogens, such as Listeria monocytogenes Scott A, Staphylococcus aureus FRI 722, Salmonella typhimurium MTCC 1251 and B. cereus F 4433 revealed that it has bactericidal effect. The scanning electron microscopy images of ABP-treated cells revealed complete disruption of the cells. In addition, the mode of action studies using FTIR, release of UV absorbing materials, cell reporter assays showed that the target of action of the ABP was on the cell wall of the pathogens. An economical production media (consisting of corn steep liquor (a byproduct for corn milling), yeast extract and NaCl) was developed for maximum production of ABP. The optimum conditions for higher yield of ABP (51200 AU/ml) in this formulated media was found to be temperature 37°C, pH 8, agitation speed 150 cycles/min and an incubation time of 24 h. The technological properties and the mode of action of the ABP indicate that it may belong to the cationic- membrane-acting lantibiotic group. In order to verify the suitability of B. licheniformis Me1 for food application the safety of the culture was evaluated in vitro and in vivo. The isolate was nonhaemolytic and non-phospholytic. The in vivo toxicological safety analysis as determined using suitable animal models revealed that the culture is safe, non toxic, non-irritant, and non-mutagenic and can be used for food industry application. The ABP produced by this culture was analysed for the biopreservative effectiveness. Two different methodologies were tested, direct application and food packaging film application. The ABP was found to be efficient in controlling the growth of L. monocytogenes Scott A in milk and other dairy products, such as cheese and paneer. Furthermore, the ABP was used to develop active packaging films (LDPE and cellulose) and their efficacy in controlling growth of pathogens in packaged food was also evaluated. Both the active (LDPE and cellulose) films with ABP showed inhibitory activity against common food-borne pathogens tested. The absorption and release of ABP to and from the cellulose film was found to be better as compared to LDPE film, since the binding of ABP in the former case was evaluated to be more stable. The dairy products, such as cheese and paneer samples inoculated with L. monocytogenes Scott A, and packed with ABP activated cellulose and LDPE films showed a reduction in the viable count of the indicator organism. In addition, a comparative evaluation of the in vitro probiotic properties of the Bacillus cultures was carried out and the culture B. licheniformis Me1 exhibited potential probiotic properties, such as acid and bile tolerance, bile salt hydrolase activity, hydrophobicity, antioxidant activity, etc. With this work, a novel food-grade isolate, B. licheniformis Me1 was obtained which can find potential application in food industry as a probiotic or as biopreservative.

Item Type: Thesis (PhD)
Uncontrolled Keywords: antibacterial peptide, food-borne pathogens
Subjects: 500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology > 02 Bacteriology
600 Technology > 08 Food technology > 10 Food Microorganisms
Divisions: Food Microbiology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 03 Mar 2014 09:56
Last Modified: 03 Mar 2014 09:56
URI: http://ir.cftri.com/id/eprint/11357

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