Screening, Characterization Of Bacteriocin-Producing Lactobacillus Strains And Preliminary Study On The Bacteriocin Characteristics

Bacteriocins are antimicrobial compounds that are ribosomally synthesized by many different bacterial species including lactic acid bacteria (LAB). In this study, for producing broad inhibition spectra and superior stabilities against heat treatments and pH variations bacteriocins, the baceriocin-producing lactic acid bacteria were isolated and identified, the properties of their baceriocins were studied, and the medium and fermentation conditions for production of baceiocins were optimized. The main results were as follows:1. A total of300lactic acid bacteria strains were isolated from chinese traditionally fermented food (pickles, yoghourt and fermented sausage). Using the spot-on-the lawn test, the strain P72with highest antagonistic activity to the tested bacteria strains was selected from the300lactic acid bacteria strains.2. Based on its morphologic, culture, physiological and biochemical characteristics, and components of its16S rRNA sequence, the strain P72was identified as Lactobacillus plantarum.3. According to the results of the catalase test and proteae tests, the active substances produced by strain P72were shown the the proteinaceous nature and indicated as bacteriocin. The bacteriocin is superior stabilities against heat treatments (100℃,20min) and pH variations (pH2-9) and has high tolerance to1%(w/v) different surfactants (Tween20, Tween80and SDS) and NaCl.4. Fermentation medium and conditions were optimized by one-factor-at-a-time experiments and orthogonal design method. The optimal medium for the production of bateriocin was composed of the following factors:fructose20g/L, peptone5g/L, concentrated yeast extract5g/L, beef extract15g/L, K2HPO42g/L, MgSO4·7H2O0.58g/L, MnSO4·4H2O0.25g/L, CH3COONa5g/L,(NH4)2·HC6H5O72g/L, Tween801mL/L.The optimal fermentation conditions for the production of bateriocin were composed of the following factors:inoculum,2%(V/V); fermentation temperature,28℃; stationary incubation time,24h; initial pH,7.0.5. The bacteriocin was purified by50%saturation of ammonium sulfate precipitated and its molecular weight was shown smaller than6.5kDa by Tricine-SDS-PAGE.6. The result that the antimicrobial activity was not changed after plasmid curing of strain P72, indicated that the bacteriocin is encoded by chromosomal gene rather than by the plasmid gene of strain P72.