Screening Of Lactic Acid Bacteria With Inhibitory Activity And The Antimicrobial Mechanisms Of Bacteriocin 63 And 90

Lactic acid bacteria,a group of microorganism,are isolate from many resources and generally regard as safety.In recent years,lactic acid bacteria are widely used in food and medicine processing industry because of the good fermentation ability and probiotic function.In dairy and meat products industry,lactic acid bacteria can accelerate the process of fermentation and improve the quality of the food.Moreover,they can inhibit the pollution of other spoilage microorganisms in the fermentation process.The inhibitory ability of lactic acid bacteria are usually potent against Gram-psitive bacteria,but weak activity against yeasts and moulds.The number of lactic acid bacteria with wide antimicrobial spectrum are very few.Therefore,it is meaningful to screen more lactic acid bacteria with wide antimicrobial spectrum to apply in many aspects in the future.The inhibitory activity of lactic acid bacteria are derived from the secretion of kinds of metabolites,such as organic acid,H₂O₂and some proteinaceous compounds(such as bacteriocins and cyclic dipeptides).The antimicrobial metabolites of different lactic acid bacteria may be different,so it is necessary to identify the antimicrobial characterization of lactic acid bacteria.At present,with the limitation of the fungicides use,researches has paid more attention on bacteriocins due to its novelty and safety.Bacteriocins exist in the fermentation broth of lactic acid bacteria.However,the fermentation broth usually includ many impurities and the content of bacteriocins are very low,so it is difficult to purify bacteriocins by using the traditional methods.Moreover,the purification process are also time-consuming and the yied of bacteriocins is too low to be used in the other researches.In recent years,heterologous expression is a good alternative method for obtaining bacteriocins,and Escherichia coli expression system has been widely used because of the advantages of low cost and high production of bacteriocins.The major antimicrobial mechanisms of bacteriocins are damaging the cell menmbrane of the bacteria by forming pore and path.Moreover,they also could change the permeability of cell membrane,casuing the leakage of intracellular substances and interfering the normal physiological metabolism of bacteria,leading the death of bacteria eventually.This study had been isolated lactic acid bacteria with inhibitory activity against Penicillium digitatum and Staphylococcus aureus from leaves,fruits and soil,investigated the inhibitory activity of cell-free supernatants of lactic acid bacteria through in vivo and in vitro tests and determined the antimicrobial mechanisms of bacteriocins which were produced by Lactobacillus sucicola JT03.The main results were as follows:(1)Sixty-eight lactic acid bacteria were isolated from all the samples.Sixteen strains were isolated from the surface of citrus fruit,six isolates were picked from citrus leaves,twenty-four strains were screened from the citrus orchards soil and the other twenty-two strains were isolated from peanut leaves.According to the in vitro test,ten strains had the good inhibitory activity against P.digitatum and S.aureus.They were identified by 16S r DNA,and the results indicated that the ten strains belonged to three species,Weissella paramesenteroides,L.sucicola and Pediococcus acidilactici,respectively.(2)In vitro,the cell-free supernatants of L.sucicola JT03,W.paramesenteroides JT13 and P.acidilactici JY03 could effectively inhibit the growth of P.digitatum,E.coli and S.aureus.Moreover,the cell-free supernatants of three lactic acid bacteria could reduce the incidence of citrus green mould and inhibit the spread of disease area.(3)After neutralization and catalase treating,the inhibitory activity of cell-free supernatants of L.sucicola JT03,W.paramesenteroides JT13 and P.acidilactici JY03 were still existed,but the inhibitory activity of the cell-free supernatants were great changed after pepsin,protease K and trypsin treating.In particular,the inhibitory activity of the three cell-free supernatants were completely lost after trypsin treating.Therefore,it was inferred that the major antimicrobial substances of the three cell-free supernatants maight be some proteinaceous compounds,such as bacteriocins.(4)Two bacteriocins(bacteriocin 63 and bacteriocin 90)were heterologously expressed in E.coli expression system successfully and they all expressed high inhibitory activity against many kinds of Gram-positive and Gram-negative bacteria.(5)The MIC of bacteriocin 63 and bacteriocin 90 were 10 AU/m L and 20 AU/m L,respectively.According to the time-killing curves and scanning electron microscope,the two bacteriocins could kill S.aureus in a short time by forming hole in cell member and changing the morphology of the cell.In conclusion,this study had been picked out three lactic acid bacteria with high inhibitory activity and investigated the inhibitory characterization of cell-free supernatants of the three lactic acid bacteria by in vivo and in vitro tests.Moreover,the antimicrobial mechanisms of bactriocins produced by L.sucicola JT03 were also determined.