Screening Of High-Yield Bacteriocin Producing Strains Induced By Mutations And The Antimicrobial Mechanism And Applicationg Of Plantaricin

Microbiological contamination is the main reason of corruption in food preservation.The prevention and control of microbial contamination in food is a major research project in the field of food safety.In present,the most widely used food additives are chemical preservatives However,improper use of chemical additives in food will endanger human health Therefore,efficient and safe natural preservatives have wide application prospects in food preservation.Bacteriocin are small molecules peptides with antimicrobial activity synthesized by ribosome.The bacteriocin of lactic acid bacteria are considered with natural,non-toxic and green characteristic.They are good natural antiseptic.Our lab isolated L.plantarum JL-A65 from fermented vegetables.plantaricin JL-A65 producted by L.plantarum JL-A65has a broad inhibitory spectrum,good thermal stability and pH stability.but the bacteriocin production by wild strain is low,which limitfurther research and the application of bacteriocin.In this study,L.plantarum JL-A65 was used as the starting strain,the yield of plantaricin JL-A65 was increased by physical and chemical mutagenesis,and the mechanism of plantaricin JL-A65 was studied.Finally,plantaricin JL-A65 was applied to the preservation of fruit and vegetable juice.The main research results are as follows:1.Quantitative detection method for plantaricin JL-A65 was established.A quantitative detection method of plantaricin JL-A65 by HPLC was established.The standard curve equation was y=28.248x+6.4541 when the content was 0.05-0.5mg/m and the correlation coefficient was 0.9982.The standard curve equation is y=30.220x+4.7010 when the content was 1-5mg/m and the correlation coefficient reaches 0.9904.2.High-yield plantaricin JL-A65 strains were obtained by physical and chemical mutagenesis.Lactobacillus plantarum JL-A65 was used as the starting stain and mutagenized with atmospheric and room temperature plasma(ARTP)The optimal irradiation time of ARTP was 40 s Two mutants named A7-10 and A8-110 were obtained.The bacteriocin production by the mutants increased by 45.1%and 48.9%compared with the starting strain.Lactobacillus plantarum JL-A65 was used as the starting stain and mutagenized with methylnitro nitroguanidine(MNNG)and the optimum concentration of MNNG was 1.5 mg/mL.Two mutants named M2-58 and M7-111 were obtained.The bacteriocin production by the mutants increased by 46.6%and 31.3%compared with the starting strain.3.The mechanism of plantaricin JL-A65 on Pseudomonas fluorescens and Bacillus cereus was studied.The plantaricin can be found to be bactericidal to two kinds of bacteria.The fluorescence intensity of two indicator bacteria were increased after treatment with plantaricin JL-A65.It indicated that the cell membrane permeability was changed and fluorescent dyes can take through the cell membrane into the cell;The conductivity of the supernatant was increased after treatment with plantaricin JL-A65,which indicated that the ions leaked from the inside of the cell to the outside of the cell.The contents of the nucleic acid and protein in the bacteria were also increased after treatment with plantaricin JL-A65.These result indicated that the bacterial cell membrane permeability was damaged after the treatment with plantaricin JL-A65,eading to the leakage of the cell contents.4.The preservative effect of bacteriocin in fruit and vegetable juice was studied.Bacteriocin and potassium sorbate have synergistic role in the preservation of watermelon juice.The total number of bacteria,yeast and mold in control watermelon juice were higher than the national standard after storing in 2 days The bacteriocin and potassium sorbate were added to the watermelon juice in the ratio of 128:500ug/mL and it could prolong the shelf life from 2d to 6d.The pH of tomato juice was low,which had a certain inhibitory effect on the growth of microorganisms.The total number of bacteria,yeast and mold in control tomato juice were higher than the national standard after storing in 6 days Bacteriocin has a inhibitory effect on yeast and mould in tomato juice after pasteurized and it could prolong the shelf life to more than 8d.5.The antimicrobial effect of plantaricin JL-A65 on tomato juice with microorganism was studied.The bacteriostasis effect of bacteriocin and potassium sorbate was better than that of namamycin onEscherichia coli.When adding 102CFU/mL and 104CFU/mL Escherichia coli,bacteriocin has better bacteriostasis than potassium sorbate.potassium sorbate has better bacteriostatic effect than bacteriocin when adding 106CFU/mL Escherichia coli.As for antimicrobial effect on yeast and fungi,bacteriocin is weaker than potassium sorbate and nalamycin.The combination of bacteriocin and nalamycin has a good antimicrobial effect on bacteria,yeast and mould.