Screening And Identification Of Bacteriocin-producing Lactic Acid Bacteria From Fermented Meat And Isolation,Purification And Characterization Of Bacteriocins

In recent years,some typical additives have been detected toxicity,the use of conventional preservatives in food has been re-evaluated,so the exploration and application of natural preservatives caused wide public concern.Bacterin is a natural preservative,which was protein or peptide substances synthesis by the ribosome.while in the medical field can also be used instead of antibiotics.In this study,lactic acid bacteria with antibacterial activity were screened from five kinds of fermented meat（Nanjing Ban duck,Hainan bacon,bacon,Xuanwei ham,Niugan）with Staphylococcus aureus and Escherichia coli as the indicator bacteria.The wells were screened and the selected strains were identified.At the same time,the bacteriocin produced by the strain was purified by column chromatography and high performance liquid phase,and the molecular weight of the purified bacteriocin was determined by SDS-PAGE.Finally,the physical and chemical properties of the obtained bacteriophage were tested and the mechanism of bacteriostasis was explored.The results of this study are as follows:1 96 strains of lactic acid bacteria were isolated and screened from 5 kinds of fermented meat products（Nanjing duck,Hainan fish,bacon,Xuanwei ham and cow）.The antibacterial strains were screened by Oxford cup test with The Gram-Staphylococcus aureus and Gram-negative bacteria Escherichia coli as the indicator bacteria,the results showed that 75 strains of bacteria have antibacterial effect,37 strains have significant bacteriostasis,but only 14 strains of bacteria not only inhibit Staphylococcus aureus and inhibit E.Coli.The fermentation characteristics test showed that Y2,Y9,Y19,Y24,B1,B18 not only meet the bacteriostatic conditions and meet the fermentation conditions.2 B18,Y19,Y24 were sensitive to protease and proved to have protein properties.The similarity between B18 and Enterococcus faecalis was 99.5%.The similarity between Y19 and Lactobacillus sakei was 99.4%.Y24 and Weissella hellenica were 99.9%.However,considering the bacteriostasis and acid production rate,Y19 was the target strain in the follow-up experiment.3 Y19 was purified by ammonium sulfate precipitation.The purified bacteriocin was dialyzed by dialysis bag.After purified by ion exchange DEAE-52,the peak 1 position was obtained when the elution buffer was citric acid phosphate buffer（pH 6..4）containing 0.1 mol/L NaCl.After purification with dextran G-50,four absorption peaks were obtained by antibacterial test.Peak 4 was the active peak.The moderately purified bacteriocin was obtained and the apparent molecular weight of the bacteriocin was determined to be 20 kDa by SDS-PAGE.The purity of bacteriocin was 97.5% after high performance liquid chromatography.4 Y19 produce bacteriocin stability for temperature after heating at 60℃,80℃,100℃,115℃,121 ℃for 10 min,20 min and 30 min respectively,heating at 121 ℃for 30 min after bacteriostatic activity only reduced by 14%.In pH 2 to 13 has good antibacterial activity,but under the condition of neutral and weak acid bacteriostatic activity is higher than the bacteriostatic activity of alkaline conditions.The addition of NaCl has obvious inhibition to the growth of bacteria,the best addition amount of 2-4%.The growth of bacteria was also affected by the addition of NaNO₂.The addition of NaNO₂ at 50 mg/mL had little effect on the growth of bacteria and and The addition of NaNO₂ at 100 mg/mL and 150 mg/mL had little effect on the growth of bacteria.Y19 bacteriocins produced for gram positive bacteria and negative bacteria have inhibition,widen the application range of bacteriocin.5 Differential scanning calorimetry were used to speculate the mechanism of bacteriostasis.Bacteriocin entered the cell through the cell membrane,thus affecting the DNA or RNA of the bacteria,so as to achieve bacteriostasis.This provides a strong basis for its use as a biological preservative.