Mechanism And Application Of Bacteriocin Produced By Enterococcus Faecium BZ2 Against Listeria Monocytogenes

Strain BZ2 is a strain screened by the research group from yogurt milk in the early stage,which can produce bacteriocin with broad-spectrum antibacterial ability.Bacteriocin AB-2 especially shows strong antibacterial activity against Listeria monocytogenes,has potential value as a natural food preservative.Therefore,in this study,the strain BZ2 was firstly identified,and the growth curves of strain BZ2 and Listeria monocytogenes were established.Secondly,the antibacterial activity and antibacterial mechanism of the crude extract of bacteriocin AB-2 were studied with Listeria monocytogenes as the indicator bacteria,and its antibacterial effect on Listeria monocytogenes in orange juice was determined.Its findings are as follows:The strain BZ2 was identified as Enterococcus faecium by 16S rRNA molecular identification technology.It was determined that the crude extract of bacteriocin AB-2 was extracted with 1:4 saturated ammonium sulfate in the precipitation method of saturated sulfuric acid solution,and the diameter of the inhibition zone of bacteriocin AB-2 was22.37 mm,the protein content was 1.6181 mg/m L,and the antibacterial titer was38036AU/m L,the minimum inhibitory concentration was 6.3207μg/m L.When the initial bacterial concentration was lower than the OD_600nmvalue of 0.0013,the bacteriocin AB-2could effectively prolong the lag phase and the initial stage of the logarithmic growth phase of Listeria monocytogenes,and delay the normal growth of Listeria monocytogenes.When the initial viable count OD_600nmwas 0.6,the growth of Listeria monocytogenes was more effectively inhibited.The lethality rate of bacteriocin AB-2 against Listeria monocytogenes reached 99.63%at 3h.Through the effect of glucose addition on the inhibition of Listeria monocytogenes by1×MIC bacteriocin AB-2,it can be seen that 10mmol/L glucose can effectively protect Listeria monocytogenes,and 1×MIC bacteriocin AB-2 can effectively protect Listeria monocytogenes.The inhibitory effect of Listeria is slowed down.According to the determination of nucleic acid and protein,the OD_260nmof the bacterial suspension treated with 10mmol/L glucose and 1×MIC bacteriocin BZ2 for 6h decreased by 0.69 compared with the control group,and the OD_280nmdecreased by 0.21 compared with the control group.Intracellular nucleic acid and protein efflux of Listeria monocytogenes was slowed.The effect of 1×MIC bacteriocin AB-2 on the membrane permeability of Listeria monocytogenes showed that the addition of 1×MIC bacteriocin AB-2 made the K⁺in the cells of Listeria monocytogenes leak out rapidly,The mass concentration of extracellular K⁺was 10.87mmol/L at 6h.Under the action of bacteriocin AB-2,the cell membrane of Listeria monocytogenes was destroyed,and the electrical conductivity of Listeria monocytogenes treated with 1×MIC bacteriocin AB-2 increased by 10.94 compared with the control group at 6 h.μS/cm,resulting in the leakage of electrolytes such as intracellular ions.The effect of 1×MIC bacteriocin AB-2 on the membrane integrity of Listeria monocytogenes showed that the addition of 1×MIC bacteriocin AB-2 resulted in a large amount of nucleic acid and protein leakage of Listeria monocytogenes.Under the action of1×MIC bacteriocin AB-2,the extracellular lactate dehydrogenase content of Listeria monocytogenes increased by 97.604 U/L at 6 h compared with 0 h,which eventually led to the permeability of Listeria monocytogenes membrane.Sexuality is increased and integrity is destroyed.The extracellular ATP content of Listeria monocytogenes treated with 1×MIC bacteriocin BZ2 increased from 0.746μmol/L to 279.05μmol/L within 0～30min,and the Listeria monocytogenes without bacteriocin BZ2 treatment,the content of extracellular ATP increased from 3.73μmol/L to 54.28μmol/L within 0～30 min.The results showed that the leakage rate of Listeria monocytogenes increased under the action of bacteriocin AB-2,and the effect of Listeria monocytogenes on Listeria monocytogenes increased.The membrane structure was severely damaged.It is inferred from electrophoresis that bacteriocin AB-2 cannot bind to the DNA of Listeria monocytogenes,and it is believed that the mechanism of action of bacteriocin AB-2 on Listeria monocytogenes does not involve attacking the DNA of Listeria monocytogenes.Scanning electron microscopy and transmission electron microscopy showed that the permeability and integrity of the cell membrane were severely damaged,resulting in a large amount of intracellular material leakage,which eventually led to the death of Listeria monocytogenes.The results of the bacteriocin stability experiment showed that the stability of bacteriocin BZ2 was poor.Bacteriocin AB-2 exhibited inhibitory effects on Listeria monocytogenes in orange juice at-20℃,4℃,30℃and 37℃;the best bacteriostatic effect was at 30℃and 37℃.It can rapidly and completely inhibit the growth of Listeria monocytogenes in orange juice within 1 day.In this thesis,we have a deeper understanding of the bacteriostatic activity of bacteriocin AB-2,clarified its mechanism of action,and provided a theoretical basis for the development and utilization of bacteriocin AB-2 as a food preservative.