Screening Of Marine Source Antimicrobial Active Lactic Acid Bacteria And Bacteriocin Isolation And Their Effects On Listeria Monocytogenes

In this study,the antibacterial activity of lactic acid bacteria isolated from the guts of marine animals near the South China Sea(Zhanjiang)was evaluated and bacteriocin producing lactic acid bacteria were screened by double agar oxford cup method,and the isolation and purification,structure identification of bacteriocin in the supernatant of fermentation of target bacteria as well as the properties of bacteriocins were studied.Finally,the inhibitory effect of bacteriocin on L.m was evaluated through the inhibition and removal of Listeria monocytogenes(L.m)biofilm formation by bacteriocin,cell membrane destruction and DNA damage experiments.The main findings were as follows:1.The antibacterial activity was detected by double oxford cup method.Bacteriocin-producing lactic acid bacteria were screened by acid-discharging,hydrogen peroxide exhaust and protease sensitivity test,and the target bacteria were identified and their biological characteristics were studied.The result showed that the fermentation supernant for 20 out of 25 strains lactic acid bacteria having antibacterial activity to 5kinds of indicator bacteria at different levels.The active strains accounted for 80 % of the total screened strains.Bacteriocin-producing strains ML-72 was screened by acid discharging,hydrogen peroxide exhaust and protease sensitivity test.The 16 S r RNA identification was Enterococcus durans.The biological characteristics research showed that the optimum growth temperature of the bacteria strain ML-72 was 32?,was able to tolerate the p H3.5 growth environment.The growth was greatly inhibited when the salt concentration was greater than 6%.2.The determination of the titer and protein concentration of inhibiting L.m were used as the evaluation indexes for the isolation and purification of bacteriocin,the supernatant of fermentation broth of strain ML-72 was separated and purified step by step through ethyl acetate extraction,Sephadex LH-20 gel column,SP-Sepharose Fast Flow cationic column and high performance liquid.The result showed that a single peak was obtained with antibacterial activity after the separation of gel column,cationic column and C18 chromatographic column.The isolate was named as bacteriocin QY-C,and its potency was improved to 2535.13 IU/m L,protein concentration was 0.25 mg/m L,the final purification fold was improved 37.67,and the yield was 7.11%.3.The LC-MS/MS analysis was conducted to the bacteriocin QY-C.The secondary structure of bacteriocin QY-C was analyzed by using infrared spectroscopy to determine functional groups,circular dichroism spectrum and the characteristics of bacteriocin were studied.The result showed that the molecular weight was 1254.63 Da,amino acid sequence was: Gly-Ala-Val-His-Asp-Val-Lys-Asp-Val-Leu-Asp-Ser.The similarity with the protein DCD-1 of the known database was relatively high;the matching-degree was82%.PYMOL software was used to simulate 3D model,it had an anastomosing spatial structure.The bacteriocin had the characteristic functional groups of hydroxyl(C-H),hydroxyl(-OH),carbonyl(C=O)etc,of which C=O was the biggest feature of bacteriocin.the MIC value of bacteriocin QY-C was 31.25 ?g/m L,MBC value was 62.50 ?g/m L.The bacteriocin QY-C had good thermal,p H stability.The bacteriocin was sensitive to protease,while its antibacterial activity would not be affected by lipase and amylase effect,and it had strong antibacterial activity to Gram-positive bacteria and Gram-negative bacteria.4.Micropore plate crystal violet staining was used to detect L.m biofilm.Cell membrane permeability was detected by measuring extracellular ultraviolet absorption value,electrical conductivity,ATP,lactate dehydrogenase and other indexes.The mycelia morphology was observed with electron microscope.The effect of bacteriocin QY-C on the L.m's genome DNA was detected by agarose gel electrophoresis.The result showed that when MIC treated for 12 h,the clearance rate of L.m biofilm formed was 24.35%,and it was improved to 39.26% at 24 h.Bacteriocin QY-C had a good cleaning effect on L.m biofilm.After the bacteriocin treated L.m for 3 h,contents of extracellular nucleic acid absorption value,protein absorption value,electrical conductivity,and lactate dehydrogenase were significantly increased compared to control group.Microscopic observation showed the indentation and slight cell contraction of the thallus,which damaged the membrane of L.m.The bacteriocin changed the permeability of the thallus.The bacteriocin QY-C also had certain damaging effect on DNA structure.