Screening Of Marine Source ?a Bacteriocin Lactic Acid Bacteria And Separation Of Bacteriocin And Antibacterial Action Mode Of Listeria Monocytogenes

The purpose of the study was to isolate lactic acid bacteria with the antibacterial activity and the multi-phase classification were identified in the intestinal tracts of eight marine animals collected in the South China Sea of Zhanjiang,by dilution coating method and double-layer agar Oxford cup punching method.Colony PCR method was used for specific primers lactic acid bacteria with antibacterial activity were screened for IIa bacteriocin;extracted with ethyl acetate,purified by Sepharose LH-20 column,SP-Sepharose FF,reversed phase semi-preparation High-performance liquid chromatography was used to purification class IIa bacteriocin.The molecular mass and amino acid sequence were determined by LC-MS/MS analysis.The purified bacteriocin was evaluated for antibacterial spectrum,pH,temperature and protease sensitivity.The electrical conductivity was observed and measured by electron microscopy to evaluate the inhibitory effect of bacteriocin on Listeria monocytogenes.The main results are as follows:(1)A total of 40 lactic acid bacteria were isolated from the intestinal tract of 8 common marine animals in the South China Sea.The molecular biology and phylogenetic analysis of 16 strains with antibacterial activity were found to belong to the thick-walled bacteria.4families,5 genera(Enterococcus,Weissella,Leuconostoc,Pediococcus,Lactobacillus),12 different species(Enterococcus lactis,Enterococcus saigonensis,Enterococcus durans,Enterococcus dispar,Lactobacillus pentosus,Lactobacillus xiangfangensis,Lactobacillus paracasei subsp.Tolerans,Leuconostoc holzapfelii,Pediococcus pentosaceus,Weissella confusa,Weissella cibaria,Weissella ghanensis)showed abundant biodiversity,of which Enterococcus(50%)was the dominant genus,and 4 strains were potential new in Enterococcus and Weissella genus kind.The isolates of 40 strains of lactic acid bacteria have antibacterial activity.The lactic acid bacteria have certain inhibitory effects on V.parahaemolytics and L.monocytogens.Among them,9 strains simultaneously belong to E.coli,V.parahaemolytics,S.aureus,L.monocytogens,B.subtilis has antibacterial activity,accounting for 22.5% of the total number of lactic acid bacteria.(2)Using strains zy-B,zy-E,zy-I,zy-F,zy-F2,zy-S6,zy-2,zy-3,zy-4 as test strains according to class IIa bacteria Colony PCR amplification of 7 pairs of specific primers O-1,O-2,O-3,O-4,O-5,O-6 and O-7 designed by class IIa bacteriocin conserved sequences.The results showed that 5 pairs of lactic acid bacteria suspected of producing class IIa were successfully screened from 26 strains of lactic acid bacteria by using 7 pairs of specific primers,and 5 strains of suspected class IIa bacteriocin lactic acid bacteria were subjected to acid exchange,hydrogen peroxide and protease sensitivity test.It was confirmed that all the 5 strains were class IIa bacteriocin-producing bacteria,and the strain zy-B had the advantages of strong antibacterial activity and wide antibacterial spectrum.(3)Extract bacteriocin from the fermentation broth with ethyl acetate,mix ethyl acetate with fermentation broth,separate and collect the organic phase,and evaporate ethyl acetate at 40 °C,reconstitute with 25 mL PBS to obtain crude bacteriocin and measure Its antibacterial activity has a titer of 6553.6 AU/mL.The crude bacteriocin extracted by ethyl acetate was sequentially subjected to gel chromatography and cation exchange respectively through LH-20 column,AKTA purifier 100 protein purification system and semi-preparative high-performance liquid phase to obtain better separation effect.The antibacterial potency was 8192 AU/mL,4096 AU/mL,5120 AU/mL,and the purified sample showed a single antibacterial activity peak at t=1.859 min,and was named bacteriocin PE-ZYB1.The molecular mass of bacteriocin PE-ZYB1 was determined by LC-MS/MS to be 2019.22 Da,and the N-terminal amino acid sequence of LDFNKFNRSMQYGNGVE was analyzed by LC-MS/MS.The Uniport protein database was found to be closest to the shape of an unidentified protein.The peptide database(ADP)was found to be similar to the antimicrobial peptide Andersonin-X1(accession number:AP01874)with only 38.09% similarity.It is speculated that this bacteriocin may be a potential novel bacteriocin.(4)The antibacterial spectrum of purified bacteriocin PE-ZYB1 was found to have broad-spectrum antibacterial activity against Gram-positive and Gram-negative food-borne pathogens and spoilage bacteria,and had high activity at high temperatures.Heat resistance and stability.The bacteriocin PE-ZYB1 can maintain good antibacterial activity at pH of 2-7.The bacteriocin PE-ZYB1 was found to be sensitive to trypsin in a protease sensitivity assay.The extracellular conductivity of Listeria monocytogenes after bacteriocin PE-ZYB1 increased rapidly after 1 hour,indicating that bacteriocin PE-ZYB1 can damage cell membrane quickly and efficiently,and enhance cell membrane permeability.The growth inhibition of bacteriocin PE-ZYB1 against Listeria monocytogenes is very obvious,and it has a killing effect on Listeria monocytogenes,and it is not a simple inhibitory effect.Scanning electron microscopy showed that the morphology of the cells was significantly changed before and after the action of Listeriamonocytogenes.After the action,the cells were deformed,depressed,and wrinkled on the cell surface,and the cells were broken and the intracellular substances were dissolved.This indicates that the damage caused by bacteriocin PE-ZYB1 to the cell membrane can cause intracellular macromolecular substances to leak,and finally lead to cell death.