Study On The Generation And Mechanism Of Class ?a Bacteriocin Resistance

The main research objective are class ?a bacteriocins and their sensitive Listeria strains.Firstly,the bioactive class ?a bacteriocins were extracted and the resistant Listeria strains were induced to compare the differences in growth metabolism between the sensitive and resistant strains,and then the mechanism of resistance was further studied.The results are helpful to elucidate the mechanism of action of bacteriocins,provide a theoretical basis for their future application in food,and provide a reference for subsequent studies.The main contents of this paper are as follows:1.Bacteriocins were extracted and their bacteriostatic activities were determined.The bacteriocins of Lactobacillus plantarum,Lactobacillus curvus and Pediococcus acidilactici were extracted by ammonium sulfate precipitation method,acid precipitation method and Yang's adsorption method,respectively.The optimal extraction method for Lactobacillus plantarum is ammonium sulfate precipitation,and the best extraction effect is achieved when the concentration of ammonium sulfate is 70%.Yang's adsorption method was used to extract the bacteriocins from Lactobacillus curvus and Pediococcus acidilactici.Listeria monocytogenes,Listeria ivanovii and Listeria innocua showed sensitivities to the above three bacteriocins.The titers of Pediococcus acidilactici,Lactobacillus curvus and Lactobacillus plantarum bacteriocins determined by Listeria monocytogenes were 640 AU/m L,320 AU/m L and 320 AU/m L,respectively.2.Induction and resistance analysis of resistant strains.Listeria monocytogenes were induced with a resistance frequency of 10^-5,and two stable resistant strains were obtained.The MIC results of the Oxford cup assay showed that the resistant bacteria were not sensitive to the neutral fermentation supernatants containing bacteriocins,but the sensitivity to nisin remained unchanged.The growth curves of the Listeria strains were determined.With the presence of pediocin,the growth rate of the wild-type Listeria strain in the logarithmic phase was higher than that of the resistant strains.The logarithmic phase of the resistant strains was delayed,and the final bacterial density was higher than that of the wild-type strain.With the presence of different concentrations of pediocin,the wild-type strain was significantly inhibited,while the resistant strains still showed a growth trend,but the cell density was lower than that without the addition of pediocin.The wild-type strain reach a stable period faster than that of the resistant strains with the presence of glucose and mannose.3.Study on resistance mechanism.The cell surface hydrophobicity,sensitivity tolysozyme,bacteriocin absorption capacity,enzyme secretion and regulation gene validation were determined.The results show that the resistant strain had increased cell surface hydrophobicity and decreased biofilm formation index.The absorption capacity of resistance cells to bacteriocin was decreased and there was no significant difference in the sensitivity to lysozyme,and no secreted enzymes that inactivated bacteriocins were detected.After the amplification of two regulatory genes,lmo0095 and res D,the sequencing results showed that the sequence of the original strain was consistent with that of the resistant strain.4.Cloning and expression of the target gene in recombinant bacteria.The mpt C and mpt D genes of the mannose-phosphate transferase system were amplified to obtain the target fragments with the sizes of about 800 bp and 900 bp,respectively.The recombinant plasmids p MD19-mpt C and p MD19-mpt D connected with the T vector were used to construct the recombinant plasmids pet32a-mpt C/pet32a-mpt D,p NZ8048-mpt C/p NZ8048-mpt D.The recombinant plasmids pet32a-mpt C/pet32a-mpt D were expressed in E.coli Bl21?DE3?.Recombinant proteins Trx A-Mpt C and Trx A-Mpt D were obtained after IPTG induction.The recombinant protein bands with a molecular weight of about 50 k Da appeared.Recombinant NZ9000?p NZ8048-mpt C/p NZ8048-mpt D?strains were successfully obtained by electrotransformation of Lactococcus lactis NZ9000 cells.The growth curve of NZ9000?p NZ8048-mpt C?strain was measured after nisin induction,and the growth rate and growth amount increased with the increase of nisin concentration.The best growth condition was obtained when the nisin induction concentration was 10 ng/m L.The biomass of NZ9000?p NZ8048-mpt D?strain attained the highest biomass when the nisin induction concentration was 5 ng/m L.In the presence of bacteriocin,the growth of recombinant strains induced by high concentration of nisin?? 5 ng/m L?was inhibited,and both showed sensitivity to bacteriocin.