Study On Lactobacillus Bacteriocin Durancin GL Targeted Inhibition Of Listeria Monocytogenes

Bacteriocins have received increasing attention,from their initial use in food preservation strategies to recently proposed biomedical strategies aimed at combating some bacterial infections.Listeria monocytogenes is a very serious pathogen,causing more than 25% mortality of listeriosis,and almost all listeriosis is caused by eating contaminated food.Compared with most foodborne pathogens,Listeria monocytogenes is more harmful to human beings because it can tolerate many harsh conditions in food processing(such as low temperature,high pressure and low pH).In recent years,there have been a number of poisoning incidents caused by Listeria monocytogenes around the world,making listeria a hot spot in the study of food safety prevention and control.Lactic acid bacteria was isolated from a Spanish cheese,Enterococcus durans 41 D,the bacteria to produce a targeted inhibition of single increase of listeria new biological active peptide(named as lactic acid bacteria bacteriocin Durancin GL),the research about lactic acid bacteria bacteriocin Durancin GL properties and inhibition of single listeria characteristics research,in order to provide new options for the development of efficient natural food preservatives,at the same time for further clear the mechanism of action of bacteriocin provided the scientific basis.The main research contents and results are as follows:By building the lactic acid bacteria bacteriocin Durancin GL recombinant E.coli implements the efficient preparation of bacteriocin,the research results show that the expression of lactic acid bacteria bacteriocin Durancin GL exists in fermented supernatant on only a few,most of the products accumulate in the cells of recombinant strains,the cell wallbreaking,affinity purification and intestinal kinase after removal of redundant sequences,preparation of lactic acid bacteria bacteriocin purity as high as 98.07%.Bacteriostatic analysis of bacteriocin Duranicn GL showed that the bacteriocin had a targeted inhibitory effect on listeria.Durancin GL has strong thermal stability and antibacterial activity over a wide pH range,and is sensitive to protease K,papain and chymotrypsin.The presence of intramolecular disulfide bond is related to its antibacterial activity.Durancin GL could inhibit the growth of Listeria monocytogenes Scott A in both pure culture and cold food substrate,and the MIC of the bacteria on Listeria monocytogenes Scott A was 2.5 ± 0.40 mg/L.The inhibitory effect of lactic acid bacteria bacteriocin Durancin GL on Listeria monocytogenes is related to the concentration of bacteriocin.With the increase of addition amount,the continuous inhibitory effect on Listeria monocytogenes is prolonged.When the bacteriocin acts on Listeria monocytogenes,it can increase the electrical conductivity of the bacteria in the extracellular environment and release intracellular proteins and nucleic acids into the extracellular environment.On the one hand,bacteriocins increase the permeability of bacterial cells,leading to the release of substances.On the other hand,with the release of intracellular substances,the cells of the bacteria will break and die,which will accelerate the release of intracellular substances,and then the morphology(intracellular and extracellular)of the bacteria will change.Bacteriocin Durancin GL molecular weight is 4.967 KDa(< 10 KDa)and N-the sequence contains conservative YYGNG,thermal stability.Durancin GL binds to the cell membrane surface action site of Listeria monocytogenes,resulting in increased cell membrane permeability,release of intracellular protein nucleic acid and other proteins to the extracellular environment,and changes in cell morphology and structure.According to the standard of classification of bacteriocin by Klaenhammer,lactic acid bacteria bacteriocin Durancin GL has these properties should be classified as Class Ⅱa bacteriocin.Study on the exogenous expression of Man-PTS element in Listeria monocytogenes.In Escherichia coli,pET32a-IIC and pGS21a-IID expression vectors were constructed by codon optimization,and Man-PTS II C and II D elements were successfully e xpressed.In eukaryotic mammalian cells(293-6E and CHO-3E7),the expression vectors pTT 5-IIC and pTT 5-IID carrying DYKDDDK tags were constructed by codon optimization.They were used to express in 293-6E and CHO-3E7 cells,respectively.The results of SDS-PAGE and Western blot analysis showed that the expressed protein was consistent with the expectation.Based on the principle of homologous recombination,single increase listeria was realized with the help of a carrier pHOSS1 mannose transferase phosphate system(Man-PTS)components in ⅡC and ⅡD related gene knockout,building a Man-PTS ⅡC and missing Ⅱ D single bacteria listeria mutations.Through the mutant strains L.monocytogenes GKC and L.monocytogenes GKD bacteriocin sensitivity,growth curve,and the use of carbon source research,clear lactic acid bacteria bacteriocin Durancin GL targeted molecular lock list to add listeria Man-PTS membrane protein components ⅡC and ⅡD is lactic acid bacteria bacteriocin Durancin GL receptor molecules.Further use yeast two hybrid technology(ubiquitin system)study found that lactic acid bacteria bacteriocin Durancin GL with single increase listeria Man-PTS ⅡC there are interactions between molecules,and the Man-the other components of the PTS ⅡD does not exist between intermolecular interactions or the effect is very weak.