Study On Physicochemical Properties And Mode Of Action Of Novel Bacteriocins BM1122 And BM1300

If the food is improperly handled during processing and storage,it may be contaminated with various food-borne pathogens and spoilage bacteria,causing spoilage and even leading to food-borne diseases.With the increasing of the public's concept of health,people are increasingly favoring foods containing natural preservatives.Therefore,the food industry urgently needs a natural,safe and efficient preservative.Bacteriocins are proteins or peptides synthesized by the ribosome,which have inhibitory effects on varieties of food-borne pathogenic bacteria,and the bacteriocins produced by lactic acid bacteria are generally considered to be safe and have potential as natural food preservative in the future.Lanhua Yi from Lab of Resources discovered two bacteriocins BM1122 and BM1300 from Lactobacillus crustorum MN047,and heterologous expressed using E.coli(Yi et al.2016;Yi et al.2018a;Yi et al.2018c).Through structural prediction and functional annotation,it is found that the structure and functional annotation of the two bacteriocins were different.BM1122 was DNA-binding protein through the function prediction,and BM1300 had rich ?-helix struture(Yi et al.2018a),indicating that the two bacteriocins may have different bacteriostatic mechanisms.However,the physicochemical properties of the two bacteriocins,the specific bacteriostatic mechanism and the bacteriostatic effect in food are still unclear.In this study,the new bacteriocin BM1300 was purified and identified,and the physicochemical properties of the bacteriocins BM1122 and BM1300 were determined;secondly,the modes of action of the two bacteriocins on Staphylococcus aureus and E.coli were investigated;finally,BM1122 and BM1300 were initially applied to inhibit the microorganisms in milk and beef.This laid a good theoretical foundation for the future application of two novel bacteriocins.Results are as follows:(1)The results of purification and identification of bacteriocin and its physicochemical properties: the crude sample of bacteriocin BM1300 was purified by anion chromatography exchange and RP-HPLC,and was identified by LC-MS / MS.The coverage of amino acid residues was 76.39% containing core amino acid.Both bacteriocin BM1122 and bacteriocin BM1300 have broad-spectrum antibacterial activity and can inhibit the growth of various Gram-positive and Gram-negative bacteria.Both BM1122 and BM1300 had good stability to temperature(60-120?),p H(2.0-11.0),enzymes(such as pepsin,trypsin,papain,and catalase),surfactants(5%EDTA?5 mmol/L SDS?5%Tween-40?5%Tween-80?5%Triton X-100).These results indicated that bacteriocins BM1122 and BM1300 had good processing stability.At the same time,the bacteriocin BM1122 and BM1300 have no obvious hemolysis circle,indicating that the two bacteriocins have no hemolytic activity.(2)The results of modes of action of bacteriocin: time-kill showed that both bacteriocins have good antibacterial effect on S.aureus and E.coli,and the antibacterial ability of E.coli is better than S.aureus for them.At the same bacteriocin concentration,BM1122 is more effective than BM1300.Scanning electron microscopy and transmission electron microscopy results showed that BM1122 caused S.aureus inward depression and separation of the plasma wall,but caused the formation of cavities on the surface of E.coli cells and the outflow of intracellular material.Differently,BM1300 can cause the destruction of S.aureus and E.coli cell membranes,and both even cell lysis and the outflow of intracellular materials can cause cell death.The damage of cell membrane integrity by two bacteriocins was demonstrated by the experiment of propidium iodide(PI)uptake and the release of lactate dehydrogenase.Moreover,BM1122 and BM1300 inhibited the biofilms formation of both S.aureus and E.coli.And the two bacteriocins cause the arrest of S phase of bacterial cell cycle.Therefore,bacteriocin BM1122 and BM1300 exerted the antibacterial activity through various modes of action.(3)The results of preservative application of bacteriocin in milk and beef meat: both BM1122 and BM1300 significantly reduced the counts of S.aureus and E.coli in milk in a concentration-dependent manner.With the increasing concentration,the bacteriostatic effect is better.At the same time,both bacteriocins could enhance the ability to inhibit viable bacteria in beef stored at 4?.Therefore,BM1122 and BM1300 significantly inhibited the increase in the number of viable bacteria in S.aureus and E.coli in milk(0-7 days)and beef(0-10 days)in a certain storage time.