| Bacteriocins have great potential as food additives for controlling foodborne diseases and extending shelf life.In our previous research,a new class II bacteriocin(bacteriocin BM1029)gene was identified from the genome of Lactobacillus MN047 and was successfully expressed in the E.coli.In this study,the novel bacteriocin BM1029 was selected as the research objest,the inhibitory activity and mechnisim of BM1029 against E.coli ATCC25922 and S.aureus ATCC25923 were explored by a series of experiments.The experimental results as follows:(1)The engineering bacteria was induced by IPTG and then destroyed by repeated freeze-thaw.The obtained supernatant was used as crude bacteriocin sample.Salt ions and impurity proteins were removed by dialysis and ultrafiltration,and then further separated and purified by exchange chromatography and high-performance liquid chromatography(HPLC).The purified sample was identified by LC-MS/MS and 14 fragments were obtained with a coverage of 88.52%.The prediction of structure and property showed that BM1029 has an α-helical structure,high stability,and the functional annotation showed that BM1029 is a possible DNA binding protein.(2)The antibacterial spectrum and stability of bacteriocin BM1029 were determined by agar diffusion method.The results showed that BM1029 possess broad antibacterial activity against both Gram-positive and Gram-negative foodborne pathogens and the minimum concentration(MIC)of the bacteriocin BM1029 against tested strains ranged from 2-32 mg/L.At the same time,bacteriocin BM1029 shows high stability to high temperature,different p H and surfactant treatment: Bacteriocin BM1029 is thermally stable after treated at 60,80,100 and 120 °C for 5,10 and 20 minutes,respectively;The bacterial inhibitory activity of BM1029 did not decrease significantly after treatment with different p H(2-11)and chemicals.What’s more,bacteriocin BM1029 had low hemolytic activity which were similar to ampicillin,so it had low toxicity to eukaryotic cells.In the analysis of growth curve and time killing kinetics,it was found that BM1029 can inhibit the growth rate of E.coli and S.aureus,and to a certain extent,it has a stronger inhibitory effect on E.coli than S.aureus.(3)In order to clarify the activity of bacteriocin BM1029 against staphylococcus aureus and Escherichia coli,the studies of bacteriostatic mechanism were carried out.Scanning electron microscopy(SEM)showed that the bacteriocin BM1029 caused morphological changes on the surface of the indicator cells.With the treatment of BM1029,E.coli and S.aureus had uneven distribution of ribosomes and DNA,and intracellular material leakage,indicating that the target of action of BM1029 may also exist in the periplasmic space.Lactate dehydrogenase(LDH)release experiments showed that BM1029 induces increased permeability of the cell membranes of E.coli and S.aureus.The release of propidium iodide(PI)was measured by flow cytometry,indicating that the integrity of the cell membrane of the indicator bacteria was damaged after treatment with BM1029.Compared with S.aureus,BM1029 caused more LDH release and PI uptake in E.coli which had a more significant cell membrane destruction effect.Gel blocking experiments indicated that BM1029 binds to the DNA of indicator bacteria,so DNA may be a target for intracellular action of the bacteriocin.What’s more,BM1029 interferes with the cell cycle of E.coli and S.aureus,causing the cell cycle to arrest in the R phase.(4)Studies on the application of bacteriocin BM1029 during storage of dairy and meat products have shown that BM1029 inhibits the growth of E.coli and S.aureus in milk and beef meet.In addition,the presence of BM1029 also reduce the rate of p H drop in the milk storage and the total number of colonies during the storage of beef refrigeration.In general,the new bacteriocin BM1029 has broad-spectrum bacteriostatic activity and good stability which can be used to alternative to take the place of antibiotics and chemical additives,and to be used as a natural food preservative in milk and meat products. |
