Screening And Identification Of Bacteriocin Strains In Honey And Study On Physicochemical Properties Of Bacteriocin

In recent years,the harm of chemical preservatives to human body has aroused people’s alert.Therefore,natural preservatives with non-toxic and harmless to human body,green environmental protection,broad-spectrum bacteriostasis and stable performance have become the focus of scholars at home and abroad.Bacteriocin is essentially a polypeptide substance,which is non-toxic and harmless to the human body and is easy to be absorbed.Bacteriocin has gradually become an important source of ideal natural antimicrobial agents.In this experiment,a bacteriocin-producing Bacillus thuringiensis,was screened from honey and a series of studies were carried out on its bacteriocin.The main results are as follows:1.Screening of bacteriocin-producing strains from honey.Fifteen strains,numbered FM-1 to FM-15,which showed inhibitory effect on Staphylococcus aureus and Escherichia coli were isolated from honey by Agar diffusion in Oxford Cup.After excluding hydrogen peroxide,organic acid and other influencing factors,FM-3,FM-5 and FM-7 still have antibacterial activity.After the exclusion test of proteins or peptides,the antibacterial activities of FM-3 and FM-7 disappeared,indicating that the bacteriostatic substances produced by these two strains were either proteins or peptides,which conformed to the properties of bacteriocins.By observing the growth status of two strains of bacteria on the plate,the better growth state of FM-3 was selected as the test strain.2.The physiological,biochemical and 16 s rDNA identification of the tested strains.The physiological and biochemical identification of the target strain was carried out by traditional methods,and the results showed that the strain belonged to Gram-positive bacteria.The total DNA template of the screened strain was extracted,and the DNA template of the strain was amplified by PCR with 16 s rDNA universal primers.By means of BLAST comparison,the similarity between FM-3 strain and Bacillus thuringiensis was over 96%.The phylogenetic tree was constructed by nucleic acid system software,analysis indicates that phylogenetic relationship of between FM-3 strain and Bacillus thuringiensis was the closest.The FM-3strain was definitely Bacillus thuringiensis.3.The bacteriocins produced by FM-3 strain was isolated and purified.By measuring the diameter of bacteriostatic zone and using coomassie brilliant blue method,the titer of bacteriocin was 276 AU/m L.Four different ion peaks were obtained from the crude bacteriocin by DEAE-52 ion exchange chromatography,and their OD values were determined and Oxford cup bacteriostatic test was carried out.It was found that peak 3 and peak 4 had better bacteriostatic effect than the other two peaks,so the peak 3 with stronger antibacterial activity was selected for further purification by dextran gel GM50 gel chromatography after comparing peak 3 and peak 4.After purification,there were obvious peaks in the chromatogram.By comparing the OD values of the two peaks and the diameter of the bacteriostatic zone,it was found that the second component had better bacteriostatic effect.The molecular weight of bacteriocins produced by FM-3 Strain was determined by Tricine-SDS-PAG gel electrophoresis,the results showed that the band was single and bright,and the molecular weight was 4.5KDa.4.Study on the characteristics of bacteriocins produced by FM-3 strain.The single factor experiments on temperature,heating time,acid-base,enzyme and bacteriostatic spectrum bacteriocin from FM-3 Strain were carried out.The results showed that with the increase of temperature,the bacteriostatic activity of the FM-3 strain against Staphylococcus aureus decreased gradually,in the temperature range of 20 ℃～100℃,bacteriocins produced by FM-3 strain all have bacteriostatic activity.The results showed that the bacteriocin produced by FM-3 strain had strong adaptability to temperature.The bacteriostatic activity of bacteriocin produced by FM-3 strain changed little with the increase of heating time,which indicated that heating time had little effect on its bacteriostatic activity.The bacteriostatic activity of bacteriocin decreased under strong acid and alkali environment,but all of them had bacteriostatic ability,which indicated that bacteriocin produced by FM-3 strain had certain acid-base tolerance.The enzyme sensitivity test showed that the bacteriocin produced by FM-3 strain was stable to trypsin,protease K,lipaseⅡ and pepsin.The bacteriocin produced by FM-3 strain had higher bacteriostatic effect on several indicator bacteria,and the size of bacteriostatic circle belonged to the normal bacteriostatic range.Bacteriostatic spectrum experiment showed that bacteriocin produced by FM-3 strain had certain inhibitory effect on bacteria and mold.