Screening And Identification Of The Bacteriocin-like-producing Bacteria And Studying On The Bacteriocin-like

Bacteriocins are ribosomally synthesized peptides exhibiting antibacterial activity. The bacteriocins have attracted significant attention because of their potential use as non-toxic and safe additives for food preservation and prevention of food spoilage by food borne pathogenic bacteria. Considering safety of producing-bacteriocin bacteria and the security of the bacteriocins, only the bacteriocin nisin, which is produced by Lactococcus lactis,is permitted using in food up to date as a food preservative. But bioproperties and biochemical characteristics of Nisin limit it used in the food industry widly Therefore, more new bacteriocins is expected. To date, screening of bacteria producing novel bacteriocins with a broad antimicrobial spectrum and identifying those novel bacteriocins have attracted the most attention. When developing food-grade bacteriocin, the safety of bacterial which produce bacteriocin must beconsided.Shanxi vinegar is one of the two most well known vinegars in China. At the baces of the previous study, the 75 strains of bacteria which isolated from the Cupei of Shanxi vinegar was screened for finding the strains which could produce antibacterial compounds.And a strain which had a broad spectrum and the most strong ability of inhibiting was studied, including determining the property of the antibacterial compounds,the identification of bacteria, Bio-characteristics and the isolation and purification of the main antibacterial et.al..This will provide theoretical basis for potential utility as a biopreservative in the food industry.The main topics of this thesis are as follows:By using cylinder-plate method,the 17 strains of bacteria producing antibacterial compounds, was screened from 75 strains of bacteria which isolated from the Cupei of Shanxi vinega. All of the 17 strains of bacteria could inhibite the growth of the Gram-positive bacteria Bacillus subtilis, Staphyloccocus and the Gram-negative bacteria Escherichia coli. The 17 strains were identified as Bacillus by means of phenotype. Strain A32 had the most strong ability of inhibiting to B. subtilis, Staphyloccocus and E. coli.By excluding the effects of organic acids, hydrogen peroxide and testing of proteinase digestion, the property of the antibacterial substance produced by strain A32 was determined,which including H2O2 and bacteriocin.Through the curves on growth of strain A32 and production of antimicrobial protein,it was induced that the antimicrobial protein was the primerary production. By the test of effect on strain A32 to antimicrobial protein,it was found that the antimicrobial protein cound not inhibite the growed of strain A32.It induced that the antimicrobial protein had immunogenicity to the strain A32.According all above the inforation on the antimicrobial protein,the conclution could be get that the antimicrobial protein was the bacteriocine-like.Strain A32 was identified by means of phenotype, physiological and biochemical characteristics as well as with 16S rDNA gene analysis. The phenotype revealed that it was Bacillus.The physiological and biochemical revealed that strain A32 was similar with the B.subtilis.16S rDNA gene of strain A32 was got and sequenced.The sequence was submitted to the genbank and the GenBank accession numbers was JX025646. The sequence was compared with the 16S rDNA gene in the genbank by the tool of basic local alignment search.The results revealed that strain A32 was similar with B. subtilis.According to the results above all, it was get strain A32 was B.subtilis, and named B.subtilis HJD.A32.This strain had applicated for patent and was stored at China general microbiological culture collection center.The antimicrobial spectrum was installed by using Double-layer cylinder-plate method, The results showed that the bacteriocin had inhibitory activity and had a broad spectrum, it could inhibite the growth of many food-spoiling bacteria and food-borne pathogens. It could inhibite the growth of yeast, but could not inhibite the growth of mold. The titers to the indicator bacteria B. subtilis SH108 was 256AU/mL.The bacteriocin had good stability against temperature and pH.The inhibitory activity was not influenced disposed under 60 ℃ to 100℃ for 20 minutes. The inhibitory activity changed little throughout the pH range of 3.0 to8.0.Some activity was lost disposed by chemical reagent,l% SDS improved of 40% antimicrobial activity,andl% tween20,tween80 and span80 made 17%,24% and 8% loss of the inhibitory activity,respectively. The bacteriocin was sensitive to trypsinase, proteinase K and pepsin. This characters suggests that bacteriocin may have a broad application as biopreservatives in the food industry.By ultrafiltration,the molecular weight of the inhibitory protein in the supernant was crudly estimate by ultrafiltration.The results showed that there were many inhibitory protein,and the Molecular weight was greater than the 3KDa.The inhibitory protein in the supernant was seperate by the by ammonium sulfate precipitation method, ethyl alcohol precipitation method、acid precipitation method, respectively.The results show that the ammonium sulfate precipitation method was the best,the acid precipitation method was better,and the ethyl alcohol precipitation method was worse in the inhibiting effects; The acid precipitation method and the ethyl alcohol precipitation method was more simple than the ammonium sulfate precipitation method in the program.In this test,the acid precipitation method was used to seprate inhibitory protein in the supernant at last. And the molecular weight of the crude bacteriocine-like was under the14.4kDa.The crude bacteriocine-like was concentrated and graded.and the results showed that not only the crude bacteriocine-like which molecular weight was up the 3kDa had the activity of inhibitity but also the matericine which molecular weight was under the 3kDa.It revealed that there were more than three kind bacteriocine-like in the crude material.This results was different with the results before.It infered that the activity of bacteriocine -like which molecular weight was under the 3kDa could be tested by cylinder-plate method becouse of the concentration of the material.The crude bacteriocine-like was puraficated by Bio-scale TM Mini Macro-Prep(?)High Q Cartridge chromatogram,and got four perk: A,B,C,D.The perk A had the activity to Bacillus SH108.The time range of elution was 5-12min, the eluate was O.lmol/L NaCl(0.02mol/L, pH5.7 phosphate buffer).The molecular weight was under the 3K in the peakA,and the bacteriocine-like which the molecular weight was up the 3K was not tested in the peak A.It was induced that the bacteriocine-like which the molecular weight was up the 3K may be absorbed by the matrix.The molecular weight of the proteins in the Peak A were 1000Da or so which estimated by MALDI-TOF-MS. It need further research on the purification of bacteriocine -like.