Isolation And Purification Of IturinA In Bacillus Amyloliquefaciens And Its Inhibition Against Saccharomyces Cerevisiae In Orange Juice

In recent years,with the appearance of food safety accidents increasing,the consciousness of food security of consumer is strengthening.Consumers' trust in food additive reduced because its excessive or exceeding the scope use.They try their best to avoid food added with additives and prefer green food which is good for health.Therefore,the development of natural addictive become a research hotspot.Microorganism is a major resources of natural food preservative,but there are some issues about it,such as low yield and less types.They have a narrow applicable range because of their specificity to bacteria,and their antibacterial effect is influenced by physical and chemical properties of food.These reasons restrict its scope of application in food industry.Based on these problems existing currently,this experiment regard the honey as starting point and try to screen bacillus with antimicrobial activity and without dangerous to our health.And then we aim to separate and identify the antimicrobial substance and enhance the yield of it by physical and chemical mutagenesis and genome shuffling.Theses can make a contribution to its industrial application.1.122 bacillus were isolated from raw honey and one of them was confirmed that its secondary metabolites can inhibit Bacillus pumilus,E.Coli and Saccharomyces cerevisiae.According to its morphological and cultural characteristics,the physiological and biochemical properties and 16s rRNA sequence analysis,it was identified as Bacillus amyloliquefacien.Its fermentation centrifugal supernatant can stay stable along with the temperature and pH changes,and its can not be decomposed by proteinase.To determine its antimicrobial spectrum,16 kinds of food spoilage organism were selected including gram-positive bacterial,gram-negetive bacterial,yeast and fungi.Experiments proved that it has broad-spectrum antimicrobial activity and can inhibit all of the bacteria mentioned above except Pseudomoas fluorescens,Pichia pastoris and Aspergillus niger.2.The main anti-yeast substance of Bacillus amyloliquefacien LZ-5 was separated by organic solvent extraction,SephadexLH-20 column chromatography and high performance liquid chromatography(HPLC).The LC-ESI-MS was adopted to determine the molecular mass of the substance,and the HPLC results was agreement with that of the standard substance of iturinA.Then the anti,yeast substance was preliminarily identified as the mixture of iturinA2,iturinA3 and iturinA6.The standard curve was set up and the yield of iturinA was determined.3.The Bacillus amyloliquefacien was treated by mutagenesis(including UV mutagenesis,NTG mutagenesis and ARTP mutagenesis)and genome shuffling to enhance the yield of iturinA.For the best mutagenic effect,the mutation conditions were as follows:UV for 120 s;NTG for 0.4 mg/ml;ARTP for 60 s.6 mutant strains were obtained from populations generated by mutagenic whose yield enhanced 23.4%compared to the wild strain.Improvements in lipopeptide yield was evident among four strains subjected to recursive protoplast fusion.3 high yield strain with the production of iturinA exhibiting 168.92 mg/mL,162.38 mg/mL and 179.22 mg/1 was obtained after two rounds of genome shuffling,which was a 2.03-fold increase compared with the wild strain.4.The MIC and MFC was determined by microdilution method.To evaluate the efficacy of iturinA for control of spoilage yeast in food,3 different levels of iturinA was added to orange juice incubated with Saccharomyces cerevisiae.The juice treated with 0.76 mg/ml iturinA showed a significant(p<0.05)control on yeast population during the storage agreeing with that of the 0.30 mg/ml natamycin.In addition,iturin A showed a subtle efficiency on chemical-physical characteristics of orange juice.the changes in total suger,total acid and soluble solids are related in the fermentation of yeast.And the changes in vitamin C were the consequences of oxidation reaction.