Isolation And Identification Of Bacillus In Wuliangye Jiupei And Their Antimicrobial Substances

In traditional liquor brewing process,the distiller's fermentation of Wuliangye Jiupei contain a variety of microorganisms,these microbes not only caused by ethanol fermentation and production of flavor substances,some of them also produced other important functional sunstances.Since that fermented grains contain a large number of Bacillus strains,the Bacillus strains in this paper were taken the famous liquor Wuliangye Liquor Products grains.Through screening of strains with antimicrobial activity,analyzing the antibacterial substances,determine the antimicrobial spectrum and the antibacterial genes,we get the biological and physicochemical properties.The strain belongs to Bacillus amyloliquefaciens based on morphological observation,biochemical and physiological tests,and the analysis of 16 S rRNA sequence,which was designated Bacillus amyloliquefaciens WY047.The culture supernatant of WY047 was found to be inhibitory to a wide spectrum of bacteria(Gram-positive and Gram-negative)and fungi.The broader spectrum and the synergistic action suggested its huge application prospect.Nine pairs of primers specific for bacillomycin D,fengycin,flagellin,iturin A,bacilysin,mersacidin,TasA,subtilin,and surfactin were designed.The presence of six genes(bmyD,fenA,hag,ituA,mrsA,and tasA)of antimicrobial substances was detected by PCR.The simultaneous production of different antibiotics and the synergistic effect of them could be associated with its broad range of antagonistic activity.Ion exchange and gel filtration chromatography were used to purify proteins.The highest fraction level of antimicrobial activity was estimated to be 35,207 Da,and it was identified as flagellin by protein sequencing.Antimicrobial peptides were extracted with methanol and identified through LC–MS.The possible TasA encoded by tas A could not be isolate in this study.Bacillus WY047 was shown to harbor six antibiotic genes and produce at least five antimicrobial compounds.The antimicrobial substance was tested for sensitivity to proteolytic and nonproteolytic enzymes,heat,pH,and various chemicals.There was no loss of antimicrobial activity upon exposure to trypsin,-amylase,and pepsin.The addition of methanol,ethanol and acetonitrile showed no appreciable effect on antimicrobial activity.The AMP mixtures produced by WY047 were shown to be stable even though extreme temperature and pH stresses.To test the distribution of antimicrobial genes in Bacillus strains,nine pairs of primers specific for bmyD(bacyllomicin),fenA(fengycin),hag(flagellin),ituA(iturin),mrsA(mersacidin),tasA(TasA),spaS(subtilin)and srf(surfactin)examined in 80 isolates of Bacillus spp.obtained from Jiuqu.Among them,61 isolates of Bacillus spp own hag gene;52 isolates of Bacillus spp own fenA gene;45 isolates of Bacillus spp own ituA gene;41 isolates of Bacillus spp own bmyD gene;while only 11 isolates of Bacillus spp own mrs gene.Most strains had between two and four AMP genes whereas strains with five genes were seldom detected and only one of the strains had six genes.The most frequent AMP gene markers were hag,fenA,ituA and bmyD,and the most frequent genotypes hag-fenA-ituA and hag-fenA-bmyD.To sum up,many Bacillus strains in liquor Jiupei have antibacterial protein or antibacterial peptide genes,produce a variety of antibacterial substances.Some strains also have a variety of antibacterial gene with a broad spectrum of antibacterial activity.Through the further research of these strains and their antibacterial substances,it is expected to develop biological pesticide or biological preservative with good application prospect.