Establishment Of Screening System And Mutation Breeding Of High-yield Antibacterial Lipopeptide Of Bacillus Strains

Aquaculture is a major industry in China.However,with the rapid development of aquacuture and increasing of high density intensive cultivation,the diseases of aquaculture occur frequently.The abuse of antibiotics also caused continually appearance of the dug-resistant bacterial strains,so searching fora safe and effective substitute of antibiotics becomes the urgent matter.Bacillus sp.can produce many kinds of antimicrobial substances whose bacteriostatic mechanism differs fundamentally from traditional antibiotics.These antimicrobial substances are less likely to cause resistance and have broad-spectrum antimicrobial activity.Application of antimicrobial peptides could not only withstand the pathogenic bacteria invasion,but also could increase growth-performance of aquatic animals.In order to improve the low yields of wild-type strains,we have established an integrated systerm of isolation,identification,optimazation of fermentation condition and mutation breeding.Main results were as following:(1)By determing the antimicrobial activities of all the strains preserved by our laboratory,strains P1 and P6 were selected as the target strains.According to the morphological,physiological and biochemical characteristicand 16 S r DNAsequence,they were identified as Bacillus amyloliquefaciens.(2)The culture medium and culture conditions was optimized to inprove the production of lipopeptide.The optimal culture conditions were that:MSM medium with 20 g sucrose,NH4NO3 2g,KH2PO4 3g,Na2HPO4 10 g,Mg SO4 0.2 g,yeast extract 0.2 g,Ca Cl2 0.7μg,Mn SO4 1μg,distilled water 1 L;fermentation temperature 30℃,natural p H,culture volume 80 m L,fermentation time 60 h,rotation speed 220 r/min and inoculation quantity 5%.The inhibition zone reached 22 mm after optimation,which increases 51.7% compared with the wide type strains.(3)The temperature tolerance,acid-alkal tolerance and UV irradiation tolerance of fementation liquid and different extraction methods of lipopeptide were studied.The results indicated that activity remained 84.76% after 30 min with 100℃ and remained above 50% after exposure to ultraviolet light for 3 h,and the p H tolerance ranged from 3~9.The antibacterium spectrum of thefermentation broth was evaluated and the results showed that it exhibited antibacterial effect to all of the 15 aquatic pathogens.(4)Bacillus sp.P1 and P6 were used as the starting strain,and UV mutagenesis was carried out to improve the yield of lipopeptide.The results showed that the total lipopeptide content of these mutant strains increased by 66.3%,92%,5.2% and 31% respectively.The results of RT-PCR showed that the expression of sfp gene,itu D gene and fen A gene in the mutant strain P1-120-15 were 1.19 times,1.09 times and 1.11 times compared to the starting strain P1.But the results were not significantly different.(5)Thetraditional and high-throughput method for screening of lipopeptide-producing strains were both studied.It was found that the traditional method has the disadvantages of low through-put and sensitivity.Two high-throughput method,namely CPC-BTB method and BTB method were further explored to establish a high-throughput method for screening of lipopeptide-producing strains.The results showed that the BTB method was more accurate and stable,thus is more suitable for screening.