Breeding On High Yield Of Bacillomycin Strains And Its Application On Rice Mildew Prevention

Bacillus subtilis fmbJ was isolated and selected by our laboratory independently.Many researches of this strain have been done including antimicrobial spectrum,fermentation process,structure identification of antimicrobial peptide and antibacterial mechanism.Bacillus subtilis fmbJ can produce multiple antimicrobial peptides including surfactin,fengycin and bacillomycin,which have important potential applications in animal husbandry,agriculture and food industry.The structure of bacillomycin is similar to iturin and its length of carbon chain is C₁₄-C₁₇.Compared with other antimicrobial peptides,bacillomycin has a strong inhibitory effect on fungi especially Aspergillus flavus,and it has been used for controling plant pathogens.However,the low bacillomycin yield of Bacillus subtilis fmbJ makes it difficult for large-scale production,which limits its industrial application.Using Bacillus subtilis fmbJ as the original strain,a mutant strain with high-yield bacillomycin was screened out through different methods of mutagenesis and protoplast breeding.Bacillomycin produced by the mutant strain was applied to prevent rice from Aspergillus flavus mildew.The results are as follows:1.Mutant strains with high-yield bacillomycin were bred using physical mutagenesis,chemical mutagenesis and combinational mutation.Strain fmbJ cells were treated with UV irradiation and the low energy N⁺ ion implantation.According to positive mutation rate and death rate,the following conditions were determined:the optimal mutagenesis time of UV irradiation and dose of N+ion were 60 s and 1000×10¹² ions/cm²,respectively.The optimal mutagenesis concentration of NTG and EMS were 800μg/mL and 1.0 mol/L,respectively.After EMS and UV irradiation treatment,the highest death rate was achieved when the mutagenesis concentration of EMS was 0.6 mol/L and the UV irradiation treating time was 60 s,which has a better mutagenic effect than single factor mutation.Three stable and high-yield mutants UV60-4,N84 and EMS-UV-43 were ultimately selected out and UV60-4 has the highest production of bacillomycin up to 306.31 mg/L,which was 1.32 times of the starting strain.2.The protoplast fusion methods of two mutagenic strains UV60-4 and N84 were studied.The optimal conditions for the preparation of protoplast were as follows:using cells in middle-later logarithmic growth stage after 6 h culture in the complete medium,the lysozyme concentration was 0.1 mg/mL,the fusion temperature was 37 ℃,the fusion time was 10 minutes.Under the optimal condition,the protoplasm formation rate was more than 95%and the renewable rate was more than 22.5%.The optimum inactivation conditions were determined:parent strains were heat-inactivated in boiling water for 90 min and inactivated by ultraviolet treatment for 120 min（30 w,15 cm）.By using the above protoplast fusion methods,a high-yielding fusion strain UV-N-61 was obtained with the maximum production of bacillomycin up to 344.71mg/L,which was 1.39 times of the starting strain and was 12.54%higher than the mutant strains.3.In the study of preventing rice mildew caused by Aspergillus flavus,the amount of bacillomycin that can effectively control mildew was determined.Rice treated with different concentration of bacillomycin,6ml water and 500 μL Aspergillus flavus（3.3×10³ cfu/mL）were stored at 37℃ for 15 days,The results showed that 0.15 mg/g bacillomycin treated rice have good sensory properties in which the amount of mold was 1.7×10⁴ cfu/g and fatty acid content was 61.34 KOH/100g dry basis.In conclusion,0.15 mg/g bacillomycin can effectively inhibit Aspergillus flavus caused rice mildew.