| The synthesis of lipopeptide by Bacillus subtilis is the combination of between the β-hydroxy fatty acid and peptide through the lactone bond. This lipopeptide is a kind of biosurfactant with the fatty acid chain as the hydrophobic group and the amino acid chain or ring as the hydrophilic group. This biosurfactant is characterized with high performance, low toxicity and easy degradation. It has great potential in terms of the biological production, medicine, materials, environmental protection, food and cosmetic product, which has already become the hot spot for new biological reagents research. This paper mainly focus on plasma at normal atmosphere and room temperature(ARTP) mutagenesis breeding, optimization of shaking flask fermentation condition, the nature of lipopeptide and identification are studied. The following is the main research of this paper:⑴ Flask fermentation conditions are optimized for the starting strain. Shaking flask fermentation medium is obtained by single factor experiment preliminary(g/L). sucrose 30, urea 4, yeast extract 1, potassium dihydrogen phosphate 10, disodium hydrogen phosphate 4, sodium chloride 5, magnesium sulfate 0.3, monosodium glutamate 1.2, p H 8.2. Cant using kinds of inoculation, 200 rpm, 37 ℃72 h, lipopeptide yield increased from 0.45 to 3.21(g/L), discharge of oil ring diameter from 3.6 to 6.5(cm).⑵ By using the ARTP plasma mutagenesis technology on the strain, the strain with high yield is slants big, middle bulge, high foam, slightly white, wrinkled edges, more sticky and picked with drawing. After repeated mutagenesis breeding,our lab get a high-yield genetic stability strain and named Bacillus subtilis SF90. Compared to the starting strains, shake flask fermentation period reduced to 60 h, the activity of biosurfactant is enhanced to 2.3. The crude yield of lipopeptide is enhanced from 1.93 to 5.1(g/L).⑶ Metal elements Zn2 +, Fe2 +, Mn2 +, Cu2 + single factor experiment showed that trace amounts of metal ions on lipopeptide synthesis has a significant role in promoting. Orthogonal experimental results show that the Zn SO4·7H2O is added on the culture medium of 1.0 mg/L, the Fe SO4·7H2O 0.2 mg/L, the Mn SO4·H2O 0.5 mg/L, about 3 times higher surface activity, lipopeptide yield increased from 6.11 to 11.12(g/L).⑷When added at earlier stage of then fermentation(0-12)h or the later stage(44-48)h and at optimized concentration(0.001-0.01)g/L of aminoglycoside antibiotics(spectinomycin, gentamycin, toxin), this can greatly improved the yield of lipopeptide, trace amounts of spectinomycin can cause late fermentation bacteria form elongate, conducive to reduce the feedback inhibition of intracellular, and make lipopeptide about 2.2 times higher surface activity, the yield increased from 3.3 to 6.02(g/L).⑸Using Bacillus subtilis SF90 fermented supernatant, the research on lipopeptide about tolerance of temperature and p H, and emulsification, flocculation and antibacterial, etc. The activity of the biosurfactant is reduced by 10% when the supernate is treated with 121 for 2 hours. It can tolerate low temperature, the ℃activity did not change when stored at 4℃for 3 months, the tolerant p H range is 2-12, when the solution of weak acid and weak alkaline p H has more excellent biological surface active. The fermentation broth have good the emulsification performance to crude oil. It has a more stable emulsification effect than detergent. Compared with the condition of alum and poly glutamic acid, lipopeptides also has good flocculation as well. Tube plate method for the determination of antimicrobial spectrum: the volume added in the Oxford Cup is 50 ul, it has the greatest antibacterial effect on Bacillus cereus、Pseudomonas aeruginosa, and the MIC is 0.1mg/ml、0.25mg/ml separately. It doesn’t have influence on accharomyces cerevisiae, it also has great effect on Ustilagomaydis corolla, and the MIC is 0.15mg/ml. At the same time, experiments are done on 4 pathogens for plants, whose result is that it has great effect on Rice sheath blight fungus, whole wheat phage, not good on Watermelon anthrax, and totally not work on Cotton Verticillium Wilt.⑹By MALDI-TOF-MS technique to identify the components of lipopeptide freeze-dried samples, the results showed that Bacillus subtilis SF90 to produce lipopeptide with Surfactin, Bacillomycin D, Fengycin composition. Among of them, Surfactin as main material, the peak time of 21.5-25(min), mass-to-charge ratio(m/z) mainly includes 1008.6615, 1022.6710, 1036.6893, 1058.6682, 1044.6682, 1030.6370, 1059.6711, etc. Followed by Bacilloycin D, peak time is 9.7 to 12(min),(m/z) 1031.5400, 1045.5541, 1059.5696. Fengycin quantity is less, the peak time is 12.5 to 14.5(min),(m/z) mainly includes 1464.8008, 1477.8131, 1485.7795, 1500.8007, 1513.8107, 1527.8230, 1513.8107. |
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