Screening And Breeding Bacillus Natto Strain With Enhanced Production Of Surfactin

Cyclic lipopeptide is a kind of biosurfactant synthesized by microorganisms, which is composed with hydrophilic peptide ring and hydrophobic fatty acid moieties. In addition to the classical application as surfactant, they also have antibacterial, antifungal, antiviral and antitumor activity because they have unique chemical form and bola-amphiphle structure, which showed wide application perspective. Thus surfactin have a very important application in medicine, food, micro-production, cosmetics and other fields.In this study, the strains Bacillus natto NS-6 producing a high-yielding surfactin can be isolated from the existing 10 natto strains in the laboratory after screeening by blood agar plate and oil plate, as surfactin can dissolve blood cells lipopeptide surfactants with, and the only strains producing surfactin emulsifying hydrocarbons shape the oil spots. Physical mutagenesis with UV and microwave and chemical mutagenesis with DES was used to develop strains of Bacillus natto (NS-6) that enhanced surfactin productions. We finally obtained the mutant NS-64 after screening with blood plate or blood agar plate as well as screening of flask fermentation. The mutant NS-64 pruduced 2.607 g/L surfactin compared with 1.12 g/L for the wild-type. It was viewed that the mutant strain had good genetic stability of generating offspring after being times without number. It was viewed that the mutant strain had good genetic stability of generating offspring after being times without number.Based on the results of single-factored and orthogonal experiments, a Plackett–Burman design was used to evaluate the influence of factors relating to fermentation medium of the strain NS-64 in the first optimization step. It can be deduced that peptone concentration, yeast concentration and CaCl2 concentration influenced surfactin production negatively. And then, Response surface methodology was used to optimize the parameters of the 3 factors above. In conclusion, the optimized result demonstrated that the best composition of fermentation medium is peptone 1.73%, saccharose 0.5%, yeast 0.063 % , MgSO4·7H2O 8.0×10-4mol/L, phosphorus 0.05mol/L(Na2HP O4·12H2O:KH2PO4 = 4:3), CaCl2 1.385×10-4 mol/L and the best pH of the medium is 7.0. The other parameters for the production of surfactin are as follows: the proper temperature is 37℃, the best volume of the liquid fermentation medium for surfactin synthesis is 60mL/250mL, the amount of inoculation is 3% and the agitation is 110 rpm. Under the optimized fermentation circumstance, the mutant strain NS-64 screened fermented in the optimized fermentation medium three times. The yield was measured in the way of dry matter. The yield was 3.210g/L, 3.187 g/L and 3.165 g/L. The strain is fit for fermentation production with steady yield.