| Bacillus subtilis can synthesize a variety of small molecule antimicrobial peptides through a non-ribosomal pathway.This type of antimicrobial peptide has the advantages of broad bacteriostasis,low drug resistance and high thermal stability,which are widely used in food,cosmetics,pharmaceutical and other industries.However,due to the current problem of low yield of metabolites of the original strain of B.subtilis,it is difficult to meet the industrialization of mass production.Therefore,it is particularly important to find a fast and efficient method to increase the production of antimicrobial peptides.The aim of this study was to use the mutagenized strains mut HS-301 and mut HS-407 obtained from B.subtilis original strain HS-A38 as the starting strain,and to screen the high-producing antimicrobial peptide strain by recursive protoplast fusion technique and analyze its metabolites for laying the foundation for industrial production applications.The main findings are as follows:1.We studied the optimal conditions for protoplast preparation,regeneration,inactivation and fusion of B.subtilis mutagenized strains mut HS-301 and mut HS-407.The results showed that:（1）the optimal culture conditions were secondary culture to collect the cells within 12-13h later in the logarithmic phase;（2）the optimal conditions for protoplast preparation and regeneration were the protoplast formation rate was over 98%when the protoplasts were treated with 0.2 mg/m L lysozyme at 37°C for 25 min and the regeneration rate was up to 15.2%when0.1 mol/L sodium succinate as the hypertonic stabilizer in regeneration medium;（3）the heat inactivation conditions and UV inactivation were determined and heat inactivation was 100°C water bath for 40 min and UV inactivation for 80 min（15 w,20 cm）;（4）the optimal fusion conditions were determined and the inactivated protoplasts were treated with 40%PEG 6000（p H 9.0）and 0.01 mol/L Ca Cl2 at 37°C for 15 min,and the fusion rate reached 3.70×10-6.2.In order to obtain high-producing antimicrobial peptide strains,we used two generations of recursive protoplast fusion technology to screen.After 9 generations of genetic stability verification,a stable high-producing fusion F2-95 was obtained.Moreover,the extract of antibacterial peptide was prepared by acid precipitation and methanol extraction,which was analyzed by RP-HPLC and LC-MS in subsequent experiments.The results revealed that the high-producing fusion strain F2-95 had the same retention time of each peak compared with the original strain HS-A38,and the peak response value of the retention time was 8-10 min.A total of 14 active peaks were detected on the LC/MS chromatogram,the molecular weight of No.1peak belongs to Bacilysin;the molecular weight of Peaks 2-6 belongs to Bacillomycin,of which Bacillomycin D is the main product and Bacillomycin L is the by-product;the molecular weight of peak 8-10 belong to Fengycin A;the molecular weights of peaks 7 and 11-15 belong to Surfactin.By calculating and comparing the peak areas of the peaks,it is concluded that the peak of Bacillomycin D has the largest peak area and the highest content,indicating that the antibacterial peptide produced by the experimental strain is mainly Bacillomycin D.3.In order to further verify the increase of yield of high-producing fusion strains,the fusion strain F2-95 and experimental strains were analyzed and compared from fermentation yield yield,bacteriostatic activity and molecular biology.The results showed that the fusion strain F2-95 increased the extract yield by 35.27%relative to the original strain HS-A38.Antibacterial activity test found that the inhibitory effect of the fusion strain F2-95 on Staphylococcus aureus was 23.58%compared with the original strain HS-A38,and the inhibitory effect on Vibrio parahaemolyticus was increased by 24.31%.The quantitative expression of BCM,a key gene of Bacillomycin D,in the fusion strain F2-95 was compared with the original strain HS-A38 by real-time PCR.The results showed that the BCM gene expression level of the fusion strain F2-95 was 11.71 times that of the original strain HS-A38.Since Bacillomycin D is currently the only substance discovered from microorganisms and confirmed to inhibit Aspergillus flavus,these experimental results can provide more valuable data for studying the metabolic engineering application research of Bacillus subtilis. |
