Screening Of Mutant Strain Muths-407 From Bacillus Subtilis And Purification And Application Of Active Substances Produced

Bacillus subtilis is a probiotic microorganism,and most of the antibacterial substances produced are low molecular antimicrobial peptides(Lipopeptides).The antibacterial peptide is a kind of small molecule polypeptide with antibacterial activity produced by the body in the process of resisting pathogenic microorganisms.It has the advantages of resistance to medicine,low toxicity,broad antibacterial spectrum,high thermal stability and biodegradability.However,the low level of antimicrobial peptides produced by Bacillus subtilis makes it impossible to be produced on a large scale,which severely limits its development and application.In order to obtain high-yield antimicrobial peptide strains,the Bacillus subtilis mutant mutHS-301,which has been obtained in the laboratory,was used as a starting strain to further carry out buy UV chemical compound mutagenesis and random screening.The oxford cup method was used for primary screening and rescreening.Finally,a mutant strain with good bacteriostatic effect and stable genetic property was obtained,named as Bacillus subtilis mutHS-407.The mutagenized strain was subjected to shake flask fermentation,and the fermentation broth was subjected to solid-liquid separation.The obtained supernatants were separated by acid precipitation and extracted with an organic solvent,and finally concentrated and dried to obtain a crude extract.The extract had a yield of 2.305 g/L,and the yield was increased by 17.3%compared to that of the original strain.The bacteriostatic experiment showed that the substances produced by the strain mutHS-407 had significant antibacterial activity against Staphylococcus aureus,Vibrio parahaemolyticus,Pseudomonas aeruginosa and Aspergillus flavus.The molecular weight of the active substances contained in the extract was found to be 3.3 Da or less by Tricine-SDS-PAGE gel electrophoresis analysis.The crude extract was further separated and purified by silica gel column chromatography and macroporous adsorption resin.As a result,a single component was obtained and it was found to have strong antifungal activity.Further analysis of the purified components by high performance liquid chromatography(HPLC)showed that the fraction produced a single peak at a retention time of 16-18 min,which was as high as 92.42%.Based on mass spectrometry(MALDI-TOF-MS)analysis and data comparison,the main component was identified as Bacillomycin D.The active extract produced by the fermentation of the strain mutHS-407 was further applied to the antifungal test of peanut.Firstly,through screening of the environmental conditions of peanut storage,the conditions for simulating the peanut resistance to aflatoxin were determined.The water content was 15 mL in 50 g peanuts,and the inoculum amount of Aspergillus flavus(1.5×10~4 cfu/mL)was 400?L,stir well and incubate at 37°C for 6 d,at which time all the peanuts are moldy.Secondly,the peanuts inoculated with Aspergillus flavus were treated with different concentrations of the antibacterial extract,and sensory evaluation was carried out 10 days after storage culture.The results showed that the addition of 120mg/mL antibacterial extract could effectively inhibit the mildew reaction of peanut aflatase.Finally,the strain mutHS-407 fermentation broth was spray-dried directly to obtain the antibacterial peptides dry powder,and the product was compared with the common anti-mold agent for commercial application.The results showed that,in addition to some chemical anti-fungal agents,The antibacterial extract of the experimentally prepared antibacterial extract was significantly higher than other biological antifungal agents.Therefore,the antibacterial extract is expected to be developed into a novel biological antifungal agent and applied to the food and feed industry.