Study On ARTP Breeding,Fermentation Conditions Optimization And Product Characteristics Of High-yield Surfactin Bacillus Subtilis

Surfactin is a kind of lipopeptide,secondary metabolite from microorganisms,and is considered to be a promising biological surfactant because of its excellent surface activity and emulsification properties.It has antibacterial,antiviral,anti-cancer and other biological activities,making it has broad potential application value in food industry,pharmaceutical industry,environmental protection and other fields.However,the surfactin producing ability of natural strains is usually very low,this limits its application in industrial production.Therefore,it is of great significance to improve the yield of surfactin by mutation breeding of strains.In this study,Atmospheric and Room Temperature Plasma?ARTP?mutagenesis technology was employed to screen the mutant Bacillus subtilis strain of high surfactin yield;and genomics and other methods were used to explore the preliminary mutagenesis mechanism.Secondly,by using the yield of surfactin as the index,single factor and response surface optimization experiments were conducted on the fermentation process of the mutant strain to obtain the optimal fermentation conditions.Surfactin produced by mutant strain under optimal fermentation conditions was extracted and identified,and its functional properties such as surface activity,emulsification,antioxidant and inhibit proliferation of cancer cells were also studied.The main research contents and results of this research were as follows:?1?Study on the mutation breeding of strain with high yield surfactin by ARTP.Bacillus subtilis CICC 10721,was selected from 6 Bacillus by comparison as the starting strain because of its highest surfactin yield in the strains.ARTP was used for mutagenesis breeding,and its treatment condition of 24 s mutagenesis time,100 W power,and 10 SLM gas capacity was choosed,due to a 92.21%dead rate was observed at this condition,which may casuse higher positive mutation rate of the strain.The mutant strain M45 was obtained by hemolytic circle primary screening and shaker fermentation rescreening,the ratio of hemolytic circle diameter to colony diameter was 6.11.The mutant strain's surfactin yield was 34.2%higher than that of the original strain,and the yield kept stability in the subculture.This result suggests that ARTP had a good effect on improving surfactin yield of Bacillus subtilis.Through the fermentation kinetics study,it was found that the maximum cell dry weight,maximum growth rate and surfactin synthesis parameters of the mutant strain M45 were all greater than those of the original strain,indicating that the growth performance of the mutant strain M45 and surfactin producing ability in fermentation were both higher than that of the original strain.?2?The preliminary mechanism of ARTP treatment on Bacillus subtilis CICC10721 was explored.It was observed with scanning electron microscopy that the morphology of the bacteria changed after ARTP treatment,and some phenomena such as bending,protrusion,depression,breakage and lengthening were also observed.In addition,the intracellular calcium ion concentration of the mutagenesis significantly increased,indicating that the ARTP treatment changed the cell membrane permeability of Bacillus subtilis.Through genome resequencing analysis,it was found that 66 SNP non-synonymous mutation sites occurred in the mutant M45 compared with the original strain,involving 30 genes.Amino acid sequences encoded by differentially expressed Genes were analyzed in GO?Gene Ontology?,KEGG?Kyoto Encyclopedia of Genes and Genomes?,eggNOG?evolutionary genealogy of Genes:Non-supervised Orthologous Groups database?,respectively.It was found that dltC,ytrC and ycbP were related to membrane composition,OTC,argF and argI were related to amino acid transport and metabolism,argF,yobL and pbpA were related to organic acid binding and metabolism,and fevC,ytrC,secDF and agG were related to membrane transport.?3?The yield of surfactin was used as the main evaluation index to optimize the fermentation process of mutant strain M45.The results of single factor experiment showed that the main factors affecting the yield of surfactin were the initial pH,inoculum and glucose concentration.After response surface optimization experiment,the optimal fermentation conditions were obtained as follows:glucose content 4.35%?W/V?,ammonium nitrate content 1%?W/V?,inoculum of 4.68%?V/V?,initial pH7.14,fermentation temperature 30?,shaking speed 180 r/min;in this condition,the yield of surfactin were 2.15 mg/mL,increased by 148%than the initial yields without optimization,respectively.?4?The structure identification and functional characteristics of surfactin produced by mutant strain M45 were studied.The purity of surfactin extracted from fermentation broth was found to be 89.3%by HPLC analysis;and the main surfactin types were identified to be C₁₂ surfactin,C₁₃ surfactin,C₁₄ surfactin,C₁₅ surfactin and C₁₆ surfactin by amino acid analysis and Liquid Chromatograph Mass Spectrometer analysis.It was observed that the critical micelle concentration of surfactin extract was about 0.1 mg/mL and the surface tension was reduced by 56%to 31 mN/m compared with the blank group;the emulsification index of surfactin extract solution reached 75%,and this index of 5 mg/mL surfactin extract solution only decreased 2.7%in 5 days.The results of antioxidant activity study showed that the IC₅₀ concentration of surfactin extract on 1,1-diphenyl-2-picrylhydrazyl?DPPH?free radical scavenging rate was 4.42 mg/mL.The inhibitory effects of surfactin extract on the proliferation of Caco-2 colon cancer cells,HepG2 liver cancer cells and Hela cervical cancer cells were studied.And the surfactin extract showed significant inhibitory effects on the proliferation of the three types of cancer cells,with IC₅₀ concentrations of 293.11,90.94 and 81.67 g/mL after 24 h,respectively.