Lipopeptide Surfactants Producing Strain Screening And Product Characteristics

Biosurfactant(BS)are compounds containing hydrophilic groups and oilophilic groups produced by microorganisms.Usually produced by the metabolism of bacteria,yeasts,and fungi in nature,especially bacteria that grow in water insoluble substrates have higher yields.Lipopeptide biosurfactants are cyclic lipopeptides produced by the metabolism of Bacillus sp.,surfactants are known for their special surface activity.The producing bacteria are widely distributed in the upper layer of soil and plant rhizosphere,and are often exposed to harsh environmental conditions.In order to adapt to the harsh environment,Bacillus subtilis promotes the adsorption,emulsification,wetting,dispersion,or dissolution of insoluble substances by the strain itself through the synthesis of surfactants.Surfactan is generally composed of a small peptide of 7amino acids linked to C13-C15 via lactone bonds β-Hydroxy fatty acids are linked to form cyclic lipopeptides.It has antibacterial,antiviral,enhanced oil recovery,hemolysis,and other functions,and is widely used and in large demand in industry,environment,agriculture,food,and daily chemical industries.However,at present,the biological yield of Surfactin is extremely low and the difficulty of extraction and purification is high.Its production cannot meet the current market demand.The main studies and their results are as follows:First of all,in this paper,using the surface activity and hemolytic of surfactant,we used cetylpyridinium chloride-brominated thymol blue(CPC-BTB)combined with blood agar plate for the screening of producing lipopeptide.The main sample was taken from the underground soil of a gas station in Foshan,Guangdong Province,and a bacterium producing surfactant was isolated from it.After physiological and biochemical experiments and 16 S r RNA sequencing analysis,the strain was identified as a Bacillus subtilis strain and named Bacillus subtilis SX-20.The products of SX-20 fermentation were first extracted by acid precipitation,methanol solution extraction,and vacuum rotary evaporation.The following methods were adopted to analyze the properties and structure of the product: ⑴oil displacement test and droplet collapse test;⑵ Fourier Transform Infrared spectroscopy(FTIR)analysis(FTIR);⑶ amino acid analysis;⑷ Liquid Chromatography-Mass Spectrometry(LC-MS);⑸ bacteriostatic experiment.The surfactant surfactin C was determined to be a ring lipid peptide consisting of 15 carbon atom fatty acid chains and amino acid(Asp: Glu: Val: Leu ratio 1:2.5:1:4).The activity analysis results indicate that the Surfactant has an inhibitory effect on Propionibacterium acnes,and similar studies have not been reported yet.Then to increase the production of surfactant,fermentation medium and fermentation conditions were optimized by single factor experiment,and PB experiment was carried out.The results showed that the composition of Landy medium had little effect on the synthesis of surfactant.The surfactin C yield reached 1.03 g/L after 36 h of fermentation at 25°C at a concentration of 5%.Finally,to further improve the production level of surfactin C by SX-20 strain,strong promoters P43,Papr E,and Pglv were amplified from B.subtilis168,and the promoter sequence of the srf A gene of SX-20 was replaced by homologous recombination methods,with a view to improving the expression level of srf A and promoting the synthesis of surfactin C.Currently,this research is still ongoing.