Optimization Of Surfactin Production And Antibacterial Effect Research

Surfactin is one of the most intensively studied biological surfactants.It not only has surface activity,but also has the characteristics of antibacterial,antiviral,anticancer,low toxicity,and easy degradation will not cause environmental pollution.With the deepening of research and the continuous improvement of separation and purification technology,the application of surfactant in the fields of medicine,food,biological control,daily necessities and petroleum industry will be more extensive,but the fermentation yield is low and the separation is difficult,which restricts the surfactant Industrial application.In this study,a high-surfactin-producing Bacillus velezensis HSB1 was obtained as a starting strain.Specific studies were carried out from the aspects of surfactant purification,structure identification,fermentation conditions optimization,and antibacterial effects.The main conclusions are as follows:1.A high performance liquid chromatograph(Waters E2695)was used to detect the content of Surfactin.The optimized liquid conditions were: column: ZORBAX SB-C18(5 um,4.6*250 mm),column temperature 35 ?,mobile phase A: pure water+0.1% trifluoroacetic acid,mobile phase B: acetonitrile +0.1% trifluoroacetic acid,detection wavelength 205 nm,flow rate 0.8 m L/min,elution conditions: 0-9 min,mobile phase A 40 %,9-25 min,Mobile phase A decreased from 40% to 7%,25-30 min,mobile phase A increased from 7% to 40%.2.The preparative high-performance liquid chromatograph(Shimadzu LC-20R)was used to separate and purify the single component of the Surfactin.Water + 0.1%trifluoroacetic acid,mobile phase B: acetonitrile + 0.1% trifluoroacetic acid,detection wavelength 205 nm,separation time 30 min,90% mobile phase A for isocratic elution,flow rate: 10 m L/min.The injection volume was 2000 u L.Using liquid chromatography-mass spectrometry to analyze the separated components by mass spectrometry,the separated singlecomponents were determined to be C12 Surfactin A,C13 Surfactin A,C14 Surfactin A,C15 Surfactin A,C16 Surfactin A and C15 Surfactin B.3.Surfactin fermentation medium screening test showed that the best fermentation medium for the production of Surfactin by strain HSB1 was Landy medium,which was cultivated under the conditions of 30 ?,150 r/min,200 m L/500 m L of liquid loading,and 5% inoculation volume.h,the output reaches 612.39 mg/L;The single factor test showed that fermentation temperature,rotation speed,filling volume and inoculation volume had a significant effect on the production of Surfactin.The optimal conditions were rotation speed 175 r/min,filling volume 175 m L/500 m L,inoculation volume 5% and temperature 33 ?;Precursor addition test showed that adding 2.5 mmol/L L-Leu to Landy fermentation medium can increase the production of Surfactin by 50.9% to 928 mg/L,and the proportion of C15 Surfactin A increased from 32.48% to 42.86 %,an increase of 10.38%,and the addition of D-Leu will inhibit the accumulation of Surfactin,when the amount of D-Leu is 1.5 mmol/L,the production of Surfactin is reduced by 66%;add 0.7 mmol/L of L to the fermentation medium of Landy-At Val,the production of Surfactin is increased to 699.2 mg/L,while at the addition of 1 mmol/L,the production of Surfactin is suppressed and the production is reduced by 24%;Response surface optimization showed that the best fermentation conditions were: adding 2.5 m M/L L-Leu to Landy fermentation medium,inoculation volume5%,fermentation temperature 35 ?,rotation speed 175 r/min,filling volume 125 m L/500 m L The output of Surfactin is the highest,which has increased by 59% from612.39mg/L before optimization to 973.52 mg/L.4.Confrontation experiments showed that the strain HSB1 had significant inhibitory effects on Micrococcus luteus,Xanthomonas,Staphylococcus aureus and fungi.The minimum inhibitory concentrations of C12 Surfactin A and C15 SurfactinA against Staphylococcus aureus are 50 ug/m L and 37.5 ug/m L,indicating that the longer the carbon chain of Surfactin,the better the bacteriostatic effect;PI staining results show that The mycelium of Fusarium graminearum treated with 80 ug/m L of C15 Surfactin A for 4 h emitted obvious red fluorescence under green excitation light,indicating that Surfactin would damage the cell membrane of Fusarium graminearum mycelium,thereby inhibiting the growth of mycelium.