Isolation And Identification Of Secondary Metabolites From Pseudomonas Protegens FD6 And Optimization Of Fermentation Condition

With the increasingly serious problem of pesticide resistance and residues,application of beneficial microorganisms to control diseases has become an ideal green prevention and control measure.Previous research showed that Pseudomonas protegens FD6 could produce 8 secondary metabolites,including 2,4-diacetyl phloroglucinol,luteolin pyocyanin,nitropyrrolidine,Pyochelin,Pyoverdine and Orfamide,which was satisfactorily efficient to control Botrytis cinerea in tomato.In addition,three unknown secondary metabolites were produced by strain of FD6.In order to further explore the secondary metabolites produced by strain of FD6,the crude extract of FD6 fermentation was isolated and purified in this study,and the structure of the isolated compounds was further identified.The production of secondary metabolites is not only affected by the strain itself,but also affected by many factors,such as carbon source,nitrogen source,temperature,trace elements and so on.In order to improve the bacteriostatic activity of FD6,the fermentation conditions were optimized by single factor test and response surface test.The detailed results are showed as follows:1.In this study,KB medium was used to select as the best fermentation for FD6 by shake bottle fermentation from six cultures:KB,LB,TSB,DMB,PB and Landy.Compared with the other five mediums,the metabolite yield of KB fermentation was the highest and up to 500μg/mL,which was as twice as that of PB fermentation and 8-times that of Landy fermentation.1H-NMR spectra showed that the hydrogen signal of KB fermented crude extract was abundant in both high and low field regions.2.100 L FD6 fermentation liquid using KB as medium was fermented in a fermenter.The fermentation was centrifuged,extracted by ethyl acetate three times,and removed organic solvent by rotary steaming under reduced pressure to obtain a total of 16 g crude extract.Then,20 components were obtained from the crude extract after separation by normal phase silica gel column chromatography.In this research,5 selected components were used to analysis by LC-MS and TLC,and were further separated and purified by comprehensive normal phase and reverse phase silica gel column chromatography,Sephadex LH-20 dextran gel column chromatography and high-performance liquid chromatography.The results showed that two compounds were separated,that compound 1 and compound 2 were identified as dibutyl phthalate(DBP)and Methyl 2-(2-hydroxyphenyl)-4-Thiazolecarboxylate respectively by LC-MS and nuclear magnetic resonance spectroscopy.3.Using KB as the base medium,the culture medium and culture conditions for FD6 were optimized by single factor test and response surface test.The results showed that the optimal culture medium and culture conditions of FD6 were as follows:glycerol 5‰,arginine 1.98%,K2HPO4 1.5‰,MgSO4·7H2O 1.5‰,pH 6.97,fermentation temperature 24.12℃,oscillating culture for 60 h.After optimization,the inhibition rate of sterile filtrate of FD6 fermentation against Botrytis cinerea was nearly doubled than that of no optimization.The fermentation crude extract contained orfamide A,a cyclic lipid peptide compound,and is more heat resistant,but more sensitive to ultraviolet radiation.These results indicated that secondary metabolites produced by P.protegens FD6 was more abundant in KB medium,and two compounds were identified from the crude extract of FD6 fermentation which provided lead compounds for the development of biogenic fungicide with high efficiency and low toxicity.The inhibition rate of the sterile filtrate against Botrytis cinerea was increased nearly double by optimizing the medium and culture conditions which improving the disease prevention effect of biocontrol by FD6.