Optimization Of Fermentation Conditions And Separation Of Active Substances Of Marine Streptomyces HS-B31

Streptomyces are a group of microorganisms,and they can produce a variety of secondary metabolites.The antibiotics isolated from Streptomyces are the most widely used and studied in practice.Marine microorganisms live in a special environment with low temperature,high salt,high pressure and oligotrophic conditions,and such a special environment enabling them to produce novel active substances.In this study,the culture medium and culture conditions were optimized to increase the production of active substances of marine Streptomyces HS-B31,and the extracts of fermentation broth were isolated,purified and preliminarily identified in order to find novel active compounds.The results are as follows:?1?On the basis of the original medium,the strain HS-B31 was used as the original strain for shaking flask fermentation.Firstly,the antimicrobial activity of fermentation broth was taken as the index,and the composition of the medium was selected by single factor experiment.The optimal carbon source was corn starch,the optimal nitrogen source was peptone,and the compound inorganic salts were K₂HPO₄·3H₂O,MgSO₄·7H₂O and CaCO₃.The above five single factors were optimized by orthogonal test.The L₁₈?3⁷?orthogonal table was used to obtain the optimal medium composition by the range analysis method.The results showed that the optimized medium were corn starch 25 g/L,peptone 1 g/L,K₂HPO₄·3H₂O0.3 g/L,MgSO₄·7H₂O 0.3 g/L and CaCO₃ 0.4 g/L.As a result,the antimicrobial activity of fermentation products of strain HS-B31 against Staphylococcus aureus increased by 29.6%,and the antimicrobial activity of fermentation products of strain HS-B31 against Vibrio parahaemolyticus increased by 35.1%.?2?On the basis of optimized media,the culture temperature of the strain HS-B31,the initial pH of the medium and the fermentation time were optimized by single factor experiment method.The optimal fermentation conditions were obtained as follows:initial fermentation broth pH 7.2,temperature 28?C,rotation rate 220 r/min,fermentation time 7days.Then the thermal stability and acid-base stability of the antibacterial active substances were studied.The results showed that the antibacterial active substances had good stability at pH 3¹1,and had good stability at 10⁶0?C.?3?The fermentation broth of the strain HS-B31 was extracted with ethyl acetate,and the organic phase was concentrated to dryness under reduced pressure,and then dissolved with a small amount of methanol to obtain the extract of the fermentation broth.Tricine-SDS-PAGE analysis of the crude extract showed that the molecular weight of the crude extract of strain HS-B31 was less than 3.3 kDa.Four components A₁,A₂,A₃ and A₄,were separated from the extract of strain HS-B31 by TLC and column chromatography.The bacteriostasis experiments were carried out on four components by the filter paper method with Vibrio parahaemolyticus as indicator bacteria.The results showed that the components A₁,A₂ and A₃ had antibacterial activity,and the effects of A₂ and A₃ were obvious.This study laid a foundation for further pilot fermentation and structural identification of active components A₁,A₂ and A₃.