Isolation,Purification And Structural Identification Of Antibacterial Active Substances Produced By Marine Steptomyces HS-B34

Streptomyces is a resource-rich microbial group capable of producing a variety of secondary metabolites.Marine Streptomyces survives in a special environment of low temperature,high salt,high pressure and low nutrition.Such special environment enables it to produce active substances with novel structures.In this study,a strain of marine Streptomyces HS-B34 obtained from the previous screening in our lab was further explored to isolated active substances and identify active single compounds with novel structures.The findings are as follows:(1)On the basis of Actinomycetes culture medium,the strain HS-B34 was used as the initial strain to optimize the shake flask fermentation conditions.The optimized culture medium composition was:corn starch 25.0 g/L,peptone 1.0 g/L,K₂HPO₄·3H₂O 0.3 g/L,Mg SO₄·7H₂O 0.3 g/L and Ca CO₃0.4 g/L.Next,the culture conditions of strain HS-B34 were optimized,and the optimal fermentation conditions were determined as the initial fermentation broth p H 7.0,shaking speed 220 r/min,fermentation temperature 28?C and fermentation time 7 d.As a result,the antibacterial effect of the fermentation product from strain HS-B34 on Vibrio parahaemolyticus increased by 33.4%.(2)An equal volume of ethyl acetate is further added to the fermentation broth of strain HS-B34 for extraction.After extraction,the organic phase is concentrated under reduced pressure and evaporated to dryness to obtain an extract of the fermentation broth.The extract was separated and purified by silica gel column chromatography to obtain four single components,namely A₁,A₂,A₃and A₄.Then each component was tested for bacteriostatic activity.The results showed that components A₁and A₂had bacteriostatic activity,and the bacteriostatic effect of A₁was the most obvious.Finally,the active components were analyzed by high performance liquid chromatography,and the results showed that the purity was high.(3)Next,ESI-MS analysis technology was used to identify the molecular mass of the active component A1 with a large amount of separation and strong activity.According to the ESI-MS(positive)spectrum,[M+H]⁺=279,[M+Na]⁺=301,which shows that the relative molecular mass of substance A₁is 278.Fourier infrared absorption spectrum was used to analyze the molecular structure of A₁.The spectrum was divided into three bands for analysis,which were 400?1200cm^-1,1200?1900cm^-1and 2500?40000cm^-1.Finally,combined with the analysis of its molecular mass,compound A₁was identified as dibutyl phthalate(DBP).(4)Take several pathogenic bacteria in the laboratory as indicator bacteria,the antibacterial spectra of DBP produced by component A₁was measured by using the filter paper method.The compound has antibacterial activity against several common pathogenic bacteria,and has the most obvious antibacterial activity against Vibrio parahaemolyticus.This study laid the foundation for the scale-up production of active substance A₁and the industrial application development of DBP.