The Fermentation Conditions And Structure Identification Of Biological Antiseptic Substances Produced By Actinomycetes

With the improvement of food safety demands among people, the exploitation of new microbial food antiseptics and their application in food industry can satisfy people’s requirement of food safety that has broad development prospects. The species diversity and metabolic diversity of soil actinomycetes are very rich. The important secondary metabolites produced by actinomycetes that have great academic and commercial value and receive worldwide attentions. The screening of microbial food antiseptic with significant antibacterial activity, which is important for guaranteeing food safety and improving the quality of the product.Guangzhou is located in the subtropical zone of rich ecosystem diversity, species diversity and genetic diversity in China, which contains huge actinomycetes resources with vast development and utilization potential. Therefore, the development and utilization of soil actinomycetes with antibacterial activity have been hot issues that always be interested by scholars.In this research, total of 64 soil samples were collected from 11 sampling positions of Guangzhou and its surrounding areas.190 strains of actinomycetes were isolated with plate dilution method. The isolation result showed that the amount and diversity of soil actinomycetes were the most abundant in vegetable garden soil, followed by orchard soil and forest soil, hardly any in river soil flower garden soil. According to the results, actinomycete content and diversity had connection with the organic matter content of soil. Soil actinomycetes for antibacterial activity were screened through antibacterial test. Preliminary screening used agar plug method with 5 kinds of indicative bacteria.134 strains were obtained in this process.7 strains with significant antibacterial activity were considered as optimal strains through screening,then 7 indicative bacterial and fungus strains were choosed, the antibacterial activity of 7 optimal strains were determined furtherly.Through the analyses of 16S rDNA sequence of 2 optimal strains compared to Streptomyces, the similarity over 99%.The result of phylogenetic position showed that the strains PY-C-21 and Streptomyces toxytricini were in the same branch,also the strain ZC-G-5 and Streptomyces albulus were in the same branch.Therefore,they were homologous and had close relationships,then the results of morphological and cultural characteristics observation, physiological and biochemical tests showed that the 2 optimal strains compared with cluster analyses of related homologous strains were basically the same, therefore, the strain PY-C-21 was identified as Streptomyces toxytricini,the strain ZC-G-5 was identified as Streptomyces albulus.In the optimization of fermentation conditions of the optimal strains, through single factor experiment, on the basis of the initial fermentation medium as Gause 1 medium, the best carbon source and nitrogen source of the fermentation medium were determined as starch and potassium nitrate respectively, the best fermentation time of the strain was 7 d, the best fermentation pH was 7, best inoculation amount was 10%, the optimal loading fluid amount to 30 ml (100 ml conical flask fermentation capacity), the best rotational speed was 160 r/min, the best fermentation temperature 28 °C.In the structure identification of active products, first of all, the fermented products of strain PY-C-21 were prepared, the crude extracts were obtained by ethyl acetate and dichloromethane extraction, then the crude products were refined through silica gel column chromatography furtherly, the pure compounds were got through high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and preparative chromatography. Lastly, the active products were identified as dibutyl phthalate, benzoic acid, phenol, 2,2’-methylenebis[6-(1,1-dimethylethyl)-4-methyl-, bis(2-ethylhexyl) phthalate by HPLC,gas chromatography mass spectrum(GC-MS) and high resolution mass spectrum(HRMS).This study on the existing bases in the lab, which has enriched the repository of antimicrobial strains that produced antimicrobial active substances and the separation and purification of antimicrobial active substances and related studies on food biological preservative furtherly, meanwhile a batch of optimal strains were obtained through screening, the optimal strains can be used as initial strains, the industrialization of objective products can have been achieved through the strain mutagenesis breeding and fermentation optimization in the future, finally which looking forward to the application in the field of microbial source food preservatives and can improve the effect and safety of food biological antiseptic constantly.