Enrichment And Purification Of Antimicrobial Substances From Streptomyces Alboflavus TD-1

Grain crops from all over the world are attacked by fungal pathogens every year,causing serious economic losses.As one of the main sources of natural antibacterial substances,active substances and antibiotics,Streptomyces spp.plays a crucial role in the prevention and control of fungal diseases,natural antimicrobial substances were used to take over traditional chemical methods,and had become a new biological control alternative,attracting attention from researchers worldwide,and became a hot topic.The present results showed that secondary metabolites of Streptomyces Alboflavus TD-1 contained active products to inhibit various pathogenic fungi of crops which include Botrytis cinerea,Pyricularia oryzae,Fusahum graminearum,and Penicillium digitatum.In order to realize large-scale and industrialized production of antibacterial substances of S.Alboflavus TD-1 activity,the effects of different industrial carbon sources and nitrogen sources on the yield of active substances from S.Alboflavus TD-1 were investigated.Meanwhile,combining with qualitative and quantitative analysis method of the Oxford cup,TLC and HPLC,we found that in industrial carbon sources,potato starch could significantly promote the production of anti-fungal metabolites from S.Alboflavus TD-1,followed by soluble starch;and the most effective nitrogen source for fermentative metabolic yield was soybean meal,followed by soybean meal.The antibacterial rate of the indicator bacteria by the Oxford cup method was 66.7%.The best industrial fermentation medium components were potato starch 10 g/L,potato starch 1 g/L,potassium hydrogen phosphate 0.5 g/L,soluble starch 20 g/L,sodium chloride 0.5 g/L,magnesium sulfate 0.5 g/L,ferrous sulfate 0.001 g/L.The optimized bacteriostasis rate reached 73.97%,which was 38%higher than that without optimization.To separate and purify the secondary metabolites of S.Alboflavus TD-1,we used High-speed Countercurrent Chromatography(HSCCC)and Solsel to confirm 9 kinds of solvent separation system,and then adjust the distribution proportion of antibacterial substances in different systems.Finally,the optimal separation conditions for separating fermentation antibacterial products of S.Alboflavus TD-1 was determined,n-hexane:ethyl acetate:methanol:water ?1:6:1:5,speed of 850 r/min,25 ?,flow rate of 5 min/mL.Under the optimal conditions,5 kinds of component were separated.No.2 components had strong antibacterial activity and higher purity by HPLC,which indicated that HSCCC could use for rapid and high-throughput sample preparation of S.Alboflavus TD-1 metabolites.What's more,No.2 components were detected and identified by Fourier transform infrared spectrometer(FI-IR)and IT-TOF liquid mass spectrometer with High resolution.We found that No.2 components didn't contain other groups of elements except C,H,0 elements,and contained four compounds,molecular weight were 633.3997,634.4000,673.3961,674.3993,respectively.We infer that the three compounds were homogeneous substance the had the similar structure,and the chemical formula could be C36H56O9.