Studies On Antimicrobial Components From Fermentation Broth Of Some Actinomycetes Strains

Microbial resources is a kind of potential renewable medicinal resource and one ofthe most important sources of lead compounds and new drug. Actinomycetes is veryclose to human life, and it is the resources of biological activity material that is moreabundant than other microorganisms.This paper is about the further research work on one actinomycete strainStreptomyces alboflavus313, including the influence of the antibacterial activity andthe content of the metabolism product by feeding precursor into the fermentation brothof the strain, purification of the active compouds and structural identification. Inaddition, it is about the research work based on three strains SC11、11D2-9and GXWl,including evaluation of antimicrobial activity, purification and structural identificationof the main active metabolite, strains identification. The primary results are as follows:1. The influence of the antibacterial activity and the content of the metabolismproduct has been studied by feeding precursor into the fermentation broth of the strainStreptomyces alboflavus313. The results showed that the content of metabolites m/z750,723,860and743was improved than the contrast by10.50times,22.48times,16.73times and12.59times respectively after feeding0.5g/L L-glutamate.2. Guided by the results of the bioassay, an antimicrobial component, novelnon-chlorinated cyclic hexapeptide NW-G12was isolated from the fermentation of S.alboflavus313. The difference between NW-G01and NW-G12is the Trp of NW-G12was not chlorinated. Comparison of the antibacterial spectra and inhibition activities ofNW-G01and NW-G12, no obvious differences were observed. It indicated that thepresence of chlorine atom at Trp was not a decisive factor for the antibacterial activity.Also, an active compound F2was separated from the fermentation broth of S.alboflavus313. According to analysis of MS,1H NMR and13C NMR spectroscopydatas, the molecular structure of F2was identified as Desertomycin A.3. The mechanism of action of compound NW-G01against Botrytis cinerea wasstudied. After treated with NW-G01at100μg/mL for12h, the mycelial reducing sugar,chitosan, soluble protein and pyruvate content, chitinase activity showed decliningtendency. 4. Guided by the results of the bioassay, an active compound F5was separatedfrom the fermentation broth of the strain SC11. Bioassay results in vitro showed that F5had strong antibacterial activity against gram-positive bacteria Bacillus cereus, Bacillussubtilis and Staphylococcus aureus with the MIC values of3.9μg/mL,7.8μg/mL and7.8μg/mL, but it had no obvious antibacterial activity against gram-negative bacteriaEscherichia coli and Pseudomonas aeruginosa with the MIC values of more than250μg/mL. It also exhibited inhibitory effect on mycelial growth against pathogenic fungiof main crop, such as Colletotrichum gloeosporioides, Verticillium dahliae, Gibberellazeae, Alternaria alternata, Fusarium oxysporum, Sclerotinia sclerotiorum and Botrytiscinerea. According to analysis of MS,1H NMR and13C NMR spectroscopy datas, themolecular structure of F2was identified as Desertomycin A.5. An analytical method for the determination of Desertomycin and its analoguesin microbial fermentation broth by LC-MS was established. The method was suitablefor rapid detecting Desertomycin and its anologues in the early-stage of antibioticsscreening from the fermentation broth to avoid repeated screening.6. Bioassay results in vitro showed that the fermentation broth of the strain11D2-9showed strong antibacterial activity against gram-positive bacteria. But had noobvious inhibitory effects against gram-negative bacteria and plant pathogenic fungi.The results of pot experiment indicated that the fermentation filtrate had69.33%protective efficacy and61.93%curative efficacy against Blumeria graminis. Guided bythe results of the bioassay, an active compound was separated from the fermentationbroth of the strain11D2-9. According to analysis of MS,1H NMR and13C NMRspectroscopy datas, the molecular structure of the compound was identified asAlborixin.7. Bioassay results in vitro showed that the fermentation broth of the strain GXWlshowed good antibacterial activity against gram-positive bacterium, gram-negativebacterium and plant pathogenic fungi. Guided by the results of the bioassay,antimicrobial protein bands3-1and3-2were separated from the fermentation broth ofthe strain GXWl. According to technology of LC-MS/MS, the bands were identified asPhosphate-binding Protein Precursor.8. According to the results of morphological characteristics, culture characteristics,physiological and biochemical measurement and analysis of16S rDNA sequence,strains SC11,11D2-9were highly homological (99%) with the type strain Streptomycesalboflavus NBRC-13196Tand GXWl was highly homological (99%) with the typestrain Streptomyces rubiginosohelvolus NBRC12912T. The strains were similar features in majority with the type strain. Therefore, the strain SC11,11D2-9and GXWlwere suggested to identify as Streptomyces alboflavus SC11, Streptomyces alboflavus11D2-9and Streptomyces rubiginosohelvolus GXWl, respectively.