Isolation And Screening For Antibacterial Endophytes And Ferment Optimization Of The Active Strain

The following items were carried through in this research: surface sterilization of plant samples, isolation and purification of endophytes, screening for antibacterial endophytes,identification and ferment optimization of the active strain,which can be resources for natural antimicrobial products.The results obtained were as follows:1. Isolation and purification of endophytesEight endophyte strains were isolated from three sorts of plants, including five fungal strains, two bacterial strains and one actinomycete strain. But three of them (two fungal strains ,one actinomycete strain) can't be cultivated in vitro.2. Screening for antibacterial endophytesEndophyte fermentation fluid was disposed by centrifugation. Supernatant liquid was used for antibacterial tests. The processed fermentation fluid of endophytic bacteria EF-B01 strain derived from Ficus microcarpa showed strong inhibition to Pseudomonas aeruginosa and Escherichia coli , and the inhibition zone dimeter of the processed fermentation fluid of endophytic bacteria EF-B01 against Pseudomonas aeruginosa was 23.28mm. The processed fermentation fluid of EF-B01 strain showed weak inhibition to Staphylococcus aureus. The processed fermentation fluid of EF-B01 strain showed strong inhibition to the growth of Aspergillus niger, and the rate of restrain reached 82.3% after 5 days. Moreover, the processed fermentation fluid of EF-B01 strain showed strong inhibition to the spore formation of Aspergillus niger.3. Identification of active endophyte strain EF-B01Antibacterial endophytes strain EF-B01 was identified by cultivation characters, morphologic observations, physiological and biochemical characters and sequence analysis of the 16S rDNA as the one of Bacillus subtilis, named as Bacillus subtilis EF-B01.4. Optimization of fermentation conditions of active endophyte strain EF-B01With the purpose of finding out the optimum fermentation conditions which could be used to increase the biomass and the yield of active substances ,optimum carbon and nitrogen source, initial pH, medium loading quantity, inoculation amount, rotary rate and fermentation temperature were researched. The results indicated that the optimal carbon source was 3% corn flour, the optimal nitrogen source was 4% bean flour, initial pH was 8.0, medium loading quantity was 30%, inoculation amount was 1%, rotary rate was 160rpm and fermentation temperature was 34℃.Under the optimal conditions, the OD value of the fermentation fluid of B. subtilis EF-B01 strain was 7.95 after 48h, increasing 52.0% comparied with the initial conditions. The inhibition zone dimeter of the processed fermentation fluid against Pseudomonas aeruginosa was 26.42mm, increasing 13.5% comparied with the initial conditions.