Isolation And Identification Of Antimicrobial Endophyte And Its Antimicrobial Substances

Recently, endophyte, a microorganism group with rich diversity and complexity, couldproduce some bioactive substances, such as antitumor substance, antibacterial andinsecticidal substances. Therefore, endophyte was a potential source for producing newnatural drugs.In this study, using Escherichia coli as the indicator bacterium, the antimicrobialendophyte was isolated from the Lusang mulberry twig and identified with themorphological, physiological and biochemical characteristics and16S rRNA genesequences analysis. Based on the single factor experiments, the fermentation conditions ofthe antimicrobial endophyte were optimized with response surface methodology. Moreover,the antimicrobial substances were isolated and purified from the antimicrobial strian withthe methods of ethyl acetate extraction and silica gel column chromatography. Theantimicrobial substances were analyzed with gas chromatography-mass spectrometry(GC-MS). Furthermore, the antimicrobial mechanism of the antimicrobial substances wasstudied using the methods of ultraviolet spectroscopy, infrared spectroscopy andtransmission electron microscope. The main conclusions were obtained as follow:75strains, which contained49fungus and26bacteria, were isolated from the Lusangmulberry twig. In this study, two antimicrobial endophytes, which were recorded as ME1and ME2, were obtained. The morphological, physiological and biochemical characteristicsshowed that strain ME1was a gram-positive bacterium with flagellum and spore, withoutcapsule,0.6-0.7μm×1.8-1.9μm long and rod; the strain could utilize D-glucose, sucrose,α-lactose and starch, and it wasn’t able to utilize casein. Strain ME2was a gram-positivebacterium with flagellum and spore, without capsule,0.6-0.7μm×1.5-1.6μm long and rod.The strain could utilize D-glucose, sucrose, α-lactose, starch and casein. According to“Bergey’s Manual of Bacterial Identification” and “Identification Manual of Commonbacterial”, strains ME1and ME2were preliminarily identified as Bacillus. Through theanalyses of16S rRNA gene sequences, strains ME1(Accession No: JQ900635) andME2(Accession No: JQ900623) were finally identified as Bacillus subtilis.In this study, the optimized components of culture medium for strain ME1were potatoextract6.04g/L, glucose38.80g/L, NH4NO310.20g/L, CaCl219.80g/L; the optimumfermentation parameters were inoculation amount2%, liquid volume50%, initial pH ofculture medium7.08, fermentation time72h, fermentation temperature27.9oC, rotationalspeed152r/min. Under this optimum condition, the antimicrobial circle diameter of fermentation products from strain ME1was up to24.27mm for Escherichia col i.The result of GC-MS analysis showed that fermentation products from strain ME1,contained some antimicrobial substances such as (2,5-Dimethyl-1-p-chlor-ophenyl-3-yl-methylene)-N-(isoquinolin-5-yl)amine;3,6-Dibutyl-1,2-dihydro-1,2,4,5-tetraz-ine;4,4a,5,6,7,8,9,9a-octa-hydro-10,10-1,4-Methano-1H-cyclohepta[d]pyridazine; Nonane d ioic acid,bis(2-methylopropyl) ester and2,4-Di[p-chlorostyryl]-5-nitr-othiazole.The extract (Extr.01) of fermentation broth from strain ME1inhibited the growth ofEscherichia coli and Staphylococcus aureus during the logarithmic phase; Moreover, theultraviolet and infrared spectroscopy of the culture medium for the indicator bacteria(Escherichia coli and Staphylococcus aureus) showed that the extract (Extr.01) leaded toleakage of cell contents. The further observation of the transmission electron microscopeshowed that the extract (Extr.01) resulted in the increasing of cell wall and membranepermeability and bacteria death because of the bacterial cell wall and membrane damage,leakage of cell contents.