Studies On Screening And Identification Of Marine Sponge-associated Actinomycetes HA01041,HA01184 With Anti-MRSA Activity And Their Fermentation Optimization

In this paper, 39 marine sponge speciments were collected from Hainan, Zhanjiang, and research was carried out on the isolation of sponge-associated actinomycetes, screening and identification of anti-MRSA strains, optimizing fermentation condition and so on. Strain HA01041 and HA01184 possessed higher stability and repeatability of activity against MRSA, and were further studied.Using seven culture media, some actinomycetes were isolated from marine sponge collected from Zhanjiang by the method of dilution. The results showed that Gause 1 containing 1% dissolubile starch, 0.1% KNO3, 0.05% K2HPO4, 0.05% MgSO₄·7H₂O, 0.001% FeSO₄, 0.005% K₂Cr₂O₇ with initial pH 7.2 was the best media for sponge-associated actinomycete isolation. Utilizing the aforesaid medium, 583 strains were isolated from 39 marine sponge samples, which were collected from island of Hainan, Zhanjiang. 77 strains with anti-MRSA active compounds were obtained by the method of Kirby Bauer.By studying the morphology, cultural characteristics, physiological and biochemical characteristics, 16S rDNA and phylogenetic tree, HA01041 was proposed as Streptomyces verne; HA01184 maybe a novel strain of Streptomyces, and more research should be carried out by DNA-DNA hybridization.The optimal liquid fermentation conditions and media of HA01041 and HA01184 were obtained. The optimum culture media of HA01041 contained sucrose 1.0%, glycerin 2.0%, (NH₄)₂SO₄ 1.0%, soybean and Indian powder 1.5%, K₂HPO₄ 0.05%, old sea water 75%, distilled water 25%. The optimum fermentation conditions of HA01041 were initial pH7.0⁷.2, seed age 48h, 10% inoculation amount, and the size 75mL in 250mL Erlenmeyer flask at 200r/min, 5d. The optimum culture media of HA01184 contained dissolubile starch 1.5%, glucose 1.5% , (NH4)2SO4 1.0%, soybean and Indian meal 1.5%, K2HPO4 0.05%, old sea water 75%, distilled water 25%. The optimum inoculation conditions of HA01184 were initial pH7.0⁷.2, seed cultural time 60h, 12% inoculation amount, and the size 75mL in 250mL Erlenmeyer flask at 200r/min, 5d.The physical and chemical properties of the antibacterial compounds have been studied, the results showed the bioactive substances of strain HA01041 and HA01184 all have heat stability, light stability, but not be stable at strong acidity (pH<3.0) or alkali (pH>9.0) condition. The bioactive substances of strain HA01041 and HA01184 all can be dissolved in chloroform, ethylacetate, n-butanol, methyl alcohol, ethanol and acetone, and can be weakly dissolved in water, but not be dissolved in benzene, petroleum ether and cyclohexane.