深海贝莱斯芽孢杆菌DH82的抑菌活性物质初步分离纯化及其抑菌谱检测

Purification of antimicrobial substance produced by deep sea Bacillus velezensis strain DH82 and its inhibition spectrum

贝莱斯芽孢杆菌(Bacillus velezensis)DH82发酵上清液经最佳硫酸铵饱和度(70%)沉淀、透析获得粗蛋白,粗蛋白经Sephadex G-75柱层析的色谱条件为柱1.6 cm×50.0 cm;上样量为5 mL;洗脱液为超纯水,流速2 mL/min;检测波长280 nm,获得两个具有抑菌活性的峰蛋白PrI和PrII。两个峰蛋白经抑菌谱检测,发现对常见水产指示病原细菌包括迟缓爱德华氏菌(Edwardsiella tarda)、嗜水气单胞菌(Aeromonas hydrophila)、坎氏弧菌(Vibrio campbellii)、溶藻弧菌(Vibrio alginolyticus)、哈维氏弧菌(Vibrio harveyi)、无乳链球菌(Streptococcus agalactiae)和金黄色葡萄球菌(Staphylococcus aureus)都具有较好抑菌活性,表明深海贝莱斯芽孢杆菌DH82产生的抑菌活性物质对水产病原细菌的抑菌谱较广,其中峰蛋白PrII对指示植物病原真菌水贼镰刀菌(Fusarium equisetum)也具有较好抑菌作用。进一步对峰蛋白进行最小抑菌浓度(MIC)检验,发现其对迟缓爱德华氏菌和溶藻弧菌的MIC都较低(为7.16μg/mL)。以上研究结果表明,深海贝莱斯芽孢杆菌DH82具有较宽的抑菌谱及较强的抑菌活性,预示该菌株在常见水产细菌性及个别植物真菌性病害防治上具有较好的开发利用价值。

The fermentation supernatant of Bacillus velezensis DH82 was precipitated with optimal 70%saturation of ammonium sulfate and the crude protein was obtained by dialysis.The crude protein was separated by Sephadex G-75 column.The chromatographic conditions were set at 1.6 cm×50.0 cm column,loading sample volume 5 mL,using ultrapure water as eluting buffer,flowing rate 2 mL/min and detecting protein at wavelength of 280 nm.Two peak proteins PrI and PrII were obtained.The result of inhibition spectrum analysis showed that the proteins had good antimicrobial activity against the aquatic pathogens such as Edwardsiella tarda,Aeromonas hydrophila,Vibrio campbellii,V.alginolyticus,V.harveyi,Streptococcus agalactiae and Staphylococcus aureus.The antimicrobial active substance of B.velezensis DH82 had a broad inhibition spectrum for some aquatic pathogenic bacteria.The protein PrII showed higher antimicrobial activity against the plant pathogen fungi of Fusarium equisetum.The minimal inhibitory concentration(MIC)of the peak proteins was determined.The MICs of the proteins to E.tarda and V.alginolyticus were lower than 7.16μg/mL.The findings in this study indicate that B.velezensis DH82 is valulble in the potential application in the prevention and control of common bacterial diseases in aquaculture and some cases of plant fungal diseases.