鳗弧菌(Vibrio anguillarum)M3菌株生长条件及其对蛋白酶产量的影响

THE IMPACT OF CULTURE CONDITIONS ON VIBRIO ANGUILLARUM STRAIN M3 GROWTH AND PROTEASE PRODUCTION

采用体外测定细菌浓度、胞外产物 (ECP)蛋白含量和蛋白酶活力的方法 ,进行了鳗弧菌M3菌株在 2 2 16E培养基中的培养条件研究。结果表明 ,该菌株用固体培养基培养至 2 4h左右 ,可得到较高的菌体浓度、ECP蛋白含量和蛋白酶活力。采用响应面分析方法设计实验 ,用SAS统计软件分析数据 ,得到NaCl浓度、pH值和温度对菌体生长及蛋白酶产量影响的回归模型。在 2 2 16E培养基的基础上 ,添加不同氮源、碳源物质以及不同浓度蛋白胨进行生长研究。结果表明 ,胰大豆蛋白胨能促进菌体生长及ECP蛋白分泌 ;NH4 Cl与酪蛋白水解物可抑制蛋白酶的产生 ;牙鲆肌肉匀浆对菌体、ECP蛋白产量和蛋白酶产生有不同程度的促进作用 ;培养基中蛋白胨浓度为 4 %时菌体量与ECP蛋白含量达最高值 ,在蛋白胨浓度为 2 %时蛋白酶分泌量已稳定 ;1%的葡萄糖、蔗糖、甘油均能显著地提高菌体及ECP蛋白产量 ,却抑制了蛋白酶的产生。

Bacteria Vibrio anguillarum strain M3,its concentration,extracellular products (ECP) and protease activity were analyzed on solid and liquid 2216E media. Our study showed that during a 24-hour period,higher values are found for the concentration of bacteria,extracellular product and protease activity on the solid media than on the liquid media. An experiment was designed with Response Surface Methodology to assess the effects of NaCl concentration,pH value and temperature on the growth of bacteria and its protease production. Two regression models were established with SAS statistical software to evaluate the data. Typical analysis of the regression models predicted that the maximal bacteria growth was achieved in the conditions of 0.58% NaCl,pH 7.94 and 23.3℃. Maximum protease production was achieved in the conditions of 0.71% NaCl,pH 8.29 and 23.04℃. In addition,bacteria growth was studied by adding different concentrations of nitrogen,carbon and peptone into 2216E media. It was found that protease activity was lower in the brain heart infusion media than in the peptone media . Compared with the peptone media,bacteria growth and ECP production were stimulated on the trypticase soy agar media,but protease production on both media showed the same level. The presence of Casamino Acids can stimulated bacterial growth and ECP production jointly with NH_4Cl. However,both NH_4Cl and Casamino Acids in the media could significantly decrease the production of bacteria proteases. Muscle homogenate of flounder Paralichthys olivaceus was able to enhance the bacteria growth,ECP and protease production on different levels. Concentration of bacteria and ECP reached a maximal value at 4% peptone level in the 2216E medium,but all dropped when the peptone concentration in the medium was higher. Protease production was at a maximum value at 2% peptone level in medium,remaining stable when the concentration of peptone rose to 4%,then dropping with further rises of peptone concentration. Bacteria growth and ECP production increased in the presence of 1% glucose,sucrose or glycerin in the medium,with sucrose giving the highest stimulation effect. However,bacteria protease production decreased dramatically when 1% glucose,sucrose or glycerin was present in the medium. The effect of all three repressors on protease production was on a similar level.