嗜热链球菌CGMCC 1.1864所产的一种新型细菌素ST9

Thermophilin ST9, a New Bacteriocin Produced by Streptococcus thermophilus CGMCC 1.1864

摘要:

本文利用琼脂扩散法测定嗜热链球菌(Streptocccus thermophilus) CGMCC 1.1864发酵上清液的抑菌效果, 结果表明此菌能够产生抑菌物质, 且在排除有机酸和过氧化氢的影响后上清液不但能抑制革兰氏阳性菌, 对革兰氏阴性菌也有抑制能力。此抑菌物质具有热稳定性, 于100°C处理 2 h及121°C处理20 min仍保留抑菌活性, 但若将其在100°C处理2 h的上清液立即置于?20°C保存, 其抑菌活性有较大损失。常温下(37°C), 该抑菌物质在pH 2.0?9.0范围内有很好的稳定性。发酵上清液经各种蛋白酶及?-淀粉酶处理后抑菌活性完全消失, 而对过氧化氢酶不敏感, 表明此抑菌物质为多肽, 属于细菌素, 本文初步将其命名为嗜热链球菌素ST9。由于ST9对其产生菌具有吸附作用, 选择pH吸附释放法对该嗜热链球菌素进行粗提, 然后经SephadexG-25凝胶层析柱除去杂蛋白, 最后冷冻干燥得纯品。通过Tricine-SDS-PAGE分析得到其分子量约为5.0 kD。

Abstract:

In this study, antimicrobial activity of the culture supernatant of Streptococcus thermophilus CGMCC 1.1864 was investigated with the agar diffusion method. The subsequent experiments results indicated the antimicrobial activity of the supernatant were not due to the existence of hydrogen peroxide and organic acid in the solution. The antimicrobial spectrum assays showed that the supernatant could inhibit the growth of both Gram-negative bacteria and Gram-positive bacteria. Heat stability test was also performed with the antimicrobial substance and no activity lost was observed after 2 h incubation at 100°C or 20 min at 121°C. However, its activity was reduced significantly if the boiling samples were transferred to ?20°C freezer for storage immediately. It was also tolerable to changes of pH levels, no significant reduction of its antimicrobial activity was observed at pH 2.0 through pH 9.0. The treatment of proteases and α-amylase could completely abolish the antimicrobial activity of the supernatant, while no activity loss was observed when treated with catalase. These results demonstrated that the antimicrobial substance belonged to bacteriocin family and was named as thermophilin ST9 tentatively. Since thermophilin ST9 could specially adsorb to the producing strain, pH-adsorption method was chosen to purify the bacteriocin, and subsequently the SephadexG-25 column chromatography was adopted to eliminate other proteins. Elution solution was further concentrated with freeze-drying method to obtain thermophilin ST9 powder. Purified thermophilin ST9 was subjected to Tricine-SDS- PAGE analysis and its molecular mass was determined to be 5.0 kD.