豆酱源枯草芽孢杆菌S1-2产细菌素分离鉴定及其稳定性研究
Isolation and Identification of Bacteriocin-producing Bacillus subtilis S1-2 from Soybean Paste and Its Stability Study
张淘崴 1乌日娜 2纪帅奇 3娄梦雪 4马颖 1武俊瑞2
作者信息
- 1. 沈阳农业大学食品学院,辽宁沈阳 110866;辽宁省食品发酵技术工程研究中心,辽宁沈阳 110161
- 2. 沈阳农业大学食品学院,辽宁沈阳 110866;辽宁省食品发酵技术工程研究中心,辽宁沈阳 110161;沈阳市微生物发酵技术创新重点实验室,辽宁沈阳 110866
- 3. 辽宁省食品发酵技术工程研究中心,辽宁沈阳 110161;沈阳市微生物发酵技术创新重点实验室,辽宁沈阳 110866
- 4. 沈阳农业大学食品学院,辽宁沈阳 110866;沈阳市微生物发酵技术创新重点实验室,辽宁沈阳 110866
- 折叠
摘要
本研究首先从东北传统豆酱中分离筛选出一株具有良好抑菌作用的菌株,结合 16S rDNA与持家基因gyrA测序分析等方法对菌株鉴定.其次,利用乙酸乙酯萃取法对其所产细菌素进行粗提,使用葡聚糖凝胶Sephadex-G50和高效液相色谱(HPLC)技术进一步纯化,结合SDS-PAGE和液相色谱-质谱联用(LC-MS/MS)技术进行鉴定.最后通过最小浓度实验与生长抑制曲线分析了细菌素对金黄色葡萄球菌的抑制效果,并探究了细菌素对热、酸碱和蛋白酶的稳定性.结果表明,枯草芽孢杆菌(Bacillus subtilis)菌株S1-2对金黄色葡萄球菌和单增李斯特氏菌抑菌效果最好,该菌株产生的细菌素经分离纯化鉴定为subtilosin A,分子量约为 3 kDa,对金黄色葡萄球菌和单增李斯特氏菌有明显的抑制效果,对金黄色葡萄球菌的最小抑制浓度为 0.625 mg/mL.该细菌素在20～80℃抑菌活性稳定,且在 100～120℃仍表现出抑菌活性;对酸碱耐受性较高,经过pH为 1、10的强酸强碱处理后仍表现出抑菌活性,在pH6.0～8.0的范围内表现出最高的抑菌活性;能够被蛋白酶K、胃蛋白酶、胰蛋白酶水解.综上,枯草芽孢杆菌S1-2与其产生的细菌素subtilosin A在食品生物防腐中具有较强的研究和应用潜力.
Abstract
In this study,a strain with good antibacterial effect was screened and identified from traditional soybean paste in Northeast China.The strain was identified using methods such as 16S rDNA and housekeeping gene gyrA sequencing analysis.Its bacteriocins were crude extracted using ethyl acetate extraction,further purified using dextran gel Sephadex-G50 and high performance liquid chromatography(HPLC)techniques,combined with SDS-PAGE and liquid chrom-atography-mass spectrometry(LC-MS/MS)for identification.The antibacterial effect of the bacteriocin on Staphylococcus aureus was analyzed by determining the minimum concentration and growth inhibition curve.Additionally,the stability of the bacteriocins was investigated by exploring its thermal stability,pH tolerance,and resistance to proteases.Results showed that the Bacillus subtilis S1-2 strain had the best antibacterial effect on Staphylococcus aureus and Listeria monocytogenes.The bacteriocin produced by the strain was identified as subtilosin A,with molecular weight of approx-imately 3 kDa,and demonstrated a significant inhibitory effect on Staphylococcus aureus and Listeria monocytogenes.The minimum inhibitory concentration against Staphylococcus aureus was 0.625 mg/mL.Furthermore,the bacteriocin exhibited stable antibacterial activity between 20～80℃and even between 100～120℃.It showed the highest antibacterial activity in the pH range of 6.0～8.0,as well as good acid and alkali tolerance with pH values of 1 and 10.Furthermore,it could be hydrolyzed by proteinase K,pepsin,and trypsin.In conclusion,Bacillus subtilis S1-2 and its bacteriocin subtilosin A have great potential in food preservation due to their strong antibacterial activities.
关键词
枯草芽孢杆菌/细菌素/分离纯化/抑菌Key words
Bacillus subtilis/bacteriocin/separation and purification/antibacterial引用本文复制引用
基金项目
国家自然科学基金面上项目(32172279)
沈阳市科技创新平台项目(21-103-0-14)
沈阳市科技创新平台项目(21-104-0-28)
出版年
2024
国家科技期刊平台
NSTL
万方数据