传统发酵蔬菜中拮抗温和气单胞菌的乳酸菌的筛选与鉴定

林洋; 马欢欢; 吕欣然; 孙梦桐; 白凤翎; 励建荣

doi:10.13386/j.issn1002-0306.2017.04.035

传统发酵蔬菜中拮抗温和气单胞菌的乳酸菌的筛选与鉴定

Screening and identification of lactic acid bacteria with antagonistic activity against Aeromonas sobria from traditional fermented vegetables

摘要

摘要: 目的:从传统发酵蔬菜中筛选对温和气单胞菌具有拮抗作用的乳酸菌。方法:采用双层琼脂扩散法筛选,通过生理生化反应和16S rRNA序列分析进行鉴定,研究蛋白酶、p H和温度等因素对乳酸菌无细胞上清液（CFS）抑菌活性的影响,采用扫描电镜分析乳酸菌无细胞上清液对温和气单胞菌细胞结构完整性的影响。结果:从腌渍酸黄瓜中筛选出对温和气单胞菌（10⁶CFU/m L）具有较强抑制活性的菌株LZH2-5,经鉴定为植物乳杆菌（Lactobacillus plantarum）。LZH2-5无细胞上清液经胰蛋白酶、木瓜蛋白酶、中性蛋白酶、碱性蛋白酶和胃蛋白酶处理后其抑菌活性分别丧失29.38%、23.93%、22.30%、19.47%和14.59%;经40、60、80℃处理30 min后其拮抗活性基本不变,在100℃和121℃处理30 min后活性分别下降了16.66%和25.40%,在p H3.0⁴.0范围内保持其抑菌活性,粗提物的最小抑菌浓度12.0 mg/m L。扫描电镜表明经LZH2-5 CFS处理使温和气单胞菌的细胞结构破坏并溶解。结论:从腌渍酸黄瓜中筛选的植物乳杆菌LZH2-5对温和气单胞菌具有较强抑制作用,初步分析抑菌活性物质为细菌素类,可作为水产品养殖过程中控制温和气单胞菌的拮抗制剂出发菌株。

Abstract: Objective: To screen lactic acid bacteria( LAB) with strong antagonistic activity against Aeromonas sobria from traditional fermented vegetables.Methods: Cell- free supernatants( CFS) from LAB with inhibitory activity against A. sobria was screened using the double agar diffusion methods. The strain was identified by biochemical tests and 16 S rRNA sequence analysis.The antibacterial substance was analyzed using protease sensitivity test,acid test and heat- treated test.The integrity of cell structure was observed by scanning electron microscope. Results: Strain LZH2- 5 with outstanding antagonistic activity against A.sobria( 10~6 CFU / m L) was screened from pickled cucumbers,and it was identified as Lactobacillus plantarum. The antagonistic activity of cell- free supernatants produced by strain LZH2- 5 was lost 29.38%,23.93%,22.30%,19.47% and14.59% by treating with trypsin,papain,neutral protease,alkaline protease and pepsin respectively.The antibacterial activity of CFS was stable after thermal treatment with 40,60,80 ℃ for 30 min,and decreased 16.66% and 25.40% after incubation at100 ℃ and 121 ℃ for 30 min,respectively,and was effective within p H3.0 ~ 4.0. The MIC of inhibitory substance was12.0 mg / m L. The scanning electron microscope images revealed that the integrity of cell of A.sobria treated with LZH2-5 CFS was damaged and dissolving. Conclusion: Lb. plantarum LZH2- 5 with strong inhibitory activity against A. sobria was screened from pickled cucumbers.The antibacterial substance produced by strain LZH2-5 was preliminarily determined as bacteriocins.It can be used as the antagonism original strain for the control A.sobria in aquaculture.