抑制嗜水气单胞菌乳酸菌的筛选、鉴定及抑菌作用

Screening,Identification,and of Lactobacteria Inhibition Effect of Aeromonas hydrophila

从健康成年尼罗罗非鱼(Oreochromis niloticus)肠道中分离纯化得到乳酸菌,采用牛津杯双层平板法筛选具有抑制嗜水气单胞菌(Aeromonas hydrophila)活性的乳酸菌菌株,借助16S rRNA分子鉴定乳酸菌种类。通过排除有机酸和过氧化氢干扰及蛋白酶敏感性等试验分析抑菌活性最佳乳酸菌的抑菌活性物质成分,并进行热、酸碱稳定性和扫描电镜观察等确定抑菌活性物质对嗜水气单胞菌的抑菌作用。结果显示,尼罗罗非鱼肠道中有3株乳酸菌对嗜水气单胞菌表现出良好的抑菌效果,经鉴定后分别为植物乳酸菌(Lactobacillus plantarum)和唾液乳杆菌(Lactobacillus salivarius)。其中,菌株LX3的抑菌活性最佳,抑菌圈直径可达到(25.25±0.23)mm且对多种蛋白酶敏感,经不同温度和酸碱性处理后抑菌活性物质仍保持较高的抑菌活性,特别是在pH 12和100℃处理后,抑菌圈直径仍达到(13.87±0.12)和(16.65±0.26)mm。此外,扫描电镜观察发现菌株LX3抑菌活性物质主要破坏了嗜水气单胞菌的细胞结构,使细胞内容物流出,致其死亡。结果表明,尼罗罗非鱼肠道中拥有较为丰富的抑制嗜水气单胞菌乳酸菌资源,尤其是菌株LX3显示出良好的抑菌活性与抑菌作用,这对于替代抗生素防治嗜水气单胞菌引发的相关鱼类疾病具有重要价值。

Isolation and purification of lactobacteria from intestine of healthy adult Nile tilapia(Oreochromis niloticus),screening of lactobacteria with inhibition of Aeromonas hydrophila by double-layer plate method,Identification of lactobacteria species with 16S rRNA molecular markers.By eliminating the interference of organic acids and hydrogen peroxide and the sensitivity of protease,the antibacterial active components of lactobacteria with the best antibacterial activity were analyzed,and thermal stability,acid-base stability and scanning electron microscopy were used to determine the antibacterial activity of the antibacterial substances against Aeromonas hydrophila.The results showed that,three lactobacteria in Nile tilapia intestines showed good bacteriostatic effect against Aeromonas hydrophila,they were identified as Lactobacillus plantarum and Lactobacillus salivarius.Among them,strain LX3 had the best inhibition activity,the diameter of inhibition zone could reach(25.25±0.23)mm,and it was sensitive to a variety of proteases.After different temperature and acid-base treatment of antibacterial active substances,still maintain high antibacterial activity.Especially after treated at pH 12 and 100℃,the diameter of the inhibition zone can still reach(13.87±0.12)and(16.65±0.26)mm.In addition,it was found under scanning electron microscopy that the antibacterial active substance of strain LX3 mainly destroyed the cell structure of A.hydrophila,cell content flows out,causing its death.The above results indicated that within Nile tilapia intestines have abundant resources of lactobacteria that inhibit A.hydrophila,especially the strain LX3 showed good inhibition activity and inhibition.In particular,the strain LX3 shows good inhibition activity and inhibition effect,which was of great value for replacing antibiotics to prevent and treat related fish diseases caused by A.hydrophila.