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从开菲尔奶中筛选出一株具有强抑菌活性的乳酸菌KF1,经16S rDNA测序鉴定为植物乳杆菌。排除有机酸、过氧化氢的干扰试验,通过蛋白酶K和胰蛋白酶水解试验,证明该菌株产生的抑菌活性物质具有蛋白质性质,是一种细菌素。该细菌素在pH值2~4范围内抑菌效果稳定,当pH值达到8时,抑菌活性完全丧失;在121℃处理60 min后,仍保留约68%的活性,稳定性较好。抑菌谱试验表明,KF1细菌素对13株奶牛乳房炎病原菌都有不同程度的抑制作用,其中,对无乳链球菌、表皮葡萄球菌、溶血葡萄球菌等革兰氏阳性菌的抑菌作用最强,抑菌直径大于18 mm,抑制效果优于G―菌。通过研究KF1细菌素对菌体细胞壁、细胞膜通透性的影响,揭示其抑菌机制为膜孔道渗漏机制。另外,本文还发现KF1细菌素对多重耐药金黄色葡萄球菌生物被膜的形成―主要的毒力因子具有明显的抑制作用,显示其在奶牛乳房炎预防、治疗方面的应用前景。
A KF1 lactic acid bacterium with strong antimicrobial activity was screened from Kefir milk and identified as Lactobacillus plantarum by 16S rDNA sequencing. Exclusion of organic acids, hydrogen peroxide interference test, through the protease K and trypsin hydrolysis test, to prove that the strain of antibacterial active substances with protein properties, is a bacteriocin. The antibacterial activity of the bacteriocins was stable at the pH range of 2 ~ 4. When the pH reached 8, the bacteriostatic activity was completely lost. After the treatment at 121 ℃ for 60 min, the bacteriocins still retained about 68% activity with good stability. The antibacterial spectrum test showed that KF1 bacteriocins had different degrees of inhibition on 13 dairy mastitis pathogens. Among them, the antibacterial activity against Gram-positive bacteria such as Streptococcus agalactiae, Staphylococcus epidermidis and Staphylococcus haemolyticus Strong, antibacterial diameter greater than 18 mm, inhibitory effect is better than G-bacteria. By studying the effect of KF1 bacteriocins on cell wall and cell membrane permeability of bacteria, the mechanism of bacteriostasis was revealed as membrane pore leakage mechanism. In addition, KF1 bacteriocin was also found to inhibit the formation of multiple drug-resistant Staphylococcus aureus biofilm, the main virulence factor, and show its potential application in the prevention and treatment of dairy mastitis.