一株具有抑制单胺氧化酶作用的干酪乳杆菌筛选

【目的】通过体外模型从健康人体粪便内分离筛选出具有抑制单胺氧化酶(MAO)活性的乳酸菌,为今后乳酸菌体内抗衰老的研究提供参考。【方法】采用单胺氧化酶体外抑制模型对乳酸菌的发酵上清及无细胞提取物进行了筛选,并对筛选出的样品进行了两种指标的测定,即样品的剂量效应,以及样品与酶的预保温时间对酶活抑制率的影响;同时利用膜分离技术对不同分子量范围的样品进行了MAO的抑制测定。以筛选出的菌株JH-23为目的菌,通过16S rDNA序列分析及API细菌鉴定系统对菌株进行鉴定。【结果】筛选出的菌株JH-23无细胞提取物对MAO的抑制率达到33.7%。样品经冻干后,在反应浓度为16 mg/mL时抑制率达到53.2%,且MAO抑制率随预保温时间的增加而上升,在30 min之后抑制效果趋于平稳;粗样品经48 h透析后,透析液中的MAO抑制率较透析前明显升高。菌株JH-23的鉴定结果显示其属于干酪乳杆菌。【结论】开发了一种以单胺氧化酶作为靶位酶的新式体外筛选模型,该模型方便快捷且灵敏性高,对之后的抗衰老体内研究有所帮助。筛选出的干酪乳杆菌JH-23细胞裂解物对MAO有抑制作用,其中起到MAO抑制作用的主要是细胞内的小分子类物质。

Screening of a Lactobacillus casei with monoamine oxidase-inhibitory activity

Abstract: Objective] To screen for monoamine oxidase (MAO)-inhibiting lactobacillus from healthy human dejecta in an in vitro model, and to provide reference for anti-ageing study in vivo of lactobacillus in the future. Methods] The monoamine oxidase inhibitory model in vitro was applied to screen fermented supernatant and cell-free extracts originated from lactic acid bacteria, and two indices based on screened samples were determined, including biological dosage-effect and the effect of pre-incubated time between MAO and inhibitor on suppression rate. Meanwhile, with membrane separation technology, MAO inhibition from different molecular weight range of samples was measured. Screened strain JH-23 was investigated as target probiotic lactobacillus, identified through the sequence analysis of 16S rDNA gene and API system. Results] The MAO inhibitory rate of cell-free extracts produced by strain JH-23 reached 33.7 %. After samples being vacuum freeze-dried, MAO inhibitory rate was up to 53.2 % when the reactive concentration was 16 mg/mL. The inhibition was significantly enhanced with the duration of pre-incubation increased, and then the inhibitory effect became steady after 30 minutes. Crude samples were dialyzed for 48 hours, MAO inhibitory effect of dialysates was significantly increased compared with pre-dialysis. The result of bacteria identification demonstrated that strain JH-23 was ascribed to Lactobacillus casei. Conclusion] A new screening model in vitro, regarding monoamine oxidase as a target enzyme, was explored in this study. This model was characterized by convenience, rapidity and high sensitivity, and it could be useful for the following anti-aging research in vivo. The cell-free extracts of Lactobacillus casei JH-23 was inhibitory to MAO, and the intracellular small-molecules played a major role in the inhibition.