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【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。
【Objective】 Lactobacillus which inhibits the activity of monoamine oxidase (MAO) was isolated and purified from healthy human feces by in vitro model, which could provide reference for the future research on anti-aging of lactic acid bacteria. 【Method】 The monoamine oxidase inhibitory model was used to screen the fermentation supernatant and cell-free extract of lactic acid bacteria. Two indexes of the selected samples were determined, that is, the dose effect of the sample and the pre-incubation of the sample with the enzyme Time on the rate of inhibition of enzyme activity; at the same time using membrane separation techniques for different molecular weight range of MAO inhibition assay. The strain JH-23 was selected as the target strain, and the strain was identified by 16S rDNA sequence analysis and API bacterial identification system. 【Result】 The results showed that the inhibitory rate of the selected strain JH-23 cell-free extract on MAO reached 33.7%. After the sample was lyophilized, the inhibition rate reached 53.2% when the reaction concentration was 16 mg / mL, and the inhibition rate of MAO increased with the pre-incubation time, and the inhibition effect tended to be stable after 30 min. The crude sample was dialyzed After the dialysis fluid MAO inhibition rate was significantly higher than before dialysis. The identification of strain JH-23 showed that it belonged to Lactobacillus casei. 【Conclusion】 A novel in vitro screening model with monoamine oxidase as target enzyme was developed. The model is convenient, rapid and sensitive, which is helpful for the subsequent study of anti-aging in vivo. Screening of Lactobacillus casei JH-23 cell lysate on MAO inhibition, which plays a MAO inhibitory effect is mainly intracellular small molecules.