Screening And Characterization Of Probiotic Lactobacillus Strains With Inhibitory Activity To Monoamine Oxidase

Ageing is one of the critial social problems human being will face in 21th century. Senescence-related diseases, such as Alzheimer's disease and Parkinsonism syndrome, etc., have become the serious threats to human health and caused increasingly heavy cost to society all the world. As the elderly population increases, functional foods providing health benefits to delay ageing and prolong health span become more desirable. Among various alternatives besides medicines with anti-ageing potentials, probiotics generally regarded as safe, are considerably worth studying in-depth,for which have demonstrated abilities to improve the host's health.Recently, some health benefits related to probiotics have been confirmed, i.e. antihypertensive effect, anti-hyperlipidemia property, cholesterol-reducing activity etc., while the anti-ageing effect of probiotics is seldomly reported. Actually, as early as in 1905, E.Metchnikoff, a famous microbiologist, found that Bulgarians who drank yogurt in their routine life generally lived with a longevous life span. Most researches with respect to anti-ageing capability of probiotics so far were focused on animal / human subject trials, whereas few in vitro models suitable for surveying the anti-ageing activity of probiotics have been reported for the complexiety of ageing.Monoamine oxidases (MAO), especially MAO-B, key enzymes envolved in ageing procedure, are primarily responsible for the catabolism of endogenous and xenobiotic monoamine substance. For the lasting elevation of its activity will result in apolexis throughout the body step by step, MAO is also regarded as symbol of ageing procedure, or ageing-correlated enzyme.Therefore, an in vitro screening and assessment model, envolving monoamine oxidase (MAO) as a target enzyme, was established in this study to select probiotic bacterial strains with inhibitory activity to MAO as candidate with anti-ageing potentials for further study in vivo.It is presumed that MAO inhibitor posses potential anti-ageing effect, and the MAO-inhibitory active constituents originated from probiotics were screened in vitro in this paper. Subsequently, the acetone extraction method and thin layer chromatogram (TLC) were applied to extract and separate active components from cells of the test strain, and the MAO inhibitory activity of the separated components were assayed, among which the component with strongest MAO-inhibitory activity was further assessed by the National Center for Drug Screening,Shanghai. Finally, the physiological functions and growth characteristics of candidate probiotic strains with MAO-inhibitory activity were investigated. Meanwhile, screened strain was identified through molecular biology methods and API carbohydrate fermentation experiment.Cell lysates of strain JH-23 cultivated in MRS broth demonstrated strong MAO inhibitory effect and the inhibitory rate reached 33.7 % in the primary screening test. When the reactive concentration was 16 mg/mL, the MAO inhibitory activity was up to 53.2 %. After the cell lysates being dialyzed by a membrane with MWCO of 1 KD, it was found the major active components existed in the dialysates. These results revealed the major active components resourced from intracellular small molecules (MW<1 KD). Therefore, acetone was selected to extract the small molecules inhibitory to MAO from the bacterial cells.Four active components from cells of JH-23 by acetone immersion was eventually separated by thin layer chromatogram (TLC), among which the componentⅠdisplayed the strongest MAO inhibitory activity, in a concentration-dependant manner, with an inhibitory rate reached 55.4% at concentration of 120μg/mL. The inhibitory activity of componentⅠto MAO was confirmed by National Center for Drug Screening.Strain JH-23 was anaerobic, Gram-positive, rod-shaped and nonsporeforming. It was identified as Lactobacillus casei (Named as Lactobacillus casei JH-23) via the full length sequence analysis of 16S rDNA gene and API system. Lactobacillus casei JH-23 showed a strong tolerance to acid, bile salt and NaCl. The optimal temperature and pH value for the growth of JH-23 were 37℃and 6.0 respectively. Lactobacillus casei JH-23 grows vigorously in MRS broth, whereas it demonstrates poor growth and non-curdy ability in skim milk.