MOLECULAR AND BIOCHEMICAL STUDIES OF PROBIOTIC PROPERTIES OF LACTOBACILLUS FERMENTUM NKN51

Abstract

Probiotics are health beneficial microbes which constitutes normal gut flora and live in a mutualistic relationship with the host. Owing to their health benefic nature probiotics are commercially available as food supplements. Most widely used Probiotic genera are Lactobacillus and Bifidobacterium. However genetic basis of the health beneficiary properties of probiotic microbes are poorly understood which leads to inconsistent results and limits attribution of measurable effect to the microbes. Understanding the molecular insights of probiotic properties will support their scientific use contributing towards more judicious applications in health and food industry. The present study involves screening of fermented dairy product and infant gut flora for isolation of lactic acid bacteria. 15 isolates obtained after screening were identified as lactic acid bacteria by biochemical analysis and 16S rRNA sequencing. Probiotic potential of all the isolates was analyzed by screening of four important attributes; phytase production, in-vitro cholesterol removal, asparaginase production and bile salt hydrolase production. Out of all isolates NKN51 have shown good phytase activity and in-vitro cholesterol removal ability. NKN52 and NKN55 were good asparaginase and bile salt hydrolase producers. Besides this NKN53 and NKN59 have also shown good asparaginase activity. NKN52 and NKN55 were identified as L. bevis and NKN53 was Weisella paramesenteroides. There are no reports of asparaginase production from any of these microbes in literature, hence the study reports novel finding of asparaginase production by L. bevis and Weisella paramesenteroides. Asparaginase is an antineoplastic enzyme and may contribute significantly to the anticancer property of these microbes. NKN51 was identified as Lactobacillus fermentum it has shown good cholesterol reducing effect in vitro from synthetic medium. Cholesterol reducing effect is a much desirable character in Lactobacilli as hypercholesterolemia is one of the biggest health threats worldwide. Along with that, high phytase activity of NKN51 made us to choose the isolate for furthes studies. Inositol hexakisphosphate (IP6 or phytate) degradation by commensal gut flora has drawn much attention in last few years because of its antinutritive properties and role in inter-kingdom signalling. Still genetic information of IP6 degradation is lacking from most of the gut flora. In present study we have cloned and characterized a novel protein tyrosine phosphatase like phytase (PTPLP) from probiotic bacterium Lactobacillus fermentumNKN51 (phyLf). phyLf shown high substrate specificity towards IP6 with Maximum enzyme activity at pH 5, temperature 60oC and 100mM ionic strength. IP6 degradation was further confirmed by II Zymography and HPLC analysis. Among various reported PTPs, phyLf exhibited exceptionally high resistance against oxidative inactivation by H2O2. Enzyme activity was not much affected by disulphide bond denaturants and chelating agent. Specific activity, Michaelis-Menten’s constant (Km) and Catalytic turnover number (Kcat) for IP6 were 174.5U mg-1, 0.7735mM and 84.31 sec-1 Further in-vitro cholesterol reducing effect of Lactobacillus fermentumNKN51 was analysed. An increase in growth of NKN51 was reported in presence of cholesterol in comparatively nutritionally scarce medium, M17-thioglycollate, which clearly indicates cholesterol assimilation or metabolization by bacteria. To further confirm the cholesterol consumption by Lactobacillus fermentumNKN51 the isolate was allowed to grow in M17 medium supplemented with fluorescent cholesterol analog, NBD-cholesterol and a distinct shift was observed in fluorescence spectrum of cells grown in presence of fluorescent cholesterol in comparison to control cells in flow cytometric analysis. The result suggests and confirms the assimilation of cholesterol by Lactobacillus fermentum NKN51. The study provides experimental proof to the concept of cholesterol assimilation by Lactobacilli for imparting hypocholesterolemic effect. For mechanistic insight of cholesterol consumption, putative 3beta hydroxy steroid dehyrogenase gene was cloned and characterized from the genome of Lactobacillus fermentum NKN51. Cholesterol dehydrogenase activity of the protein product was confirmed by zymography and biochemical analysis. These findings give a way to explore the complete mechanism of cholesterol consumption in Lactobacilli. And provide scientific basis to the concept of hypocholesterolemic effect of Lactobacilli. respectively. Bioinformatic and Phylogenetic analysis have shown that except for active site, it does not share much homology with previously reported PTPLPs and lies at interphase of PTPLP and MptpB like proteins, hence constitutes a new subclass of PTPLPs. Thus far phyLf represents the first phytase gene characterized from genera Lactobacillus. Widely accepted probiotic properties of this genus enable its phytase to be more acceptable for human consumption In fourth part of the study a facile single step green method of synthesizing silver nanoparticles functionalized with an antibacterial peptide from a food-grade lactic acid bacterium has been reported. The synthesized enterocin coated silver nanoparticles show broad spectrum inhibition against a battery of food borne pathogenic bacteria without any detectable toxicity to red blood cells. The present results evince that a new category of biocide based on silver nanoparticles coated with food-grade antibacterials can be developed using simple methods.