Screening Of Strains For Converting Ellagitannin To Produce Urolithin And Study On Its Anti-Aging Effect

Healthy mitochondria are critical to cellular energy production and the body metabolism.During the aging process,the ability of mitophagy decreases,and dysfunctional mitochondria accumulate continuously,which may lead to many diseases,such as sarcopenia and Parkinson’s disease.Therefore,targeted intervention of mitophagy may help alleviate these diseases.It is known that urolithin A can effectively induce mitophagy,resulting in the improvement of various cell and body aging phenotypes.However,little is known about the bacterial species involved in the production of urolithin A from ellagic acid by gut microbiota.At present,people have discovered a Bifidobacterium pseudocatenulatum with the ability to convert ellagic acid to produce urolithin A,but the production and application based on this strain are greatly limited because it does not belong to the list of edible fungi and the substrate ellagic acid is insoluble in water.In addition,no edible strains that can convert ellagitannins(which exist widely in nature and have good water solubility)to produce urolithin A have been described so far.Based on the above background,this project aims to screen edible strains that can transform dietary ellagitannins to produce urolithin fermentation metabolites represented by urolithin A.The anti-aging effects of the prepared urolithin fermentation metabolites were analyzed by Caenorhabditis elegans model.And the dose-effect relationship of oral intake of urolithin fermentation metabolites was further clarified,so as to provide theoretical reference for the development of anti-aging products in the future.The main research contents and results are as follows:(1)The strains with the ability to convert ellagitannins to produce urolithin A were screened by protein sequence homology comparison and in vitro fermentation.The reported enzymes that may be involved in the metabolic process of ellagitannin into urolithin A were investigated.The protein sequences of these enzymes were compared with database of laboratory microorganisms,and microorganisms that might express the target enzymes were screened out.Through pomegranate juice fermentation,strains with the ability to transform ellagitannin to produce urolithin A were screened out.The results showed that three Lactobacillus plantarum,including CCFM1286,CCFM1290,and CCFM1291,with the ability to transform ellagitannin into urolithin A were screened from 168 candidate strains.And among the urolith metabolites prepared by fermentation of these three strains,the contents of urolithin A were 15.90±1.46 μM,24.70±0.82 μM,and 32.01±0.97 μM respectively,and the total contents of other urolithin were 234.07±2.31 μM,179.10±3.41 μM,and 234.07±2.31 μM respectively.(2)Based on the urolithin fermentation metabolites prepared by Lactobacillus plantarum CCFM1286,Lactobacillus plantarum CCFM1290,and Lactobacillus plantarum CCFM1291 respectively,the anti-aging effects were studied by the model of Caenorhabditis elegans.The results showed that urolithin fermentation metabolites prepared by Lactobacillus plantarum CCFM1286,Lactobacillus plantarum CCFM1290,and Lactobacillus plantarum CCFM1291 respectively could prolong the lifespan of Caenorhabditis elegans by 26.04±0.12%,32.05±0.14% and 46.33±0.12% by improving the mitochondrial function and/or reducing the level of reactive oxygen.Among them,the anti-aging effect of urolithin fermentation metabolites prepared by Lactobacillus plantarum CCFM1291 is the best.(3)This paper used D-galactose-induced aging mouse model to explore the dose-effect relationship of urolithin metabolites prepared by Lactobacillus plantarum CCFM1291 through oral intake and urolithin A standard release through the colon to exert anti-aging effects and the possible mechanism.The results showed that subcutaneous injection of 1000mg/kg/d D-galactose to Babl/c mice for 6 weeks can effectively induce aging.80 mg/kg/d urolithin fermented metabolites can effectively exert anti-aging effect,while 400 mg/kg/d urolithin fermented metabolites have the better anti-aging effect.The main results are as follows: compared with the model group,80 mg/kg/d and 400 mg/kg/d urolithin fermentation metabolites can increase exercise ability by 52.18% and 75.08% respectively,and cognitive ability by 204.91% and 341.19% respectively.At the same time,they can also inhibit the increase of oxidative stress level in the body,the accumulation of aging-related molecules,and improve tissue inflammation or atrophy and other pathological injuries.3.0 mg/kg/d may be the lowest effective dose for the colon release of urolithin A standard to exert anti-aging effects,and urolithin A exhibits dose-dependent anti-aging effects.In addition,both urolith fermentation metabolites and urolith A standard can down-regulate the AGEs/RAGE/NFκB signaling pathway,and may also down-regulate the m TOR signal,ultimately alleviating Dgalactose-induced inflammation and autophagy dysfunction.These findings highlight the potential application of these fermented metabolites in the subsequent development of antiaging products.