Explorating Lipid-lowering Mechanism On 3T3-L1 Cells And Obese Rats Via Supernatant Of Enterococcus Fermented Lotus Leaf

Medicinal and food homologous ingredients improve efficacy and reduce side effects through microbial fermentation,and that is urgently need to be studied.There have been a lot of reports on the research of lotus leaf as a medicine and food homologous ingredient for lipid-lowering and weight loss.However,using lactic acid bacteria and other microbial resources to ferment the lotus leaf to improve its function is rarely studied,especially the mechanism of Enterococcus fermented lotus leaf improves lipid metabolism on adipocytes and the lipid-lowering on rats has not been reported yet.Therefore,in this paper,supernatant of two probiotic Enterococci(Enterococcus faecium WEFA23 or Enterococcus hirae WEHI01)fermented lotus leaf was systematically studied,and the composition changes of supernatant of Enterococcus fermented lotus leaf were analyzed and their effects in adipocyte model(3T3-L1 preadipocytes)and lipid-lowering mechanisms in obese SD rat models.The full text is divided into five chapters.The first chapter,introduction,summarized the research progress of medicinal and food homologous ingredient,E.faecium,E.hirae,plant fermentation broth,and lipidlowering of lotus leaf,and outlooked the biological activity of fermentation supernatants of lotus leaf by Enterococcus in lipid-lowering.The second chapter studied the composition changes of supernatant of Enterococcus fermented lotus leaf,and carried out separation,purification and composition analysis.First,the p H value changes of supernatant of Enterococcus fermented lotus leaf were determined with the fermentation time.The results show that the p H of low and high concentrations(50,100 mg/m L)lotus leaves can be reduced after fermentation,and p H of high concentration of supernatant of E.hirae WEHI01 fermented lotus leaf was reduced to 4.14.Subsequently,the content of total flavonoid,total phenol,crude polysaccharide,short-chain fatty acid and protein of supernatant of two Enterococci fermented lotus leaf were determined.The results showed that,on the one hand,supernatant of Enterococcus fermented lotus leaf can significantly reduce the content of total flavonoid,total phenol and protein content in the supernatant;on the other hand,significantly increase the content of crude polysaccharides(P < 0.05).And supernatant of E.hirae WEHI01 fermented lotus leaf can significantly increase shortchain fatty acids.The third chapter explored the in vitro lipid-lowering effects of supernatant of Enterococcus fermented lotus leaf and their ability to reduce adipogenesis of 3T3-L1 preadipocytes.First,the CCK-8 method was used to analyze the cytotoxicity of supernatant of two Enterococci fermented lotus leaf on 3T3-L1 preadipocytes.The results showed that the supernatant was non-toxic to cells.Secondly,3T3-L1 preadipocytes were induced to differentiate and stained with Oil Red O staining.The effect of supernatant of Enterococcus fermented lotus leaf and unfermented supernatant on inhibiting adipogenesis was evaluated,and it was found that supernatant of two Enterococci fermented lotus leaf could inhibit adipocyte adipogenesis;among them,fermentation supernatant of high-concentration lotus leaf by two Enterococci have the best inhibitory effect,with the inhibition rates being 56.17% and 56.57%,respectively.Finally,the RNA of the differentiated mature adipocytes was extracted,and the m RNA expression of adipose differentiation-related and adipogenesis-related transcriptional factors was detected.The results showed that supernatant of Enterococcus fermented lotus leaf can significantly down-regulate adipocytes expression of adipogenesisrelated genes(P < 0.05).The fourth chapter explored the lipid-lowering effect of f supernatant of E.hirae WEHI01 fermented lotus leaf through the high-fat obesity model of SD rats.Supernatant of E.hirae WEHI01 fermented lotus leaf can significantly alleviate weight gain of obese rats,reduce epididymal fat and liver weight,oral glucose tolerance,insulin resistance index,content of total cholesterol,total triglycerides,and low-density cholesterol in serum,content of total cholesterol and total triglyceride in liver.And it was found that supernatant of E.hirae WEHI01 fermented lotus leaf can reduce lipid accumulation in liver and reduce the size of adipocytes in adipose tissue through HE staining and Oil Red O staining.And RT-q PCR showed it can significantly downregulate the m RNA levels of genes related to adipose differentiation and adipogenesis(P< 0.001).The analysis of short-chain fatty acids showed it can significantly increase the acetic acid content in the cecum(P < 0.01).The fifth chapter systematically summarized the components of supernatant of two Enterococci fermented lotus leaf and their lipid-lowering effects in 3T3-L1 preadipocytes and SD obese rats,and further looked forward to the in-depth study of mechanism of bacterial action in the future and development of supernatant of E.hirae WEHI01 fermented lotus leaf into a natural lipid-lowering health care product.In summary,this article analyzed the composition of the supernatant of E.hirae WEHI01 and E.faecium WEFA23 fermented lotus leaf;explored in vitro lipid-lowering effect of supernatant of E.hirae WEHI01 and E.faecium WEFA23 fermented lotus leaf in 3T3-L1 preadipocyte and in vivo lipid-lowering effect of supernatant of E.hirae WEHI01 fermented lotus leaf in SD obese rats.And it was confirmed that the supernatant of E.hirae WEHI01 fermented lotus leaf has a good lipid-lowering effect in vitro and vivo.The above results lay the foundation for finding natural lipid-lowering medicinal and food homologous ingredients and expanding its application in food and medicine.