Mechanism Of Quality Formation Of Eucommia Ulmoides Leaves Fu Tea And Its Hypolipidemic Effect

Eucommia ulmoides Olive.belongs to rare relict tree species and is widely planted in China.Eucommia ulmoides bark,a classical and valuable traditional Chinese medicine,has distinct pharmacological actions,including providing necessary nourishment for the liver and kidney,as well as increasing bone density and enhancing muscle function.According to the scientific finding,the active components of Eucommia ulmoides leaves are similar to those of Eucommia ulmoides bark.Moreover,Eucommia ulmoides leaves has the same pharmacological effects as Eucommia ulmoides bark.Thus,Eucommia ulmoides leaves is valuable and potential development resources.However,Eucommia ulmoides leaves have been always used and managed as raw materials for Chinese medicinal materials and health foods,which limits the application of Eucommia ulmoides leaves for foods.In 2018,based on the traditional dietary habits and local demand,the National Health Commission proposed that Eucommia ulmoides leaves should be managed as medicine and food homologous products.Then,the pilot project of Eucommia ulmoides leaves that was managed as medicine and food homologous products was carried out in 2019,which provided a policy basis for the development of Eucommia ulmoides leaves-based foods.Nowdays,Eucommia ulmoides leaves tea is commercially available,including Eucommia ulmoides leaves green tea,Eucommia ulmoides leaves Pu-erh tea,Eucommia ulmoides leaves tea powder,Eucommia ulmoides leaves tea drinks and Eucommia ulmoides leaves blended tea.Unfortunately,the soup of Eucommia ulmoides leaves tea maintains the heavy green color,and tastes bitter,which results in a low market acceptance.In this research,Eucommia ulmoides leaves tea were used as raw materials and processed to produce Eucommia ulmoides leaves Fu tea using the manufacturing technology of traditional Jingyang Fuzhuan tea.The resulting Eucommia ulmoides leaves Fu tea is decorated with the "golden flower",with a smell of the mixed fragrance of tea and soft flowers,as well as pure fungus flavor.Further,the soup color of tea is brown and red with a mellow taste.Then the material basis and mechanism of quality formation of Eucommia ulmoides leaves Fu tea were elucidated,and the effect of Eucommia ulmoides leaves Fu tea on hypolipidemic were examined.The results provided a theoretical basis for scientifically driving healthy consumption of Eucommia ulmoides Fu tea and efficient utilization of Eucommia ulmioides leaves.The main research contents and results are as follows:(1)The technology of Eucommia ulmoides leaves Fu tea by solid-state fermentation was thoroughly optimized.The effects of moisture content and time of pile,temperature,humidity and time of flowering on sensory quality of Eucommia ulmoides leaves Fu tea were systematically investigated,respectively,and the dynamic alternations of active components during the manufacturing process of Eucommia ulmoides leaves Fu tea were analyzed.The results showed that the optimal fermentation conditions were as following:the moisture content of pile was 30%,the time of pile was 12 h,the temperature of flowering was 28℃,the humidity of flowering was 70%,and the total time of flowering was 18 days.After drying and aging for one year,Eucommia ulmoides leaves Fu tea has some advantage properties such as smooth appearance,dry surface,moderate tightness,and dark brown color.Also,inside the tea golden flowers were generally lush without undesired microbes.The soup color is brownish red and bright with a smell of the pure fungus fragrance.The soup tastes a mellow smooth and round palate.Leaf base was brown,and leaves were intact with soft and bright characteristics.At the same time the contents of Eucommia ulmoides leaves Fu tea including water extract,caffeine,polysaccharide,total flavonoids,free amino acids,polyphenols,chlorogenic acid,geniposide,geniposidic acid,aucubin and soluble proteins of Eucommia ulmoides leaves Fu tea decreased significantly during the manufacturing process,whereas the contents of total proteins and tea pigments showed an increasing trend during the manufacturing process.(2)The microbiota structure of Eucommia ulmoides leaves Fu tea samples was analyzed using 16Sr DNA and ITS method during the manufacturing process.The results showed that the fungus which were detected in the samples of Eucommia ulmoides leaves Fu tea at different processing stages could be classified into 3 phyla fungus and 23 genera,and the bacteria could be classified into 7 phyla and 47 genera.The compositions and diversities of microbial community in Eucommia ulmoides leaves Fu tea changed obviously at early stage,and then gradually stabilized.Specifically,the compositions and diversities of fungal community reached a stable state after 8 days of flowering,while the composition and diversity of bacterial community reached a stable state after 12 days of flowering.Furthermore,Aspergillus was the dominant fungus in the processing of Eucommia ulmoides leaves Fu tea,and its relative abundance ranged from 56.34%to 95.58%in the stage from raw materials to 4 days of flowering,while its relative abundance was above 99.99%in the stage from 8 days of flowering and all subsequent stages,Aspergillus was the microbial basis that endowed Eucommia ulmoides leaves Fu tea with the characteristic of "lushly golden flowers".The overwhelmingly dominant position of Aspergillus in Eucommia ulmoides leaves Fu tea can effectively avoid the contamination of undesired microbes during the manufacturing production process,which is conducive to the directional regulation of fermentation process.(3)The gas chromatography-ion mobility spectrometry(GC-IMS)and headspace solid phase microextraction and gas chromatography-mass spectrometry(HS-SPEM-GC-MS)technique were used to analyze the changes of volatile compounds during Eucommia ulmoides leaves Fu tea processing.The results found that the volatile components detected by GC-IMS were mainly small molecules with carbon chains between C2 and C10,while those detected by HS-SPEM-GC-MS were mainly large molecules.The combination of the two methods was employed to comprehensively exhibit the changes of volatile components in Eucommia ulmoides leaves Fu tea.Overall,a total of 51 volatile components were identified by GC-IMS,including 16 aldehydes,10 alcohols,9 ketones,5 esters,4 pyrazines and 7 others.Moreover,a total of 152 volatile constituents were identified by HS-SPEM-GC-MS,including 13 acids,20 esters,18 ketones,22 alcohols,17 aldehydes,24 alkanes,15 aromatics,7 furans,10 alkenes and 6 others.GC-IMS analysis showed that,during the progress of processing of Eucommia ulmoides leaves Fu tea,the contents of pyrazines,aldehydes and ketones substances with green and earthy odor gradually decreased,while the contents of alcohols,aldehydes and esters substances with medicinal,fruit and alcohol odor gradually increased.The analysis of HS-SPEM-GC-MS detection results showed that during the progress of the processing process of Eucommia ulmoides leaves Fu tea,the content of some volatile components with pungent,green and pungent odor,such as propionic acid,acetic acid,2-hexenaldehyde,etc.,decreased significantly,while the volatile components with fungus flower fragrance,wood fragrance,flower fruit fragrance,frankincense,etc.such as linalool,lauric acid,myristic acid,ethyl palmitate,chicory ketone,acetaldehyde,etc.have been generated or their relative content increased.The alternations of these components might improve the aroma quality of Eucommia ulmoides leaves Fu tea.The comprehensive analysis showed that the flowering and aging stages were the main stages that formed the aroma characteristics of Eucommia ulmoides leaves Fu tea with "slight and pure fungus fragrance".(4)Ultra high performance liquid chromatography-quadrupole-exactive orbitrap mass spectrometry(UHPLC-Q-Exactive/MS)was used to determine the non-volatile components during the manufacturing process of Eucommia ulmoides leaves Fu tea.The results showed that a total of 166 lipid compounds and 133 non-lipid compounds were identified by UHPLC-Q-Exactive/MS.Specificly,the non-lipid compounds of 133 compounds mainly include 34 flavonoids and flavonoid glycosides,5 iridoids,18 phenolic acids,24 amino acids,31 organic acids,5 alkaloids,4 triterpene saponins,7 nucleosides and 5 other compounds.The results of principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)showed that flowering and aging were the key stages that formed the "mellow" taste quality of Eucommia ulmoides leaves Fu tea.The results also found that starch and sucrose metabolism,limonene and pinene degradation,and α-linolenic acid metabolism were the key metabolic pathways in the anterior segment of flowering(0 d to 8 d),the posterior segment of flowering(8 d to 25 d),and the aging stage,respectively.The contents of caffeic acid,chlorogenic acid,quinic acid and other phenolic acids with bitter taste decreased by 35.81%,46.67%and 24.89%,respectively,in the Eucommia ulmoides leaves Fu tea at aging for one year compared with the raw materials of Eucommia ulmoides leaves tea,indicating that the content reducation of caffeic acid,chlorogenic acid,quinic acid and other phenolic acid might play a major role in improving the taste of tea.In addition,the decrease of L-phenylalanine,L-tyrosine and other bitter amino acids contributed to the formation of the "mellow" taste of Eucommia ulmoides leaves Fu tea.(5)The model of hyperlipidemia rats was established.The experimental animals such as lipid metabolism physiological and biochemical indicators,intestinal flora structure changes and fat metabolism related gene expression changes were thoroughly investigated.The results showed that Eucommia ulmoides leaves Fu tea could perform the hypolipidemic effect.Eucommia ulmoides leaves Fu tea can significantly prevent the weight gain of rats fed with high-fat diet,reduce the levels of triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and enchance the level of high-density lipoprotein cholesterol(HDL-C)in serum of rats.The results of high-throughput sequencing showed that Eucommia ulmoides leaves Fu tea reversed the increase of the ratio of Firmicutes and Bacteroidota in the intestinal flora in rats caused by high-fat diet,which was conducive to the recovery of intestinal flora in rats,thereby reducing blood lipid by regulating lipid metabolism in rats.Addtionanly,the results of expression analysis of genes related to fat metabolism showed that Eucommia ulmoides leaves Fu tea could down-regulate the relative expression levels of adipocyte differentiation genes(C/EBPa,C/EBPβ and PPARy)and adiposynthesis genes(ACC and FASN)in liver of rats with hyperlipidemia induced by high-fat diet.Furthermore,the relative expression levels of lipolysis genes such as ACLY,ATGL,CPT-1A,PGC-1α,HSL,TLR4,Pla2g2a and Apoa4 were up-regulated,thereby alleviating the symptoms of hyperlipidemia and fatty liver and the oxidative stress state of liver in rats.