混菌发酵绿茶产脂肪酶及其对酯型儿茶素的水解作用分析
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摘要
微生物的发酵作用导致黑茶加工过程中儿茶素的生物转化和总茶多酚含量的下降,这一转化过程的代谢途径和调控机制尚不清楚。通过添加冠突曲霉(Aspergillus cristatum)菌株PE-1和黑曲霉(A. niger)菌株PE-4混合发酵绿茶,采用鉴别培养基平板检测、酶活性测定和酶蛋白分离纯化,研究脂肪酶活性及其对酯型儿茶素的水解作用。两个菌株单菌发酵和混合菌种发酵绿茶条件下均可以检测到脂肪酶活性,混菌发酵酶活力高于两个菌株单菌发酵的酶活力。混菌发酵条件下获得一个分子量约为37 kDa的脂肪酶,对EGCG和ECG有不同的生物转化作用。UPLC检测结果表明,该脂肪酶对EGCG有较好的底物选择性,EGCG的水解率为94.46%,EGC的产率为63.33%;ECG的水解率为15.45%,EC的产率为4.15%。脂肪酶对EGCG和ECG的生物转化将为儿茶素代谢途径的研究和单体儿茶素的生产起到促进作用。
In the process of dark tea manufacture, microbial fermentation leads to the biotransformation of catechins and the decrease of the total polyphenol contents. Up to date, the metabolic pathways and regulatory mechanisms of this transformation process were still unclear. The green tea was fermented by mixed fermentation of Aspergillus cristatum strain PE-1 and A. niger strain PE-4. Lipase activity was detected by a differential culture medium plate test and enzyme activity assay. The results indicate that lipase activities in mixed strain fermentation were stronger than that of signal strain fermentation. In mixed fermentation, a lipase protein with a molecular weight of approximately 37 kDa was obtained by protein separation and purification, which had different biotransformation efficiency for EGCG and ECG. HPLC results show that the lipase had a better substrate selectivity for EGCG than that for ECG. The efficiency of EGCG hydrolysis and the yield of EGC were 94.46% and 63.33%. While the efficiency of ECG hydrolysis and the yield of EC were 15.45% and 4.15%. The biotransformation of EGCG and ECG by lipase would promote the study of the catechin metabolism and the production of monomeric catechins.
In the process of dark tea manufacture, microbial fermentation leads to the biotransformation of catechins and the decrease of the total polyphenol contents. Up to date, the metabolic pathways and regulatory mechanisms of this transformation process were still unclear. The green tea was fermented by mixed fermentation of Aspergillus cristatum strain PE-1 and A. niger strain PE-4. Lipase activity was detected by a differential culture medium plate test and enzyme activity assay. The results indicate that lipase activities in mixed strain fermentation were stronger than that of signal strain fermentation. In mixed fermentation, a lipase protein with a molecular weight of approximately 37 kDa was obtained by protein separation and purification, which had different biotransformation efficiency for EGCG and ECG. HPLC results show that the lipase had a better substrate selectivity for EGCG than that for ECG. The efficiency of EGCG hydrolysis and the yield of EGC were 94.46% and 63.33%. While the efficiency of ECG hydrolysis and the yield of EC were 15.45% and 4.15%. The biotransformation of EGCG and ECG by lipase would promote the study of the catechin metabolism and the production of monomeric catechins.
引文
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[3]Li Q,Huang J,Li Y,et al.Fungal community succession and major components change during manufacturing process of Fu brick tea[J].Scientific Reports,2017,7:6947.doi:10.1038/s41598-017-07098-8.
[4]Zhao M,Zheng D L,Su X Q,et al.An integrated metagenomic/metaproteomic investigation of the microbial communities and enzymes in solid-state fermentation of Pu-erh tea[J].Scientific Reports,2015,5:10117.Doi:10.1038/srep10117.
[5]舒正玉,杨江科,闫云君.黑曲霉F044脂肪酶的分离纯化及酶学性质研究[J].生物工程学报,2007,23(1):96-100.
[6]陈桂梅,邓永亮,黄亚亚,等.冠突散囊菌生长过程中几种胞外酶活性变化[J].茶叶科学,2013,33(4):306-310.
[7]黄建安,刘仲华,施兆鹏.茯砖茶制造中主要酶类的变化[J].茶叶科学,1991,11(增刊1):63-68.
[8]杨金梅,严蒸蒸,陈佳佳,等.散囊菌属真菌胞外水解酶和菌株LP-7分泌蛋白分析[J].菌物学报,2017,36(6):691-704.
[9]Sang S,Lambert JD,Ho C-T,et al.The chemistry and biotransformation of tea constituents[J].Pharmacological Research,2011,64(2):87-99.
[10]夏涛,高丽萍,刘亚军,等.茶树酯型儿茶素生物合成及水解途径研究进展[J].中国农业科学,2013,46(11):2307-2320.
[11]Qin J-H,Li N,Tu P-F,et al.Change in tea polyphenol and purine alkaloid composition during solid-state fungal fermentation of postfermented tea[J].Journal of Agricultural&Food Chemistry,2012,60(5):1213-1217.
[12]Xu J,Hu F-L,Wang W,et al.Investigation of biochemical compositional changes during the microbial fermentation process of Fu brick tea by LC-MS based metabolomics[J].Food Chemistry,2015,186:176-184.
[13]Zhong K,Zhao S-Y,Jonsson L J,et al.Enzymatic conversion of epigallocatechin gallate to epigallocatechin with an inducible hydrolase from Aspergillus niger[J].Biocatalysis and Biotransformation,2008,26(4):306-312.
[14]Zhang S,Gao X,He L,et al.Novel trends for use of microbial tannases[J].Preparative Biochemistry&Biotechnology,2015,45(3):221-231.
[15]聂志银,刘亚军,刘莉,等.茶树酯型儿茶素水解酶鉴定及其检测体系的建立[J].茶叶科学,2011,31(5):439-446.
[16]Aguilar C N,Rodríguez R,Gutiérrez-Sánchez G,et al.Microbial tannases:advances and perspectives[J].Applied Microbiology&Biotechnology,2007,76(1):47-59.
[17]Chávez-González C,Rodríguez-Durán L V,Balagurusamy N,et al.Biotechnological advances and challenges of tannase:an overview[J].Food and Bioprocess Technology,2011,5(2):445-459.
[18]Chen H,Sang S.Biotransformation of tea polyphenols by gut microbiota[J].Journal of Functional Foods,2014,7(1):26-42.
[19]Hocking A D,Pitt J I.Dichloran-glycerol medium for enumeration of xerophilic fungi from low-moisture foods[J].Applied and Environmental Microbiology,1980,39(3):488-492.
[20]Gramss G,Gunther T,Fritsche W.Spot test for oxidative enzymes in ectomycorrhizal wood and litter decaying fungi[J].Mycological Research,1998,102(1):67-72.
[21]徐德阳,王莉莉,杜春梅.微生物共培养技术的研究进展[J].微生物学报,2015,55(9):1086-1096.
[22]Bertrand S,Bohni N,Schnee S,et al.Metabolite induction via microorganism co-culture:a potential way to enhance chemical diversity for drug discovery[J].Biotechnology Advances,2014,32(6):1180-1204.
[23]Jayabalan R,Marimuthu S,Swaminathan K.Changes of content of organic acids and polyphenols during kombucha tea fermentation[J].Food Chemistry,2007,102(1):392-398.