茶叶微生物固态发酵中咖啡碱降解途径初探

郑城钦; 马存强; 张正艳; 李肖宏; 吴婷婷; 周斌星

doi:10.13305/j.cnki.jts.2020.03.009

茶叶科学 >

2020 , Vol. 40 >Issue 3: 386 - 396

DOI: https://doi.org/10.13305/j.cnki.jts.2020.03.009

茶叶微生物固态发酵中咖啡碱降解途径初探

郑城钦 ,
马存强 ,
张正艳 ,
李肖宏 ,
吴婷婷 ,
周斌星

展开

1.云南农业大学龙润普洱茶学院,云南昆明 650201;
2.昆明大朴茶业有限公司,云南昆明 650224

郑城钦,男,硕士,主要从事制茶工程与质量控制方面的研究。

收稿日期: 2019-11-26

修回日期: 2019-12-23

网络出版日期: 2020-06-09

基金资助

云南农业云南省现代茶叶产业体系（2017KJTX007）、国家自然科学基金项目（31960617）、国家自然科学基金项目（31760225）、国家自然科学基金项目（31560221）

收起

A Preliminary Study on the Degradation Pathway of Caffeine in Tea Microbial Solid-state Fermentation

ZHENG Chengqin ,
MA Cunqiang ,
ZHANG Zhengyan ,
LI Xiaohong ,
WU Tingting ,
ZHOU Binxing

Expand

1. Longrun Pu-erh Tea College, Yunnan Agricultural University, Kunming 650201, China;
2. Kunming Dapu Tea Industry Company Limited, Kunming 650224, China

Received date: 2019-11-26

Revised date: 2019-12-23

Online published: 2020-06-09

Fold

摘要

为探究微生物作用下咖啡碱的降解产物与途径,将普洱茶发酵中筛选鉴定的Aspergillus sydowii NRRL250（聚多曲霉）、Aspergillus pallidofulvus NRRL4789、Aspergillus sesamicola CBS137324和Penicillium mangini CBS253.31等优势菌株分别接种至晒青毛茶进行单菌种固态发酵,并采用高效液相色谱（HPLC）测定咖啡碱、可可碱、茶碱的含量,探究微生物对咖啡碱代谢的影响;另外,基于UHPLC-QTOF-MS代谢组学技术,以灭菌处理组（ST组）和原料组（RM组）为对照,对聚多曲霉接种发酵样进行代谢组学分析。结果表明,A. pallidofulvus NRRL4789、A. sesamicola CBS137324和Penicillium mangini CBS253.31等优势菌株对咖啡碱等嘌呤类碱代谢均无显著影响,而在聚多曲霉接种发酵中,咖啡碱含量显著下降（P<0.05）,降幅达83.89%;茶碱含量显著增加（P<0.05）,发酵末期含量为(25.03±1.17) mg·g^-1;而可可碱保持基本稳定。由此可知,聚多曲霉对咖啡碱降解代谢有显著影响。采用UHPLC-QTOF-MS方法检出茶碱、3-甲基黄嘌呤、1,7-二甲基黄嘌呤等9种与咖啡碱降解相关的代谢物。在聚多曲霉作用下,茶碱、3-甲基黄嘌呤、1,7-二甲基黄嘌呤、7-甲基黄嘌呤含量显著提高（P<0.05）。茶碱、3-甲基黄嘌呤、1,7-二甲基黄嘌呤和1-甲基黄嘌呤与咖啡碱及其相关代谢物的N-脱甲基化途径相关。1,7-二甲基尿酸、1-甲基尿酸与咖啡碱相关代谢物的氧化途径相关。由此可知,聚多曲霉为降解普洱茶咖啡碱的优势菌株,且具有将咖啡碱转化为茶碱的潜在能力;在咖啡碱降解代谢过程中,存在聚多曲霉作用下的N-脱甲基化和氧化,并以N-脱甲基化为主。

关键词： 普洱茶; 固态发酵; 聚多曲霉; 咖啡碱; 降解途径

本文引用格式

郑城钦 , 马存强 , 张正艳 , 李肖宏 , 吴婷婷 , 周斌星 . 茶叶微生物固态发酵中咖啡碱降解途径初探[J]. 茶叶科学, 2020 , 40(3) : 386 -396 . DOI: 10.13305/j.cnki.jts.2020.03.009

Abstract

In order to explore caffeine degradation products and pathways under the action of microorganisms, the dominant strains including Aspergillus sydowii NRRL250, Aspergillus pallidofulvus NRRL4789, Aspergillus sesamicola CBS137324 and Penicillium mangini CBS253.31 were screened and identified during pu-erh tea fermentation. Strains were inoculated into sun-dried green tea leaves for solid-state fermentation. High performance liquid chromatography (HPLC) was used to determine caffeine, theobromine and theophylline contents to explore the effect of microorganisms on caffeine metabolism. UHPLC-QTOF-MS was used for the metabonomic analysis of Aspergillus sydowii inoculated fermentation with sterilization treatment group (ST group) and raw material group (RM group). The results show that the dominant strains such as A. pallidofulvus NRRL4789, A. sesamicola CBS137324 and Penicillium mangini CBS253.31 had no significant effects on the metabolism of caffeine and other purine alkaloids. However, caffeine content was decreased significantly (P<0.05) with a great reduction about 83.89% during the inoculated fermentation of Aspergillus sydowii. Additionally, theophylline content was increased significantly (P<0.05) and arrived to (25.03±1.17) mg·g^-1 at the end of fermentation. While theobromine content remained stable. Therefore, Aspergillus sydowii has a profound effect on caffeine degradation metabolism. Nine metabolites related to caffeine degradation were detected by UHPLC-QTOF-MS during the inoculated fermentation, Among them, theophylline, 3-methylxanthine, 1,7-dimethylxanthine and 7-methylxanthine contents were significantly increased (P<0.05) under the action of Aspergillus sydowii which were related to N-demethylation pathway of caffeine and its related metabolites. 1,7-dimethyluric acid and 1-methyluric acid were related to the oxidation pathway of caffeine-related metabolites. It can be seen that Aspergillus sydowii is the dominant strain that can degrade caffeine and has the potential ability to convert caffeine into theophylline. Under the action of spergillus sydowii, both N-demethylation and oxidation were found in caffeine degradation metabolism, and the former was the dominant.

Key words： Pu-erh tea; solid-state fermentation; Aspergillus sydowii; caffeine; degradation pathway

参考文献

[1] Zhang L, Zhang Z Z, Zhou Y B, et al.Chinese dark teas: Postfermentation, chemistry and biological activities[J]. Food Research International, 2013, 53(2): 600-607.
[2] 全国原产地域产品标准化工作组. 地理标志产品普洱茶: GB/T 22111—2008 [S]. 北京: 北京标准出版社, 2008.
National regional product standardization working group of the People's Republic of China. Geographical indication products Pu-erh tea: GB/T 22111—2008 [S]. Beijing: Beijing Standard Press, 2008.
[3] 陈华红, 李雪玲, 岩燕, 等. 顶头孢霉对普洱茶品质的影响[J]. 食品科技, 2011, 36(10): 53-56.
Chen H H, Li X L, Yan Y, et al.The effects of a strain of Cephalosporium acremonium on the quality of Pu-er tea[J]. Food Science and Technology, 2011, 36(10): 53-56.
[4] Lv H P, Zhang Y J, Lin Z, et al.Processing and chemical constituents of Pu-erh tea: A review[J]. Food Research International, 2013, 53(2): 608-618.
[5] Zhang L, Li N, Ma Z Z, et al.Comparison of the chemical constituents of aged Pu-erh tea, ripened Pu-erh tea, and other teas using HPLC-DAD-ESI-MSn[J]. Journal of Agricultural and Food Chemistry, 2011, 59(16): 8754-8760.
[6] Wang D, Xu K L, Zhong Y, et al.Acute and subchronic oral toxicities of Pu-erh black tea extract in Sprague-Dawley rats[J]. Journal of ethnopharmacology, 2011, 134(1): 164. doi: 10.1016/j.jep.2010.11.068.
[7] Wang D, Xiao R, Hu X T, et al.Comparative safety evaluation of Chinese Pu-erh green tea extract and Pu-erh black tea extract in Wistar rats[J]. Journal of Agricultural and Food Chemistry, 2010, 58(2): 1350-1358.
[8] Hollingsworth R G, Armstrong J W, Campbell E.Caffeine as a repellent for slugs and snails[J]. Nature, 2002, 417(6892): 915-916.
[9] 栗娜, 籍保平, 李博, 等. 可降解咖啡碱菌株的筛选与鉴定[J]. 食品科学, 2010, 31(21): 218-221.
Li N, Ji B P, Li B, et al.Screening and identification of aPseudomonas luteastrain capable of degrading caffeine[J]. Food Science, 2010, 31(21): 218-221.
[10] Gokulakrishnan S, Chandraraj K, Gummadi S N.A preliminary study of caffeine degradation byPseudomonassp. GSC 1182[J]. International Journal of Food Microbiology, 2007, 113(3): 346-350.
[11] Zhou B X, Ma C Q, Wang H Z, et al.Biodegradation of caffeine by whole cells of tea-derived fungiAspergillus sydowii,Aspergillus nigerand optimization for caffeine degradation[J]. BMC Microbiology, 2018, 18(1): 53. doi: 10.1186/s12866-018-1194-8.
[12] Scharbert S, Hofmann T.Molecular definition of black tea taste by means of quantitative studies, taste reconstitution, and omission experiments[J]. J Agric Food Chem, 2005, 53(13): 5377-5384.
[13] He R, Chen L, Lin B, et al.Beneficial effects of oolong tea consumption on diet-induced overweight and obese subjects[J]. Chinese Journal of Integrative Medicine, 2009, 15(1): 34-41.
[14] 顾陈菲, 宝丽, 谢果, 等. 狭叶茶提取物对小鼠氧化应激性肝损伤的保护作用[J]. 茶叶科学, 2008, 28(2): 115-122.
Gu C F, Bao L, Xie G, et al.Protective effects ofCamellia angustifoliachang on oxidative stress-induced liver damage in mice[J]. Journal of Tea Science, 2008, 28(2): 115-122.
[15] Kurihara H, Fukami H, Asami S, et al.Effects of oolong tea on plasma antioxidative capacity in mice loaded with restraint stress assessed using the oxygen radical absorbance capacity (ORAC) assay[J]. Biological & Pharmaceutical Bulletin, 2004, 27(7): 1093-1098.
[16] Nawrot P, Jordan S, Eastwood J, et al.Effects of caffeine on human health[J]. Food Additives and Contaminants, 2003, 20(1): 1-30.
[17] 黄丽凤, 刘友平, 黎代余. 茶多酚提取纯化工艺研究进展[J]. 中国食品添加剂, 2010(1): 69-72.
Huang L F, Liu Y P, Li D Y.Advances on research of purification of tea polyphenols from green tea leaves[J]. China Food Additives, 2010(1): 69-72.
[18] 姚永宏, 柴友荣, 李中林, 等. 降低茶叶咖啡碱的研究进展[J]. 西南农业学报. 2009, 22(6): 1799-1802.
Yao Y H, Cai Y R, Li Z L, et al.Research progress for tea decaffeination[J]. Southwest China Journal of Agricultural Sciences, 2009, 22(6): 1799-1802.
[19] Iancu I, Strous R D. Caffeine intoxication: history, clinical features, diagnosis and treatment [J]. Harefuah, 2006, 145(2): 147-151, 163-164.
[20] Wang Q P, Gong J S, Chisti Y, et al.Fungal isolates from a Pu-erh type tea fermentation and their ability to convert tea polyphenols to theabrownins[J]. J Food Science, 2015, 80(4): M809-M817.
[21] 马存强, 王洪振, 周斌星, 等. 普洱茶固态发酵中转化咖啡碱为茶碱菌株的鉴定与应用[J]. 食品工业科技, 2018, 39(15): 119-124.
Ma C Q, Wang H Z, Zhou B X, et al.Identification and application of fungal converting caffeine to theophylline from Pu-erh tea solid-state fermentation[J]. Science and Technology of Food Industry, 2018, 39(15): 119-124.
[22] 马存强, 周斌星, 王洪振, 等. 普洱茶渥堆发酵中可降解咖啡碱真菌菌株的筛选和鉴定[J]. 茶叶科学, 2017, 37(2): 211-219.
Ma C Q, Zhou B X, Wang H Z, et al.Screen and identification of fungi strain degrading caffeine in Pu-erh tea during solid-state fermentation[J]. Jounal of Tea Science, 2017, 37(2): 211-219.
[23] Wang X G, Wan X C, Hu S X, et al.Study on the increase mechanism of the caffeine content during the fermentation of tea with microorganisms[J]. Food Chemistry, 2008, 107(3): 1086-1091.
[24] Ivanisevic J, Elias D, Deguchi H, et al.Arteriovenous blood metabolomics: a readout of intra-tissue metabostasis[J]. Sci Rep, 2015, 5(1): 12757. doi: 10.1038/srep12757.
[25] Liang Y R, Zhang L Y, Lu J L.A study on chemical estimation of Pu-erh tea quality[J]. Journal of the Science of Food and Agriculture, 2005, 85(3): 381-390.
[26] Ashihara H, Gillies F M, Crozier A.Metabolism of caffeine and related purine alkaloids in leaves of tea (Camellia sinensisL.)[J]. Plant and Cell Physiology, 1997, 38(4): 413-419.
[27] Woolfolk C A.Metabolism of N-methylpurines by a Pseudomonas putida strain isolated by enrichment on caffeine as the sole source of carbon and nitrogen[J]. Journal of bacteriology, 1975, 123(3): 1088-1106.
[28] Ashihara H, Sano H, Crozier A.Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering[J]. Phytochemistry, 2008, 69(4): 841-856.
[29] Mazzafera P.Catabolism of caffeine in plants and microorganisms[J]. Frontiers in Bioscience, 2004(9): 1348-1359.
[30] Mccarthy A A, Mccarthy J G.The structure of two N-methyltransferases from the caffeine biosynthetic pathway[J]. Plant Physiol, 2007, 144(2): 879-889.
[31] Hakil M, Denis S, Viniegra-González G, et al.Degradation and product analysis of caffeine and related dimethylxanthines by filamentous fungi[J]. Enzyme and Microbial Technology, 1998, 22(5): 355-359.
[32] Cornish H H, Christman A A.A study of the metabolism of theobromine, theophylline, and caffeine in man[J]. J Biol Chem, 1957, 228(1): 315-323.

Options

文章导航

模态框（Modal）标题

摘要

本文引用格式

Abstract

参考文献