Novel Oxidation Oligomer of Chlorogenic Acid and (-)-Epigallocatechin and Its Quantitative Analysis during the Processing of Keemun Black Tea.

Not only do flavan-3-ols participate in the formation of chromogenic oxidation products such as theaflavins, but chlorogenic acid (3-caffeoylquinic acid, CQA) is also involved in the enzymatic oxidation during black tea processing. The critical oxidation product of CQA and (-)-epigallocatechin (EGC) were identified as an adduct containing benzobicyclo[3.2.2]nonenone structure, which was named as the dichlorogeniccatechin (DCGC) oligomer. It was composed of two molecules of CQA and one molecule of EGC. The effects of the initial reactant ratio and reaction time on the generation of DCGC were also analyzed. A high proportion of CQA promoted the production of DCGC, but a high proportion of EGC inhibited the DCGC formation. In addition, the content of DCGC in Keemun black tea during processing was determined. The content of DCGC highly increased after withering but decreased after drying. This study provides a new perspective for the investigation of other oxidation oligomers in black tea.

Amadori Reaction Products of Theanine and Glucose: Formation, Structure, and Analysis in Tea.

Amadori rearrangement products (ARPs) derived from the Maillard reaction between theanine and glucose (ARP 1), as well as pyroglutamic acid and glucose (ARP 2), were identified by liquid chromatograph tandem mass spectroscopy methods. The effects of initial reactant ratio, temperature, pH, and heating time on ARP generation were analyzed. The formation of both ARPs was most favored under 100 °C, while an alkaline environment slightly promoted the generation of ARP 1 and acidic conditions contributed more to ARP 2 formation. The decomposition of ARP 1 was suggested to be the predominant formation mechanism of ARP 2. Preparation, purification, and structure identification of ARP 1 were conducted, with its structure confirmed as 1-deoxy-1-l-theanino-d-fructose. The contents of ARP 1 in green, black, dark, white, yellow, and Oolong teas were quantitatively determined, of which black teas contained the highest levels of ARP 1, possibly due to the high glucose content and processing techniques.

Free, soluble conjugated and insoluble bonded phenolic acids in Keemun black tea: From UPLC-QQQ-MS/MS method development to chemical shifts monitoring during processing.

Phenolic acids, including benzoic acid and hydroxycinnamic acid derivatives, are the main compounds of black tea. An efficient and accurate analytical method to quantify ten phenolic acids was established and validated by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS). The chemical shifts during the processing of Keemun black tea were analyzed and the phenolic acids were quantified. Compared with fresh tea leaves, after processing, the contents of free phenolic acids, including gallic acid, salicylic acid, p-coumaric acid, and ferulic acid, increased markedly. Still, the contents of soluble conjugated phenolic acids, including p-coumaroylquinic acid isomers and chlorogenic acid isomers, decreased. Furthermore, the total contents of lignin, and insoluble bonded phenolic acids decreased. The adduct of (-)-epigallocatechin gallate and 3-caffeoylquinic acid was detected in tea samples, and its content increased highly after fermentation. The developed and validated analytical method can be used to monitor the manufacturing process of black tea.

Identification of low-molecular-weight color contributors of black tea infusion by metabolomics analysis based on UV-visible spectroscopy and mass spectrometry.

Nine black tea samples with different color intensity were firstly determined by chromatic difference analyzer. The color characteristics were secondly quantitatively described by UV-visible spectroscopy. Thirdly, liquid chromatography tandem mass spectrometry (LC-MS) based metabolomics analysis was applied in low-molecular-weight compounds. Finally, the color contributors were identified by the correlation analysis of color, spectrometry and mass data. UV-visible based metabolomics analysis revealed that the wavelength at 380-520 nm (VIP > 1.50) was the critical absorbance band for distinguishing different color of BT infusions, while LC-MS based metabolomics analysis indicated that there were 48 main marker compounds responsible for the classification of different BT infusions. Correlation analysis results showed that the coefficients of theaflavins, thearubigins, theabrownins, flavonoid glycosides, and some hydroxycinnamoyl acids were > 0.7, which suggested they were main color contributors of BT infusion. The present study expanded a new vision on the color analysis of BT infusion.

Study on In Vitro Preparation and Taste Properties of N-Ethyl-2-Pyrrolidinone-Substituted Flavan-3-Ols.

N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs) were prepared by an in vitro model reaction, and the taste thresholds of EPSFs and their dose-over-threshold factors in large-leaf yellow tea (LYT) were investigated. The effects of initial reactant ratios, reaction temperatures and time, pH values, and water addition on the yield of EPSFs were explored. The contents of EPSFs during roasting were determined by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). When the initial ratio of (-)-epigallocatechin gallate (EGCG) to theanine was 1:2 and roasted under 120 °C for 120 min, the contents of EPSFs were the highest. The bitterness and astringency thresholds of four EPSF isomers were measured by the half-tongue method, of which EPSF2 and EPSF3 had higher thresholds than EGCG. In LYT, four EPSFs had lower bitterness and astringency dose-over-threshold factors than EGCG. This study suggested that the reduction of bitterness and astringency of tea after roasting may be mainly due to the formation of EPSFs.

Model Studies on the Reaction Products Formed at Roasting Temperatures from either Catechin or Tea Powder in the Presence of Glucose.

During tea processing, roasting significantly affects the transformation pathway of catechins. When (-)-epigallocatechin gallate (EGCG) and glucose were roasted at different pH values, the degree of degradation and isomerization of EGCG was the lowest at pH 7 and the highest at pH 8. Thirty-five products were found in the model reaction of EGCG and glucose under high temperatures, of which four EGCG-glucose adducts were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). In addition, catechins, gallic acid, and theanine in tea with added glucose were significantly reduced during roasting. The contents of four EGCG-glucose adducts were increased significantly at 150 °C after 30 min and dropped gradually after 60 min. Therefore, based on the present study, EGCG could form crosslinks with glucose under high temperatures in a short time, which provides insight for tea processing and synthesis of catechin-sugar adducts.