ABSTRACT Qingzhuan tea (QZT), a post-fermented tea, has been reported to have anti-obesity and anti-hyperglycemic effects, perhaps due to bioactive compounds that inhibit lipase and

α-amylase. It is unknown what chemical constituents’ changes and what bioactive compounds occur during the manufacture of QZT. The aim of this study was to determine the secondary

metabolites changes that occur during post-fermentation and how these changes affect the ability of QZT to inhibit the activities of lipase and α-amylase. During the processing steps,

metabolites levels and their inhibitory effects on lipase and α-amylase were assessed. Changes in content and activities suggested that the first turn over or the second turn over was

compounds were altered during pile fermentation of QZT and tentatively identified the bioactive compounds formed during QZT manufacture. SIMILAR CONTENT BEING VIEWED BY OTHERS CHANGES IN

AMINO ACIDS, CATECHINS AND ALKALOIDS DURING THE STORAGE OF OOLONG TEA AND THEIR RELATIONSHIP WITH ANTIBACTERIAL EFFECT Article Open access 07 May 2024 Β-GLUCOSIDASE-PRETREATED BLACK GOJI

BERRY TEA REDUCES GLUCOSE RELEASE AND ENHANCES BILE ACID BINDING CO-DIGESTION WITH HIGH-FAT MEALS IN SIMULATED DIGESTION Article Open access 11 April 2025 KLUAI HIN (_MUSA SAPIENTUM_ LINN.)

PEEL AS A SOURCE OF FUNCTIONAL POLYPHENOLS IDENTIFIED BY HPLC-ESI-QTOF-MS AND ITS POTENTIAL ANTIDIABETIC FUNCTION Article Open access 09 March 2022 INTRODUCTION Qingzhuan tea (QZT) is an

important Chinese dark tea, mainly produced in Hubei province. It has long been an indispensable beverage for people in high-fat and high-calorie diet areas, such as the Mongolia, Xinjiang,

Qinghai and Gansu provinces of China1. QZT is made through five processing steps. Briefly, one bud and five leaves are plucked, fixed at high temperature, rolled, dried in the sun, then

fermented in a large pile, which is turned three times and aged for several months. After this pile fermentation, the tea is steamed, pressed and dried to obtain the Qingzhuan dark tea

(QZT)2,3. Like other dark teas, pile fermentation is critical for the formation of the characteristic flavor and quality of QZT2,4. During this process, oxidation, condensation, degradation,

and polymerization of polyphenols and amino acids occur through the joint actions of damp heat and microorganisms to form the pure aroma and mellow taste of QZT3,5,6. The current research

on QZT fermentation has mainly focused on fluorine removal, leaf structural changes, polysaccharide content, and microbial sequencing3,7,8,9. However, little research has been done on the

chemical changes during pile fermentation of QZT, which has led to the process not being accurately controlled and thus varied quality of QZT. Tea manufacturing is an indispensable condition

for the formation of quality components and functional ingredients. Different tea types have unique quality characteristics and health effects due to different processing techniques10. By

adjusting and optimizing the tea processing techniques, metabolic flux could be altered, quality ingredients and functional ingredients transformations can be purposefully activated, thereby

improving tea quality components formation and health effects11. Therefore, the analysis of the variation in chemical constituents during pile fermentation of QZT processing may help to

elucidate the formation mechanisms of tea quality and health functions, and also provide a theoretical basis for QZT quality control. Obesity is a major public health problem, and highly

related to metabolic diseases, such as hyperlipidemia, hyperglycemia and cardiovascular disease12. Excessive intake of carbohydrates and lipids is the leading cause of obesity. Some

digestive enzyme inhibitors, such as acarbose or orlistat, have been used to treat diabetes or obesity by limiting the absorption of carbohydrates or lipids13,14. However, these chemically

synthesized inhibitors have some gastrointestinal side effects. Therefore, there is an ongoing search for natural digestive enzyme inhibitors with fewer side effects for prevention or

mitigation the symptom of obesity or diabetes. Previous researches have shown that some types of tea exhibited pronounced inhibitory effects towards the activities of α-amylase and

pancreatic lipase15,16,17,18. There are few reports on whether QZT, produced under a different process, inhibits pancreatic α-amylase and lipase activities _in vitro_, and it is unknown what

components within QZT contribute to anti-obesity and anti-hyperglycemic bioactivities. In studies on other types of tea, both targeted and untargeted metabolomics approaches have been

followed19,20,21. Applying liquid chromatography tandem mass spectrometry (LC-MS) and multivariate statistical analysis together, the complexity and variability of the metabolites present in

tea can be revealed22,23,24. In this study, we used high-performance liquid chromatography (HPLC) and a non-targeted metabolomics approach to determine the dynamic metabolic changes that

occur during QZT processing and to identify the bioactive compounds found during processing and in the final tea product. Tea extracts from different processes was tested for inhibition of

pancreatic α-amylase to determine whether the anti-obesity effect of QZT is mainly caused by slowing down the digestion of starch through inhibiting α-amylase activity. In addition, it was

suggested that QZT might prevent fat absorption by inhibiting pancreatic lipase similar to Pu’er tea25. To explore this hypothesis, water extract of each pile fermentation step samples were

investigated by an _in vitro_ assay of pancreatic lipase inhibition. To sum up, the purpose of this study was to clarify the effect of pile fermentation on the chemical content and the

anti-obesity and anti-hyperglycemic health effects of QZT. MATERIALS AND METHODS TEA PROCESSING Fresh tea leaves (FL) comprising one bud with five leaves were harvested from the cultivars

grown around Zhaoliqiao tea factory at Chibi City (Fig. 1). The leaves were fixed at 300 °C for 2 min, rolled for 10 min, then dried in the sun to less than 13% water content to obtain the

raw tea (RT). The RT was wetted to 30% humidity and piled into 3 m tall, 2 m wide piles on the floor of an warehouse for continuous fermentation with three turnings and samples taken at 7

days (first turn over, FT), 14 days (second turn over, ST), 21 days (third turn over, TT), 51 days (aged for 1 month, A1), and 111 days (aged for 3 month, A3). The A3 samples were also

steamed, pressed and dried to obtain the Qingzhuan tea (QZT). In order to ensure relative consistency, representativeness, and accuracy of the experiments, five experimental samples (5 kg)

were collected from the same batch of QZT, immediately freeze-dried (FreeZone® plus Freeze dryer, LABCONCO, America), and stored at −20 °C until analysis. SAMPLES AND CHEMICALS Standards of

18 amino acids and L-theanine (purity >99%), caffeine (CAF, >98%), (−)-gallocatechin (GC, >98%), (−)-epicatechin (EC, >98%), (+)-catechin (C, >98%), (−)-epigallocatechin (EGC,

>98%), (−)- gallocatechin gallate (GCG, >98%), (−)-epicatechin gallate (ECG, >98%), (−)-epigallocatechin gallate (EGCG, >98%), gallic acid (GA, >98%), Pancreatic α-amylase

and pancreatic lipase were bought from Yuanye Biotechnology Company (Shanghai, China). DL-4-chlorophenylalanine was bought from Sigma Chemical Co. (St. Louis, MO). Methanol, acetonitrile and

formic acid were bought from Thermo Fisher (Thermo Scientific, USA). SAMPLE PREPARATION For LC-MS analysis, the freeze-dried tea powder (50 mg) was weighed into a 1.5 mL centrifuge tube.

After the addition of 1 mL of extract solvent (methanol-acetonitrile-water, 2:2:1), the samples were mixed with ultrasonication at 60 Hz and 25 °C for 20 min. The supernatants were gained

after centrifuging at 12000 rpm at 4 °C, and transferred to a sample vial for LC-MS analysis. For pancreatic α-amylase and lipase inhibition assays, the freeze-dried tea samples were ground

into powder. Tea powder (0.1 g) was extracted with 5 mL of distilled water at 100 °C for 45 min while stirring, yielding a tea infusion. The supernatants were sieved through a 0.22 μm

aqueous filter (Jinteng, Tianjin, China) before analysis. DETERMINATION OF POLYPHENOLS AND CAFFEINE Tea polyphenols and purine alkaloids were extracted using the method described by Ma _et

al_., with minor modifications26. In brief, 3 mL of 70% methanol was added to tea powder (0.1 g) in a 5 mL centrifuge tube and vortexed at room temperature for 10 min. After centrifugation

at 6000 rpm for 5 min, the sediment was extracted twice using the same method. The supernatants were collected together and diluted in 5 mL 70% methanol. The combined samples were filtered

using a 0.22 μm organic membrane (Jinteng, Tianjin, China) before performing HPLC. The HPLC (Waters 2695, Milford, America) was coupled with Agilent ZORBAX SB-Aq C18 (250×4.6 mm, 5 μm)

column, UV detector (set at 280 nm) and a 2998 PDA detector. Column temperature was 40 °C. Mobile phase A was 2% (_v_/_v_) acetic acid, and mobile phase B was acetonitrile. The elution

gradient was set as follows: 0 min, 93.5% A; 16 min, 15% A; 16–25 min, 25% A; 30 min, 93.5% A; and 40 min, 93.5% A. The injection volume and the flow rate were 10 μL and 1.0 mL/min. The

contents of theaflavins (TFs), thearubigins (TRs) and theabrownin (TBs) were determined using spectrometric methods27. DETERMINATION OF FREE AMINO ACIDS Amino acids concentrations were

assayed following the method of Ma _et al_., but with minor changes26. In brief, a 0.1 g tea sample was extracted at 100 °C for 45 min in 10 mL of water; the mixture was shaken once every 15

 min. The filtrates were sieved by a 0.22 μm aqueous filter (Jinteng, Tianjin, China) and analyzed by HPLC. Free amino acids were detected using a Waters 2695 HPLC system coupled with a 2998

PDA detector. Mobile phase A, B, C was Waters AccQ•Tag Eluent A, acetonitrile and water, respectively. The column temperature and wavelength were 37 °C and 248 nm. The elution gradient was

set as follows: 0 min, 100% A; 0.5 min, 99% A, 1% B; 18 min, 95% A, 5% B; 19 min, 91% A, 9% B; 10.1 min, 1% B; 29.5 min, 83% A, 17% B; 33 min, 60% B, 40% C; 36 min, 100% A; and 45 min, 100%

A. The injection volume and the flow rate were 10 μL and 1.0 mL/min. Soluble sugar was assayed following the sulfuric acid-anthrone colorimetric method28. LC-MS ANALYSIS LC-MS analysis was

conducted following Wang _et al_., with some modifications20. LC-MS analysis was conducted using a Ultimate 3000 (Dionex, Sunnyvale, CA) coupled with an Orbitrap Elite™ hybrid ion

trap-orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Hyper Gold column (1.9 μm, 2.1×100 mm) was used to separate tea samples. Mobile phases A and B was 0.1% (_v_/_v_) formic acid

and acetonitrile. The elution gradient was set as follows, 0 min, 5% B; 2 min, 40% B; 7 min, 80% B; 11 min, 95% B; 15 min, 95% B; 15.5 min, 5% B; 20 min, 5% B. The injection volume and the

flow rate were 4 μL and 0.3 mL/min. The MS was conducted under both positive and negative modes with HESI spray voltage at 3.8 and 3.2 KV, successively. The sheath gas pressure, auxiliary

gas pressure, capillary temperature was 35 arb, 10 arb and 350 °C, respectively. The full-scan MS mode with resolution 60,000 and scan range _m_/_z_ 50–1000 was used for LC-MS running.

METABOLOMICS ANALYSIS The raw data collected from the LC-MS was analysed using the Qualitative Analysis Software (Thermo SIEVE 2.1) to get the peaks information of retention time,

mass-to-charge ratio (_m/z_), and intensity of MS. For each chromatogram, each ion intensity was calibrated using the internal standard. The obtained data was processed by multivariate

statistical analyses, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), to determine the tea metabolites variations induced

by the steps of the manufacturing process. PANCREATIC Α-AMYLASE AND LIPASE INHIBITION ASSAYS The ability of each sample (FT through QZT) to inhibit pancreatic α-amylase was analysed using an

iodine-starch kit29. Briefly, the 0.1 g tea power were extracted in 5 mL water, 50 μL sample and 1.0 mL starch substrate (0.4 g/L) were added to pH 7.0 PBS solution (incubation at 37 °C for

5 min), then 50 μL α-amylase (1 mg/mL) was mixed together. After incubated for 7.5 min, 1.0 mL iodine diluent (0.01 M) and 3.0 mL water was added for measuring at 660 nm. All samples were

analysed with six independent repetitions. The inhibitory rate of samples on α-amylase was calculated by the following equation: $${\rm{Inhibition}}\,{\rm{rate}}\,( \%

)=({1-A}_{{\rm{sample}}}{/A}_{{\rm{blank}}})\times 100 \% $$ The ability of tea samples to inhibit pancreatic lipase was assayed by a kit following the published method12. Briefly, 50 μL tea

sample and 1.0 mL substrate (0.4 g/L) were added to 100 mM pH 8.2 Tris-HCl buffer (incubation at 37 °C for 5 min), then 50 μL lipase (1 mg/mL) was mixed together and immediately measured

the absorbance (A1) at 420 nm. After 20 min, the absorbance (A2) was measured again. All samples were analysed in six biological duplications. The inhibition rate of samples on lipase was

calculated as the following equation. $${\rm{Inhibition}}\,{\rm{rate}}\,( \%

)=[1\,-\,({{\rm{A}}}_{{\rm{1sample}}}\,-\,{{\rm{A}}}_{{\rm{2sample}}})/({{\rm{A}}}_{{\rm{1blank}}}\,-\,{{\rm{A}}}_{{\rm{2blank}}})]\times 100$$ STATISTICAL ANALYSES Results were expressed as

mean ± SD, with three or six separate determinations. One-Way ANOVA analysis and correlation analysis were performed by SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Values in Table 1 that were

labeled with different letters represent a significant difference of (_P_ < 0.01). The significance level between groups in Table 2 are represented by ** for _P_ < 0.01 and by * for

_P_ < 0.05. Heat-map analysis was analysed using Multi Experiment Viewer software (version 4.8.1). RESULTS AND DISCUSSION CHANGES IN TEA CATECHINS, CATECHINS’ OXIDATION PRODUCTS AND

CAFFEINE CONTENTS DURING QZT PROCESSING Tea polyphenols and caffeine are important ingredients that affect the taste and aroma of tea. While most free amino acids have a refreshing taste,

L-theanine, which accounts for about 60% of the free amino acids in tea, has an umami taste. After black tea and dark tea making critical steps, fermentation or post fermentation, the

content of L-theanine is dramatically decreased or not detectable30,31. Therefore, it is important to detect the changes in L-theanine and other amino acids content that occur during QZT

manufacturing processes (Table 1). Tea polyphenols and alkaloids are the main contributors to the taste and quality of tea infusion. The products of catechins oxidation, including

theaflavins (TFs), thearubigins (TRs) and theabrownin (TBs), are the primary pigments directly or indirectly associated with the color of tea liquor and dry tea32,33. 8 flavan-3-ols (EGCG,

EGC, ECG, GCG, EC, C, GC and CG), GA and caffeine were detected and quantified by HPLC (Table 1). In the FL, EGCG, ECG, EGC, GC and EC were the main polyphenols, while significantly

decreased after the FT. Conversely, gallic acid (GA), a building block of the other catechins, increased to 8.79 ± 0.11 mg/g and remained relatively high through the following processes. TFs

and TRs gradually decreased during the pile fermentation processes, and had a slightly increased in the FT process, followed by relatively stable content through the following processes.

The content of TBs increased gradually during the pile fermentation processes. The caffeine content remained relative stability through the whole manufacturing processes. These results

indicated that the FT had the greatest effect on catechin, TFs, TRs and GA levels. FREE AMINO ACIDS CONTENTS DURING QZT PROCESSING In the FL, the main amino acids were L-theanine, alanine,

glutamic acid, and aspartic acid. Among them, the content of aspartic acid, serine, glutamic acid, glutamine, arginine and L-theanine decreased sharply after the first turn over (FT) and

remained stable after subsequent processes. The largest change was the decrease in L-theanine, which was 5.75 ± 0.02 mg/g in the raw tea (RT) but decreased to 0.17 ± 0.01 mg/g after the FT.

Interestingly, the contents of valine and leucine increased dramatically after the FT and remained high after subsequent processes. The content of soluble sugar gradually decreased from FT

process, followed by relatively stable content through the following pile fermentation processes. These results suggested that the FT is the critical step that due to the oxidation

polymerization of catechins and that the increased GA and amino acids levels (valine and leucine) play an significant role in the unique chemical profile of QZT. NON-TARGETED METABOLOMICS

ANALYSIS To further reveal the dynamic changes in the chemical profile during QZT processing, non-targeted metabolomics analysis using LC-MS was applied, with the FL and QZT samples as the

controls. 2709 ion features were determined after the peak alignment and the exclusion of ion features (Table S1). The raw data were analysed using a multivariate statistical software

(SIMCA-P 14.1). The chemical profile after each of the eight manufacturing steps were well distinguished, all replicates from each process clustered together and separated from other samples

from the other steps [Fig. 2 (PC1 = 46.3% and PC2 = 14.1%)]. The eight samples classified into five types (Fig. 2), The FL, RT, FT and ST samples all stood alone, while the other 4 samples

(TT, A1, A3 and QZT) clustered together. The two initial steps, FL to RT, are similar to green tea manufacturing processes. During the FL and RT stage, with high-temperature and

high-moisture, the polyphenols of fresh leaves undergo isomerization, hydrolysis and oxidation polymerization, accompanied by breakdown of starch, pectin, proteins and chlorophyll34.

Therefore, the chemical feature of RT sample has a significant difference with the FL sample. The RT sample was the starting point of pile fermentation, during which the chemical

constituents underwent major changes from RT to A3. The chemical profiles in the later fermentation stages (TT, A1 and A3) were similar to those of the final QZT, indicating that the early

fermentation stages (FT and ST) were critical steps for the formation of QZT quality and taste. Therefore, the temperature, humidity and duration of the early pile fermentation steps (FT and

ST) should be accurately controlled to ensure consistent quality of QZT. In addition, the contents of the TT, A1, A3 and QZT samples were similar and without significant changes, these

processing steps might be shortened to decrease the production cost and improve the production efficiency of QZT. We intend to optimize these steps coupled with e-nose and taste-panel

testing for further studies. HEAT-MAP ANALYSIS OF THE RELATIVE VARIATION IN CONSTITUENTS DURING QZT PROCESSING To analyse the constituents responsible for the samples clustering after each

manufacturing process, OPLS-DA analysis was performed on a random combination of tea samples to find components with variable importance in the projection (VIP) scores >1 and T-test

values of _P_ < 0.05. A total of 91 VIPs was obtained (Fig. 3). Through comparison with standards, MS2 spectra characteristics, or matching by a combination of accurate mass and retention

time, metabolomic databases, and the literatures, the VIP compounds in the samples were identified. To gain an overview of the changes in metabolites during the fermentation processes,

heat-map analysis was used to reveal the relative changes of the chemical constituents. The analytical compounds were identified into 6 categories, namely catechins, amino acids, flavonoids

and flavone glycosides, alkaloids, phenolic acids and other compounds, among which flavonoids were the most numerous metabolites (Fig. 3). As displayed in the heat-map, manufacturing

processes either dramatically decreased or increased the content of many compounds. The following portion discuss several significant variation in each chemical category. Tea polyphenols,

such as EGCG, C, GC and their oxidation products, are the main components determining the taste of tea35. Tea polyphenols can be oxidized into dimeric, oligomeric and polymeric compounds

during fermentation36. In this study, simple catechins were higher in the FL and RT samples, and were significantly reduced after the FT process, while C, EGC and GC levels gradually

declined after the FT or ST process. Almost all the flavonoids and flavone glycosides decreased due to dark tea specific processing steps (after step FT), while levels of

epicatechin-3-glucoside, kaempferol-3-rhamnoside, kaempferol, apigenin, quercetin, morin and malvidin increased during pile fermentation. The content of caffeine was relatively stable, while

content of theobromine and theophylline increased, mainly result from microbial fermentation. _Aspergillus niger_ (van Tieghem) has been identified to produce theophylline and theobromine4.

Meanwhile, the content of the catechins and theaflavins decreased sharply after the FT process. Some studies have pointed out that the complex of caffeine and theaflavins improves the taste

and freshness of tea. Reduced caffeine and theaflavins may form complexes that are involved in forming the unique, refreshing taste of QZT. Free amino acids are vital for tea quality,

contribute to the umami taste, and also contribute to the health benefits of tea, such as L-theanine and tryptophan37,38. Some amino acids, such as L-theanine, aspartic acid and arginine,

were significantly decreased after the FT, while leucine, valine and other branched amino acids and tryptophan and other aromatic amino acids were increased after the FT and retained a

relatively high content during the later fermentation processes (Fig. 3). Some studies have pointed out that branched amino acids can be quickly decomposed into glucose, so it was speculated

that increases in essential amino acids, such as leucine, valine, and tryptophan, are sources for the taste and aroma in QZT38,39. Several essential amino acids exhibit special functions in

the human body. For example, leucine had been proven to reduce blood glucose39, while tryptophan contributes to the production of melatonin, both of which are current hotspots of

exploration for mechanisms contributing to obesity. Furthermore, phenolic acids, such as xanthurenic acid, salicylic acid, guanine, xanthine, cytosine, uracil, 3-methylxanthine, 16:0/0:0 and

18:2/0:0 fatty acids increased significantly after pile fermentation, and the changes in the levels of these components might have a certain correlation with the quality and beneficial

effects of QZT. Moreover, the contents of phaeophorbide B, pheophorbide A and pyropheophorbide A were significantly increased after fermentation. These three compounds might contribute to

the color of dry tea and tea infusion40. These results suggested that, of all the manufacturing steps, the pile fermentation steps were most critical for changes in the levels of the

chemical constituents. The abundant components along with the higher moisture in the early fermentation steps are conducive to a series of oxidation, condensation, degradation and

polymerization reactions of tea ingredients under the joint action of humidity and microorganisms. The contents changes of these compounds before and after RT process formed the metabolic

characteristics of QZT, which in turn formed the characteristics pure aroma, mellow taste and color of tea liquor and healthy active ingredients of QZT. INHIBITORY EFFECT OF SAMPLES

FOLLOWING PILE FERMENTATION STEPS ON _IN VITRO_ PANCREATIC Α-AMYLASE ACTIVITY AND PANCREATIC LIPASE (PL) ACTIVITY A few reports have suggested that QZT possesses anti-obesity and

anti-hyperglycemic effects3,9. It has been shown that inhibiting pancreatic lipase (PL) effectively reduces triglycerides absorption in the intestinal tract, thus preventing hyperlipidemia

and obesity41. Inhibiting α-amylase can suppress the digestion of carbohydrates, consequently delaying the absorption of glucose and regulating the post-prandial blood glucose42,43. The

inhibitors of α-amylase and lipase are considered to be effective in diabetes control18. Previous researches have shown that tea exhibited pronounced inhibitory effects on α-amylase15,16 and

pancreatic lipase3,17. In our study, _in vitro_ lipase and α-amylase inhibition assays were applied to reveal how the different QZT steps alter the potential hypolipidemia and hypoglycemic

effects of QZT (Fig. 4). The lipase and α-amylase inhibitory ability of tea powders prepared after each step of QZT processing were tested. Results showed that all samples following each of

the steps had inhibitory effects on lipase and α-amylase (Fig. 4). Samples after the TT, A1, A3 and QZT steps exerted greater inhibitory effects than those after FL, RT, FT and ST,

suggesting that the FT or ST step was an important period for formation of the bioactive compounds responsible for the health benefits of QZT, including the anti-obesity and

anti-hyperglycemic effects. In addition, the content or composition of the active ingredients responsible for anti-obesity and anti-hyperglycemic effects in the TT, A1, A3 and QZT samples

were significantly different from those in the FL, RT, FT and ST samples. The conversion of active ingredients or changes in content after pile fermentation may lead to different effects on

anti-obesity and anti-hyperglycemic effects and are key to the formation of quality and health effects of QZT. Furthermore, we analyzed the correlation between the inhibition of lipase and

of α-amylase activities and VIP compounds (Table 2). Valine, leucine, kaempferol-3 -rhamnoside, morin, malvidin, theophylline, pyropheophorbide A and pheophorbide A showed significant

positive correlations with lipase and α-amylase inhibition, while 3-_O_-methyl-L-DOPA, apigenin-7-glucuronide, and the aspirane showed significant negative correlations. The results were

consistent with other research studies, which have shown that flavonoids25,44,45 and leucine46,47 have positive effects on inhibiting metabolic syndrome-associated enzymes such as α-amylase,

pancreatic lipase and α-glucosidase. More work is required to validate the relationship between these compounds and α-amylase and pancreatic lipase. CONCLUSION Overall, our findings

suggested that pile fermentation, more specifically the early fermentation stages (FT and ST), are the critical steps for formation of the constituents determining QZT quality and

pharmacological functions. These changes in chemical composition were correlated to changes in an _in vitro_ assay of obesity-preventing bioactivities (inhibition of lipase and α-amylase).

Based on this study, we propose that amino acid and flavonoids might be responsible for the anti-obesity and anti-hyperglycemic effects of QZT, possibly resulting from the prevention of

dietary fat absorption by inhibiting pancreatic α-amylase and lipase. Based on these results and previous studies, it seems to be worthwhile to optimize pile fermentation conditions which

maximize production of these compounds while preserving desirable quality and favor profiled. Future studies are also needed to elucidate the unannotated chemical phenotypes of QZT and to

determine if QZT containing higher levels of these compounds are acceptable to consumers or have greater health benefits. REFERENCES * Liu, P. P. _et al_. Analysis of aromatic components in

Qingzhuan bark tea. _Food Sci._ 38, 164–170 (2017). Google Scholar  * Zhang, L., Zhang, Z. Z., Zhou, Y. B., Ling, T. J. & Wan, X. C. Chinese dark teas: Postfermentation, chemistry and

biological activities. _Food Res. Int._ 53, 600–607 (2013). Article  CAS  Google Scholar  * Gao, W. Q. _et al_. Qing brick tea (QBT) aqueous extract protects monosodium glutamate-induced

obese mice against metabolic syndrome and involves up-regulation transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) antioxidant pathway. _Biomed. Pharmacother._ 103,

637–644 (2018). Article  CAS  PubMed  Google Scholar  * Zheng, W. J., Wan, X. C. & Bao, G. H. Brick dark tea: a review of the manufacture, chemical constituents and bioconversion of the

major chemical components during fermentation. _Phytochem Rev._ 14, 499–523 (2015). Article  CAS  Google Scholar  * Lv, H. P., Zhang, Y. J., Lin, Z. & Liang, Y. R. Processing and

chemical constituents of Pu-erh tea: A review. _Food Res. Int._ 53, 608–618 (2013). Article  CAS  Google Scholar  * Xu, A. Q. _et al_. Fungal community associated with fermentation and

storage of Fuzhuan, brick-tea. _Int. J. Food Microbio._ 146, 14–22 (2011). Article  CAS  Google Scholar  * Chen, Y. L. _et al_. Investigation on the isolation, identification and the

biological characteristic of _Eurotium_ fungi in the Kangzhuan and Qingzhuan brick tea. _J. Tea Sci._ 3, 232–236 (2006). Google Scholar  * He, J. G. _et al_. Comparative study on leaf tissue

morphologic changes of Chang-Sheng-Chuan green brick tea before and after fermentation. _Agric. Sci. Technol._ 03, 114–117 (2017). Google Scholar  * Chen, Y. Q. _et al_. Study on the

hypolipidemic effect and antioxidative activity of Hubei Qingzhuan tea. _J. Tea Sci._ 30, 124–128 (2010). CAS  Google Scholar  * Wang, Y. J. _et al_. Impact of six typical processing methods

on the chemical composition of tea leaves using a single _Camellia sinensis_ cultivar, Longjing 43. _J. Agric. Food Chem._ 67, 5423–5436 (2018). Article  PubMed  CAS  Google Scholar  *

Chen, K. J., Ma, J. Q., Apostolides, Z. & Chen, L. Metabolomics for a millenniums-old crop: tea plant (Camellia sinensis). _J. Agric. Food Chem._ 67, 6445–6457 (2019). Article  PubMed 

CAS  Google Scholar  * Ruby, M. A. _et al_. Human carboxylesterase 2 reverses obesity-induced diacylglycerol accumulation and glucose intolerance. _Cell Rep._ 18, 636–646 (2017). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Mohapatra, S. _et al_. _In silico_ investigation of black tea components on α-amylase, α-glucosidase and lipase. _J. Appl. Pharm. Sci._ 5(12),

42–47 (2015). Article  CAS  Google Scholar  * Raz, I. _et al_. Diabetes: insulin resistance and derangements in lipid metabolism. Cure through intervention in fat transport and storage.

_Diabetes-Metab Res._ 21, 1 (2005). Article  CAS  Google Scholar  * Sun, L. J., Gidley, M. J. & Warren, F. J. The mechanism of interactions between tea polyphenols and porcine pancreatic

alpha-amylase: Analysis by inhibition kinetics, fluorescence quenching, differential scanning calorimetry and isothermal titration calorimetry. _Mol. Nutr. Food res._ 61, 1700324 (2017).

Article  PubMed Central  CAS  Google Scholar  * Wu, S. Y. _et al_. Interactions between α-amylase and an acidic branched polysaccharide from green tea. _Int. J. Biol. Macromol._ 94, 669–678

(2017). Article  CAS  PubMed  Google Scholar  * Martins, F. _et al_. Maté tea inhibits _in vitro_ pancreatic lipase activity and has hypolipidemic effect on high-fat diet-induced obese mice.

_Obesity._ 18, 42–47 (2010). Article  CAS  PubMed  Google Scholar  * Guo, X. M., Long, P. P., Meng, Q. L., Ho, C. T. & Zhang, L. An emerging strategy for evaluating the grades of Keemun

black tea by combinatory liquid chromatography-orbitrap mass spectrometry-based untargeted metabolomics and inhibition effects on α-glucosidase and α-amylase. _Food Chem._ 246, 74–81

(2018). Article  CAS  PubMed  Google Scholar  * Daglia, M., Antiochia, R., Sobolev, A. P. & Mannina, L. Untargeted and targeted methodologies in the study of tea (_Camellia sinensis_

L.). _Food Res. Int._ 63, 275–289 (2014). Article  CAS  Google Scholar  * Dai, W. _et al_. Characterization of white tea metabolome: Comparison against green and black tea by a nontargeted

metabolomics approach. _Food Res. Internat._ 96, 40–45 (2017). Article  CAS  Google Scholar  * Zhou, J. _et al_. LC-MS-based metabolomics reveals the chemical changes of polyphenols during

high-temperature roasting of large-leaf yellow tea. _J. Agric. Food Chem._ 67, 5405–5412 (2019). Article  CAS  PubMed  Google Scholar  * Dai, W. _et al_. Nontargeted analysis using

ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry uncovers the effects of harvest season on the metabolites and taste quality of tea (_Camellia sinensis_

L.). _J. Agric. Food Chem._ 63, 9869–9878 (2015). Article  CAS  PubMed  Google Scholar  * Lee, J. E. _et al_. Metabolomic unveiling of a diverse range of green tea (_Camellia sinensis_)

metabolites dependent on geography. _Food Chem._ 174, 452–459 (2015). Article  CAS  PubMed  Google Scholar  * Chen, S. _et al_. Metabolite profiling of 14 Wuyi Rock tea cultivars using

UPLC-QTOF-MS and UPLC-QQQ-MS combined with chemometrics. _Molecules._ 23(2), 104 (2018). Article  PubMed Central  CAS  Google Scholar  * Chen, T. Y. _et al_. Pancreatic lipase inhibition of

strictinin isolated from Pu’er tea (_Cammelia sinensis_) and its anti-obesity effects in C57BL6 mice. _J. Funct. Foods._ 48, 1–8 (2018). Article  CAS  Google Scholar  * Ma, L., Liu, Y., Cao,

D., Gong, Z. & Jin, X. Quality constituents of high amino acid content tea cultivars with various leaf colors. _Turk. J. Agric. For._ 42(6), 383–392 (2018). Article  CAS  Google Scholar

  * Obanda, M., Owuor, P. O. & Mangoka, R. Changes in the chemical and sensory quality parameters of black tea due to variations of fermentation time and temperature. _Food Chem._ 75,

395–404 (2001). Article  CAS  Google Scholar  * DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. Colorimetric method for determination of sugars and related

substances. _Anal. Chem._ 28, 350–356 (1956). Article  CAS  Google Scholar  * Al-Dabbas, M. M., Kitahara, K., Suganuma, T., Hashimoto, F. & Tadera, K. Antioxidant and alpha-amylase

inhibitory compounds from aerial parts of _Varthemia iphionoides_ Boiss. _Biosci., Biotechnol., Biochem._ 70, 2178–2184 (2006). Article  CAS  Google Scholar  * Sari, F. & Velioglu, S. Y.

Changes in theanine and caffeine contents of black tea with different rolling methods and processing stages. _Eur. Food Res. Technol._ 237, 229–236 (2013). Article  CAS  Google Scholar  *

Syu, K. Y., Lin, C. L., Huang, H. C. & Lin, J. K. Determination of theanine, GABA, and other amino acids in green, Oolong, black, and pu-erh teas with dabsylation and high-performance

liquid chromatography. _J. Agric. Food Chem._ 56, 7637–7643 (2008). Article  CAS  PubMed  Google Scholar  * Obanda, M., Owuor, P. O., Mangoka, R. & Kavoi, M. M. Changes in thearubigin

fractions and theaflavin levels due to variations in processing conditions and their influence on black tea liquor brightness and total colour. _Food Chem._ 85, 163–173 (2004). Article  CAS

  Google Scholar  * Ghosh, A., Tudu, B., Tamuly, P., Bhattacharyya, N. & Bandyopadhyay, R. Prediction of theaflavin and thearubigin content in black tea using a voltammetric electronic

tongue. _Chemom. Intell. Lab. Syst._ 116, 57–66 (2012). Article  CAS  Google Scholar  * Naldi, M. _et al_. UHPLC determination of catechins for the quality control of green tea. _J. Pharm.

Biomed. Anal._ 88, 307–314 (2014). Article  CAS  PubMed  Google Scholar  * Chaturvedula, V. S. P. & Prakash, I. The aroma, taste, color and bioactive constituents of tea. _J. Med. Plants

Res._ 5, 2110–2124 (2011). CAS  Google Scholar  * Tan, J. F. _et al_. Study of the dynamic changes in the non-volatile chemical constituents of black tea during fermentation processing by a

non-targeted metabolomics approach. _Food Res. Int._ 79, 106–113 (2016). Article  CAS  Google Scholar  * Wang, K. & Ruan, J. Y. Analysis of chemical components in green tea in relation

with perceived quality, a case study with Longjing teas. _Int. J Food Sci. Tech._ 44, 913–920 (2009). CAS  Google Scholar  * Yu, Z. M. & Yang, Z. Y. Understanding different regulatory

mechanisms of proteinaceous and non-proteinaceous amino acid formation in tea (_Camellia sinensis_) provides new insights into the safe and effective alteration of tea flavor and function.

_Criti. Rev. Food Sci_. 1-15 (2019) * Bowne, J. B. _et al_. Drought responses of leaf tissues from wheat cultivars of differing drought tolerance at the metabolite level. _Mol. Plant._ 5,

418–429 (2012). Article  CAS  PubMed  Google Scholar  * Yu, X. L. _et al_. Chlorophyll metabolism in postharvest tea (_Camellia sinensis_ L.) leaves: variations in color values, chlorophyll

derivatives, and gene expression levels under different withering treatments. _J. Agric. Food Chem._ 67, 10624–10636 (2019). Article  CAS  PubMed  Google Scholar  * Sugiyama, H. _et al_.

Oligomeric procyanidins in apple polyphenol are main active components for inhibition of pancreatic lipase and tri-glyceride absorption. _J. Agric. Food Chem._ 55, 5906–5906 (2007). Article

  CAS  Google Scholar  * Zhang, B. W. _et al_. Dietary flavonoids and acarbose synergistically inhibit alpha-glucosidase and lower postprandial blood glucose. _J. Agric. Food Chem._ 65,

8319–8330 (2017). Article  CAS  PubMed  Google Scholar  * Stanley, T. H., Van Buiten, C. B., Baker, S. A. & Elias, R. J. Impact of roasting on the flavan-3-ol composition,

sensory-related chemistry, and, _in vitro_, pancreatic lipase inhibitory activity of cocoa beans. _Food Chem._ 255, 414–420 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Boath, A. S., Grussu, D., Stewart, D. & McDougall, G. J. Berry polyphenols inhibit digestive enzymes: A source of potential health benefits? _Food Dig._ 3, 1–7 (2012). Article  CAS 

Google Scholar  * Burgos-Edwards, A., Jiménez-Aspee, F., Thomas-Valdés, S., Schmeda-Hirschmann, G. & Theoduloz, C. Qualitative and quantitative changes in polyphenol composition and

bioactivity of _Ribes magellanicum_ and _R. punctatum_ after _in vitro_ gastrointestinal digestion. _Food Chem._ 237, 1073–1082 (2017). Article  CAS  PubMed  Google Scholar  * Liu, K. _et

al_. Relationships between leucine and the pancreatic exocrine function for improving starch digestibility in ruminants. _J. Dairy Sci._ 98, 2576–2582 (2015). Article  CAS  PubMed  Google

Scholar  * Guo, L. _et al_. Leucine affects α-amylase synthesis through _PI3K/Akt-mTOR_ signaling pathways in pancreatic acinar cells of dairy calves. _J. Agric. Food Chem._ 66, 5149–5156

(2018). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by grants from the China Postdoctoral Science Foundation (2018M632821), the Open

Fund of State Key Laboratory of Tea Plant Biology and Utilization (SKLTOF 20180105), National Natural Science Foundation of China (31902081), and the Earmarked Fund for China Agriculture

Research System (CARS-23). We thank Yijun Wang for his technical assistance. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Institute of Fruit and Tea, Hubei Academy of Agricultural Sciences,

430064, Wuhan, China Lin Feng, Panpan Liu, Pengcheng Zheng, Ziming Gong, Shiwei Gao, Lin Zheng & Xueping Wang * State Key Laboratory of Tea Plant Biology and Utilization, School of Tea

and Food Science & Technology, Anhui Agricultural University, 230036, Hefei, China Lin Feng, Liang Zhang, Yongchao Yu & Xiaochun Wan * College of Horticulture, Northwest A&F

University, 712100, Yangling, Shanxi, China Jie Zhou Authors * Lin Feng View author publications You can also search for this author inPubMed Google Scholar * Panpan Liu View author

publications You can also search for this author inPubMed Google Scholar * Pengcheng Zheng View author publications You can also search for this author inPubMed Google Scholar * Liang Zhang

View author publications You can also search for this author inPubMed Google Scholar * Jie Zhou View author publications You can also search for this author inPubMed Google Scholar * Ziming

Gong View author publications You can also search for this author inPubMed Google Scholar * Yongchao Yu View author publications You can also search for this author inPubMed Google Scholar *

Shiwei Gao View author publications You can also search for this author inPubMed Google Scholar * Lin Zheng View author publications You can also search for this author inPubMed Google

Scholar * Xueping Wang View author publications You can also search for this author inPubMed Google Scholar * Xiaochun Wan View author publications You can also search for this author

inPubMed Google Scholar CONTRIBUTIONS This study was conceived by G.Z.M. and W.X.C.; Z.P.C. and L.P.P. collected the samples during Qingzhuan tea fermentation; F.L. performed LC-MS and HPLC

analysis experiments, Z.L. and G.S.W. performed α-amylase and lipase inhibition assays analysis, data were analyzed and manuscript were revised by Z.L., Z.J. and L.P.P.; F.L. supervised the

project, formulated the data including figures and tables as well as writing the manuscript. All authors read and provided helpful discussions and approved the final version. CORRESPONDING

AUTHORS Correspondence to Ziming Gong or Xiaochun Wan. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer

Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION TABLE S1. TABLE S2. RIGHTS AND PERMISSIONS OPEN ACCESS

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as

long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third

party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the

article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright

holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Feng, L., Liu, P., Zheng, P. _et

al._ Chemical profile changes during pile fermentation of Qingzhuan tea affect inhibition of α-amylase and lipase. _Sci Rep_ 10, 3489 (2020). https://doi.org/10.1038/s41598-020-60265-2

Download citation * Received: 29 May 2019 * Accepted: 06 February 2020 * Published: 26 February 2020 * DOI: https://doi.org/10.1038/s41598-020-60265-2 SHARE THIS ARTICLE Anyone you share the

following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer

Nature SharedIt content-sharing initiative