Abstract Lu'an Guapian (LAGP) tea, a traditional Chinese green tea exclusively produced from mature leaves without buds or stems, is known for its mellow taste and distinct appearance. Subjective (sensory evaluation) and objective assessment (colorimeter and E-tongue) were conducted to provide a deeper insight into the differences between LAGP made from summer and autumn leaves and spring leaves. A total of 52 key differential metabolites were screened by metabolomics. Catechins and flavonoids, especially flavonol glycosides, exhibited significant seasonal variation. Pathway enrichment analysis indicated that flavonoid biosynthesis was the key factor that caused the quality changes of LAGP tea in different seasons. Additionally, preliminary explorations revealed that appropriate addition of odorants, especially hexanal (12.0–14.4 μg/L) and (Z)-3-hexen-1-ol (4.88–5.85 μg/L), could effectively mask the bitterness of EGCG and improve sensory acceptability. These findings provide theoretical insights into the seasonal effects of LAGP tea and guide the optimization of optimization of summer-autumn tea production. Keywords: Lu'an Guapian tea, Harvest season, Multi-sensory evaluation, Metabolomics, Odor-taste interactions Graphical abstract [39]Unlabelled Image [40]Open in a new tab Highlights * • Seasonal changes significantly increased the bitterness and astringency of LAGP tea. * • The umami amino acid content of SPT was significantly higher than SUT and AUT. * • A total of 52 metabolites were identified as key seasonal markers of LAGP tea. * • Flavonoid biosynthesis pathway was significantly correlated with seasonal variation. * • Addition of hexanal or (Z)-3-hexen-1-ol can effectively mask the bitterness of EGCG. 1. Introduction Tea is one of the three most common non-alcoholic drinks in the world, and is consumed for thousands of years for its unique flavor and health benefits ([41]Tang et al., 2019; [42]Zhai et al., 2022). Among them, green tea has the longest history of production and drinking. Due to the different raw materials and processing methods of tea leaves, Chinese green tea (e.g., Lu'an Guapian, Huangshan Maofeng, Xihu Longjing) has a wide variety of appearance and taste. Lu'an Guapian (LAGP), a non-fermented green tea originating from Lu'an City, Anhui Province, China. Its production process includes spreading, fixing, rolling, shaping, preliminary drying and deep baking. Unlike most Chinese green teas that are made from young buds, LAGP tea is processed from mature single tea leaves (Camellia sinensis) without any buds or stems ([43]Su et al., 2019). Therefore, the unique picking standards and special processing give LAGP tea its a flat, melon seed-like appearance and a rich, robust taste ([44]Yu et al., 2024). Seasonal variation is a critical factor influencing tea quality. Fluctuations in temperature, humidity, and light intensity across different seasons significantly affect the chemical composition, sensory attributes, and yield of tea. In spring, lower temperatures and softer light conditions promote nitrogen metabolism in tea plants, leading to elevated levels of free amino acids, such as theanine, glutamic acid, and aspartic acid. These compounds contribute to the umami of spring tea. In contrast, during summer and autumn, higher temperatures and stronger light suppress nitrogen metabolism while enhancing carbon metabolism, which promotes the synthesis of tea polyphenols, such as catechins and flavonoid glycosides, and the bitterness and astringency of tea is enhanced ([45]Xu et al., 2012). This reduces consumer acceptance, leading to lower economic benefits and large quantities of summer and autumn tea are not harvested and utilized. Therefore, a comprehensive understanding of the seasonal variations in chemicals and their impacts on sensory characteristics is essential. Currently, researchers have studied the impact of seasonal changes on the quality of different teas. Dai et al., He et al. and Yin et al. investigated the differences in metabolite contents of green tea in different seasons in Zhejiang, Sichuan, and Henan Provinces, China, respectively, and found that the contents of catechins, procyanidins, quercetin-O-glycosides, apigenin-C-glycosides, and amino acids showed sharp seasonal fluctuations ([46]Dai et al., 2015; [47]He et al., 2022; [48]Yin et al., 2022). Liu et al. and Ye et al. explored the changes in the sensory and physicochemical qualities of black teas in different seasons, and demonstrated that glutamic acid might be the reason for the differences in the tastes, and analyzed the color of black tea infusion ([49]Liu et al., 2023; [50]Ye, Guo, et al., 2022). In a recent metabolomics study of non-volatile flavor compounds in tea leaves from different seasons through LC-MS, it was reported that more theanine, theogallin and gallotannins that contribute to umami were found in spring ([51]Chen et al., 2025). Recently, the quality of tea has been often evaluated through the integration of sensory evaluation, metabolomics, and other analytical techniques ([52]Wen et al., 2023). These approaches effectively characterize flavor profiles for tea and evaluate nuanced quality differences. However, existing studies on summer and autumn teas have predominantly focused on conventional green teas processed from one bud with two or three leaves. In contrast, LAGP tea is a distinctive Chinese green tea that adopts a unique single leaf plucking standard. This requirement for a higher degree of leaf maturity before harvesting leads to a delay of approximately 15–20 days in the spring plucking period compared to conventional green teas, thereby significantly narrowing the harvest window for spring tea and limiting the efficient utilization of tea resources. To date, investigations into the seasonal variation of LAGP tea remain limited. Owing to its unique harvesting method, the accumulation patterns of key metabolites in LAGP tea may differ markedly from those in traditionally harvested teas. Therefore, comprehensive studies are needed to elucidate the factors contributing to its bitterness in summer and autumn LAGP teas. The taste of tea is a complex sensory attribute, determined by a multitude of chemical compounds. The key substances that contribute to tea taste have been widely studied in previous reports, mainly including catechins, alkaloids, flavonoids and flavone glycosides, amino acids, sugars and phenolic acids ([53]Zhang et al., 2020). Catechins are the principal contributors to the bitterness and astringency of green tea, and EGCG being the most abundant and significant catechin ([54]Xu et al., 2018; [55]Ye, Ye, et al., 2022). Apart from catechins, flavonoids and flavone glycosides were also identified as critical factors contributing to the bitterness and astringency of tea infusion, owing to their extremely low taste thresholds (0.001–19.80 μmol/L) ([56]Scharbert & Hofmann, 2005). Caffeine is the main alkaloid in tea, accounting for 1 % to 4 % of the dry weight of tea. It has a strong bitterness and acts synergistically with catechins (e.g., EGCG) to enhance bitterness and astringency while reducing umami and sweetness ([57]Xu et al., 2018). Additionally, taste quality was affected by odorants. The simultaneous presence of odorants and tastants can interfere with the perception of taste, thus altering the intensity of taste through masking, inhibiting, and neutralizing effects ([58]Shepherd, 2006; [59]Spence, 2022). Actually, odor-taste interactions were also present in tea infusion. In Sichuan green tea systems, the exogenous addition of sweet aroma compounds, such as octanol (0.0054 mg/L) and furaneol (0.015 mg/L) enhanced the sweetness and reduced the bitterness of the tea infusion, and their bitterness-masking effects increased with rising concentrations. In contrast, 2-hexenal exhibited a concentration dependence, suppressing bitterness below 0.19 mg/L, but may increase the bitterness of tea at higher concentrations ([60]Zhao et al., 2024). In another study on green tea, phenylacetaldehyde (3.6–10.0 μg/L) significantly enhanced the sweetness of tea infusion with increasing addition concentration, while 1-octen-3-ol (2.8–8.4 μg/L) enhanced the umami perception through its mushroom-like aroma characteristics ([61]Wei et al., 2022). Additionally, [62]Yu et al. (2021) achieved the highest sweetness and overall acceptability in Keemun black tea infusion by adding 108 μg/L of geraniol and 1.03 μg/L of β-ionone. It indicates that when aroma characteristics correspond with taste characteristics, the presence of specific aroma can enhance the perception of the associated taste. Previous studies reported that hexanal and (Z)-3-hexen-1-ol have been identified as important aroma attributes in LAGP tea ([63]Li, Zhou, et al., 2024; [64]Yu et al., 2023; [65]Yu et al., 2024). They were products of lipid degradation and typically exhibit a higher concentration of green odor in fresh tea leaves. As processing progresses, their concentrations decrease, transforming into a fresh and green apple-like aroma, contributing to the fresh aroma of the tea ([66]Zhang et al., 2023). Coincidentally, [67]Caporale et al. (2004) investigated the taste-odor interactions between bitterness (quinine hydrochloride) and the cut grass odorant ((Z)-3-hexen-1-ol) in an olive oil model system, and found that (Z)-3-hexen-1-ol had a positive and significant effect on the perception of bitterness in olive oil. A novel perspective on the perception of bitterness was provided. Inspired by these findings, considering the significant role of hexanal and (Z)-3-hexen-1-ol in shaping the fresh aroma of LAGP tea, as well as the importance of EGCG as the major bitter and astringent substance in tea, an exploration into the impact of hexanal and (Z)-3-hexen-1-ol on the taste of EGCG is warranted. This study aimed to investigate the material basis of quality differences in LAGP tea during seasonal changes through multi-sensory evaluation and metabolomics analysis. Meanwhile, through combining multiple analytical techniques, the potential relationships of metabolites with tea quality were analyzed and discussed. Additionally, the interaction of odorants (hexanal and (Z)-3-hexen-1-ol) with the major bitter and astringent compound (EGCG) in tea was preliminarily explored. The findings not only provide insights into the seasonal effects on LAGP tea quality, but also lay the foundation for its processing and in-depth utilization in summer and autumn. 2. Materials and methods 2.1. Samples Fresh tea leaves (Camellia sinensis) were obtained from the same tea plantation in Lu'an, Anhui, China, during the spring (April), summer (August), and autumn (September) of 2023. As shown in [68]Fig. 1A, spring tea (SPT), summer tea (SUT) and autumn tea (AUT) were processed following the production process of LAGP tea. Specifically, fresh untreated tea leaves were first spread at room temperature for 4 h. During this period, fresh leaves lost water moderately and the compounds underwent slight hydrolysis and oxidation. Then, the enzymatic activity was intentionally terminated through high-temperature fixation (87–95 °C) using the 6CSL-200-12ZD equipment (Songyang Bomei Machinery Co., Zhejiang, China) to ensure complete inactivation of endogenous enzymes (e.g., polyphenol oxidase and peroxidase) and preserve the non-fermented characteristics of LAGP tea. Following this, the leaves were rolled with a 6CR-Z45 rolling machine (Chunjiang Tea Machinery Co., Zhejiang, China) for 30 min. The leaves were then shaped with a heatable 6CLZ-80 shaping machine (Shangyang Tea Machinery Co., Zhejiang, China), maintaining the tea leaves temperature at 60–70 °C for 270 s. After shaping, the leaves were preliminarily dried in a bamboo roasting basket by pulley liquefied gas drying at 80–90 °C for 30 min. Finally, deep baking was performed using the 6CST-50D machine (Chunjiang Tea Machinery Co., Zhejiang, China) at 95–105 °C for 40 min to keep the moisture content below 5 %. Tea samples were stored in sealed aluminum foil pouches at −20 °C until analysis. Fig. 1. [69]Fig. 1 [70]Open in a new tab Processing flow and sensory quality of Lu'an Guapian tea in different seasons. (A) Processing flow charts of Lu'an Guapian tea. Spring tea (SPT), Summer tea (SUT), Autumn tea (AUT). (B) The colorimetric values of SPT, SUT, and AUT tea infusions. Errors bars represent mean ± SD. Different letters indicate a significant difference at the 0.05 level, as determined by one-way ANOVA and Tukey's post-hoc test. (C) Radar chart for quantitative descriptive analysis of taste and E-tongue analysis. 2.2. Chemicals and materials Gallic acid (GA), caffeine, epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC), catechin (C), gallocatechin gallate (GCG), gallocatechin(GC), anthrone and ninhydrin were purchased from Yuanye Bio-technology Co., Ltd. (Shanghai, China). Folin-Ciocalteu phenol reagent and EDTA-2Na were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). 4-Chloro-DL-phenylalanine was purchased from MedChemExpress (Shanghai, China). Formic acid, acetonitrile and methanol were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Acetic acid, Sodium chloride (NaCl) were purchased from Sinopharm Chemical Reagent Co.,Ltd. (Shanghai, China). Distilled water was purchased from Wahaha Group Co., Ltd. (Hangzhou, China). Deionized water was made by water purification system (Ulupure, Sichuan, China). Sulfosalicylic acid was purchased from Guangfu Technology Development Co., Ltd. (Tianjing, China). Quinine, Tannins, Sucrose, Sodium glutamate, Hexanal, (Z)-3-Hexen-1-ol and EGCG required for sensory experiments were food grade. All the standard compounds are at least 95 % pure. 2.3. Tea infusion color measurement The colorimetric parameters of tea infusion, namely the Lab values, were measured by a colorimeter (CHN SPEC CS-820, Hangzhou, China), which provided readings for L* (brightness, 0–100), a* (green-red, −100-100), and b* (blue-yellow, −100-100). The tea infusion was prepared according to the Chinese National Standard GB/T 23776–2018. Specifically, 3 g of LAGP tea samples were infused in 150 mL of boiling water (100 °C) for 4 min, and then the tea infusion was immediately filtered into a tea evaluation bowl. The tea infusion was measured using a quartz cuvette in the visible light range of 360–780 nm. 2.4. Sensory evaluation 2.4.1. Traditional sensory evaluation 15 experienced tea evaluators, ranging in age from 20 to 45, made up the professional sensory evaluation team. The sensory evaluation protocol strictly followed Chinese National Standard GB/T 23776–2018 (Methodology for sensory evaluation of tea). Specifically, tea infusion was prepared by brewing 3.0 g of LAGP tea with 150 mL of boiling water (100 °C) for 4 min. After filtration, aroma evaluation was conducted at cup temperatures of approximately 75 °C, 45 °C, and 25 °C, respectively, taste evaluation was carried out by taking 5 mL of the tea infusion into the mouth and circulating it to ensure full contact with all regions of the tongue, when the tea infusion temperature was approximately 45–50 °C. Evaluations of appearance, infusion color, and brewed leaves were performed at room temperature. 2.4.2. Quantitative evaluation of taste Tea infusion for quantitative evaluation of taste was prepared as described in [71]Section 2.3. The bitterness, astringency, umami and sweetness of LAGP tea were evaluated using quinine, tannin, sodium glutamate and sucrose as reference compounds, respectively. Quinine concentrations ranged from 0 g/L to 0.070 g/L, tannin from 0 g/L to 2.20 g/L, sodium glutamate from 0 g/L to 1.00 g/L, and sucrose from 0 g/L to 18.00 g/L. Corresponding scores for each attribute ranged from 0 to 10. In addition, regarding the acceptance evaluation: “0–2 points” indicated a very dislike attitude, “3–5 points” represented a neutral attitude, “6–8 points” indicated a favorable attitude, and “8–10 points” indicated a very favorable attitude (Table S1). To ensure the professionalism of evaluators and the reliability of data, evaluators were trained for at least two weeks and familiarized with the standard scales of taste compounds. The training method followed that described by [72]Huang et al. (2022). During the evaluation process, the panel rated the tea samples on a ten-point scale for four taste attributes and overall acceptability. Password evaluation was employed to ensure that evaluators remained blinded to the sample information. All flavor substances used were food-grade to ensure safety. Evaluators' rights and privacy were fully protected throughout the process. Participation was voluntary, and evaluators were fully informed and retained the right to withdraw at any time. 2.5. E-tongue measurements for the taste intensity The taste intensity of tea infusions was assessed using an electronic tongue (TS-5000Z, INSENT, Tokyo, Japan) following the methodology outlined by [73]Wu et al. (2024). The preparation of tea infusion was carried out as detailed in [74]Section 2.3, which was then filtered through 400-mesh gauze. After cooling to room temperature, 35 mL of tea infusion was transferred to a measuring cup for measurement. The testing procedure consisted of a 120 s sample test, a 40 s taste test, and a 10 s cleanup. Four repetitions of each tea sample were performed and stable data from the last three trials were chosen for subsequent analysis. 2.6. Analysis of tea polyphenols (TPs) and soluble sugar by UV spectrophotometry The content of TPs was analyzed in accordance with the international standard ISO 14502-1. In brief, 0.2 g of tea powder was extracted with 5 mL of 70 % methanol in a water bath at 70 °C for 10 min, with occasional shaking. After cooling, the mixture was centrifuged at 3500 r/min for 10 min, and the supernatant was carefully transferred to a volumetric flask. The extraction process was repeated and the supernatants were mixed into a 10 mL stock solution. After 100-fold dilution of the stock solution, 1 mL of the diluted solution was transferred to a 10 mL graduated test tube. Subsequently, 5 mL of Folin-Ciocalteu phenol reagent was added, followed by the addition of 4 mL of Na[2]CO[3] solution within 3 to 8 min. Following a reaction time of 1 h, the absorbance at 760 nm was measured, and the concentration of TPs was determined based on the gallic acid standard curve (y = 9.88× + 0.005, R^2 = 0.999). Analysis of soluble sugar content was conducted with modifications based on the method reported by [75]Mao et al. (2018). Finely ground tea sample (0.5 g) was extracted with 8 mL of boiling deionized water for 30 min. The mixture was filtered and diluted to 250 mL after several thorough washes of the residue with boiling water. The anthrone reagent was prepared by mixing 0.6 mL of anthrone with 100 mL of H₂SO₄ and then slowly adding 33 mL of distilled water. To conduct the assay, 1 mL of the test solution was combined with 8 mL of anthrone reagent. The mixture was boiled for 7 min and cooled to room temperature. Then the absorbance at 620 nm was measured for the extract, and the glucose standard curve was used to calculate the soluble sugar concentration (y = 4.26× + 0.003, R^2 = 0.999). 2.7. Determination of catechins and caffeine by high performance liquid chromatography (HPLC) The samples were prepared and measured according to the international standard ISO 14502-2. The stock solution was prepared as described in [76]Section 2.6. 2 mL of the stock solution was taken and transferred to a 10 mL volumetric flask, then diluted to the mark with a stabilizing solution composed of ascorbic acid, EDTA-2Na, acetonitrile, and water. Finally, the mixture was filtered through a 0.22 μm Millipore filter and prepared for HPLC analysis. Analyses were performed with the Waters E2695 HPLC system (Milford, MA, USA) equipped with a UV/Vis detector, column oven, quaternary solvent manager, and empower software for chromatographic data acquisition, processing, and reporting. The chromatographic column was a Phenomenex C18 column (5 μm, 250 mm × 4.6 mm, Phenomenex, Los Angeles, USA). The oven temperature was 35 °C. The mobile phase consisted of Phase A (9 % acetonitrile, 2 % acetic acid, 0.2 % EDTA-2Na solution and 88.8 % H[2]O) and Phase B (80 % acetonitrile, 2 % acetic acid, 0.2 % EDTA-2Na solution and 17.8 % H[2]O). The gradient elution was as follows: 0–10 min, 100 % A; 10–15 min, 100–68 % A; 15–25 min, 68 % A; 25–40 min, 68–100 % A, with a flow rate of 1.0 mL/min. The sample was injected into 10 μL, and the wavelength was 278 nm. 2.8. Measurement of free amino acids by high-speed amino acid analyzer 18 free amino acids were quantified by a high-speed amino acid analyzer (L-8900, Hitachi, Tokyo, Japan), following the method of [77]Huang et al. (2024). In brief, 0.10 g of tea powder was ultrasonically extracted with 5 mL of 4 % sulfosalicylic acid for 30 min, with intermittent shaking every 5 min. The solution was allowed to stand for 10 min. Then, 1.5 mL of supernatant was centrifuged for 10 min at 12,000 rpm and filtered using a 0.22 μm Millipore filter before analysis. 2.9. Non-targeted metabolomics analysis based on LC-MS Metabolite extraction was performed according to the following method outlined by [78]Wang et al. (2018), with slight modifications. Specifically, 0.25 g of tea powder was combined with 4 mL of methanol and subjected to ultrasonic extraction at 60 kHz for 30 min. The mixture was subsequently centrifuged at 4 °C for 15 min at 15000 rpm. 100 μL of the supernatant and 50 μL of the internal standard (DL-4-chlorophenylalanine) were transferred into 10 mL centrifugal tubes. The samples were filtered through a 0.22 μm Millipore filter before analysis. An equal volume of each sample was taken to prepare a quality control (QC) sample for monitoring instrument stability and correcting data deviations. The LC-Orbitrap-MS determination conditions were set as follows: The chromatographic separation was performed on a Hypersil GOLD C18 column (100 × 2.1 mm, 1.9 μm, Thermo Fisher, USA) with a constant flow rate of 0.3 mL/min. The mobile phase consisted of solvent A (H₂O containing 0.075 % formic acid) and solvent B (acetonitrile containing 0.075 % formic acid). The elution program: 0–2 min, 95 %–60 % A, 2–7 min, 60 %–20 % A, 7–11 min, 20 %–5 % A, 11–15 min,5 % A, 15–15.5 min,5 %–95 % A, 15.5–20 min, 95 % A. The injection volume was 4 μL, and the scanning mode was set to full MS. Mass spectrometric detection was conducted using a heated electrospray ionization (HESI) source in both positive and negative ion modes. The spray voltages were 3.8 kV and 3.2 kV, the capillary temperatures were 350 °C and 300 °C, the resolution was set at 60000, and scanning range was 50 to 1000 m/z. 2.10. Extraction and analysis of hexanal and (Z)-3-hexen-1-ol 2.10.1. Headspace-solid phase microextraction (HS-SPME) Headspace solid-phase microextraction (HS-SPME) was employed to capture highly volatile compounds ([79]Wang et al., 2022). For the extraction process, 1 g of tea powder was placed in a 20 mL vial and the internal standard was added at a certain concentration, specifically 2–10 μL of ethyl decanoate (30–40 μg/L, dissolved in dichloromethane), to ensure reliable quantification. Then, a magnetic stirrer was inserted, and the vial was sealed. The sample was incubated at 60 °C for 15 min. Subsequently, stabilized flexible HS-SPME fibers (65 μm, PDMS/DVB, Oakville, Canada) mounted on a manual SPME holder and exposed to the headspace for 30 min, with the vials maintained at 60 °C. The fiber was quickly placed into the GC–MS injector (7890B, Agilent, CA, USA) for thermal desorption at 250 °C for 5 min. 2.10.2. Gas chromatography-mass spectrometry (GC–MS) A 7890B gas chromatograph, paired with a 5977B mass spectrometer (Agilent, CA, USA) and a DB-5MS column (30 m × 0.25 mm × 0.25 μm, Agilent, CA, USA), was employed for identification. The mass spectrometer was operated in electron impact mode (MS-EI) at 70 eV, scanning masses from 30 to 350 m/z. A homologous series of n-alkanes (C[7]-C[40]) was employed to determine the linear retention indices. Volatile compounds were identified based on mass spectra, retention indices, and comparison with authentic reference standards ([80]Yu et al., 2023). The temperature program for analysis using the DB-5MS column began at 35 °C (held for 3 min), then increased to 160 °C at a rate of 4 °C/min (held for 2 min), and finally ramped up to 250 °C at 10 °C/min (held for 5 min). 2.10.3. Quantitation of volatiles in tea and calculation of OAVs The relative amount of volatile compounds was quantified by calculating the peak area ratio of the target compound to the internal standard (ethyl decanoate). Then, the odor activity values (OAVs) were calculated to assess the contribution of aroma. Additionally, the definition of OAV is the ratio of odorant concentrations to their odor thresholds in water. 2.11. Sensory evaluation of the interaction of hexanal/(Z)-3-hexen-1-ol with EGCG This experiment was designed to investigate the effect of odorants (hexanal and (Z)-3-hexen-1-ol) on the taste of EGCG model solutions. A 0.5 μg/mL stock solution was prepared by dissolving hexanal and (Z)-3-hexen-1-ol in anhydrous ethanol, followed by dilution with pure water. Subsequently, five hexanal/(Z)-3-hexen-1-ol-EGCG solutions were formulated separately using EGCG model solution as solvent ([81]Table 1). The concentrations of hexanal and (Z)-3-hexen-1-ol in these solutions were determined based on their respective concentration ranges in SPT, SUT and AUT. The average concentration of EGCG in the three LAGP samples measured in [82]Section 2.8 was 2 mg/mL, which served as the EGCG model solution and was also the blank group. In the experiment, the blank group solution was given a score of 5 for bitterness, astringency, and acceptability. Panelists were asked to sip the sample, keep it in their mouths for 30 s, then spit it out and rate the sample on a ten-point scale for intensity of bitterness, astringency and acceptability. In order to minimize potential interactions between samples, evaluators were instructed to rinse their mouths with purified water after each tasting session and wait for a period of 5 min before proceeding to evaluate the subsequent sample. Table 1. Concentration gradients in hexanal/(Z)-3-hexen-1-ol-EGCG solutions[83]^a. Solution __________________________________________________________________ Hexanal-EGCG solution __________________________________________________________________ (Z)-3-hexen-1-ol-EGCG solution __________________________________________________________________ Sample 1 2 3 4 5 6 7 8 9 10 Concentration (μg/L) 9.60 10.80 12.00 13.20 14.40 1.95 2.93 3.90 4.88 5.85 [84]Open in a new tab ^a The 2 mg/mL EGCG model solution, with its concentration determined from the average concentration of EGCG in SPT, SUT and AUT measured in [85]Section 2.8, was used as the solvent. 2.12. Statistical analysis All experiments were conducted with at least three replicates, and the results are presented as the mean ± standard deviation (SD). Analysis of variance (ANOVA) and post-hoc tests were performed using SPSS 27.0. Data visualizations were generated with Origin 2022, GraphPad prism 9.5 and Chiplot ([86]https://www.chiplot.online/). Metabolomics data were subjected to noise filtering, peak extraction and peak alignment and peak filling with Xcalibur and MS-DIAL. Data were screened based on the fill rate exceeding 50 %, signal-to-noise ratio greater than 10, and QC relative standard deviation (RSD) below 30 %. Metabolites were identified by MS-FINDER and metabolomics public databases (PubChem, MONA, UNDP, ChEBI). Multivariate data analysis was performed with SIMCA 14.1. 3. Results and discussion 3.1. Color and taste characteristics of LAGP tea in SPT, SUT, and AUT 3.1.1. Colorimetric values of tea infusions The color of tea infusions serves as a critical indicator of tea quality, reflecting its relationship with seasonal variations. The color measurements of LAGP tea infusions across different seasons were presented in [87]Fig. 1B. In terms of brightness (L*), SPT exhibited the highest L* value (98.50 ± 0.07), followed by AUT (97.18 ± 0.17), with SUT showing the lowest value (96.86 ± 0.11). The a* and b* values showed that the color of the LAGP tea infusion was yellowish-green, and further analysis revealed that the color of the tea infusion tended to deepen and the yellow color became more pronounced with the change of seasons. Compared with SPT, the greenness of SUT and AUT decreased by 1.21 and 1.17 times, the yellowness increased by 1.25 and 1.19 times, respectively. These changes can be reflected not only visually, but also analyzed for statistically significant differences by ANOVA and Tukey's post-hoc test (Table S2). The main contributors to the color of tea infusions were water-soluble pigments, including catechins, anthocyanidins, flavonoids ([88]Wan, 2003). These compounds not only affect the color but also influence the sensory properties of tea infusions. Previous research showed that, within certain concentration ranges, higher levels of rutin, quercetin-3-O-rutinoside and quercetin-3-O-glucoside result in decreased brightness and increased yellowness ([89]Li, Zhu, et al., 2024). This is due to the specific color properties of flavonoids in aqueous solutions, where their concentration directly impacts the tea's color. Thus, the decrease in brightness and increase in yellowness observed in SUT and AUT teas likely reflect changes in the concentrations of these compounds. 3.1.2. Quantitative description analysis of taste In order to further elucidate the taste characteristics of LAGP tea infusions across different seasons, quantitative description analysis of taste were conducted on three LAGP samples after the traditional sensory evaluation (Table S3). The results presented in [90]Fig. 1C. SUT exhibited the strongest bitterness and astringency, which were 1.54 - fold and 1.25 - fold higher than those of SPT respectively. In contrast, SPT demonstrated superior umami (5.17 ± 1.59) and sweetness (3.06 ± 0.90), both of which were significantly higher than those of SUT and AUT. The prominent bitterness and astringency of SUT and AUT contributed to a significantly lower acceptance by the evaluators. Furthermore, no significant differences in bitterness, astringency, umami, sweetness, and acceptability were observed between SUT and AUT. 3.1.3. E-tongue characterization of LAGP tea taste To further validate the accuracy of the quantitative description analysis and minimize potential subjective bias, E-tongue technology was employed to objectively analyze the taste characteristics of LAGP tea ([91]Fig. 1C). By simulating human taste perception using specialized sensors, the E-tongue quantifies flavor compounds and provides reliable data. This objective approach confirmed the flavor differences observed in LAGP tea. The E-tongue data revealed significant seasonal variations (Table S4). Bitterness increased significantly from spring tea (3.24 ± 0.07) to summer tea (3.56 ± 0.09) and autumn tea (3.62 ± 0.22). For astringency, both summer tea (6.72 ± 0.31) and autumn tea (5.88 ± 0.28) were significantly higher than spring tea (4.85 ± 0.60) (p ≤ 0.05), with no significant difference between summer and autumn tea. Additionally, umami scores for spring tea were significantly higher than those for summer tea and autumn tea (p ≤ 0.05). In terms of sweetness, spring tea was significantly higher than summer tea, while autumn tea exhibited an intermediate value. In conclusion, the results from both the E-tongue and quantitative description analysis were consistent for the four main flavor indicators — bitterness, astringency, umami, and sweetness. This result further confirms the superiority of SPT in terms of taste qualities, while also validating the relatively higher bitterness and astringency of SUT and AUT. 3.2. Effect of seasonal variations on non-volatile compounds in the LAGP 3.2.1. Tea polyphenols and soluble sugar Tea polyphenols (TPs), abundant in tea, were key components influencing its taste and were widely recognized for their health benefits, including antioxidant and anti-inflammatory properties. TPs were highly accumulated in SUT (22.42 %) and AUT (21.93 %), which were significantly higher than SPT. The increased TPs content contributed to the pronounced bitterness and astringency of tea infusions (Table S5). This accumulation is likely linked to high temperatures and strong sunlight in summer and autumn, which accelerate carbon metabolism in tea and promote the synthesis of secondary metabolites ([92]Jiang et al., 2021). Soluble sugars were the primary sweeteners in tea infusions. Studies demonstrated that the content of soluble sugars in SPT was significantly higher than that in SUT and AUT (Table S5). Although the proportion of soluble sugars in green tea is relatively low, these sweet substances can partially alleviate the bitterness and astringency of the tea ([93]Zhang et al., 2020). 3.2.2. Catechins and caffeine Seven catechins were quantified by HPLC ([94]Table 2), revealing differences among the three samples. Catechins are categorized into galloylated catechins and non-galloylated catechins based on molecular structures ([95]Xu et al., 2018). Galloylated catechins, including EGCG, ECG and GCG, significantly affect the bitterness and astringency of tea. The contents of EGCG (112.55 mg/g) and ECG (22.51 mg/g) in SUT were significantly higher than those in SPT (p ≤ 0.05), which were 1.18- and 1.14- fold higher than those in SPT, respectively, explaining the enhancement of bitterness and astringency in the SUT. Non-galloylated catechins, such as EGC, C, EC and GC exhibit milder bitterness. Among these, seasonal differences were significant for EC, which increased from 12.25 mg/g in SPT to 14.74 mg/g in SUT and 13.92 mg/g in AUT, with all differences being statistically significant (p ≤ 0.05). However, no significant differences were observed for EGC and C among the samples. The total catechin content averaged 171.85 mg/g, 200.32 mg/g, and 195.14 mg/g in SPT, SUT, and AUT, respectively. Previous studies have reported that light intensity and temperature synergistically regulate catechin synthesis ([96]Xiang et al., 2021). During summer, high light intensity increases the photosynthetic rate, thereby might stimulat the production of galloylated catechin precursors, which function as antioxidants to protect tea plants from light-induced oxidative damage ([97]Liu et al., 2023). Additionally, high summer temperatures could enhance the activity of enzymes in the phenylpropanoid biosynthesis pathway, which might lead to increased catechin levels. Consequently, teas harvested in summer and autumn generally have higher catechin levels, resulting in stronger bitterness and astringency. Caffeine is one of the important contributors to the bitterness of tea, and its content mainly depends on the metabolism of tea ([98]Ye, Ye, et al., 2022). The content of caffeine was the highest in SUT (40.89 mg/g), followed by AUT (37.23 mg/g) and SPT (33.10 mg/g), and SPT was significantly lower than that in SUT and AUT (p ≤ 0.05). Table 2. The contents of 7 catechins, 18 amino acids and caffeine in LAGP teas of different seasons. Compounds[99]^a __________________________________________________________________ Content (mg/g)[100]^b __________________________________________________________________ SPT SUT AUT EGCG 95.07 ± 1.30b 112.55 ± 1.99a 110.42 ± 5.24a ECG 19.64 ± 0.99b 22.51 ± 0.48a 21.3 ± 0.74b EGC 42.12 ± 0.98a 43.14 ± 0.88a 43.61 ± 1.34a EC 12.25 ± 0.49c 14.74 ± 0.20a 13.92 ± 0.29b C 0.79 ± 0.54a 1.26 ± 0.67a 1.09 ± 0.52a GCG 0.86 ± 0c 2.89 ± 0.19a 1.30 ± 0.27b GC 1.12 ± 0.09b 3.24 ± 0.15a 3.51 ± 0.18a Galloylated catechins 115.58 ± 1.83b 137.95 ± 2.51a 133.01 ± 5.69a Non galloylated catechins 56.27 ± 2.01b 62.37 ± 0.50a 62.13 ± 1.28a Total catechins 171.85 ± 3.39b 200.32 ± 2.96a 195.14 ± 6.97a Caffeine 33.10 ± 1.53c 40.89 ± 0.51a 37.23 ± 1.25b Theanine[101]^c 6.39 ± 0.12a 2.41 ± 0.08c 3.06 ± 0.03b Glutamine[102]^c 0.20 ± 0.02a 0.12 ± 0.00b 0.22 ± 0.01a Glutamic acid[103]^c 2.96 ± 0.02a 1.93 ± 0.05c 2.39 ± 0.06b Aspartic acid[104]^c 2.45 ± 0.06a 1.2 ± 0.04c 1.88 ± 0.03b Threonine[105]^d 0.15 ± 0.00c 0.17 ± 0.01a 0.25 ± 0.01b Serine[106]^d 0.46 ± 0.00b 0.32 ± 0.01c 0.58 ± 0.02a Glycine[107]^d 0.02 ± 0.00b 0.01 ± 0.00c 0.02 ± 0.00a Alanine[108]^d 0.16 ± 0.01c 0.19 ± 0.01b 0.24 ± 0.00a Cysteine[109]^d 0.25 ± 0.01a 0.05 ± 0.00b 0.06 ± 0.00b Proline[110]^d 0.04 ± 0.00c 0.14 ± 0.01b 0.20 ± 0.00a Valine[111]^e 0.07 ± 0.00c 0.14 ± 0.00b 0.23 ± 0.00a Isoleucine[112]^e 0.03 ± 0.00c 0.10 ± 0.00b 0.18 ± 0.00a Leucine[113]^e 0.06 ± 0.00c 0.11 ± 0.00b 0.17 ± 0.00a Tyrosine[114]^e 0.09 ± 0.00c 0.34 ± 0.01a 0.31 ± 0.00b Phenylalanine[115]^e 0.11 ± 0.01c 0.21 ± 0.01b 0.38 ± 0.01a Lysine[116]^e 0.05 ± 0.00c 0.16 ± 0.01b 0.25 ± 0.01a Histidine[117]^e 0.01 ± 0.00c 0.02 ± 0.00b 0.06 ± 0.00a Arginine[118]^e 0.07 ± 0.01b 0.06 ± 0.00b 0.49 ± 0.02a Umami amino acids 12.00 ± 0.15a 5.66 ± 0.14c 7.54 ± 0.13b Sweet amino acids 1.07 ± 0.01b 0.89 ± 0.03c 1.35 ± 0.04a Bitter amino acids 0.49 ± 0.02c 1.14 ± 0.04b 2.07 ± 0.04a [119]Open in a new tab ^a Epigallocatechin gallate (EGCG), Epicatechin gallate (ECG), Epigallocatechin (EGC), Epicatechin (EC), Catechin (C), Gallocatechin gallate (GCG), Gallocatechin (GC), Gallic acid (GA). ^b Mean values of triplicates with standard deviations (SDs). Different letters (a-c) indicate a significant difference at the 0.05 level. ^c This amino acid exhibits umami taste. ^d This amino acid exhibits sweet taste. ^e This amino acid exhibits bitter taste. 3.2.3. Amino acids Eighteen free amino acids were quantified and listed in [120]Table 2, comprising 17 proteinaceous amino acids and 1 non-proteinaceous amino acid (theanine). Proteinaceous amino acids were categorized as umami, sweet, and bitter amino acids based on their taste characteristics. Theanine, the most abundant free amino acid in tea (SPT, 6.39 mg/g; SUT, 2.41 mg/g; AUT, 3.06 mg/g), constitutes over 50 % of the total amino acids in green tea and serves as the primary contributor to the umami flavor of tea infusions. Glutamic acid and aspartic acid, which were key umami-contributing amino acids, exhibited significant seasonal variations. The glutamic acid content in SPT was 53.4 % and 23.8 % higher than that in SUT and AUT, respectively, while the aspartic acid content was 104.2 % and 30.3 % higher than that in SUT and AUT, respectively. In contrast, the total content of sweet amino acids followed a different trend, with AUT showing the highest level (1.35 mg/g), followed by SPT (1.07 mg/g), and SUT having the lowest (0.89 mg/g). Although individual bitter amino acids (valine, leucine, tyrosine, phenylalanine and arginine) were generally considered to contribute minimally to bitterness due to their low concentrations and high taste thresholds (dose-over-threshold, Dot <0.1) ([121]Liu et al., 2023; [122]Scharbert & Hofmann, 2005), an increase in their total concentration may exert a synergistic effect. Moreover, seasonal variations in their relative proportions could also play a critical role in explaining flavor differences. Quantitative analysis showed that the total content of bitter amino acids was highest in AUT (2.07 mg/g), followed by SUT (1.14 mg/g), and lowest in SPT (0.49 mg/g). Notably, the autumn season exhibited significant increases in valine, leucine, phenylalanine, lysine, and arginine, which collectively altered the infusion's taste profile. In SPT, umami amino acids accounted for 88.5 % of the total amino acids, whereas bitter amino acids constituted only 3.6 %, resulting in a umami taste. In SUT, the proportion of bitter amino acids rose to 14.8 %, and umami amino acids declined to 73.6 %, producing a noticeably more bitter profile. In AUT, bitter amino acids further increased to 18.8 %, while umami amino acids dropped to 68.8 %, contributing to a more bitter taste. In addition, the total free amino acid content in SPT was significantly higher than that in SUT and AUT. This discrepancy may be attributed to the increased light intensity during summer and autumn, which promotes carbon metabolism while inhibiting nitrogen metabolism, thereby reducing the synthesis of protein amino acids and limiting protein hydrolysis ([123]Deng et al., 2022). 3.3. Non-targeted metabolomics analysis based on LC-MS 3.3.1. Multivariate statistical analysis based on LC-MS Non-targeted metabolomics was employed to comprehensively investigate the variations in non-volatile metabolites of LAGP tea across different harvest seasons. The raw LC-MS data were processed according to the method described by [124]Wang et al. (2018), and multivariate statistical analyses were performed. Hierarchical cluster analysis (HCA) grouped the samples into two clusters, with SPT forming a distinct group while SUT and AUT clustering together, indicating similarity in chemical profiles between SUT and AUT compared to SPT (Fig. S1A). Principal component analysis (PCA) further separated the chemical phenotypes of the three samples, and showed tightly clustered QC samples near the center, demonstrating the reliability and reproducibility of the analysis. The first two principal components explained 80 % of the total variance (Fig. S1B). Orthogonal partial least squares discriminant analysis (OPLS-DA) exhibited clearer clustering, showing a strong separation between SPT and SUT along the first principal component and between SPT and AUT along the second (Fig. S1C). The OPLS-DA model was reliable (R^2 = 0.205, Q^2 = −0.774) through 200 cross-validation permutation tests. Building on the previous analysis, key differential metabolites were identified by ranking the variable importance in projection (VIP) values of all metabolites, with the criteria of VIP > 1 and p ≤ 0.05. A total of 52 differential metabolites were identified, comprising 12 flavonoids and flavone glycosides, 13 catechins, 7 free amino acids, 9 phenolic acids, and 11 other compounds ([125]Fig. 2 & Table S6). Catechins and amino acids followed the same trends as described in the previous sections. Flavone glycosides have a variety of structures due to the variation of sugar attachment sites and sugar types ([126]Bozzo & Unterlander, 2021; [127]Wan, 2003). LC-MS results indicated significant differences in flavone glycoside content among SPT, SUT and AUT samples. Specifically, kaempferol 4′-rhamnoside, vitexin 2’-O-rhamnoside, isovitexin 2”-O-glucoside, and kaempferol 3-neohesperidoside were significantly higher in SUT and AUT than in SPT. This variation was consistent with previous studies, the accumulation of flavonoids exhibited specific temporal patterns and was closely linked to temperature and light, which regulate the expression of structural genes in flavonoid biosynthesis ([128]Zhao et al., 2021; [129]Zhu et al., 2020). Phenolic acids are aromatic compounds with carboxyl and hydroxyl groups, characterized by high water solubility ([130]Wan, 2003). Despite their relatively low concentrations, phenolic acids play a crucial role in enhancing the complexity and modulating the sensory balance of tea infusions ([131]Kaneko et al., 2006; [132]Zhang et al., 2020). The results showed that phenolic acids exhibited seasonal variations, probably due to the activation of phenylpropanoid metabolism under environmental stresses in summer and autumn. The m-coumaric acid, 4,5-di-p-cis-coumaroylquinic acid, and GA were abundantly accumulated in SUT, while salicylic acid and 4-hydroxycinnamic acid were more prevalent in AUT. Fig. 2. [133]Fig. 2 [134]Open in a new tab (A) Category and percentage of SPT, SUT and AUT differential metabolites (VIP > 1, p ≤ 0.05) derived by LC -MS. (B) Heatmap analysis of 52 differential metabolites with normalization. 3.3.2. Metabolic pathway analysis of differential metabolites To elucidate the metabolic pathways affecting the seasonal variation of LAGP tea, pathway analysis was performed for 52 differential metabolites ([135]Fig. 3A & Table S7). A darker color of the bubble indicates a smaller p value and a more significant enrichment of the pathway. The highly enriched pathways include phenolic metabolic pathways (e.g. flavonoid biosynthesis, flavone and flavonol biosynthesis), amino acid metabolic pathways (e.g. alanine, aspartate and glutamate metabolism, arginine biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism), butanoate metabolism and glutathione metabolism. Notably, flavonoid biosynthesis exhibited the most significant relationship among all metabolic pathways. Fig. 3. [136]Fig. 3 [137]Open in a new tab (A) Pathway analysis of the effect of seasonal variation on differential metabolites of LAGP. The bubbles represent metabolic pathways, bubble size represents the pathway influence value obtained from the topological analysis, and bubble color indicates the p-value from the pathway enrichment analysis, expressed as -log10(p). (B) Simplified model of the flavonoid biosynthesis in LAGP, and the heatmap of the content of relevant compounds in SPT, SUT, and AUT. A simplified model of flavonoid biosynthesis and heatmaps of the relevant compound contents in SPT, SUT and AUT were shown in [138]Fig. 3B. In the flavonoid biosynthesis pathway, phenylalanine (the initial substrate) is converted to chalcone via enzymatic reactions. Chalcone undergoes isomerization to form flavanones, which are then catalyzed to produce dihydrokaempferol, dihydroquercetin, and dihydromyricetin, and further to generate structurally diverse flavonoid compounds (including flavan-3-ols, flavonols, and flavonoid glycosides, etc.) ([139]Wan, 2003). Compared to SPT, the contents of downstream products, such as EGCG, ECG, apigenin glycosides, quercetin glycosides, kaempferol glycosides, and vitexin 2”-O-rhamnoside were significantly higher in SUT and AUT. This indicates that seasonal variations have a significant influence on the flavonoid biosynthesis pathway in LAGP tea. Elevated metabolic activity during summer and autumn led to the accumulation of flavonoid metabolites, which enhanced the bitterness and astringency of SUT and AUT infusions. 3.3.3. Correlation analysis of differential metabolites with taste characteristics To further elucidate the contributions of metabolites to the taste of samples, Pearson's correlation analysis was employed to investigate the correlation between E-tongue results and key non-volatile compounds. Additionally, a correlation network analysis was conducted to identify taste-related compounds ([140]Fig. 4). The results revealed that catechins, flavonoids and flavone glycosides, as well as caffeine, exhibited significant positive correlations with bitterness and astringency. Specifically, the bitter and astringent compounds included EGCG, ECG, EGC, caffeine, epigallocatechin-(4β- > 8)-epicatechin 3-O-gallate, vitexin 2”-O-rhamnoside, GA, kaempferitrin, quercetin 3-rutinoside 7-galactoside and m-coumaric acid, which independently and synergistically enhanced the bitterness and astringency of tea infusions. In contrast, compounds positively correlated with umami and sweetness included L-aspartic acid, L-theanine, L-glutamic acid and soluble sugars. While these correlation results provide valuable insights into the potential contributors to taste attributes, it is important to recognize that they reflect statistical associations rather than direct causality. Nevertheless, several compounds identified in this study, such as EGCG, vitexin 2”-O-rhamnoside, and L-theanine, have been well-documented in prior sensory and biochemical research to contribute to bitterness, astringency, and umami, respectively ([141]Deng et al., 2022). Therefore, the observed correlations not only align with their established sensory roles but also offer supporting evidence for their involvement in shaping the taste profile of tea infusions. Fig. 4. [142]Fig. 4 [143]Open in a new tab (A) Pearson correlation analysis between E-tongue results and differential metabolites. (B) Association networks of bitterness and astringency with metabolites. Oval nodes represent metabolites and different colors indicate different types of metabolites, yellow for flavonoids and flavone glycosides, green for catechins, purple for amino acids and derivatives, pink for phenolic acids, and blue for others. Solid lines indicate positive correlations and dotted lines indicate negative correlation. (For interpretation of the references to