Comprehensive multi-omics analysis of Mengding bud yellow tea in the intangible cultural heritage: insights into taste formation

Abstract

Mengding bud yellow tea (MDBYT) is a renowned Chinese yellow tea characterized by its mellow, umami, and sweet taste. This study integrated flavoromics and metabolomics to investigate dynamic changes in non-volatile metabolites and sensory attributes during traditional “three yellowings and three fryings” processing. Sensory evaluation revealed progressive quality improvement, with the yellowing critical for enhancing umami and sweetness. Untargeted metabolomics identified 156 differentially abundant compounds. Theanine glucoside emerged as a potential contributor to the characteristic taste. KEGG pathway analysis highlighted amino acid biosynthesis and flavonoid metabolism as significantly enriched. Comparative metabolomic analysis with green tea identified 19 potential biomarkers, prominently including N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs), which were the most prominent. Correlation analysis between biomarkers and sensory attributes revealed distinct patterns in yellow and green teas, supporting their potential functional roles. These findings provide systematic and novel insights into the taste characteristics and underlying formation mechanisms of yellow tea.

Introduction

Yellow tea is one of the six major tea categories in China and is classified as a lightly fermented tea that evolved from green tea. As the second oldest type of tea, it has a history of more than 1000 years¹. Produced through specialized processing techniques, yellow tea contains abundant bioactive compounds, including high levels of tea polyphenols, amino acids, soluble sugars, and vitamins. These constituents contribute to diverse biological activities such as antioxidant, anticancer, anti-inflammatory, antibacterial, gastric protective, and lipid-lowering effects^1–5. Consequently, yellow tea possesses considerable nutritional and pharmacological values^6,7. Compared with green tea, yellow tea is characterized by a mellower, smoother, and less astringent taste, which has contributed to its growing popularity worldwide and its emergence as a new growth segment in the tea industry⁵.

Among the different yellow tea categories, bud yellow tea is particularly favored in the market. Mengding bud yellow tea (MDBYT), the earliest representative form of yellow tea, has historically been regarded as the ‘gold of tea’ and served as a tribute tea for imperial ceremonies to worship heaven and ancestors in China since the Tang Dynasty. MDBYT is distinguished by its characteristic “three yellows”: golden-yellow dry leaves, bright-yellow infusion, and tender-yellow infused leaves, as well as its unique sensory profile of sweet aroma, mellow taste, and refreshing flavor. The exceptional quality of MDBYT is closely associated with the unique terroir of Mengding Mountain in Ya’an, Sichuan Province, which is recognized as the birthplace of the world tea culture. The traditional manufacturing technique of MDBYT has been listed as part of the second batch of representative items of Sichuan Provincial Intangible Cultural Heritage.

Processing techniques play a key role in the formation of MDBYT quality, with yellowing step representing the core operation. During yellowing, fixed tea leaves undergo controlled moist-heat treatment, triggering a series of chemical transformation. The traditional processing procedure of MDBYT consists of fixing, first yellowing, first frying, second yellowing, second frying, third yellowing, and third frying, commonly referred to as the “three yellowings and three fryings” process (Fig. 1). During this process, thermo-hydrolytic reactions drive extensive biochemical transformations³, leading to a decrease in esterified catechins associated with bitterness and astringency, an increase in theaflavins, the hydrolysis of starch into simple sugars, the degradation of proteins into amino acids, and the breakdown of chlorophyll^5,7,8. Collectively, these reactions enhance sweetness and refreshness while shaping the unique flavor profile of MDBYT.

Fig. 1. The processing procedure of tea.

A Mengding bud yellow tea (MDBYT). B Green tea (GT). FL fresh tea leaves, SD spreading, FX fixing, FY first yellowing, SY second yellowing, TY third yellowing, PS piled-spreading, YT finished yellow tea, GT green tea.

Despite its long history and distinctive processing technology, the production process of MDBYT remains highly complex, and systematic studies on the metabolite changes are still limited. Especially, the biochemical transformations occurring during the ‘three yellowings and three fryings’ stages, as well as the formation mechanisms underlying the characteristic ‘three yellows’ appearance and the taste attributes of ‘mellow, sweet and umami taste’, remain insufficiently understood.

In this study, the metabolic profile changes and taste variations during MDBYT processing were comprehensively investigated using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-Q-Exactive/MS) combined with high-performance liquid chromatography (HPLC) and quantitative descriptive analysis (QDA). Key differential metabolites during processing and between bud yellow tea and green tea were analyzed using principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and heat map clustering. Absolute quantification data for catechins and amino acids were integrated into multivariate models to assess their contribution to overall variance. In addition, metabolic pathways of the key differential metabolites were annotated and enriched using the KEGG database to elucidate their potential derivation mechanisms. Correlation analysis between 19 differential compounds and sensory attributes in yellow and green teas was conducted to provide sensory validation of potential biomarkers. This study provided new insights into the relationships between flavor substances and sensory characteristics in MDBYT, offering a scientific basis for flavor quality control and processing optimization of bud yellow tea.

Results

Sensory quality analysis

Sensory evaluation based on Chinese national standards

Sensory evaluation is the most commonly used method for assessing tea quality, and analysis of variance of sensory evaluation scores can effectively distinguish differences among tea samples. As shown in Supplementary Information, Table S1, the sensory evaluation scores of tea samples exhibited a gradual increase throughout the processing stages. From fresh leaves to finished yellow tea, the total sensory score and taste score significantly increased by 21.82%, 26.72%, respectively (p < 0.05). Among the processing stages, the yellowing process had the most significant effect on the sensory score of bud yellow tea. Compared with the fixing stage, the sensory scores increased more substantially after yellowing, with the total sensory score increasing by 8.30% and the taste score rising by 9.76%, both showing significant differences. Notably, the first two of the three yellowing stages contributed most substantially to the improvement in quality scores.

As shown in Fig. 2A, the sensory characteristics of the tea samples changed markedly during processing. The appearance color shifted from green to yellow, the aroma evolved from grassy to umami and sweet, and the taste transitioned from grassy and astringent to sweet and mellow. The most notable changes occurred during the yellowing stages. The color of dry tea, brewed leaves, and infusion all transformed from the tender green of the fixed leaves to a delicate yellow after the third yellowing stage. The most obvious color transition occurred between the first and second yellowing stages, indicating that the initial two yellowing stages played a key role in the development of the characteristic coloration of MDBYT.

Fig. 2. Sensory evaluation results of samples during the MDBYT processing.

A Sensory evaluation mapping. B Radar chart of taste attributes. C Loading plot. D Hierarchical clustering analysis.

Fresh leaves and spreading leaves exhibited a pronounced astringent and grassy taste. After fixation, the grassy taste and astringency gradually decreased, while the sweetness and mellowness gradually increased. After the second yellowing stage, the umami, sweet, mellow, and brisk taste became notably enhanced, and the characteristic sensory profile of yellow tea gradually developed. Subsequent triple yellowing and drying further stabilized the distinctive umami-sweet-mellow-thick taste of finished yellow tea, accompanied by a marked improvement in overall sensory quality.

In contrast, the green tea produced from the same batch of fresh leaves but without the yellowing process retained a green appearance and exhibited an umami and brisk taste, but with lower levels of sweetness, mellowness and thickness, distinctly differing from the bud yellow tea. Previous studies have demonstrated that the yellowing process is the most crucial step in determining yellow tea quality, as the combined effects of heat and humidity promote the transformation and degradation of key substances, resulting in the characteristic sweet aroma, mellow taste, and refreshing sensation of yellow tea⁹. Consistent with these findings, the present results demonstrate that the yellowing process is the most crucial step in forming the characteristic taste profile of MDBYT, particularly by enhancing umami, sweetness, and mellowness while reducing astringency and grassy notes.

Quantitative descriptive analysis (QDA) and flavor profile construction

Establishment of MDBYT flavor descriptors

To enhance the accuracy and consistency of sensory description, quantitative descriptive analysis was conducted to characterize the taste attributes of the tea samples. A total of ten taste descriptors were identified, namely sweetness, umami, astringency, bitterness, mellowness, thickness, acceptability, fired flavor, grass taste, and overcooked taste. Significant differences were observed in these attributes, as well as in the overall taste intensity among the samples at different processing stages, indicating that each stage exhibited distinct sensory characteristics. The definitions and reference standards for each taste descriptor are presented in Supplementary Information, Table S2 and Section “Quantitative descriptive analysis (QDA)”. These descriptors effectively capture the dynamic changes in the taste profile of MDBYT during processing, providing a comprehensive framework for sensory evaluation.

Construction and analysis of taste profile

Compared with traditional sensory evaluation, flavor profiling provides a more intuitive visualization of sensory characteristics and differences among samples. Evaluators scored each sample based on the defined descriptors and reference standards, and the resulting data were converted into a radar map. The overall variations in QDA taste profiles are presented in Fig. 2B, while the flavor profiles of individual samples are shown in Fig. S1.

As illustrated in Fig. 2B, fresh leaves and spreading leaves exhibited a pronounced grassy taste, with scores of 2.24 and 2.36, respectively, indicating relatively lower acceptability. The fixation-processed leaves developed a noticeable umami flavor but were still accompanied by unpleasant grassy and smoky notes. With the progression of the yellowing stages, the sweet and mellow characteristics gradually increased. In particular, the first two yellowing stages significantly enhanced sweetness, umami, mellowness, and overall acceptability. In contrast, the umami and sweetness of the third yellowing leaves did not change significantly compared with the piled-spreading leaves. During the final drying stage, the overcooked flavor became more pronounced, leading to a decrease in overall acceptability, while umami intensity increased. The taste profile of the fresh leaves was similar to that of spreading leaves, whereas the fixation stage exhibited greater variability in flavor attributes. The profiles of the first and second yellowing leaves were comparable, although the second yellowing leaves exhibited higher scores for umami, sweetness, and mellowness. Compared with the third yellowing leaves, the finished yellow tea exhibited reduced dull notes and improved acceptability, thickness, and mellowness.

Overall, MDBYT is characterized by a composite flavor profile dominated by umami and sweetness, complemented by thickness and mellowness, with balanced levels of astringency and bitterness. These results demonstrate that the characteristic flavor profile of MDBYT is gradually established and stabilized during the processing stages.

The QDA method effectively distinguished the flavor characteristics at each stage of processing. When combined with the results of sensory evaluation, it is evident that the taste quality of MDBYT undergoes significantly change at each processing stage. These findings confirm that the yellowing process plays a crucial role in the development of characteristic flavor profile of MDBYT.

Differential analysis of sensory characteristics

To further validate the effectiveness of the QDA method in distinguishing the taste characteristics among the processing samples, the sensory evaluation scores of 10 taste descriptors were standardized and analyzed using SIMCA statistical software for principal component analysis (PCA) and hierarchical cluster analysis (HCA) (Fig. 3A–C). In the PCA score plot (Fig. 3C), the spatial distance between samples reflects the degree of difference in taste attributes, with closer proximity indicating greater similarity. The first two principal components, PC1 and PC2, explained 60.8% and 11.5% of the total variance, respectively, accounting for 72.3% of the overall variation, indicating good discrimination among samples. The fresh leaves and spreading leaves are located close to each other in the second and third quadrants, indicating similar taste characteristics dominated by grassy notes. The fixation leaves are mainly distributed in the fourth quadrant and are positioned farther from the spreading leaves but closer to the origin, suggesting that fixation significantly affected taste attributes. The first yellowing leaves are located closest to the origin, indicating that this stage produced a marked shift in sensory characteristics relative to the previous processing stages. The second and third yellowing leaves, together with the finished yellow tea, are mainly distributed in the first and fourth quadrants and clustered relative closely, reflecting similar taste attributes characterized by enhanced umami-sweetness and mellowness-thickness. The piled-spreading leaves are positioned in the first quadrant at a greater distance from the origin, associating with sweet, dull, and cooked flavor attributes. Collectively, the attributes of sweetness, umami, mellowness, thickness, and acceptability gradually increased during processing, while the grass taste and fired flavor progressively decreased.

Fig. 3. Profile analysis of non-volatile components.

A Principal component analysis (PCA) with integrated absolute quantification data. B Partial least squares-discriminant analysis (PLS-DA) with integrated absolute quantification data. C Permutation test analysis. (R²X = 0.549, R²Y = 0.571).

The results of the HCA of taste attributes are shown in Fig. 2D. Based on the taste scores, the samples are grouped into 6 sub-clusters and 2 major categories. The yellowing-related stages (the first, second, and third yellowing) form one category, including fixation leaves, yellowing leaves, piled-spreading leaves, and finished yellow tea. The fresh leaves and spreading leaves are grouped into another category, indicating that fixation and yellowing stages substantially influence the taste characteristics of MDBYT.

Overall, these multivariate statistical analyses demonstrate that PCA combined with cluster analysis effectively summarizes the changes in flavor attributes at each processing stage. The results further confirm that fixation and yellowing, particularly the yellowing process, are the key stages driving the formation of flavor profile of MDBYT.

Quantitative analysis of key taste components

To more accurately characterize changes in major taste-related compounds, quantitative analysis of catechins, caffeine, and amino acids was carried out using HPLC. As presented in Table 1, the total catechin content showed a decreased trend of 10.11% throughout processing, with a decrease of 4.85% observed during the yellowing stages. Ester-type catechins showed a decreasing trend, with epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG) showing decreases of 13.86% and 11.11%, respectively. Non-ester-type catechins also declined, with catechin (C) and epicatechin (EC) decreasing by 12.50% and 3.28%, respectively.

Table 1.

Changes in epicatechin, caffeine, and amino acid content in MDBYT processing

Unit	Compounds	FL	SD	FX	FY	SY	TY	PS	YT
g/100 g	EGC	0.83 ± 0.01bc	0.80 ± 0.01c	0.93 ± 0.09abc	0.99 ± 0.04a	0.93 ± 0.01abc	0.94 ± 0.03ab	0.83 ± 0.04bc	0.85 ± 0.07bc
	C	0.56 ± 0.01ab	0.55 ± 0.01abcd	0.59 ± 0.05a	0.56 ± 0.03abc	0.48 ± 0.02 d	0.54 ± 0.02abcd	0.49 ± 0.01 cd	0.49 ± 0.03bcd
	EC	0.61 ± 0.01ab	0.57 ± 0.00b	0.69 ± 0.07a	0.65 ± 0.06ab	0.62 ± 0.01ab	0.63 ± 0.02ab	0.56 ± 0.03b	0.59 ± 0.05ab
	EGCG	6.35 ± 0.11a	6.33 ± 0.04a	6.83 ± 0.86a	6.77 ± 1.18a	5.98 ± 0.13a	6.56 ± 0.26a	5.81 ± 0.21a	5.47 ± 0.59a
	GCG	0.36 ± 0.06abc	0.39 ± 0.00abc	0.47 ± 0.03ab	0.49 ± 0.04a	0.43 ± 0.13abc	0.33 ± 0.01bc	0.31 ± 0.01c	0.32 ± 0.02c
	ECG	0.83 ± 0.01bc	0.80 ± 0.01c	0.93 ± 0.09abc	0.99 ± 0.04a	0.93 ± 0.01abc	0.94 ± 0.03ab	0.83 ± 0.04bc	0.85 ± 0.07bc
	CG	0.05 ± 0.00c	0.05 ± 0.00bc	0.06 ± 0.00ab	0.06 ± 0.00a	0.05 ± 0.00c	0.06 ± 0.00abc	0.05 ± 0.00c	0.05 ± 0.00bc
	Total	9.61 ± 0.16ab	9.50 ± 0.02ab	10.49 ± 1.18a	10.49 ± 1.35a	9.41 ± 0.17ab	9.98 ± 0.34ab	8.88 ± 0.33b	8.61 ± 0.82b
	CAF	4.58 ± 0.02a	4.48 ± 0.00b	4.46 ± 0.01b	4.44 ± 0.01b	4.37 ± 0.02c	4.29 ± 0.01 d	4.24 ± 0.02e	4.19 ± 0.01 f
mg/g	Asp	0.65 ± 0.04b	1.89 ± 0.38a	1.91 ± 0.03a	2.06 ± 0.19a	1.91 ± 0.56a	1.80 ± 0.05a	1.95 ± 0.56a	2.37 ± 0.05a
	Ser	1.92 ± 0.02a	1.75 ± 0.28ab	1.63 ± 0.08ab	1.57 ± 0.01ab	1.48 ± 0.08ab	1.35 ± 0.03ab	1.4 ± 0.29ab	1.12 ± 0.55b
	Glu	0.39 ± 0.07 d	0.46 ± 0.05 d	1.27 ± 0.15c	2.44 ± 0.04abc	2.27 ± 0.03ab	2.44 ± 0.03a	1.63 ± 0.14bc	1.27 ± 0.71c
	Gly	2.46 ± 0.14a	2.27 ± 0.08a	1.60 ± 0.10b	1.62 ± 0.10b	1.55 ± 0.13bc	1.24 ± 0.03c	1.38 ± 0.19bc	1.23 ± 0.04c
	His	0.99 ± 0.43bc	0.89 ± 0.06c	1.09 ± 0.11bc	1.54 ± 0.15ab	2.00 ± 0.35a	1.61 ± 0.04ab	1.75 ± 0.17a	1.91 ± 0.18a
	Arg	0.59 ± 0.03c	0.96 ± 0.21a	0.85 ± 0.04ab	0.89 ± 0.05ab	0.83 ± 0.05abc	0.68 ± 0.02bc	0.68 ± 0.07bc	0.69 ± 0.01bc
	Thr	0.24 ± 0.01 d	0.41 ± 0.07bc	0.51 ± 0.03ab	0.51 ± 0.04a	0.51 ± 0.04ab	0.42 ± 0.02abc	0.41 ± 0.04c	0.46 ± 0.01abc
	Ala	0.29 ± 0.02b	0.31 ± 0.01b	0.47 ± 0.03a	0.47 ± 0.04a	0.49 ± 0.03a	0.41 ± 0.01a	0.41 ± 0.05a	0.45 ± 0.01a
	Pro	0.16 ± 0.15b	0.36 ± 0.22ab	0.63 ± 0.02a	0.58 ± 0.04a	0.60 ± 0.02a	0.52 ± 0.02a	0.56 ± 0.09a	0.56 ± 0.02a
	The	13.21 ± 0.93ab	14.12 ± 0.49a	13.17 ± 0.67ab	12.99 ± 0.86abcd	13.05 ± 0.75abc	10.77 ± 0.39 d	11.56 ± 1.39 cd	10.88 ± 0.32 cd
	Tyr	0.21 ± 0.02a	0.24 ± 0.00a	0.26 ± 0.05a	0.20 ± 0.01a	0.19 ± 0.02a	0.22 ± 0.01a	.23 ± 0.04a	0.23 ± 0.05a
	Val	0.15 ± 0.00a	0.23 ± 0.00b	0.25 ± 0.01b	0.25 ± 0.01b	0.25 ± 0.00b	0.24 ± 0.01b	0.24 ± 0.03b	0.25 ± 0.00b
	Met	2.58 ± 0.01a	3.36 ± 0.08a	3.39 ± 0.22a	3.66 ± 0.20a	3.71 ± 0.25a	3.25 ± 0.09a	2.03 ± 1.53a	2.12 ± 1.61a
	Lys	0.12 ± 0.01 d	0.4 ± 0.02c	0.50 ± 0.02ab	0.49 ± 0.03ab	0.51 ± 0.03a	0.44 ± 0.01bc	0.41 ± 0.01c	0.45 ± 0.04abc
	Ile	0.18 ± 0.01e	0.7 ± 0.02bc	0.85 ± 0.04ab	0.86 ± 0.06a	0.90 ± 0.05a	0.75 ± 0.02abc	0.59 ± 0.13 cd	0.5 ± 0.02 d
	Leu	0.40 ± 0.01e	1.11 ± 0.04c	1.38 ± 0.05ab	1.38 ± 0.09ab	1.42 ± 0.08a	1.18 ± 0.04bc	0.97 ± 0.16 cd	0.86 ± 0.03 d
	Phe	0.26 ± 0.02 d	0.97 ± 0.04c	1.20 ± 0.06ab	1.21 ± 0.08ab	1.24 ± 0.07ab	1.07 ± 0.03bc	1.05 ± 0.17bc	1.32 ± 0.05a
	Total	24.81 ± 1.32 d	30.45 ± 1.43ab	30.95 ± 1.18ab	32.12 ± 1.94a	32.90 ± 2.16a	28.39 ± 0.67bc	27.24 ± 1.47 cd	26.67 ± 2.79 cd

Different letters (a, b, c, …) indicate statistically significant differences at p < 0.05.

The contents of 7 catechin components increased during the fixing stage, followed by a marked decrease during the yellowing and piled-spreading stages, while no notable changes were observed during the drying process. This change pattern may be attributed to the increased enzyme activity in fresh tea leaves during the early stages of processing. When the temperature reaches a certain point, the enzyme activity is deactivated by heat, reducing the biotransformation of catechins. During the subsequent yellowing process, non-enzymatic wet-heat reactions become dominant. Under high temperature and humidity, catechins may undergo oxidization, thermally degradation, or isomerization into compounds such as theaflavins and quinones, or be hydrolyzed into smaller molecules such as EC and C¹⁰. The prolonged piled-spreading stage may further promote catechin oxidation. Consequently, the reduction in catechin content during the yellowing and piled-spreading stages contributes to the reduced bitterness and astringency of yellow tea compared with green tea.

Caffeine content showed a slight decreasing trend during MDBYT processing (Table 1), which may be associated with partial degradation or sublimation of caffeine under high-temperature and moist-heat conditions.

The variation of amino acid content followed trends similar to those observed in the non-targeted analysis. The total amino acids increased by 7.54% from fresh leaves to finished yellow tea. The increase was primarily observed during spreading, first yellowing, and piled-spreading stages, whereas a decline was observed after fixation and drying. These results suggest that amino acids accumulate in the early stages of processing and may be consumed during later stages, possibly through Maillard reactions or thermal degradation at elevated temperatures. Amino acids associated with umami and sweet tastes, including Asp, Glu, Pro, Val, Lys, and Phe, showed increasing trends during processing. The quantitative data suggest that the accumulation of umami- and sweet-tasting amino acids during processing may contributes to the enhanced umami and sweet taste characteristics of the finished MDBYT.

Metabolomic profiling and alterations during MDBYT processing

Profiling of non-volatile components during MDBYT processing

To comprehensively understand the relationship between quality formation and changes in non-volatile metabolites during MDBYT processing, a metabolomics approach based on UHPLC-Q-Exactive/MS was used to analyze non-volatile metabolites in samples collected at different processing stages. As shown in Fig. S1, the total flow ionogram (TIC) exhibited a stable baseline, and all non-volatile substances were detected within 18 min. After peak matching and calibration, 3734 metabolite characteristic ions were detected in the positive ion mode, and 156 nonvolatile compounds, including 15 amino acids, 13 alkaloids, 5 organic acids, 11 flavanols, 10 phenolic acids, 10 dimeric catechins, 4 flavonols, 26 flavonoid glycosides, 13 EPSFs, 11 lipids, and 6 other substances (Fig. 4A), were structurally identified (Supplementary Information, Table S3), representing a relatively high level of compound identification in tea metabolomics studies. Statistical analysis revealed that all 156 compounds exhibited significant differences in content among processing stages (p < 0.05) (Supplementary Information, Table S4).

Fig. 4. Analysis of key differential non-volatile metabolites of MDBYT.

A Non-volatile metabolites category chart. B Heatmap analysis of differential non-volatile metabolites with VIP > 1 and p value < 0.05. C VIP value analysis of differential non-volatile metabolites. D Correlation analysis between non-volatile components and taste properties. E Chemical structure of 13 EPSFs.

To visualize the variation pattern of non-volatile substances during the processing of MDBYT, we carried out unsupervised principal component analysis, and the results are shown in Fig. 3A. The triplicate samples at each stage clustered tightly together, indicating good analytical reproducibility. Meanwhile, samples from different stages were distinctly separated, demonstrating significant metabolic differences throughout processing. Fresh leaves and spreading leaves were located in different quadrants, while fixation leaves, yellowing leaves, and piled-spreading leaves were located in the second, first, and fourth quadrants, respectively, with a relatively large distance between clusters, indicating substantial metabolic changes among processing stages.

To further characterize metabolite differences among processing stages, supervised PLS-DA was conducted. Model reliability was evaluated using a 200-time permutation test. The resulting validation parameters (R² = 0.453 and Q² = −0.657 at intercept), indicating that the model was not overfitted (Fig. 3C). The PLS-DA score plot (Fig. 3B) showed a separation pattern similar to that observed in the PCA analysis, although the replicate samples were more tightly clustered and group separation was clearer. The consistency between PCA and PLS-DA results further confirms the robustness and reliability of the observed metabolite variations during MDBYT processing.

Dynamic changes in major non-volatile components

The differences in non-volatile metabolite profiles during MDBYT processing were mainly associated with variations in the levels and proportion of amino acids, catechins and dimeric catechins, flavonols and their glycosides, phenolic acids, alkaloids, organic acids, EPSFs, lipids, and other substances at different processing stages. These metabolites collectively influence the flavor characteristics of MDBYT (Fig. 4A). A Heat map was constructed to visualize the dynamic changes of these characteristic metabolites throughout processing (Fig. 6A).

Fig. 6. Differences in non-volatile components between finished yellow tea and green tea.

A Heatmap of non-volatile compounds in MDBYT and control green tea. B Volcano plot of differential compounds. C Heatmap of differential non-volatile compounds. D Correlation heatmap analysis between green tea and taste attributes. E Correlation heatmap analysis between MDBYT and taste attributes.

Amino acids

Amino acids are among the most important determinants of tea quality, and the ratio of amino acids to tea polyphenols often used to evaluate processing suitability and flavor quality¹¹. Among the 21 amino acids present in tea, those with relatively high concentrations and characteristic umami and sweet tastes include theanine, glutamic acid, aspartic acid, and asparagine. As shown in Fig. 6A, 15 amino acids showed significant changes during MDBYT processing, with total content exhibiting a overall fluctuating increase. Compared with fresh leaves, the total amino acid content in the finished yellow tea increased by 4.57% (Supplementary Information, Table S4). The content rose by 11.19% during the spreading stage, but declined during the fixing stage⁶, and increased by 5.84% during the first two yellowing stages, and then decreased in the third yellowing stage. During the piled-spreading stage, the content increased by 6.60%, but declined again in the drying stage.

During spreading, fresh leaves undergo moisture dissipation and enhanced protease activity promotes protein hydrolysis, resulting in increases in the contents of most amino acids except glutamine and serine. Theanine, proline, alanine, and threonine, which are associated with umami and sweet tastes, exhibited increasing trends during the yellowing stages. Proline and theanine may synergistically enhance umami taste. Alanine serves as a substrate for alanine decarboxylase, which catalyzes its decarboxylation via the ethylamine synthesis pathway to form theanine¹¹. The sustained high-temperature environment during yellowing promotes protein degradation into free amino acids, which likely explains the enhancement of umami and sweet tastes during this stage. During the piled-spreading stage, proline and theanine, which are associated with umami and sweet tastes, continued to rise, while the content of bitter-tasting arginine also increased. These results suggest that the accumulation of umami- and sweet-tasting amino acids during yellowing contributes substantially to the characteristic taste of MDBYT¹², while the increase in bitter amino acids during later stages may enhance thickness and flavor complexity.

Catechins

Catechins are the main component of tea polyphenols (>70%) and the primary contributors to bitter and astringent taste. Dimeric catechins formed through oxidative polymerization influence tea color, bitterness, and astringency. The total catechins and dimeric catechins showed an overall decreasing trend during MDBYT processing, with an 11.58% reduction observed from fresh leaves to finished yellow tea. Significant decreases occurred during the fixing (11.60%) and yellowing (9.47%) (Table S4), while slight increases were observed during spreading and piled-spreading.

Among the 11 catechins and their dimer, the contents of C, EC, EGC, ECG, GCG, EGCG-4’-O-Me, epiafzelechin, proanthocyanidins, and polymerized catechins increased during spreading. This increase may be associated with enhanced hydrolytic and catechin-related enzymatic activity, leading to the hydrolysis of ester-type catechins or the conversion among catechin forms^13,14. The contents of C, EC, EGC, epiafzelechin ECG, theaflavin, procyanidins, and polymerized catechins decreased significantly during fixing and yellowing, likely due to isomerization, oxidation or thermal degradation under high-temperature and high-humidity conditions. Most catechins decreased after drying, which is consistent with the previous findings of Fan et al.¹⁵. The substantial reduction in catechins, particularly during the yellowing stage, directly explains the reduced bitterness and astringency and mellower taste of MDBYT compared with green tea.

Flavonols and flavonol glycosides

Flavonols and flavonol glycosides, the primary flavonoids in plants, contribute to bitter taste and infusion color¹⁶. Flavonol glycosides play an important role in the formation of the yellow-green infusion color in green tea. The flavonol and flavonol glycoside contents generally decreased during MDBYT processing, with the main reduction occurring during the yellowing and piled-spreading stages (Supplementary Information, Table S4).

Among the 4 flavonols and 26 flavonol glycosides identified, the contents of kaempferol, quercetin, vitexin, isovitexin, kaempferol-3-glucopyranoside, and apigenin-6,8-C-diglucoside increased during yellowing, whereas the contents of most other compounds decreased. During the piled-spreading stage, myricetin, kaempferol-7-pyran-β-glucoside, and quercetin-3-rutinoside increased, whereas the contents of other flavonoids decreased. Flavonol glycosides are relatively stable due to their glycosidic structure and possess extremely low taste threshold, making them important contributors to bitterness and astringency in tea infusion¹⁷. Additionally, they have a positive enhancing effect on the bitterness of caffeine¹⁸. Therefore, the reduction in flavonol glycoside content during processing, particularly during yellowing, likely contributes to the reduced bitterness and astringency and the enhanced mellow taste of bud yellow tea.

Alkaloids

Alkaloids are characteristic components of tea, among which caffeine is the most abundant purine alkaloid and a major contributor to the bitterness of tea. The total alkaloid content significantly decreased during MDBYT processing, with notable reductions occurring during spreading, fixation, and yellowing stages, followed by a slight increase during the piled-spreading stage. Among the 13 alkaloids showing significant differences, most of them decreased during the yellowing stage, except thymine and (S)-5’-deoxy-5’-(methylsulfinyl)adenosine,. Caffeine and theobromine are the main bitter alkaloids in tea¹⁹. Although caffeine remains relatively stable throughout processing, theobromine decreased markedly during the yellowing process. The decrease in theobromine content during yellowing may partially contribute to the reduced bitterness of MDBYT.

Phenolic acids

Phenolic acids, including gallic acid, chlorogenic acid, caffeic acid, and ellagic acid²⁰, are aromatic compounds containing both carboxyl and hydroxyl groups. During tea manufacturing, ester catechins may hydrolyze to produce phenolic acids, which contribute to taste and color and often impart sour and astringent sensations. The phenolic acid content increased significantly by 100.61% compared to fresh leaves (p < 0.05) (Supplementary Information, Table S4) in MDBYT processing. The increase mainly occurred during the spreading and fixation stages, whereas no significant increases were observed during yellowing, piled-spreading, and drying stages. This increase may be attributed to enhanced hydrolytic enzyme activity during spreading and fixation, promoting the hydrolysis of ester catechins into phenolic acids. In contrast, during the subsequent yellowing and drying processes, high temperatures and humidity likely inactivate the hydrolytic enzymes, reducing further catechin hydrolysis.

Chlorogenic acid, cinnamic acid, and methoxysalicylic acid significantly increased during spreading. Chlorogenic acid possesses multiple biological activities and is a strong antioxidant. Cinnamic acid contributes balsamic aromatic notes. The substantial increase in phenolic acids during early processing stages may contribute to both the complex flavor profile and potential health benefits of MDBYT.

Organic acids

Organic acids influence tea aroma, acidity, and mellowness¹⁶. During MDBYT processing, the total organic acid content increased by 21.07%, with increases observed during fixation, yellowing, piled-spreading, and drying, whereas a decrease occurred during spreading (Supplementary Information, Table S4). Pyroglutamic acid content showed a continuous increasing trend throughout processing except during spreading. Pyroglutamic acid is a non-enzymatic reaction product of glutamic acid and possesses a characteristic sour taste. Under high-temperature conditions, glutamic acid may undergo intramolecular cyclization to form pyroglutamic acid.

EPSFs

Ethyl-2-pyrrolidinone-substituted flavanols (EPSFs) are formed by the Strecker degradation and cyclization reactions involving L-theanine, in which 1-ethyl-5-hydroxy-2-pyrrolidone substitutes catechin at the 8-C or 6-C position²¹. These compounds have been identified in black tea and white tea^22–24. EPSFs exhibit various biological activities, including anti-inflammatory effects and inhibition of acetylcholinesterase (AChE)²⁵.

In the present study, a total of 13 EPSF compounds were identified in the MDBYT samples. Their total content increased significantly throughout the processing, showing a 17.37-fold increase compared with fresh leaves. Notably, the content rose significantly during the fixation, yellowing, and drying stages. The EPSF content in the fixed leaves was 5.18 times higher than that in the spreading leaves, while the yellowing stage resulted in an 85.21% increase, and drying further elevated the content by 36.07% (Supplementary Information, Table S4). Previous studies have demonstrated that the formation of EPSFs is closely related to processing temperature²⁶. High temperatures promote Strecker degradation and Maillard reactions to form EPSFs. The results of the present study are consistent with these findings—EPSF content increased significantly during the high-temperature stages of fixation, yellowing, and drying.

Lipids

Lipids are important precursors of tea aroma components, and their degradation into fatty acids influences the aroma profile of finished tea^27,28. During MDBYT processing, the lipid content increased significantly by 89.00%, with notable increases during the spreading, yellowing, and drying stages (p < 0.05) (Table S4), whereas a marked decrease occurred after fixation. It is noteworthy that the contents of LysoPC compounds and pheophytin b increased during the processing. The increase of pheophorbide b is associated with the conversion of chlorophyll to brown pheophytin through magnesium removal under moist-heat conditions. The elevation in LysoPCs content may result from high-temperature-induced rupture of tea leaf cell membranes, which leads to the release of lipids from the phospholipid bilayer²⁹. The changes in lipid composition during processing are closely associated with the transformation of tea color from green to yellow and the development of sweet aroma during the yellowing stage.

Other compounds

Among the remaining substances, sucrose showed an overall declining trend but increased during the first yellowing and piled-spreading stages. 1-Ethyl-5-hydroxy-2-pyrrolidone significantly increased during the fixation and yellowing stages. As a direct precursor of EPSFs, the accumulation of this compound during high-temperature stages provides the substrate necessary for EPSF formation, supporting the observed increase in EPSF content during MDBYT processing.

Screening of key non-volatile metabolites

Metabolites with VIP > 1 and p < 0.05 were considered significantly different. As shown in the bar plot (Fig. 4C), 25 key differential compounds were identified. These included 6 amino acids (theanine, tryptophan, glutamine, proline, arginine, tyrosine), 5 alkaloids (choline, theobromine, phosphorylcholine, 5’-methylthioadenosine, glycerophosphocholine), 4 flavonols (EC, EGC, ECG, EGCG), 2 phenolic acids (cinnamic acid, galloyl glucose), 1 catechin dimer (procyanidin B4), 4 lipids (phytosphingosine, diglyceride (36:6), chlorophyll b, palmitic acid), and 3 additional compounds (sucrose, 1-ethyl-5-hydroxy-2-pyrrolidinone, theanine glucoside). Among these metabolites, amino acids, flavanols, and alkaloids constituted the major classes contributing to metabolic differentiation. The heatmap of key differential metabolites (Fig. 4B) further illustrated their dynamic variation patterns during processing. Umami-related compounds showed increasing trends during yellowing, while most bitterness-related compounds decreased during fixation and yellowing. Additionally, lipid compounds related to aroma formation increased after drying. These results suggest that yellowing is the major factor driving changes in the metabolite profile of bud yellow tea and is the critical processing step for the formation of its characteristic taste.

Correlation analysis between key chemical compositions and taste characteristics

To further explore relationships between non-volatile components and taste attributes³⁰, correlation analysis was performed between 13 key differential metabolites (VIP > 1, FC > 1.5) and five taste attributes (sweetness, umami, mellowness, thickness, and grassy flavor). A correlation coefficient with an absolute value > 0.3 was considered indicative of a meaningful association.

As shown in Fig. 4D, sweetness was positively correlated with proline, palmitic acid, phytosphingosine, theanine glucoside, diglyceride (36:6), pheophytin b, and 1-ethyl-5-hydroxy-2-pyrrolidinone. Umami showed significant positive correlations with proline, cinnamic acid, diglyceride (36:6), theanine glucoside, tryptophan, 5’-methylthioadenosine, and phosphocholine. Mellowness demonstrated notable positive correlations with proline, cinnamic acid, pheophytin b, theanine glucoside, and phosphocholine, while thickness was mainly associated with proline, cinnamic acid, theanine glucoside, and phosphocholine. None of 13 key compounds showed strong correlations with grassy flavor, indicating that these compounds primarily contribute to desirable taste attributes rather than grassy notes.

Importantly, theanine glucoside, a novel theanine derivative identified through chromatographic retention time, mass spectrometry, and comparison with synthetic standards, showed positive correlations with sweetness, umami, mellowness, and thickness. Its content increased continuously during processing, especially during the yellowing and drying stages. Previous studies have suggested that theanine may undergo glycosylation during tea processing to form theanine glucoside, which has also been detected in black tea and oolong tea^31,32. However, its detailed structure, functional properties, and specific contribution to tea quality remain unclear. These findings indicate that theanine glucoside may represent a characteristic compound contributing to the distinctive “mellow and umami-sweet” taste of MDBYT. Nevertheless, the unique taste profile likely arises from the synergistic interactions among numerous chemical constituents rather than the action of a single compound.

Metabolic pathway analysis of non-volatile metabolites

Compared with green tea processing, yellow tea production typically involves larger leaf loads, relatively lower fixation temperature, and longer fixation times, which may allow partial retention of enzyme activity in the leaves. To elucidate dynamic changes and formation mechanisms, KEGG pathway enrichment analyses were performed based on the 156 differential metabolites identified.

The results of the metabolic pathway enrichment analysis are shown in Fig. 5A. The bubbles represent KEGG metabolic pathways, with the horizontal coordinate and the size of the bubbles indicating the impact value, where larger bubbles represent pathways with greater influence. The vertical coordinate and color intensity represent the significance level (p value) of enrichment, with darker colors indicating lower p values. Overall, the differential metabolites identified during MDBYT processing were enriched in 16 metabolic pathways, involving 63 metabolites. Among them, 2 key metabolic pathways were identified as particularly important: amino acid biosynthesis and flavonoid metabolism.

Fig. 5. Metabolic pathways of key non-volatile metabolites during MDBYT processing.

A Metabolic pathway enrichment analysis of 156 differential compounds. B Typical amino acid metabolic pathways. C Biosynthesis of flavanols and flavonoids.

Within the amino acid biosynthesis pathway, the biosynthesis of alanine, aspartic acid, and glutamic acid was enriched during processing, while choline, serine, and threonine were formed through enzymatic or thermal degradation reactions related to amino acid metabolism (Fig. 5B). Flavonoids and flavonols were enriched in the flavonoid biosynthesis pathway (Fig. 5C). Kaempferol can be synthesized from naringenin, apigenin, and vitexin through flavonoid hydroxylase-mediated reactions, or converted into myricetin, and quercetin via flavonoid hydroxylases.

Further KEGG pathway analysis of the 13 key taste-related metabolites showed enrichment in 4 metabolic pathways, namely proline, arginine, tryptophan, and tyrosine metabolism, which were all associated with amino acid biosynthesis. For example, γ-Aminobutyric acid can be generated by glutamate decarboxylase, through transamination reactions involving succinaldehyde in the tricarboxylic acid cycle, or via the metabolism of arginine and proline. The synthesis of cinnamic acid involves coumarin and its derivatives, which originate from tyrosine or phenylalanine metabolism. In sphingolipid metabolism, plant sphingosine serves as a backbone for the formation of sphingolipids. Meanwhile, palmitic acid and diglyceride (36:6) are long-chain fatty acids involved in glycerophospholipid metabolism. Both sphingolipid and glycerophospholipid metabolism are important components of lipid metabolism and may contribute to the formation of aroma precursors and structural membrane changes during tea processing.

Overall, the KEGG analysis results, together with the observed changes in the main non-volatile component content, indicate that MDBYT processing significantly altered metabolite composition and relative abundance of metabolites. Particularly, the fixing and yellowing stages appear to be critical phases during which metabolite transformations drive the development of the characteristic “sweet, umami, mellow, and thick” taste profile of MDBYT, ultimately shaping its overall taste quality.

Comparative analysis of non-volatile components between finished yellow tea and green tea

Identification of differential compounds as potential biomarkers

Comparative analysis between finished yellow tea and control green tea produced from the same batch of fresh leaves identified 19 differential compounds as potential markers (FC > 1.5, p < 0.05). Most of these compounds were significantly enriched in finished yellow tea compared to the control green tea, including thymine, EGC-3,5-digallate, (S)-5’-Deoxy-5’-(methylsulfinyl) adenosine, 2’-(3-methylcyclohexane-1,1-diyl) diacetic acid, theasinensin A, theasinensin B, kaem-7-glucopyranoside, kaempferol, 7 EPSFs, 1-Ethyl-5- hydroxy-2-pyrrolidinone, L-(-)-methionine, theasinensin F, and EC-(4 bata->8)-EGCG. Conversely, several compounds, including L-(1)-methionine, theasinensin F, and EC-(4beta->8)-EGCG, were present at lower levels in finished yellow tea than in the control green tea.

Correlation analysis between differential compounds and taste attributes

To further elucidate the potential role of the aforementioned differential metabolites in shaping the sensory quality, Pearson correlation analysis was conducted between the 19 differential metabolites and 10 key sensory attributes (Fig. 6D, E). In green tea, the analysis revealed relationships between metabolite classes and specific taste attributes. Alkaloids ((S)-5’-Deoxy-5’-(methylsulfinyl)adenosine), organic acids (2,2’-(3-methylcyclohexane-1,1-diyl)diacetic acid), flavonoids (Kaem-7-glucopyranoside, Kaempferitrin), dimeric catechins (Theasinensin F), and flavanol derivatives (EC-(4beta->8)-EGCG) showed significant positive correlations with astringency, thickness, grass taste, fired flavor, and overcooked notes. These compounds collectively contribute to the characteristic mellow and thick taste profile of green tea. Meanwhile, compounds such as theasinensin A, 8-R-EGCG-cThea, and 6-C-R-ECG-cThea were positively correlated with sweetness, umami, and bitterness. Notably, bitter compounds may enhance the perception of umami and sweetness through flavor synergy, consistent with the observations reported by Zhang et al.³³.

In MDBYT, correlation analysis unveiled a distinct chemical basis for its characteristic flavor. Compounds such as theasinensin A, 8-C-S-EGCG-cthea, 1-ethyl-5-hydroxy-2-pyrrolidinone, and kaempferitrin showed positive correlations with sweetness, bitterness, thickness, grass taste, and fired flavor, indicating their involvement in forming the complex flavor matrix of yellow tea. On the other hand, compounds including 2,2’-(3-methylcyclohexane-1,1-diyl)diacetic acid, 6-C-R-ECG-cThea, 8-C-R-EGCG-cThea, and EC-(4bata-8)-EGCG were significantly associated with umami, mellowness, and overall acceptability, suggesting that these substances are important contributors to the mellow and refreshing sensory characteristics of MDBYT. Furthermore, the dual correlation of L-(-)-methionine with both sweetness and bitterness further implies that bitter components may participate in flavor harmonization through synergistic interactions.

Notably, theasinensin A and B were markedly enriched in finished yellow tea. Theasinensins are a class of catechin dimers formed through oxidative coupling reactions and are important flavor compounds in fermented tea, such as black tea and oolong tea^34–36. Theasinensins have been gained more attention in recent years, due to their potential biological activities and stability³⁷. Among the known theasinensin derivatives, theasinensin A (a dimer of EGCG linked through an R-biphenyl bond) and theasinensin B (a dimer of EGCG and EGC) are the most abundant forms³⁸. Previous studies have shown that the antioxidant activity of theasinensin A exceeded that of its precursor, epigallocatechin gallate (EGCG)³⁸. In addition to the high levels of theasinensin A, theasinensin B, and theasinensin F in the ‘Fuan Dabai Tea’ cultivar, the formation of theasinensins is strongly influenced by processing conditions. For instance, hot-air drying has been reported to preserve higher levels of theasinensins than sun drying due to oxidative coupling reactions at elevated temperatures²⁶. These findings are consistent with the present results, in which the yellowing process promoted enrichment of theasinensin A and B. Their higher levels in yellow tea may partially explain the stronger antioxidant activity often reported for this tea type compared with green tea³⁹.

EPSFs also showed notable differences between the two tea types. Among the 13 EPSF compounds detected in the processing samples, seven displayed significantly higher contents in finished yellow tea than in green tea. EPSFs are formed through condensation reactions between L-theanine-derived intermediates and catechins. The present results suggest that galloylated flavanols, such as ECG and EGCG, are more prone to form EPSFs with theanine than non-galloylated flavanols (EC and EGC) during MDBYT processing. Because galloylated flavanols (ster-type catechins) are major contributors to bitterness and astringency, their conversion into EPSFs can alleviate these undesirable taste attributes while enhancing the mellow and smooth characteristics of yellow tea. This observation is consistent with the sensory evaluation analysis showing that bud yellow tea exhibited a more mellow and smooth taste than green tea. Additionally, the formation of EPSFs introduces bioactive compounds that may further enhance the health benefits of yellow tea.

The formation of EPSF is closely associated with yellowing conditions. During yellowing, moist-heat conditions promote the Strecker degradation of L-theanine, generating 1-ethyl-5-hydroxy-2-pyrrolidinone, which subsequently undergoes nucleophilic substitution with the catechin A-ring, particularly of galloylated catechins, to form EPSFs^25,40. Repeated yellowing stages provide prolonged exposure, enhancing EPSF accumulation. In contrast, green tea processing lacks yellowing; thus, the moist-heat environment required for EPSF formation is absent. This mechanistic difference highlights the critical role of yellowing parameters—moderate temperature, high humidity, and sufficient duration—in driving the non-enzymatic reactions responsible for the formation of these characteristic biomarkers.

Similarly, theasinensins A and B were significantly enriched in finished yellow tea. The yellowing process facilitates theasinensin formation via both enzymatic and non-enzymatic oxidation. High humidity maintains a hydrated environment favoring oxidative coupling of catechins, particularly between EGCG and EGC or EGCG dimers^38,41. Extended yellowing time allows gradual oxidation, whereas green tea processing lacks this step. Thus, the combined effects of moderate temperature, high humidity, and prolonged duration during yellowing create favorable conditions for theasinensin biosynthesis, explaining their higher abundance in MDBYT.

Discussion

This study systematically investigated the dynamic changes and conversion mechanisms of non-volatile metabolites during the processing of MDBYT and compared the metabolite profiles of finished yellow tea and green tea produced from the same batch of fresh leaves. The results revealed that the yellowing stage was the critical period for the accumulation of most non-volatile components, with significant increases in amino acids, certain organic acids, and EPSFs, whereas catechins and several alkaloids showed a decreasing trend during processing. The reduction of galloylated catechins (EGCG, ECG) directly correlates with decreased bitterness, as these compounds are potent agonists of T2R bitter taste receptors, with the galloyl moiety being essential for high-affinity binding⁴². Conversely, the enrichment of theanine and theanine glucoside may enhance umami and sweetness through interactions with T1R1/T1R3 and T1R2/T1R3 receptors, respectively, with glycosylation providing additional hydrogen-bonding sites that strengthen receptor binding⁴³. Notably, theanine glucoside content increased progressively during processing—from undetectable levels in fresh leaves to significant levels in finished yellow tea—and this increase coincided with the emergence and intensification of “umami” and “sweet” attributes in the QDA sensory analysis. Although this temporal concordance does not establish causation, it provides suggestive evidence for the potential sensory relevance of this compound in MDBYT. Specifically, umami- and sweet-related amino acids, such as Asp, Glu, Pro, Val, Lys, and Phe, were enriched during processing, contributing to the characteristic taste profile of MDBYT.

Comparative analysis identified 19 differential compounds that could serve as potential markers to distinguish MDBYT from green tea. Among these, theasinensins A and B, along with 7 EPSFs derived from galloylated flavanols (ECG and EGCG) and theanine, were substantially enriched in MDBYT. The conversion of catechins into EPSFs introduces a pyrrolidinone moiety that alters receptor-binding geometry, thereby reducing bitterness while preserving bioactivity²⁵. Similarly, the formation of theasinensins through oxidative coupling generates rigid biphenyl structures that may modify interactions with salivary proteins and consequently reduce perceived astringency³⁸. Based on recent literature, we further hypothesize that theanine glucoside may contribute to sweetness through mechanisms analogous to those observed in glycosylated flavonoids, where glycosylation provides additional hydrogen-bonding sites that enhance binding affinity to sweet taste receptors⁴⁴. However, this hypothesis remains speculative and requires future investigation for validation.

Overall, these findings not only provide insights into the metabolic basis underlying the characteristic flavor and potential health benefits of MDBYT but also highlight the importance of specific processing steps, particularly yellowing, in shaping its chemical composition. The enrichment of bioactive compounds such as theasinensins and EPSFs may also contribute to the superior health-promoting properties of yellow tea compared with green tea. Nevertheless, it should be noted that the current study primarily establishes correlations between metabolite changes and sensory attributes. Future studies incorporating taste reconstitution experiments, receptor-binding assays, and human sensory validation will be necessary to confirm the sensory contributions of theanine glucoside, EPSFs, and other key metabolites identified in this work. Overall, this research advances our understanding of the chemical transformation occurring during MDBYT processing and provides a theoretical basis for optimizing yellow tea manufacturing processes and improving product quality.

Methods

Processing of bud yellow tea

Fresh tea leaves (single bud, Fuxuan No.9 cultivar, Camellia sinensis) were plucked from Mengding Mountain, Ya’an City, Sichuan Province, China, in March 2023. MDBYT was produced by experienced tea artisans using traditional processing techniques, with an additional piled-spreading step introduced while maintaining original flavor characteristics in Sichuan Chuanhuang Tea Industry Group Co., Ltd. (Sichuan, China). All other processing parameters followed the conventional manufacturing procedure. After being plucked, the fresh leaves were evenly spread in a spreading trough at a thickness of 2–3 cm and maintained at approximately 20 °C for 7–8 h. Following spreading, fixation was carried out using a 6CCH-63 fixation machine (Fuyang Tea Machinery General Factory, Hangzhou) at approximately 200 °C for 6 min, until the leaves became soft, the grassy odor dissipated, and the characteristic tea aroma emerged. Immediately after fixation, the hot leaves were wrapped in straw paper and placed in an electric forced-air convection oven (Tianjin Tester Instrument Co., Ltd.) with the air convection system turned off for the first yellowing step at 55 °C for 2 h. Afterwards, the leaves were removed and subjected to a second frying process at 150 °C for 6 min. The leaves were then wrapped again in straw paper while hot and placed back into the convection oven for the second yellowing at 55 °C for 2 h.

Following the second yellowing stage, the leaves underwent a third frying process at approximately 110 °C for 7 min, and repeated the above steps for a third yellowing treatment. After completion of the third yellowing, the tea leaves were subjected to a second frying step and then spread naturally on bamboo trays for piled-spreading at room temperature for about 30 h. Finally, the tea samples were dried by using a DL-16 drying machine (Shanfeng Tea Machinery Factory, Ya’an) at 80 °C until thoroughly dried.

For comparison, green tea samples used as the controls were produced from the same batch of fresh leaves according to the standard manufacturing process for Sichuan green tea.

The entire manufacturing procedure was repeated three times independently using fresh tea leaves harvested on three separate days in March 2023, generating three biological replicates for each processing stage. Representative samples (200 g per sampling point) were collected after each processing stage using a standardized five-point sampling method. The process was repeated three times. Sampling points included fresh leaves (FL), spreading (SD), fixing (FX), first yellowing (FY), second yellowing (SY), third yellowing (TY), piled-spreading (PS), drying, finished yellow tea (YT), and green tea (GT). All samples were immediately freeze-dried and stored in a refrigerator at −80 °C prior to analysis.

Reagents

Catechin standards (C, EC, ECG, GC, EGCG, CG, EGC, GCG, GA, and CAF), amino acid standards (Asp, Ser, Glu, Gly, His, Arg, Thr, Ala, Pro, The, Tyr, Val, Met, Lys, Ile, Leu, and Phe) (≥98%), and caffeine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Ethyl decanoate (≥99%), forchlorfenuron, ninhydrin, basic lead acetate, stannous chloride, aluminum chloride, and 2,6-dinitrophenol, all of analytically pure, were obtained from Hobo Bio Co., Ltd. (Chengdu, China). Methanol and acetonitrile (chromatographic grade) were purchased from Fisher Scientific (Bridgewater, NJ, USA). Ultrapure water (18.2 MΩ cm) was prepared using a Milli-Q water purification system (Merck Millipore, USA).

Traditional sensory evaluation of tea samples

The tea samples were subjected to sensory evaluation according to the Chinese national standards GB/T 23776-2018 (method of sensory evaluation of tea) and GB/T 21726-2018 (yellow tea). Briefly, 3.0 g of tea samples were brewed with 150 mL of boiling water for 5 min. The resulting tea infusion was then poured into tea evaluation cup. The sensory evaluation team consisted of five professional tea experts. The expert panellists evaluated the tea samples using a password evaluation method. The evaluation results were composed of scores and comments. The appearance, infusion color, aroma, taste, and brewed leaves were scored on a 100-point scale. The overall evaluation score was calculated using the following formula:

Total sensory score = Appearance × 25% + Aroma × 25% + Infusion color × 10% + Taste × 30% + Brewed leaves × 10% (1).

Quantitative descriptive analysis (QDA)

Quantitative descriptive analysis was performed to assess the flavor profiles of processing samples and finished teas. The sensory evaluation team consisted of 6 tea evaluators (4 females and 2 males, aged 22–55). All evaluators were professionally trained in accordance with the National Professional Standards for Tea Sensory Evaluation (Profession Code: 6-02-06-11, China).

To establish the flavor profile of MDBYT, evaluators first developed a set of taste descriptors based on the National Tea Sensory Evaluation Methodology and Evaluation Nomenclature, combined with the characteristic flavor attributes of samples obtained during MDBYT processing. Only descriptors with a usage frequency exceeding 80% were retained. The final set of 10 taste descriptors included sweetness, umami, astringency, bitterness, mellowness, thickness, acceptance, fired flavor, grassy taste, and overcooked taste. The definitions and reference substances for each descriptor were established as follows: bitterness was defined as the sensation of bitterness in the mouth or aftertaste, and referenced using 0.5 mg/mL caffeine solution; astringency was defined as the feeling of astringency on the surface of the mouth and tongue, and referenced using 0.06 mg/mL EGCG solution; sweetness and umami were referenced by tasting a mixed aqueous solution containing 0.03 g/mL sucrose and 0.3 mg/mL monosodium glutamate; and overcooked taste was defined as a dull and unpleasant steamed-overcooked taste generated during the wet yellowing process. These reference standards were used to train the panellists and ensure consistency in sensory evaluation.

Tea infusion was prepared according to GB/T 23776-2018. Briefly, 3.0 g of tea samples were placed into an evaluation cup, 150 mL of boiling water was added to the tube. After 5 min, the tea infusion was poured into the evaluation bowl to evaluate the taste sub-attributes and intensity. The tea infusion (30 mL) was labeled with a three-digit random code and presented to each evaluator in randomized order. All team members scored the samples independently without discussion. Taste intensity was scored on a 7-point scale, where “0 to 0.5” indicates imperceptible, “0.5 to 1.0” extremely weak, “1.0 to 1.5” detectable but not distinct, “1.5–2.0” perceptible, “2.0–2.5” clearly perceptible, and “2.5–3.0” extremely strong. Each biological replicate was evaluated three times. The average score from six evaluators was used as the final result.

The necessary sensory evaluation of tea samples was carried out by trained evaluators. Before participation, each evaluator provided informed consent by responding affirmatively to the statement: “I am aware that my responses are confidential, and I agree to participate in this sensory evaluation.” Participants could withdraw at any time without giving a reason. The tea products evaluated are safe for human consumption. The study was conducted in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). Under current national regulations, formal ethical approval and an approval number are not required for sensory-evaluation studies involving commercially available, food-grade teas; no institutional human-ethics committee or documentation process for such studies is available.

Quantification analysis of catechins, caffeine, and amino acids

Absolute quantitative analysis of catechin components and caffeine was conducted according to the Chinese national standard GB/T 8313-2018 (Determination of tea polyphenols and catechins in tea). Briefly, 0.2 g of ground sample was extracted with 70% methanol aqueous solution in a water bath at 70 °C. The extract was analyzed using high-performance liquid chromatography (HPLC). Chromatographic separation was carried out on a Titan-C18 column (250 mm × 4.6 mm, 5 μm, Phenomenex, USA) with gradient elution. Detection was performed at a wavelength of 278 nm. Quantification was carried out using an external standard method with catechin and caffeine reference standards.

Non-target metabolomics analysis using UHPLC-Q-Exactive/MS

Metabolite extraction

The metabolite extraction was performed based on a previously reported method with slight modifications⁶. Briefly, tea samples were ground into powder, and 1.0 g of the powder was transferred immediately into a 50 mL centrifuge tube. The sample was extracted with 15 mL of 70% methanol and incubated in a water bath at 70 °C for 30 min. The centrifuge tube was shaken every 10 min to improve extraction efficiency. After cooling to room temperature, the tube was centrifuged at 8000 r/min for 10 min. The supernatant was filtered through a 0.22 μm membrane, and 1.5 mL of filtrate was transferred into an injection vial. Three parallel replicates were prepared for each sample for subsequent UHPLC-Q-Exactive/MS analysis.

UHPLC-Q-Exactive/MS analysis

The samples were analyzed using UHPLC-Q-Exactive/MS system (Thermo Fisher Scientific, USA), following a previously reported method by Chen et al.⁴⁵, The UHPLC system (Ultimate 3000, Dionex, Sunnyvale, CA, USA) equipped with a T3 column (100 mm × 2.1 mm, 1.7 μm, Waters, UK), coupled to a mass spectrometer (Q-Exactive Focus, Thermo Fisher Scientific, Waltham, MA, USA). The column temperature was kept at 40 °C with a flow rate of 0.4 mL/min, and the injection volume was 3 μL. The mobile phase consisted of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The gradient elution program was as follows: 0 min, 2% B; 0.5 min, 2% B; 8 min, 15% B; 13 min, 35% B; 15 min, 70% B; 16 min, 85% B; 16.5 min, 2% B; 20 min, 2% B.

MS conditions: ESI+ positive ion mode, 3.5 kV capillary voltage, 300 °C capillary temperature, 350 °C auxiliary gas temperature, 10 L/min auxiliary gas flow rate, and m/z 80–1200 scanning range.

Screening of differential non-volatile metabolites

The metabolites with significant differences among the processing durations were screened by VIP > 1 and absolute fold change (FC) > 1.5. Variable Importance in Projection (VIP) is derived from supervised regression analysis based on partial least squares.

KEGG pathway analysis

The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for the pathway analysis of the identified metabolites (https://www.kegg.jp/kegg/compound/). Enrichment analysis of significantly regulated non-volatile metabolites was mapped using the online platform MetaboAnalyst 6.0. Metabolite identification and annotation were conducted based on the KEGG and PubChem databases of the NCBI.

Absolute quantitative analysis of free amino acids

Absolute quantitative analysis of free amino acids was performed using high-performance liquid chromatography according to the Chinese national standard GB/T 30987-2020 (Determination of free amino acids in plants). Briefly, the tea samples were extracted with a 70% methanol aqueous solution in a water bath at 70 °C. Chromatographic separation was performed using a Titank-C18 column (4.6 mm × 250 mm, 5 μm, Waters, USA) using gradient elution. Detection was conducted at a wavelength of 254 nm. Quantification of amino acids was performed using an external standard method.

Statistical analysis

The means and standard deviations were calculated using Excel 2019. The data were analyzed by one-way ANOVA using SPSS software (version 22.0), and the Tukey HSD method was used to analyze the significance of differences (p < 0.05). The raw UHPLC-Q-Exactive/MS data files were processed using Compound Discoverer 3.2 software (Thermo Fisher Scientific, USA) for peak matching and peak area extraction. Multivariate statistical analysis was performed using SIMCA-P 14.1 (Umetrics, Sweden). Heat map analysis was performed by using MeV 4.7.4 (Oracle, USA).

Author contributions

Jingyi Xu: conceptualization, writing—original draft, project administration, funding acquisition. Peida Luo: writing—review & editing. Mingji Xie: writing—review & editing, formal analysis, data curation. Cheng Liu: validation, investigation. Xin Li: formal analysis. Jianping Zhao: writing—review & editing, data curation. Murong Zheng: formal analysis, investigation. Changhui Chen: methodology. Bo Sun: data curation. Chunlei He: methodology. Jinlin Bian: methodology, resources. Xiao Du: funding acquisition, supervision.

Data availability

The datasets analyzed during the current study are available in the supplementary information fi les of this article.Additional raw data are not publicly available due to their large volume, but are available from the correspondingauthor on reasonable request.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41538-026-00930-4.