Key sweet-aroma compounds and formation mechanisms in Mengding bud yellow tea using HS-SPME-GC-MS and sensomics

Abstract

Mengding Bud Yellow Tea (MDBYT) is distinguished by its characteristic sweet and umami aroma. This study integrated quantitative descriptive analysis, HS-SPME-GC-MS, and UPLC-QQQ-MS to systematically elucidate, for the first time, the dynamic evolution of MDBYT aroma and its formation mechanisms. Sensory evaluation revealed that, among the 13 aroma descriptors, sweetness, umami, and mellowness were the primary attributes positively correlated with overall aroma quality. The yellowing process was the pivotal stage driving the aroma transition from grassy to sweet and umami notes. A total of 38 differential metabolites were identified, of which β-ionone, linalool, n-pentanal, n-heptanal, n-octanal, and 1-octen-3-ol, emerged as key contributors. These metabolites were primarily involved in modulating the sweetness, umami, and mellowness attributes of MDBYT. Quantitative profiling of 10 classes of GBVs demonstrated significant accumulation of linalyl and geranyl glycosides during the fixing and yellowing stages, providing strong evidence that glycoside hydrolysis constitutes a major release pathway for aroma-active compounds. Collectively, these findings demonstrate that the unique aroma of MDBYT is established through a coordinated, processing-driven biochemical network involving carotenoid degradation, glycoside hydrolysis, and lipid oxidation. This study provides a robust scientific foundation for the targeted regulation of flavor quality and the optimization of yellow tea processing strategies.

Introduction

Yellow tea is one of China’s most historically significant traditional teas, with a long-standing legacy of cultivation and consumption, and was once presented as a tribute to the imperial court during the Tang dynasty¹. It is particularly enriched in bioactive compounds, including amino acids, soluble sugars, and vitamins, and has been reported to exert a wide spectrum of health-promoting effects, including antioxidant, anticancer, anti-inflammatory, antibacterial, gastroprotective, and lipid-lowering effects^2–5. Compared with green tea, yellow tea demonstrates a notably smoother, less astringent, and more mellow sensory character, rendering it especially suitable for habitual consumption, as recommended by tea experts⁶. Owing to its distinctive flavor and diverse health benefits⁷, yellow tea has garnered increasing consumer interest in recent years and has rapidly emerged as the fastest-expanding category among the six major tea types, becoming a prominent focus of both the tea industry and academic research⁸.

Mengding Bud Yellow Tea (MDBYT), produced in the Mengding Mountain region of Ya’an, Sichuan Province, is widely regarded as the earliest and most representative variety of yellow tea in China and is frequently honored as the “Immortal Tea”⁹. Historically, MDBYT functioned as a tribute tea during the Tang dynasty (618–907 CE) and was subsequently recognized among the “Top Ten Famous Teas of China” following the founding of the People’s Republic¹⁰. Its exceptional quality is largely attributed to the traditional “third yellowing and third frying” manufacturing protocol, which confers a bright yellow liquor, pronounced sweetness, and a characteristically mellow taste^1,11. This elaborate procedure integrates fresh bud fixing, cotton paper wrapping for controlled yellowing, and prolonged low-temperature drying and constitutes the decisive stage in shaping its unique flavor profile⁶. The extended yellowing stage, conducted under conditions of elevated temperature and humidity with restricted enzymatic activity, provokes intricate biochemical remodeling of endogenous compounds¹². Despite broad recognition of the importance of processing, the precise chemical mechanisms and key compounds underlying the distinctive sweet aroma of MDBYT remain inadequately elucidated. Furthermore, prior investigations into tea aroma have predominantly focused on green or black tea or have examined isolated processing steps, thereby leaving a substantial gap in the systematic and dynamic comprehension of aroma formation across the entire and highly complex processing chain of yellow tea^13–15.

The present study seeks to comprehensively elucidate the mechanistic basis underlying the formation of the characteristic sweet aroma of MDBYT. To accomplish this, we implemented an integrated multiomics analytical strategy that combines headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC-MS)-based volatilomics, quantitative descriptive analysis (QDA), and targeted ultra-high-performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-QQQ-MS) quantification of glycosidically bound volatiles (GBVs). This comprehensive approach enables the dynamic monitoring of both chemical transformations and sensory evolution throughout all critical stages of the traditional “third yellowing and third frying” process. The principal innovation of our study lies in its holistic and stage-resolved design, which not only identifies key odorants and their temporal trajectories but also clarifies the functional role of glycosidic precursors in aroma release¹⁶, thereby enabling the construction of an integrated aroma formation model. Collectively, these findings deliver novel mechanistic insight into the flavor chemistry of yellow tea and establish a rigorous scientific foundation for targeted process optimization and advanced quality control.

Results

Changes in sensory quality during processing and construction of the flavor profile of MDBYT

According to the Standard for Sensory Evaluation of Yellow Tea (GB/T 21726-2018) and the Methods for Sensory Evaluation of Tea (GB/T 23776-2018), the sensory quality of MDBYT was systematically evaluated at each major processing stage. Table S1 shows the detailed results.

The sensory scores increased progressively throughout processing, with the most pronounced improvements observed in aroma and taste. Compared with fresh tea leaves, the final product showed a 26.33% increase in aroma score and a 26.72% increase in taste score (p < 0.05). The fixing and yellowing steps were identified as the most critical quality-forming stages. Although fixing is well recognized as essential for flavor development in green tea¹⁷, yellowing plays a decisive role in defining the quality of yellow tea by promoting extensive metabolic transformation and aroma formation^18,19. Following three rounds of yellowing, the total sensory score increased by 8.30% compared with fresh tea leaves, with aroma and taste scores increasing by 14.10% and 9.76%, respectively. These results underscore the pivotal role of yellowing in establishing the characteristic sensory profile of yellow tea.

In terms of appearance, the dry tea, tea infusion, and infused leaves gradually shifted from green to yellow, ultimately forming the distinctive “three yellows”: tender yellow dry tea, bright yellow tea infusion, and moist yellow infused leaves (Fig. 1A). This color transition was most evident during the yellowing stages, particularly between the first and second yellowing, and approached stability after the third yellowing. With respect to aroma, the fixing step effectively eliminated the grassy odor while initiating a fresh and delicate aroma. Subsequent yellowing further enhanced sweet and tender aromatic attributes, resulting in a more rounded and harmonious aroma profile. Taste evaluation indicated that following the fixing step, the tea infusion exhibited a fresh and brisk character but lacked perceptible sweetness, which subsequently evolved into a mellow-sweet profile during the yellowing process. These observations are consistent with previous findings²⁰, which demonstrated that the high-temperature and high-humidity conditions prevailing during yellowing accelerate the degradation, transformation, and recombination of key endogenous leaf compounds, processes that are fundamental to the development of the characteristic sweet aroma and mellow-sweet taste of yellow tea.

Fig. 1. Changes in sensory quality and flavour profile development of MDBYT during processing.

A Representative sensory images and extracted colour profiles of tea samples at different processing stages; B QDA radar chart; C PCA loadings plot based on standardized QDA scores. Note: fresh tea leaves (FL), spreading (SD), fixing (FX), first yellowing (FY), second yellowing (SY), third yellowing (TY), piled-spreading (PS), and the finished yellow tea (YT). Figure 2B and C were generated using Origin 2024.

Compared with conventional sensory evaluation, flavor profiling enables a more comprehensive and objective visualization of aroma attributes across samples²¹. To trace the trajectory of aroma development in MDBYT during processing, QDA was used to construct a detailed aroma profile (Fig. 1B). Based on consensus evaluation by the sensory panel, 13 high-frequency attributes were retained, encompassing both aroma qualities (e.g., sweet aroma and flowery and fruity aroma) and perceptual dimensions (e.g., aroma intensity, aroma persistence, and aroma acceptance), thereby enabling a holistic characterization of aroma evolution.

At the early stages, fresh tea leaves and spreading leaves exhibited dominant clean and refreshing aroma and grass odor, with sensory scores of 2.38 and 2.18 for clean and refreshing aroma and 2.60 and 2.30 for grass odor, respectively, values that were significantly higher than those of other attributes. Following the fixing step, grass odor was markedly reduced, and new attributes such as fired aroma and chestnut aroma emerged, accompanied by an increase in aroma persistence to 2.08, indicating substantial structural remodeling of aroma compounds during this stage.

Yellowing was identified as the pivotal stage for aroma transformation. From the first to the third yellowing round, the scores for sweet aroma, tender aroma, and aroma acceptance increased continuously, whereas clean and refreshing aroma, aroma intensity, and aroma persistence exhibited slight declines. During the piled-spreading stage, a pronounced increase in corn aroma was observed (score: 1.32), together with further enhancement of sweet aroma. In the final product, sweet aroma (score: 2.02), tender aroma (score: 1.62), fresh aroma (score: 1.44), and flowery and fruity aroma (score: 1.66) were dominant, accompanied by strong performance in aroma intensity, aroma persistence, and aroma acceptance.

These results indicate that aroma reconstruction initiates during fixing; however, the yellowing process reinforces sweet and tender aroma attributes and ultimately shapes the high-fresh, mellow-sweet, and elegant aroma style of MDBYT. The flavor profile generated by QDA effectively discriminated aroma attributes across different processing stages, supporting the organoleptic evaluation results and reinforcing the critical role of processing in aroma development.

To further validate the discriminative capacity of the QDA approach, principal component analysis (PCA) was performed on standardized scores of the 13 aroma descriptors. As shown in Fig. 1C, the first two principal components (PC1 and PC2) explained 56.2% and 26.6% of the total variance, respectively, jointly accounting for 82.8% of the total variability and enabling clear differentiation among samples.

The PCA results revealed that fresh tea leaves and spreading-stage samples formed a compact cluster, reflecting a high degree of similarity in their aroma profiles. In contrast, samples collected after the fixing step were located far from those of all other processing stages, indicating pronounced divergence in aroma composition. Samples from the third yellowing stage were positioned away from fixing-stage samples and in proximity to those from the piled-spreading and drying stages, in strong agreement with the QDA-derived aroma profiles, confirming that yellowing plays a central role in shaping the aroma of MDBYT. Although the critical role of yellowing in yellow tea flavor development has been previously recognized²², the present sensory omics data provide stage-resolved evidence that yellowing specifically drives the progressive transition from grassy toward sweet and tender aroma profiles, thereby offering a more refined understanding of this essential process.

Changes and mechanism of volatile compounds during MDBYT processing

The characteristic “sweet” aroma of MDBYT is closely associated with the dynamic remodeling of volatile composition throughout its characteristic “third yellowing and third frying” processing sequence. To systematically decipher the biochemical basis underlying this aroma development, volatile profiles of samples harvested at eight critical processing stages, namely fresh tea leaves (FL), spreading (SD), fixing (FX), first yellowing (FY), second yellowing (SY), third yellowing (TY), piled-spreading (PS), and finished tea (YT), were comprehensively interrogated using headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC-MS). The resulting data demonstrated pronounced, stage-specific effects of processing on both the qualitative composition and quantitative abundance of aroma-active compounds.

In total, 89 volatile compounds were identified, encompassing aldehydes, alcohols, organic acids, alkenes, ketones, esters, heterocyclic compounds, and phenolics (Fig. 2A; Table S2). As depicted in Fig. 2B, significant differences in both the types and relative abundance of volatiles were observed across processing stages. Fresh tea leaves (FL) contained 57 detectable volatiles, with alcohols predominating at 73.93%. Following the spreading stage (SD), the total number of volatiles declined to 45, whereas the proportion of alcohols further increased to 79.65%, corresponding to the pronounced clean and refreshing sensory character at this stage. By contrast, the fixing step (FX), conducted under elevated temperature, was a key turning point in volatile composition: alcohol content declined precipitously to 16.72%, while esters (34.51%) and alkenes increased significantly. This shift likely results from thermal volatilization and chemical transformation of green-leaf volatiles.

Fig. 2. Dynamic changes in volatile compounds during the processing of MDBYT.

A Total number and types of volatile compounds identified at different stages; B Stacked bar chart showing the relative proportions of eight volatile categories across processing stages; C Venn diagram illustrating shared and unique volatiles among key processing. Note: fresh tea leaves (FL), spreading (SD), fixing (FX), first yellowing (FY), second yellowing (SY), third yellowing (TY), piled-spreading (PS), and the finished yellow tea (YT). Figure 3A-C were created with Origin 2024.

As processing progressed, particularly during the yellowing stages (FY, SY, and TY), the combined effects of high temperature, high humidity, and low oxygen induced substantial alterations in volatile composition. Alkenes increased steadily, reaching 29.19%, 33.74%, and 32.61% at the FY, SY, and TY stages, respectively, likely reflecting enhanced carotenoid degradation. Esters reached their maximum proportion during the piled-spreading stage (PS), accounting for 53.77%, but declined sharply in the finished tea (YT). Notably, aldehydes increased markedly in YT (14.06%), approximately fourfold higher than in the preceding stage, potentially due to lipid thermal cleavage and Maillard reactions during drying. In parallel, heterocyclic compounds increased to 5.47%, contributing to the complexity of the final aroma.

To identify stage-specific volatile markers, five representative processing stages—SD, FX, SY, PS, and YT, were selected for Venn diagram analysis (Fig. 2C). A total of 32 volatile compounds were shared among these stages. Six compounds were unique to the SD stage, whereas only one compound was exclusive to the FX stage. No stage-specific compounds were detected for SY or PS stages. In contrast, four unique compounds were identified in YT, suggesting their potential roles as key contributors to the characteristic aroma of the finished product.

Throughout processing, the relative proportions of alcohols, esters, alkenes, aldehydes, and heterocyclic compounds underwent complex dynamic changes. Among these transformations, the fixing and yellowing steps were particularly critical for the conversion of aroma precursors, while the drying process facilitated further accumulation and formation of specific volatile compounds. Collectively, these findings provide important insights into the aroma formation mechanism of MDBYT.

PCA and orthogonal partial least squares discriminant analysis (OPLS-DA) analysis of volatile compounds

To further delineate the dynamic remodeling of volatile composition during MDBYT processing, multivariate statistical analyses, including PCA and OPLS-DA, were applied to the dataset comprising 89 identified volatile compounds. The PCA results (Fig. 3A) demonstrated high reproducibility and analytical stability of the extraction and metabolomics data, confirming the reliability of the analysis. Clear clustering of samples according to processing stage was observed, indicating distinct volatile profiles. The volatile profiles of fresh tea leaves and spreading-stage samples clustered tightly within the first quadrant and were clearly separated from those of the fixing and yellowing stages, which were positioned in the third quadrant. Although the volatile profiles of the fixing and first yellowing stages remained relatively proximal, samples from successive yellowing stages exhibited progressive divergence from the fixing-stage cluster, illustrating the cumulative impact of prolonged yellowing on volatile remodeling. In contrast, piled-spreading and finished tea samples were distributed within the second quadrant and were distinctly separated from yellowing-stage samples, underscoring the substantial contributions of piled-spreading and drying to aroma development. Overall, each processing step significantly altered the volatile profile of MDBYT.

Fig. 3. Multivariate analysis of volatile compounds in MDBYT processing samples.

A PCA score plot; B OPLS-DA score plot; C Permutation test; D Heatmap analysis. Note: fresh tea leaves (FL), spreading (SD), fixing (FX), first yellowing (FY), second yellowing (SY), third yellowing (TY), piled-spreading (PS), and the finished yellow tea (YT). Figure 4A-C were analyzed with SIMCA-P 14.1. Figure 4D was generated using MeV 4.7.4.

Supervised OPLS-DA was subsequently used to further discriminate volatile profiles among different processing stages, with model validity rigorously evaluated by permutation testing (Figs. 4B, C). The OPLS-DA model exhibited strong explanatory and predictive performance (R²Y = 0.973, Q² = 0.953) and achieved clear discrimination among sample groups. Permutation testing over 200 iterations yielded R² and Q² intercepts of 0.127 and −0.403, respectively, indicating the absence of overfitting and confirming model robustness. These findings provide compelling evidence for significant stage-dependent variation in aroma composition and support the identification of key volatile compounds governing aroma formation.

Fig. 4. Screening of key volatile compounds during MDBYT processing.

A OPLS-DA-based selection of volatiles with VIP > 1; B Heatmap showing dynamic changes of key volatiles (VIP > 1) across processing stages. Note: Differential compounds were screened based on VIP > 1 and p < 0.05. Note: fresh tea leaves (FL), spreading (SD), fixing (FX), first yellowing (FY), second yellowing (SY), third yellowing (TY), piled-spreading (PS), and the finished yellow tea (YT). Figure 5A was analyzed with SIMCA-P 14.1. Figure 5B was generated using MeV 4.7.4.

Heatmap analysis (Fig. 3D) further revealed distinct temporal accumulation patterns among major classes of volatile compounds. Aldehydes increased markedly during the spreading stage, declined sharply following fixing, and subsequently increased again during drying. Notably, nonanal accumulated to 522.65 μg/kg in the finished tea, contributing greasy, rose-like, and citrus notes at low concentrations. Alcohols reached maximal levels during the spreading stage but decreased substantially during fixing and yellowing. Fresh tea leaves and spreading-stage samples were particularly enriched in grass-odor alcohols, which diminished markedly after fixing, whereas fatty alcohols emerging at later stages likely originated from carotenoid and lipid metabolic pathways and contributed to the characteristic sweet aroma of MDBYT.

Alkenes displayed a pronounced overall increase throughout processing, particularly during the yellowing stages, indicating that their formation may be attributed to multiple biochemical pathways. This accumulation is potentially linked to carotenoid degradation, a well-documented source of volatile alkenes in tea leaves^23,24. In parallel, lipid degradation pathways, which are known to generate specific alkenes, are also likely to contribute to the observed profile, collectively shaping the characteristic aroma of yellow tea. In contrast, ketone levels declined during the fixing and yellowing stages but increased again at later processing stages. Ester content exhibited substantial variability, generally decreasing during fixing and yellowing while remaining detectable in the finished tea following drying.

Acids, phenols, and heterocyclic compounds were present at comparatively low concentrations. Acid levels decreased during fixing and yellowing, whereas phenolic compounds accumulated predominantly during the yellowing stage. Heterocyclic compounds increased mainly during fixing and yellowing. For instance, phenylalanine derivatives generated via the shikimic acid pathway act as important precursors of volatile phenylpropanoids and phenylcyclic compounds that impart fruity aroma characteristics. Di-tert-butylphenol has been associated with cellulase-related enzymatic activity, whereas indole, a ubiquitous tea volatile and structural isomer of indolizine, contributes substantially to aroma complexity.

Collectively, aldehydes increased while alcohols decreased throughout processing, with both classes primarily concentrated in fresh tea leaves and during the spreading stage. Alkenes and heterocyclic compounds increased markedly during fixing and yellowing, whereas esters and ketones declined predominantly during these stages. The combined PCA and OPLS-DA analyses not only validated the magnitude of volatile profile remodeling but also corroborated the QDA-based flavor stratification, thereby providing a robust analytical framework for screening key aroma compounds in MDBYT. Consistent with established observations that the fixing stage exerts a profound impact on volatile profile^15,25 the present OPLS-DA model quantitatively confirms fixing as the most disruptive processing step while uniquely highlighting the progressive and cumulative chemical evolution driven by successive yellowing stages.

Screening of key volatile compounds

To identify key aroma-active compounds with significant variation and contribution to flavor formation during processing, variable importance in projection (VIP) scores were calculated for the 89 volatile compounds based on the OPLS-DA model. A VIP score of >1 is widely accepted as the threshold indicating a compound’s substantial contribution to group discrimination in OPLS-DA. Accordingly, compounds with a VIP score of >1 and a p-value of <0.05 were selected as differential key volatiles. A total of 38 key volatile compounds were identified (Fig. 4A), most of which exhibited strong aroma activity and pronounced fluctuations across processing stages.

Heatmap clustering analysis (Fig. 4B) revealed that these key aroma compounds could be broadly categorized into two major groups. One group was dominated by green and floral aroma characteristics and was primarily abundant in fresh tea leaves and spreading stages, with representative compounds including cis-3-hexenol and linalool. The second group consisted mainly of fruity, sweet, creamy, baked, and fatty aroma types and was predominantly expressed during the later processing stages of MDBYT, with typical representatives including β-ionone, 1-octen-3-ol, and decanal. The emergence of these aroma profiles is closely associated with the elevated temperature, high humidity, and oxidative conditions prevailing during tea processing, which promote complex biochemical transformations.

Collectively, these findings demonstrate the distinct temporal distribution of key volatile compounds that contribute to the unique sweet and mellow aroma of MDBYT and emphasize the critical influence of processing conditions on aroma development.

Correlation analysis of volatile compounds with QDA

To bridge chemical composition with sensory perception and identify the key odor-active compounds responsible for the characteristic aroma of MDBYT, correlation analysis was conducted between odor-active volatiles (odor activity values, OAVs ≥1) and quantitative descriptive sensory attributes. OAVs, defined as the ratio of a compound’s concentration to its odor threshold, were calculated to assess the direct contribution of individual volatiles to aroma perception. An OAV of ≥1 is widely accepted as the criterion indicating that a compound’s concentration exceeds its sensory threshold and is therefore likely to contribute directly to the overall aroma profile. Based on this criterion, 24 compounds exhibited OAVs of ≥1 (Table S3), among which β-ionone and linalool displayed exceptionally high OAVs (>5000 and >200, respectively), highlighting their dominant contributions to aroma intensity. In addition, n-pentanal, n-heptanal, n-octanal, and 1-octen-3-ol accumulated predominantly during the later processing stages, indicating their important roles in forming the characteristic sweet aroma.

Seven aroma-active compounds meeting both a VIP score of >1 and an OAV of ≥1 criteria were subsequently subjected to Pearson correlation analysis with ten sensory aroma descriptors (Fig. 5A) to elucidate their perceptual relevance. The results revealed that n-heptanal displayed strong positive associations with fired aroma, tender aroma, corn aroma, and aroma intensity, whereas nonanal showed significant positive correlations with clean and refreshing aroma as well as flowery and fruity aroma. Nerolidol was strongly linked with fresh aroma. In addition, 1-octen-3-ol exhibited robust positive correlations with sweet aroma, tender aroma, flowery and fruity aroma, and corn aroma. Notably, β-ionone demonstrated pronounced positive associations with fired aroma, sweet aroma, tender aroma, and corn aroma. Although β-ionone has long been recognized as a key aroma constituent of tea^25,26, the present analysis uniquely establishes its direct and specific perceptual contribution to sweet aroma in MDBYT.

Fig. 5. Screening of key aroma compounds and their correlation with sensory attributes and glycosidic precursors.

A Pearson correlation heatmap between key aroma volatiles and sensory aroma attributes; B Metabolic pathway enrichment analysis of differential volatiles; C Dynamic changes of 10 GBVs during MDBYT processing. Note: fresh tea leaves (FL), spreading (SD), fixing (FX), first yellowing (FY), second yellowing (SY), third yellowing (TY), piled-spreading (PS), and the finished yellow tea (YT). Statistical analyses were performed using SPSS 22.0 software. Data in Fig. 6C were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) post hoc test for multiple comparisons. Different lowercase letters (a, b, c, d, e, f, g) above the bars indicate statistically significant differences at p < 0.05. Figure 6A and B were plotted using Origin 2024. Figure 6C was created with GraphPad Prism 9.

Collectively, these results indicate that these seven volatiles likely function as core contributors to the fresh and sweet aroma profile of MDBYT, with their concentration trends closely paralleling changes in sensory attributes.

Metabolic pathway enrichment analysis of the 38 differential volatile compounds (Fig. 5B) revealed significant enrichment across 11 metabolic pathways, primarily involving carbonyl compounds, aliphatic alcohols, sesquiterpenes, and phenylpropanoids. These results indicate that the dominant aroma compounds of MDBYT are principally derived from lipid metabolism, carotenoid degradation, and aromatic amino acid–related pathways.

Association between key aroma compounds and glycosidic precursors

Fresh tea leaves contain abundant GBVs, which function as important precursors of fruity, floral, and fresh aroma compounds and are progressively released during processing²⁷. To determine whether the key aroma compounds identified in this study originate from glycosidic precursors, UHPLC-QQQ-MS was used for absolute quantification of 10 representative GBVs. The corresponding calibration curves are provided in Table S4, and their dynamic variation patterns across processing stages are illustrated in Fig. 5C.

Phenylmethyl glucoside exhibited substantial accumulation during the spreading stage, followed by a marked decrease during fixing and yellowing, and then a gradual increase during the late yellowing stage. Phenylmethyl primeveroside accumulated predominantly during the fixing and yellowing stages, whereas phenylmethyl glucoside displayed continuous accumulation throughout processing. In contrast, phenylmethyl primeveroside showed pronounced accumulation during the fixing stage but was subsequently consumed during yellowing.

Conversely, (Z)-3-hexenyl glucoside decreased consistently throughout processing, with further consumption during yellowing. (Z)-3-hexenyl primeveroside accumulated during greening and was subsequently consumed during yellowing. Geranyl glucoside declined mainly during spreading, whereas geranyl primeveroside increased by 97.8%, from 260.35 μg/g in fresh tea leaves to 515.07 μg/g in finished tea, with accumulation occurring primarily during fixing. Linalool glucoside exhibited an overall decreasing trend, particularly during fixing, but rebounded slightly prior to yellowing. In contrast, linalyl glucoside increased by 39.8%, from 280.14 μg/g in fresh tea leaves to 391.50 μg/g in finished tea, accumulating mainly during fixing.

Overall, phenylmethyl glycosides and phenylmethyl primeveroside exhibited net increases, whereas (Z)-3-hexenyl and geranyl glycosides declined during MDBYT processing. Phenylmethyl glucoside showed a pronounced increase during the fixing and yellowing stages. Consistent with the concept that GBVs function as aroma reservoirs²⁵, the present targeted quantification reveals distinct accumulation and depletion patterns among specific precursor classes across the successive processing stages of MDBYT.

The characteristic sweet aroma of MDBYT is formed through the coordinated action of multiple key aroma compounds throughout the complex processing sequence. The integration of OPLS-DA, OAV analysis, sensory correlation, and GBV quantification clarifies the origins and accumulation mechanisms of these core volatiles and provides a robust scientific framework for understanding sweet aroma formation in yellow tea.

Integrated pathways for aroma formation during MDBYT processing

This study integrates comprehensive datasets describing the dynamic evolution of sensory attributes, volatile compounds, and their glycosidic precursors to construct a mechanistic framework for the formation of the characteristic sweet aroma of MDBYT. The observed aroma profile is generated through the combined action of multiple major biochemical reaction pathways activated throughout processing.

First, the pronounced increase in total alkenes during processing, particularly the substantial accumulation of β-ionone, which exhibits an exceptionally high OAV ( >5000), during the yellowing stages, provides strong evidence for activation of the carotenoid degradation pathway. This pathway constitutes a primary biochemical source of sweet and floral aroma notes in tea²⁸. Second, quantitative profiling of 10 representative GBVs revealed a persistent decline in (Z)-3-hexenyl glycosides and extensive structural transformation of linalool and geranyl glycosides. These results provide direct confirmation of an active glycoside hydrolysis pathway, through which key free aroma compounds are progressively liberated during processing¹⁶. In addition, the pronounced accumulation of straight-chain aldehydes and alcohols, exemplified by n-heptanal and 1-octen-3-ol, during the later processing stages, together with their high OAVs, is indicative of sustained lipid oxidation activity, further enhancing the complexity of the aroma profile²⁹.

Collectively, the characteristic sweet aroma of MDBYT arises from the combined contributions of multiple pathways, including carotenoid degradation, glycoside hydrolysis, and lipid oxidation. The analytical framework established in this study directly links the observed chemical transformations with the specific physicochemical conditions of yellow tea processing, thereby providing mechanistic insight into aroma formation. Building upon established knowledge of carotenoid degradation and glycoside hydrolysis in tea aroma development^12,30, the present integrated analysis proposes a sequential reaction network for MDBYT that explicitly associates individual processing steps with activation of these pathways and their synergistic contribution to the final sweet aroma.

Discussion

This study demonstrates that the yellowing stage represents the decisive transition point in the transformation of the aroma profile from grassy to sweet and tender. Comprehensive volatile analysis revealed pronounced and stage-dependent reorganization of major chemical classes, with the fixing and yellowing processes exerting dominant influence on the formation and accumulation of aldehydes and alkenes. Seven key aroma compounds, including β-ionone and linalool, were identified as principal determinants of the characteristic sweet aroma based on their elevated OAVs and strong correlations with sensory attributes. Furthermore, targeted quantification of GBVs confirmed that glycoside hydrolysis constitutes a central biochemical pathway governing aroma release throughout processing. Overall, tea processing, and the yellowing stage in particular, drives fundamental remodeling of both aroma type and volatile composition in MDBYT.

The novelty of this work resides in the application of an integrated multi-omics approach to dynamically analyze the special “third yellowings and third fryings” processing chain. This allowed us to go beyond static profiling and propose a sequential reaction network, directly associating specific unit operations (fixing, yellowing, and drying) with the activation of three synergistic pathways: carotenoid degradation, glycoside hydrolysis, and lipid oxidation.

Our findings offer both mechanistic understanding and practical significance to the field. We present a novel process–driven framework that clarifies how the unique processing protocol of yellow tea orchestrates chemical transformations to develop its signature sweet aroma. Additionally, the identified key odorants and their formation pathways establish a scientific basis for the targeted optimization of processing parameters, thus enhancing the flavor quality and consistency in yellow tea production.

Methods

Tea samples

Fresh single buds of Camellia sinensis cv. Fuxuan No. 9, measuring approximately 25–30 mm in length and 3–4 mm in width, was harvested on March 16, 2023, from a commercial tea garden in the Mengding Mountain region of Ya’an City, Sichuan Province, China. Tea processing was conducted at Yanghe Tea Factory (Yucheng District, Ya’an) by experienced tea masters using the traditional manufacturing procedure for MDBYT. The processing sequence comprised fresh tea leaves (FL) → spreading (SD) → fixing (FX) → first yellowing (FY) → first frying → second yellowing (SY) → second frying → third yellowing (TY) → third frying → piled-spreading (PS) → drying → finished yellow tea (YT). A schematic of the processing flow is presented in Fig. 6.

Fig. 6.

Traditional processing workflow of Mengding Bud Yellow Tea.

At each key processing stage (FL, SD, FX, FY, SY, TY, PS, and YT), ~200 g of tea material was collected using a five-point sampling method. All processing was performed in triplicate to ensure reproducibility. The collected samples were immediately freeze-dried under vacuum and stored at −80 °C until subsequent sensory evaluation, physicochemical analysis, and metabolomics measurements.

Reagents

Analytical-grade reagents were used throughout the study. Ethyl decanoate (internal standard) was purchased from Sigma-Aldrich (Shanghai, China). Diethyl ether was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). A C7–C30 saturated n-alkane standard mixture was purchased from SUPELCO (Bellefonte, PA, USA). Geraniol and benzyl alcohol were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). (Z)-3-Hexen-1-ol, 2-phenylethanol, and a mixture of linalool oxides I and II were purchased from TCI (Tokyo, Japan). Linalool was obtained from Alfa Aesar (Heysham, UK). Benzyl β-D-glucoside and 2-phenylethyl β-D-glucoside were purchased from Toronto Research Chemicals (Toronto, Canada). Ultrapure water (18.2 MΩ·cm) was prepared using a Milli-Q purification system (Merck Millipore, USA).

Ethical statement

This study involved human participants for sensory evaluation of commercially available, food-grade tea products. The study protocol was reviewed and approved by the Ethics Committee of Sichuan Agricultural University. All participants were trained evaluators who provided informed consent by affirming a standardized statement guaranteeing confidentiality and voluntary participation. They were informed of their right to withdraw at any time without reason. The study adhered to the ethical principles of the World Medical Association’s Declaration of Helsinki. According to current national regulations, sensory evaluations of commercially available food products are exempt from formal ethical approval requirements. All tea samples used are safe for human consumption.

Sensory quality analysis

Quantitative descriptive analysis (QDA) was conducted to characterize the dynamic aroma profile of Mengding Bud Yellow Tea (MDBYT) across its processing stages. The evaluation was performed by a trained panel of six assessors (four females and two males, aged 22–55 years) with extensive professional experience in tea sensory evaluation. Prior to formal assessment, all panelists underwent systematic training to calibrate their understanding of aroma descriptors, scoring standards, and the use of the intensity scale.

Samples from eight key processing stages—fresh tea leaves (FL), spreading (SD), fixing (FX), first yellowing (FY), second yellowing (SY), third yellowing (TY), piled-spreading (PS), and the finished yellow tea (YT), were independently evaluated by QDA. Aroma intensity was quantified using a seven-point category scale, which was further refined into a three-level scoring system: 0–0.5 (not perceived), 0.5–1.0 (extremely weak), 1.0–1.5 (perceptible but faint), 1.5–2.0 (clearly perceptible), 2.0–2.5 (distinct), and 2.5–3.0 (very strong). All panelists scored independently, and the mean values were taken as the final results.

Aroma descriptors were initially screened with reference to the National Standard Terminology for Tea Sensory Evaluation and the characteristic odor profiles observed in the MDBYT samples, leading to the establishment of a specific flavor wheel for this tea type. Through the evaluation process, descriptors were retained only if they were consistently used by more than 80% of the panelists. This procedure culminated in the final selection of thirteen key aroma attributes: fresh aroma, sweet aroma, flowery and fruity aroma, tender aroma, dull odour, chestnut aroma, corn aroma, aroma intensity, aroma persistence, aroma acceptance, grass odour, clean and refreshing, and fired aroma. Detailed sensory evaluation results are provided in Supplementary Table S5.

Volatile composition analysis

Volatile compounds were extracted using headspace solid-phase microextraction (HS-SPME) with a 100 µm polydimethylsiloxane (PDMS) coated fiber as described by Deng et al.³¹, with minor modifications. Freeze-dried tea samples were ground using a ball mill and sieved through a 40-mesh screen. A 0.100 g aliquot was placed into a 15 mL headspace vial, and 5 μL of ethyl decanoate (1 μg/mL) was added as an internal standard. The vial was incubated at 80 °C for 5 min, followed by 50 min of adsorption. The volatiles were subsequently analyzed via gas chromatography–mass spectrometry (GC-MS).

The GC-MS conditions were slightly modified from the method of³² using a Thermo Fisher Trace1300 GC (Thermo Fisher Scientific, Waltham, USA) equipped with an IFX3000 (Thermo Fisher Scientific, Waltham, USA) mass selective detector. Fisher Scientific, Waltham, USA) for the analysis of tea volatiles. The injector was used with a TG-5SIMMS capillary column (30 m × 0.25 mm × 0.25 μm, Thermo Fisher Scientific, Waltham, USA).

Gas Chromatography (GC) Conditions: The inlet temperature was set at 250 °C. Volatile compounds were thermally desorbed from the SPME fiber in the injection port for 10 min. Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min in splitless injection mode. The oven temperature program was as follows: initial temperature 45 °C held for 3 min; increased to 90 °C at 3 °C/min and held for 1 min; then to 102 °C at 2 °C/min and held for 1 min; then to 130 °C at 3 °C/min and held for 2 min; then to 150 °C at 5 °C/min and held for 1 min; then to 220 °C at 10 °C/min and held for 1 min; and finally to 240 °C at 15 °C/min and held for 5 min.

Mass Spectrometry (MS) Conditions: Electron ionization (EI) was used with the ion source temperature set at 230 °C. The electron energy was 70 eV. Data were acquired in full-scan mode over a mass-to-charge ratio (m/z) range of 41–300.

Absolute quantitative analysis of GBVs by UHPLC-QQQ-MS

Accurately weigh 0.100 g of tea powder into an Eppendorf (EP) tube, add 10 mL of 70% (v/v) methanol solution, and mix thoroughly. Subject the mixture to water bath at 70 °C for 30 min, followed by centrifugation at 5000 r/min for 10 min. Collect the supernatant, filter it through a 0.22 μm membrane, and store the filtered supernatant for subsequent use.

Analysis of GBVs, the assay was performed according to the method described by Li et al.,¹⁴ using an ultra-high-performance liquid chromatography system (UHPLC Infinity 1290, Agilent Technologies, USA) coupled with a quadrupole time-of-flight mass spectrometer (Q-TOF 6540, Agilent Technologies, USA). The separation was achieved on a Zorbax Eclipse Plus C18 column (100 × 2.1 mm, 1.8 μm, Agilent, USA).

Column temperature was maintained at 35 °C, and the injection volume was set at 10 μL. The mobile phase consisted of phase A (0.1% formic acid aqueous solution, v/v) and phase B (pure methanol), with a constant flow rate of 0.4 mL/min. The gradient elution program was as follows: 10% B at 0 min; linearly increased to 15% B within 0–4 min; ramped up to 25% B from 4–17 min; elevated to 32% B during 7–9 min; increased to 40% B in the period of 9–16 min; raised to 55% B from 16–22 min; further increased to 95% B within 22–28 min; held at 95% B for 2 min (28–30 min); then linearly decreased to 10% B in 1 min (30–31 min); and finally maintained at 10% B for 4 min (31–35 min).

Electrospray ionization (ESI) in positive ion mode was employed with the following parameters: capillary voltage of 3500 V; drying gas temperature and flow rate set at 300 °C and 8.0 L/min, respectively; nebulizer pressure of 35 psi; sheath gas temperature and flow rate maintained at 300 °C and 11 L/min, respectively; mass scan range of 100–1000 m/z; and cone voltages set at 0, 5, 10, 15, 20, 25, 30, 35, 40 and 45 V, respectively.

Data statistics and analysis

Raw data were processed in Excel 2019 and expressed as means ± standard deviations. One-way ANOVA was performed using SPSS 22.0, with significance set at p < 0.05. SIMCA-P 14.1 (Umetrics, Sweden) was used for orthogonal partial least squares discriminant analysis (OPLS-DA), and Tukey’s HSD test was applied for post-hoc comparisons. Volatile compounds were identified using NIST8.LIB and NIST8s.LIB spectral libraries, retaining compounds with similarity scores >70%. Retention indices (RI) were calculated using C8–C23 n-alkanes and confirmed with reference standards and published values³³. Odor activity values (OAVs) were calculated by dividing the quantified concentration of each volatile compound by its sensory threshold, with threshold values obtained from literature on related tea types^34–40. Heatmaps were generated using MeV 4.7.4 (Oracle, USA).

Author contributions

Jingyi Xu: Conceptualization, Writing-original draft, Project administration, Funding acquisition. Mingji Xie: Writing-original draft, Writing—review and editing. Xinyao Yang: Formal analysis, Validation, Investigation. Chuhan Zhang: Formal analysis, Investigation. Mingjia Li: Formal analysis, Investigation. Peida Luo: Formal analysis, Investigation. Jinlin Bian: Data curation. Lei Guo: Data curation. Yao Zou: Methodology. Bo Sun: Methodology. Qian Tang: Funding acquisition, Supervision.

Data availability

The datasets generated and/or analyzed during the current study are not publicly available due to commercial confidentiality and intellectual property considerations, but are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41538-026-00737-3.