2.2. Chemicals

Theanine (Thea), lysine (Lys), leucine (Leu), alanine (Ala), glycine (Gly), glutamine (Gln), aspartic acid (Asp), valine (Val), serine (Ser), histidine (His), threonine (Thr), proline (Pro), glutamic acid (Glu), cysteine (Cys), arginine (Arg), tyrosine (Tyr), and phenylalanine (Phe) (Wako Pure Chemical Industries, Ltd. Kanagawa, Japan). D-arabitol, xylitol, L-rhamnose, L-fucose, d-fructose, D-galactose, glucose, D-sorbitol, myo-inositol, sucrose, lactose, maltose, and trehalose standards (purity >99%) (Shanghai Anpu Experimental Science and Technology Co., Ltd. Shanghai, China). methyl tert-butyl ether (MTBE), ethyl caprate, methanol, isopropanol (chromatographic grade, Merck,). BSTFA (99%, Aladdin, USA), methoxyamine hydrochloride, pyridine (99%, Sigma-Aldrich, USA), n-hexane (chromatographic grade, CNW, Shanghai, China), anhydrous ethanol, and sodium chloride (China National Pharmaceutical Group Chemical Reagents Co., Ltd., Shanghai, China); N-alkanes (C₇–C₄₀) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.3. Sensory description analysis

An 8-member sensory evaluation panel consisting of 4 males and 4 females, all of whom possess tea sensory evaluation capabilities and have obtained national intermediate tea taster certificates, was selected from the State Key Laboratory of Tea Biology and Utilization at Anhui Agricultural University. According to GB/T 23776–2018, the sensory evaluation of tea mainly included the aroma of tea infusion. The panelists selected six aromas— chestnut, faint scent, sweet, floral, fired beans, and aroma intensity—after obtaining a preliminary understanding of the aromatic profiles of green tea. During the initial assessment, odor attributes were recorded and rated on a scale of 1 to 10. The final score for each sample was calculated by averaging the scores given by each evaluation panel member. The sensory aroma profile was measured using the average scores, and all sensory experiments were conducted more than three times.

2.4. Ethical statements

Ethical permission, to conduct a human sensory study, was granted by our institution. Participants gave informed consent via the statement “I am aware that my responses are confidential, and I agree to participate in this sensory evaluation” where an affirmative reply was required to enter the sensory evaluation. They were able to withdraw from the sensory evaluation at any time without giving a reason. The tea products evaluated were safe for consumption.

2.5. Extraction of volatiles by HS-SPME

Weighed 10 μg of ethyl caprate and diluted with ethanol to 10 mL to prepare a stock solution at 1000 ppm as the primary internal standard. This solution was stored in a freezer at −20 °C. Then, the internal standard was diluted to 5 ppm for use in analysis. Weighed 6 g of tea sample and place it into a 500 mL conical flask. Then, add 300 mL of boiling water to the flask to steep the tea for 5 min. Filter the tea infusion into a 500 mL conical flask using a cloth and then cool the flask in an ice water bath. Use a pipette to transfer 10 mL of the cooled tea infusion into a headspace vial. Add 3 g of NaCl and 2 μL of the internal standard to the vial and shake the mixture to ensure thorough mixing. Place the headspace vial in a water bath set at 30 °C to equilibrate for 15 min. After equilibration, insert the extraction needle into the vial, exposing the SPME fiber to the headspace above the liquid. Allow the fiber to adsorb the volatile compounds for 30 min (Huang et al., 2022).

2.6. Separation and extraction of volatiles by SE-SPE

Weighed 6 g of tea sample and brew with 300 mL boiling water for 5 min. Then filter the tea infusion into a 500 mL conical flask with a filter cloth, and cool it to room temperature in an ice-water bath. Add 1 μL of 1000 ppm internal standard to the cooled tea broth, and SPE extraction was performed according to (Feng et al., 2019).

GC–MS analysis conditions: The GC–MS system was consistent with SPME-GC–MS, and an Agilent 7890B gas chromatograph was connected to an Agilent 5975B mass spectrometer, and an Agilent Masshunter was used to control and operate the GC–MS system. Compounds were separated using a DB-5MS (30 m × 0.25 mm × 0.25 μm) capillary column. The carrier gas was pure helium with a purity of 99.999%. The ion source temperature was 230 °C and the GC inlet temperature was 250 °C. The following temperature program was used: hold at 40 °C for 5 min, increase to 120 °C at a rate of 5 °C / min keep for 3 min, increase to 250 °C at a rate of 8 °C / min keep for 5 min, and increase to 280 °C at a rate of 10 °C / min keep for 3 min.

2.7. Separation and identification of volatile compounds by GC–MS

Volatile compounds in the samples were identified by matching the retention indices (RI) of the mass spectra and spectral components to reference standards in the NIST 20 MS database. AMDIS was used for deconvolution (Huang et al., 2022). When SPME-GC–MS and SPE-GC–MS detected the same volatile compounds, the quantitative results from SPE-GC–MS were used as the primary results. Semi-quantification of the identified volatile compounds was performed using ethyl decanoate as an internal standard.

2.8. Characterization of key aroma of tea by GC–MS/O and AEDA techniques

Aroma-active compounds in the samples were analyzed using an Agilent 7890B gas chromatograph and olfactometry system (Sniffer 9100, Brechbühler, Switzerland). GC-O analysis was performed on a DB-5MS column with instrumental parameter settings consistent with those of SPME-GC–MS and SPE-GC–MS. SPE-extracted samples were diluted using a mixture of pentane and MTBE (1:1) (dilutions of 2, 4, 8.... 0.128) with an injection volume of 4 μL. Each diluted sample was described by sniffing by three sensory evaluators with more than six months of odor training until no odorous compounds were detected in the GC-O experiments. The flavor dilution factor (FD) was determined for each volatile compound, and the final dilution at which an odor could be smelled was the final FD value (Wu et al., 2022).

2.9. Calculation of relative odor activity values

The rOAV of each volatile compound was calculated using the ratio of the concentration of each volatile compound to its corresponding odor threshold in water (Liu et al., 2023). ROAV is referenced in the formula (Li et al., 2024). Volatile compounds with an rOAV >1 are considered to be aroma-active compounds contributing to the overall aroma of the sample (Xiao et al., 2022).

2.10. Determination of free amino acids by high-speed amino acid analyzer

Sample preparation was modified according to (Ren et al., 2021). Accurately weighed 0.100 g of ground tea powder, added 4 mL of 4% sulfosalicylic acid was fully extracted by ultrasonication for 30 min, centrifuged at 12000 r/m for 30 min, and the supernatant was assayed by a high-speed amino acid analyzer (L-8900, Hitachi). The mobile phase was lithium citrate with UV–Vis detection at 570 nm and 440 nm. The flow rate of the mobile phase was 0.35 mL/min, and the flow rate of the derivatization reagent was 0.3 mL/min. The column oven was set at 38 °C, and the post-column development equipment was kept at 130 °C. The injection volume of both standards and samples was 20 μL. The peak areas of the compounds were compared with the peaks of each amino acid standard. The experiment was repeated three times for each sample.

2.11. Determination of soluble sugars

The freeze-dried materials were crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. 20 mg of powder was diluted to a 500 μL with methanol: isopropanol: water (3:3:2, v/v/v), vortexed for 3 min and ultrasound for 30 min. The extract was centrifuged at 12,000 rpm under 4 °C for 3 min. 50 μL of the supernatant was mixed with 20 μL internal standard (1000 μg/mL) and evaporated under a nitrogen gas stream. The evaporated sample was transferred to the lyophilizer for freeze-drying. The residue was used for the further derivatization.

The derivatization method was as follows: the sample was mixed with a 100 μL solution of methoxyamine hydrochloride in pyridine (15 mg/mL). The mixture was incubated at 37 °C for 2 h. Then 100 μL of BSTFA was added into the mixture and kept at 37 °C for 30 min after vortex-mixing. The mixture was analyzed by GC–MS after diluting to an appropriate concentration (Gómez-González et al., 2010; Zeng et al., 2022; Zheng et al., 2016).

Agilent 8890 gas chromatograph coupled to a 5977B mass spectrometer with a DB-5MS column (30 m length × 0.25 mm i.d. × 0.25 μm film thickness, J&W Scientific, USA) was employed for GC–MS analysis of sugars. Helium was used as a carrier gas, at a flow rate of 1 mL/min. Injections were made in the split mode with a split ratio 5:1 and the injection volume was 1 μL. The oven temperature was held at 160 °C for 1 min and then raised to 200 °C at 6 °C/min, raised to 270 °C at 10 °C/min, raised to 300 °C at 5 °C/min, raised to 320 °C at 20 °C/min and held at the temperature for 5.5 min. All samples were analyzed in selective ion monitoring mode. The ion source and transfer line temperature were 230 °C and 280 °C, respectively.

3.2. Analysis of volatile compounds in green tea before and after puffing

By combining SPE and SPME extraction methods to extract volatile compounds, followed by analysis with GC–MS, 155 volatile compounds were identified from the samples using AMDIS deconvolution (Table S1). These volatile compounds were categorized into ten types, including 27 alcohols, 26 aldehydes, 27 ketones, 10 esters, 9 acids, 6 furans, 27 pyrroles and pyrazines, 15 others, 2 sulfides, and 6 phenols (Fig. 2a). Notably, the total concentration of volatile compounds in green tea significantly increased after puffing treatment. The total volatile compounds content in green tea was 426.91 μg/L, while that in puffed tea was 985.16 μg/L. The concentration of total volatile compounds in puffed tea was 2.25 times higher than that before puffing. During this process, the content of ketones and sulfides decreased, while the contents of alcohols, aldehydes, esters, acids, pyrazines, pyrroles, and furans significantly increased. It is worth noting that pyrazine compounds, which were not detected in green tea, constituted 18.65% of the total content in the puffed samples. After puffing treatment, the concentration of most compounds tended to increase, especially alcohols, esters, acids, pyrazines, and pyrroles, indicating that puffing treatment plays a significant role in enhancing the aroma of tea.

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Fig. 2

Compound classification contents and multivariate statistical analysis charts before and after puffing.

To better screen for differential compounds, multivariate analysis was performed on the identified volatile compounds. OPLS-DA, as a supervised discriminant analysis method, is widely used in food research (Su et al., 2022). To further distinguish the differences in volatile compounds before and after puffing, the Variable Importance in Projection (VIP) values of volatile compounds were calculated based on the OPLS-DA regression model. A VIP value >1 indicates that the volatile compound has contribution to the tea. VIP ≥ 1 reflects the differences between samples and screens for key volatile compounds with significantly different concentrations in the samples. Based on VIP > 1 and p < 0.05, 92 differential volatile compounds were selected.

To visually present the changes in volatile components of the sample before and after puffing treatment, this study used heatmap analysis, and the results are shown in Fig. 3. The heatmap results revealed significant changes in the content of volatile compounds after puffing treatment. The trends in the concentration changes of volatile compounds can be divided into two categories: the first category includes compounds whose contents increased after puffing, and the second category includes compounds whose contents decreased. The first category primarily consists of degradation products of carotenoids, terpenoid glycoside degradation products, and Maillard reaction products. For example, 2,5-dimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 3-ethyl-2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2-ethylfuran, 2-acetylpyrrole, benzyl alcohol, linalool, phenylacetaldehyde, 3-methylbutanal, 2-methylbutanal. Compounds dominated by fired beans, sweet, and floral aromas increase. Carotenoid degradation primarily occurs through non-enzymatic oxidative degradation pathways, and thermal degradation is the main method by which non-enzymatic oxidative degradation contributes to the aroma of green tea. The increase in compounds such as pyrazines is mainly due to Maillard reaction products obtained after the puffing treatment of the tea leaves (Ho et al., 2015). Linalool, as a terpenoid glycoside present in green tea, increases during the heating process through thermal decomposition (Sasaki et al., 2017). The second category of volatile compounds mainly includes aldehydes and alcohols, such as hexanal, heptanal, octanal, nonanal, (E)-2-heptenal, (Z)-4-heptenal, (E)-2-octenal, (E, E)-2,4-heptadienal, 1-penten-3-ol, etc., which contribute to the green, grassy, and fatty odors of the aldehydes and alcohols composition (Zhu et al., 2015). These compounds significantly decreased during the puffing process, possibly due to the thermal effects involved in puffing. The transformation of these volatile compounds reduced the intensity of the floral and faint scent in the green tea, which is consistent with the results of the sensory evaluation.

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Fig. 3

Heatmap of volatile compounds in green tea before and after puffing. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.3. GC-O combined with AEDA to analyze key aroma compounds

GC-O (gas chromatography-Olfactometry) is a method that combines instrumental analysis with sensory analysis to detect aroma-active compounds In this technique, gas chromatography is combined with olfaction to effectively identify aroma compounds. AEDA (Aroma Extract Dilution Analysis) is one of the commonly used methods in GC-O technology for preliminary evaluation of the importance of aroma compounds (Egea et al., 2014).

By employing GC-O to olfactively analyze the aroma-active substances in SPE extracts and HS-SPME, important information about aroma characteristics can be obtained. SPE and HS-SPME are common techniques for extracting aroma components, which can effectively enrich and concentrate aroma-active compounds in the sample (Shen et al., 2023). The aroma compounds co-eluted with solvent in SE-SPE are analyzed through HS-SPME-GC-O olfactometry to complement the impact of low molecular weight, highly volatile aroma-active compounds on the aroma of tea.

The FD factor for each aroma active compound was obtained by AEDA analysis. The higher the FD factor value, the stronger the activity of the aroma-active compound at the same level, meaning it is more significant to human olfaction (Kang et al., 2019).

To identify aroma-active compounds, olfactory analysis of SPE extracts and HS-SPME samples was performed using GC-O, and 54 aroma-active compounds were identified (Table S2). The FD factors of these aroma-active substances were obtained through AEDA. The FD factors ranged from 2 to 1024. The FD values of the aroma-active compounds in the two samples were significantly different. These aroma-active compounds are primarily formed due to the hydrolysis of glycosides, degradation of carotenoids, degradation of lipids, and the Maillard reaction that occurs during the processing (Ho et al., 2015).

In the CK sample, the highest FD factors were found for α-ionone (floral aroma) and β-ionone (violet aroma), both with an FD factor of 64. This was followed by hexanal (grass, green), cis-4-heptenal (fishy), heptanal (green), and β-Damascenone (honey, fruity) with an FD factor of 32. These compounds are likely key sources of the floral and green odors in the CK. In the PT (puffed tea) samples, β-ionone (violet aroma) had the highest Flavor Dilution (FD) factor (1024), followed by β-Damascenone (516, honey, fruity), 2,6-dimethyl-Pyrazine (256, earthy, roasted nut), 3-ethyl-2,5-dimethyl-Pyrazine (256, roasted potato, nutty), Linalool (256, floral, citrus-like), 2,3-diethyl-5-methyl-Pyrazine (256, earthy, roasty), Indole (256, floral, animal-like), and α-Ionone (256, floral, violet-like). Compared to the CK samples, the PT samples contain more compounds with sweet, floral, and roasted aromas, and these compounds often have higher FD factors, which is consistent with the results of sensory evaluation. Additionally, two unknown aroma compounds, primarily characterized by spicy and green pepper odors, were detected in the PT samples; however, their structures are unclear. Subsequently, the samples were preliminarily screened for aroma-active compounds with FD ≥ 32, a total of 38 aroma-active compounds were screened (Table 1).

3.4. Screening of key differential compounds

Generally, rOAV value >1 indicates that the volatile compound makes a significant contribution to the aroma characteristics of the product (Xiao et al., 2022). The rOAV values were calculated for compounds with FD values ≥32. The analysis revealed that there were 16 volatile compounds with an rOAV >1 (Table 2), among which heptanal (rOAV = 140.9), dimethyl sulfide (rOAV = 97.46), cis-4-heptenal (rOAV = 20.62), etc., had higher rOAV values in CK compared to PT. In contrast, β-Ionone (rOAV = 124.76), 2,5-diethylpyrazine (rOAV = 70.39), 2,3-diethyl-5-methylpyrazine (rOAV = 53.06), β-damascenone (rOAV = 36.66), 3,5-diethyl-2-methylpyrazine (rOAV = 18.29), linalool (rOAV = 15.87), 2-ethyl-3,5-dimethylpyrazine (rOAV = 15.57), 3-methylbutanal (rOAV = 14.26), and 2-methylbutanal (rOAV = 14.29) had relatively higher rOAV values in PT, making them key compounds in PT. β-Ionone and β-damascenone are volatile compounds formed from the degradation of carotenoids during tea processing (Ho et al., 2015) and are key odorants of green tea aroma. Linalool, existing in green tea in the form of terpenoid glycosides, increases through thermal decomposition during the heating process (Sasaki et al., 2017). β-Ionone, linalool, and β-damascenone mainly contribute to floral and sweet aromas (Magagna et al., 2017; Wu et al., 2022), which are compounds that significantly contribute to the floral aroma. Heptanal and cis-4-heptenal provide fresh, green aromas, while dimethyl sulfide contributes to the formation of the faint scent of green tea (Flaig et al., 2020). 2,3-Diethyl-5-methylpyrazine, 2-methylbutanal, and 3-ethyl-2,5-dimethylpyrazine make significant contributions to the roasted bean and roasty aromas of green tea (Wang et al., 2022). 3-Methylbutanal and 2-ethyl-3,5-dimethylpyrazine are also key aroma components in Longjing tea (Gong et al., 2017). Heptanal, β-ionone, dimethyl sulfide, 2,5-diethylpyrazine, 2,3-diethyl-5-methylpyrazine, β-damascenone, linalool, (Z)-4-heptenal, 3,5-diethyl-2-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, 2-methylbutanal, and 3-methylbutanal were key differential aroma compounds in CK and PT samples.

This study was supported by the National Key Research and Development Program of China (No.2021YFD1601103), the Natural Science Foundation of Anhui Province (No. 2108085MC122), the Natural Science Research Program of Anhui Universities (No. KJ2020A0136), and the Research Program on Teaching Reform of Graduate Education in Anhui Province (No. 2022jyjxggyj190).