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To assess the variation in non-volatile metabolites during the enzyme reaction stage of Oriental Beauty, untargeted analysis based on LC/MS was employed. A total of 2394 mass/retention time figures were detected in the ESI− modes, which were reduced to 1201 single molecular features after filtering. PLS-DA was applied to distinguish different processing stage (Supplemental Fig. S1a, b). The key compounds with variable importance in projection (VIP) > 1 in SIMCA P were screened out. The importance of the ten key substances was evaluated by factoring in information about corresponding authentic standards, the Human Metabolome Database, and published literature (Supplemental Table S1).
The result is shown in Fig. 1a, b. There were a total of ten substances, which were assigned to two groups. One group (Fig. 1a) of compounds increased during enzymatic reaction, including GA, oolongtheanin, (2'E,4'Z,8E)-colneleic acid, and theaflavin-3,3′-gallate. Theaflavins and oolongtheanin were products of oxidation of catechins during enzymatic reactions. Theaflavins have an effect on the astringency, brightness, color, and briskness of the black tea, and also have some health functions, such as anti-cancer activity[17], improvement of memory impairment and depression-like behavior[18]. The content of theaflavin-3,3′-gallate decreased in ST-OB stage, which may be caused by the oxidation and degradation of theaflavin to thearubigins[19]. Oolongtheanin was the characteristic dimer detected in oolong tea, and it was expected to have varieties of bioactivities[20]. Treatment of EGCG with CuCl2 produced its related polymer oolongtheanin-3-O-gallate[21]. The content of GA showed a marked increase during the enzyme reaction. GA is a precursor of catechin and has strong antifungal activity against tea plant disease[22].
Figure 1.
Metabolite (compounds with variable importance in projection > 1) variations during enzyme reaction stage analyzed by LC/MS and GC/MS. (a) These non-volatile metabolites accumulate gradually in the enzymatic reaction stage. (b) These non-volatile metabolites decrease gradually in the enzymatic reaction stage. (c) The score scatter plots of PLS-DA of volatile metabolites. (d) Validation of the PLS-DA model. (e) Heatmap of differential volatile substances during enzyme reaction stage. VIP: variable importance projection, GA: gallic acid, EGC:(–)-epigallocatachin, ECG: (–)-epicatechin gallate, CG: (–)-catechin gallate, EC: (–)-epicatechin. Different lower case letters following the number indicate significant differences during the processing (p < 0.05). * Represents compounds that have not been validated by available standards.
The other group showed a decrease during the enzyme reaction stage, including EGC, ECG/CG, EC, and punicafolin. The decrease in EGC, ECG/CG, and EC were due to the oxidative condensation of catechins into theaflavins and thearubigins. In the enzymatic reaction stage, catechins in tea leaves were oxidized and condensed under the action of enzymes to form theaflavins and oolongtheanin. As a result, the catechin content was greatly reduced during processing, which leads to an improvement in the taste quality of the tea leaves.
Alteration of volatile compounds during the endogenous enzyme reaction stage
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Comparative analysis of the initial stage (FTL, Wi-OB) and the end stage (5S-OB, ST-OB) of the enzyme stage reaction showed that the two types of tea samples could be well distinguished (Fig. 1c). The vector value from 200 permutations suggested that this PLS–DA model was not outfitted (Fig. 1d). Subsequently, 76 volatile substances of variable importance projection (VIP) > 1 were filtered out and classified by aroma type to perform a heat-map analysis (Fig. 1e). It can be seen that the aroma was characterized by roasted, green, floral, and fruity notes, which increased with the enzyme reaction stage, and the abundance of some chemical notes compounds and unknown aroma compounds decreased. Therefore, the aroma quality of Oriental Beauty was improved.
During the enzyme reaction stage, the abundance of geraniol increased the most (103 fold), followed by 3-methyl-2-butenal (34 fold). Geraniol, an acyclic isoprenoid monoterpene with sweet rose notes, was shown to possess various pharmacological functions, including antioxidant, anti-inflammatory, and antitumor activities[23,24]. In industry, geraniol and nerol were obtained by selective hydrogenation of citral. Notably, the abundance of citral increased 25 fold during the enzymatic reaction stage. 3-Methyl-2-butenal is a natural product with almond and mild-buttery notes. In conclusion, the content of aromatic compounds of green, roasted, fruity, and floral showed an increasing trend in the enzymatic reaction stage, especially citral with strong lemon notes and geraniol with rose-like notes. The changes of these volatile compounds laid a formation of aroma quality of Oriental Beauty.
Nonenzymatic reactions stage
Alteration of non-volatile constituents during the nonenzymatic reaction stage
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The nonenzymatic reaction stage is mainly the fixing process, which uses high temperature to stop the enzyme activity and fermentation, fix the quality of tea, and facilitate storage. Untargeted analysis (Supplemental Fig. S1c, d) results show that the abundance of EGCG, punicafolin, and EGC increased in the process of FX-OB (Fig. 2a), and these compounds showed no significant difference before and after non-enzymatic reaction. The content of theaflavin, theaflavin-3-gallate, and theaflavin-3, 3′-gallate significantly increase during the nonenzymatic reaction, and these compounds increase after FX-OB was perhaps due to polyphenol oxidase, which was also active and oxidized to form theaflavins[25]. The abundance of quinic acid and oolongtheanin decreased significantly during the nonenzymatic reaction stage. In past research, the abundance of quinic acid has been correlated with the grade and quality of tea[26,27].
Figure 2.
Metabolite (compounds with variable importance in projection > 1) variations during nonenzymatic reaction stage analyzed by LC/MS and GC/MS. (a) These non-volatile metabolites accumulate gradually in the nonenzymatic reaction stage. (b) These non-volatile metabolites decrease gradually in the nonenzymatic reaction stage. (c) The score scatter plots of PLS-DA of volatile metabolites. (d) Validation of the PLS-DA model. (e) Heatmap of differential volatile substances during nonenzymatic reaction stage. VIP: variable importance projection, EGCG: (–)-epigallocatechin gallate, EGC: (–)-epigallocatechin. Different lower case letters following the number indicate significant differences during the processing (p < 0.05). * Represents compounds that have not been validated by available standards.
Alteration of volatile constituents during the nonenzymatic reaction stage
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High temperature also removed inferior odor, creating caramel and floral notes. During the nonenzymatic reaction stage, the total volatile-compound content was reduced from 564.52 μg/L to 274.74 μg/L. Application of PLS-DA was used to differentiate metabolite content differences between tea samples of different processing (Fig. 2c, d). The key compounds with VIP > 1 were screened out. Fifty-five different compounds were screened out, their contents were made into heat maps, and cluster analysis was conducted (Fig. 2e). The differentiated compounds between the different processes were divided into two groups. The content of compounds in group-a increased after fixing, while that of group-b decreased. Pentanal, 3-methylfuran, 3-octen-2-one, cis-2-penten-1-ol, and hotrienol were the top five metabolites contributing to the difference. 3-Methylfuran and 3-octen-2-one increased by 5–6 fold after fixing, and it had a roasted odor. This was mainly because the Maillard reaction occurs at high temperature to produce heterocyclic compounds, such as pyrrole and furan[28]. The content of cis-2-penten-1-ol and pentanal increased by 4.44 and 7.05 fold, respectively. The abundance of hotrienol increased by 3.89 fold after fixing. Dehydration of the 8-hydroxy linalool isomer afforded 3,7-dimethylocta-1,7-dien-3,6-diol. The allylic rearrangement and dehydration of this diol yielded hotrienol[29]. In this experiment, linalool content was reduced by 0.32 fold after fixing.
Dynamic changes of the main metabolites during processing
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Using targeted analysis with LC/MS, we performed a quantitative test for flavor metabolites, including GC, EGC, C, EC, EGCG, GCG, ECG, GA, and caffeine (Table 1). The results indicated that the content of catechins significantly decreased (p < 0.05) before ST-OB and did not significantly change after FX-OB. The procedure before ST-OB was withering, shaking and rocking, and stacking. The decrease in catechin content during withering was caused by the change in flavonoid transcription induced by tea dehydration[30]. At the shaking and rocking stage, catechins were catalyzed by PPO-POD, resulting in theaflavins, theasinensins, thearubigins, and theabrownines[31]. Catechins also had some biological functions, such as anti-fungal and anti-insect activities, and induced plant defense[32,33]. Caffeine content fluctuated little while the GA content increased significantly during the whole process. Methyl gallate can be produced by GA, except for galloylated catechins[22]. The accumulation of GA content may be due to the lower conversion rate than the synthesis rate. GA add to the astringency of red wine and had antioxidant activity[34]. The flavonol O-glycosides of myricetin, quercetin and kaempferol were perceived to be astringent at very low levels[35]; the dynamic changes in the contents of these compounds are shown in Table 1. There were compounds with different trends; the content of vitexin-2-O-rhamnoside, luteolin, and kaempferol increased with the processing. Meanwhile, the content of quercetin-3-O-rutinoside, quercetin-3-O-galactoside, cynaroside, and quercetin 3-O-rhamnoside decreased with the processing. These metabolites were the main substances of bitterness and astringent taste in tea, and the reduction of these metabolites was beneficial to the improvement of tea quality.
Table 1. Dynamic variation of major taste compounds.
Compounds
(mg/g)FTL Wi-OB 1S-OB 2S-OB 3S-OB 4S-OB 5S-OB ST-OB FX-OB OB GC 8.730 ± 1.539a 6.874 ± 0.341b 6.394 ± 0.386b 4.147 ± 0.363c 3.829 ± 1.028c 2.941 ± 0.754d 1.445 ± 0.677e 0.670 ± 0.514f 0.927 ± 0.453ef 1.126 ± 0.341ef EGC 49.248 ± 2.943a 39.766 ± 3.377b 32.811 ± 4.212c 17.511 ± 3.132d 16.029 ± 8.675d 11.673 ± 6.057e 4.117 ± 3.220f 1.577 ± 1.380f 2.800 ± 2.116f 3.122 ± 1.482f C 34.730 ± 12.072a 25.859 ± 4.542b 25.784 ± 4.899b 21.133 ± 3.301c 20.225 ± 1.656c 17.996 ± 1.792ce 14.164 ± 0.725ef 10.011 ± 1.040f 11.188 ± 2.066f 10.487 ± 1.299f EC 24.382 ± 5.110a 22.508 ± 3.291ab 21.541 ± 2.957b 18.028 ± 2.620c 15.915 ± 0.365cd 13.624 ± 0.336d 9.521 ± 0.851e 6.231 ± 1.347f 7.345 ± 0.583ef 6.689 ± 0.306f EGCG 250.087 ± 3.465a 230.995 ± 7.142ab 214..127 ± 12.789b 166.138 ± 12.478c 143.142 ± 37.211d 113.768 ± 33.531e 55.881 ± 29.028f 21.082 ± 14.399g 29.749 ± 14.769g 33.393 ± 7.166g GCG 0.901 ± 0.082a 0.928 ± 0.082a 0.858 ± 0.096a 0.658 ± 0.059b 0.581 ± 0.160b 0.407 ± 0.131c 0.211 ± 0.084d 0.081 ± 0.037e 0.135 ± 0.055de 0.214 ± 0.0254d ECG 37.392 ± 7.412a 34.177 ± 4.864b 33.653 ± 3.649b 31.376 ± 3.937bc 28.058 ± 1.001cd 25.238 ± 1.621d 20.064 ± 0.703e 14.346 ± 0.896f 14.381 ± 1.357f 13.458 ± 0.861f Total catechins 405.470 ± 30.134a 361.106 ± 15.296b 335.169 ± 13.269b 258.990 ± 17.723c 227.780 ± 44.836e 185.647 ± 37.863f 105.401 ± 33.025e 53.940 ± 17.880g 66.526 ± 14.339g 68.489 ± 10.870g GA 0.680 ± 0.108f 1.440 ± 0.277e 1.988 ± 0.254e 3.475 ± 0.271d 3.584 ± 0.707d 4.027 ± .0.730d 5.398 ± 0.437c 5.821 ± 0.218c 6.755 ± 0.891b 8.187 ± 1.247a Caffeine 68.601 ± 4.509ac 71.635 ± 6.686abc 71.447 ± 4.596abc 73.500 ± 5.649c 71.140 ± 4.084abc 71.147 ± 5.983abc 70.789 ± 5.863abc 73.469 ± 6.177ce 74.056 ± 4.884bc 67.746 ± 2.673a Vitexin-
2-O-rhamnoside0.205 ± 0.077bc 0.338 ± 0.205b 0.347 ± 0.210b 0.354 ± 0.205b 0.336 ± 0.198b 0.320 ± 0.190b 0.317 ± 0.196b 0.325 ± 0.212b 0.360 ± 0.320b 0.418 ± 0.247ab Quercetin-
3-O-rutinoside1.873 ± 0.339ab 1.776 ± 0.427b 1.746 ± 0.418abc 1.783 ± 0.426ab 1.688 ± 0.509abc 1.524 ± 0.419abc 1.508 ± 0.349abc 1.366 ± 0.246c 1.417 ± 0.367bc 1.362 ± 0.345c Quercetin-
3-O-galactoside0.853 ± 0.684a 0.808 ± 0.659a 0.798 ± 0.656a 0.808 ± 0.661a 0.784 ± 0.636a 0.743 ± 0.582a 0.722 ± 0.514a 0.709 ± 0.485a 0.735 ± 0.508a 0.696 ± 0.472a Cynaroside 0.046 ± 0.007acd 0.052 ± 0.011cd 0.050 ± 0.013acd 0.052 ± 0.010acd 0.048 ± 0.012acde 0.044 ± 0.009acd 0.040 ± 0.009abe 0.036 ± 0.007b 0.034 ± 0.006b 0.034 ± 0.006b Quercetin
3-O-rhamnoside0.022 ± 0.010acd 0.022 ± 0.006acd 0.021 ± 0.005cde 0.025 ± 0.008d 0.019 ± 0.004abce 0.019 ± 0.003abce 0.016 ± 0.002e 0.016 ± 0.001be 0.015 ± 0.003be 0.015 ± 0.002be Luteolin 0.001 ± 0.000ac 0.001 ± 0.000ac 0.001 ± 0.001ac 0.002 ± 0.001cde 0.002 ± 0.001de 0.002 ± 0.001e 0.004 ± 0.001f 0.006 ± 0.001h 0.005 ± 0.001g 0.009 ± 0.001b Quercetin 0.053 ± 0.029a 0.047 ± 0.027a 0.052 ± 0.032a 0.051 ± 0.031a 0.050 ± 0.029a 0.048 ± 0.027a 0.044 ± 0.024a 0.045 ± 0.025a 0.069 ± 0.048a 0.068 ± 0.041a Kaempferol 0.046 ± 0.010a 0.042 ± 0.014a 0.046 ± 0.018a 0.047 ± 0.019a 0.045 ± 0.016a 0.43 ± .015a 0.045 ± 0.016a 0.050 ± 0.021a 0.066 ± 0.035a 0.077 ± 0.040b Vitexin 0.037 ± 0.008a 0.043 ± 0.006bcd 0.046 ± 0.006cd 0.046 ± 0.008d 0.043 ± 0.004bcd 0.041 ± 0.006abcd 0.040 ± 0.006ab 0.039 ± 0.006ab 0.041 ± 0.005abcd 0.046 ± 0.004bcd Different lowercase letters following the number indicate significant differences during the processing (p < 0.05). The results were presented in the form of mean values followed by the standard deviation. Three main trends in metabolites (Fig. 3) were observed: (1) content decreases during processing (catechins, quercetin-3-O-rutinoside, and vitexin); (2) during the process, the content initially decreases and subsequently increases (nerolidol, α-farnesene, and indole); and (3) an initial increase and then decrease in content during processing (methyl salicylate, β-trans-ocimene, benzaldehyde, benzyl alcohol, linalool, and (E)-2-hexenal). The total catechin content decreased by 0.15–0.18 fold, quercetin content decreased by 0.5–0.82 fold, vitexin increased by 0.8–1.6 fold.
Figure 3.
Dynamic variation of major metabolites. DMAPP: dimethylallyl pyrophosphate, PEP: phosphoenolpyuvate, E4P: erythrose 4-phosphate, FPP: farnesyl pyrophosphate, GPP: geranyl pyrophosphate, IPP: isopentenyl pyrophosphate, Phe: phenylalanine, IGP: indole-3-glycerol phosphate, GLVs: green leaf volatiles, the methylerythritol phosphate (MEP) pathway, the mevalonic (MVA) acid pathway, lipoxgenase (LOX) pathway.
The oxidation of tea flavanols by catechol oxidase remarkably affects the degradation of carotenoids[28]. The volatile carotenoid derivative β-trans-ocimene increased by 1.12–2.47 fold during the whole process. The content of benzyl alcohol was 3.5–53.7 fold that as was in FTL, and the content of benzaldehyde was 2.9–5 fold more in OB than in FTL. This was mainly caused by the protein degradation in the fermentation process that formed phenylalanine. Phenylalanine underwent further degradation to benzaldehyde, phenylacetaldehyde, benzyl alcohol, and phenylethyl alcohol[36]. Benzaldehyde and benzyl alcohol respectively contribute floral and almond notes in black tea[37]. The indole content decreased by 0.05–0.17 fold, and (E)-nerolidol decreased by 0.49–0.87 fold. Most noteworthy was that the characteristic volatiles of oolong tea are thought to be indole, jasmine lactone, and (E)-nerolidol[8], which had a low relative content in this experiment. This may be because the tea leaves of Oriental Beauty had high tenderness and easy fermentation, resulting in formation of monoterpene compounds such as linalool and geraniol[16].
Volatile secondary metabolites of tea were divided into three major groups according to their biosynthetic source: terpenoids (monoterpenes, homoterpenes, and sesquiterpenes), phenylpropanoid/benzenoid (indole), and fatty acid derivatives (green leaf volatiles)[38]. The dynamic changes of these compounds during processing are shown in Fig. 3. The total aroma relative content reached the highest value in 4S-OB–ST-OB, which was 530–584 μg/L. The abundance of all compounds decreased significantly during the processing steps of FX-OB. It may be that the high temperatures (200 °C) during the fixation process inactivated the enzymatic activity, and thus evaporated/degraded the volatile compounds. Before the FX-OB procedure, the variation trends of green leaf volatiles (GLVs) and monoterpenes were the highest in 2S-OB and 3S-OB, and then decreased. GLVs were mainly C6 aldehydes and alcohols in tea, and they had major inferior components of the flavor of tea (green grassy notes)[39]. Tea leaves could release a large amount of GLVs in a short time when stimulated by external stress, which was consistent with our results. Before ST-OB, monoterpene content increased by 2.93 fold, sesquiterpene content basically remained unchanged, and homoterpene increased by 5.61 fold.
Differences of main metabolites between JX and TGY varieties during processing
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Tea varieties have a greater impact on tea quality, such as the composition, content and enzyme activity of biochemical components, which depend on the variety. The excellent characteristics of tea varieties can be further exerted under appropriate processing technology[40]. The changes of the main metabolites in JX and TGY during processing were compared and analyzed, and the results are shown in Fig. 4. The main nonvolatile compounds of the two varieties showed the same trend of change (except for caffeine and quercetin-3-O-galactoside) (Fig. 4a, b). The total catechin content of JX was lower than that of TGY, but the content of theaflavins in JX was higher, which may be the reason why the Oriental beauty tea processed by TGY has a strong taste. Comparison of the content of terpenoids, phenylpropanoid/benzenoid, and fatty acid derivatives during processing of two varieties. Figure 4c and d shows that the indole and sesquiterpenes contents of the two varieties have the same trend, and the GLVs and monoterpenes have relatively large changes in JX. In TGY varieties, the content of homoterpenes increased sharply in 3S-OB and decreased after ST-OB, and this phenomenon did not appear in JX varieties. This may be due to differences between varieties.
Figure 4.
Dynamic variation of major compounds between JX and TGY during processing. (a) Dynamic changes in the content of major non-volatile metabolites in JX varieties. (b) Dynamic changes in the content of major non-volatile metabolites in TGY varieties. (c) Dynamic changes in the content of major volatile metabolites in JX varieties. (d) Dynamic changes in the content of major volatile metabolites in TGY varieties. GLVs: green leaf volatiles (hexanal, (E)-2-hexenal, cis-3-hexen-1-ol,1-hexanol, cis-3-hexenyl acetate); homoterpenes ((3E)-4,8-dimethyl-1,3,7-nonatriene); monoterpenes (β-myecene, D-limonene, β-trans-ocimene, cis-β-ocimene, linalool, hotrienol, trans-linalool 3,7-oxide, β-cyclocitral, cis-geraniol, and citral); sesquiterpenes (α-cubebene, β-bourbenene, β-cubebene, caryophyllene, α-farnesene, δ-Cadinene, nerolidol, cubenol); JX: Jinxuan, TGY: Tieguanyin.
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In this study, untargeted metabolomics based on LC/MS and GC/MS was used to comprehensively compare the characteristics of taste and aroma metabolites in Oriental Beauty during the whole production process (Fig. 5). During the enzyme reaction stage, the content of GA significantly increased, and catechin was oxidized and degraded into oolongtheanins and theaflavins, leading to an increase in GA content. The total relative content of aroma increased and reached the maximum value at the ST-OB stage. Different from other oolong teas, monoterpenes such as linalool and geraniol were dominantly synthesized through the MEP pathway during the processing of Oriental Beauty. During the nonenzymatic stage, the content of theaflavin, theaflavin-3-gallate, and theaflavin-3,3′-gallate significantly increased. The increase after FX-OB was perhaps because polyphenol oxidase was also active and oxidized to form theaflavins. The total content of aroma decreased after FX-OB, and linalool heat treatment converted to hotrienol. These finding will provide an important theoretical basis for the quality changes in the processing of Oriental beauty.
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About this article
Cite this article
Zeng L, Jin S, Fu Y, Chen L, Yin J, et al. 2022. A targeted and untargeted metabolomics analysis of 'Oriental Beauty' oolong tea during processing. Beverage Plant Research 2:20 doi: 10.48130/BPR-2022-0020
A targeted and untargeted metabolomics analysis of 'Oriental Beauty' oolong tea during processing
- Received: 19 September 2022
- Accepted: 05 October 2022
- Published online: 15 November 2022
Abstract: Oriental Beauty, a deeply fermented variety of oolong tea, is famous for its fruity aroma and sweet taste. A targeted and untargeted metabolomics was used to comprehensively analyze the dynamic changes of taste and aroma metabolites during the processing stage. During the enzyme reaction stage, the catechin components were oxidized and degraded into theaflavins and oolongtheanins. The total abundance of aroma increased from 259.24 to 564.52 μg/L, and mainly monoterpenoids formed. During the nonenzymatic reaction stage, the total abundance of aroma decreased from 564.52 to 274.74 μg/L, and linalool was thermally converted to hotrienol. In this study, metabolomics changes were conducive to better control of tea quality.
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Key words:
- Oriental Beauty /
- Oolong tea /
- Metabolomics /
- Processing