3.1. Analysis of the Number and Classification of Volatile Compounds in Dahongpao Fresh Leaves and Gross Tea
In this study, GC-MS was used to analyze the effect of aviation mutagenesis on volatile compounds in fresh leaves and gross tea from Dahongpao. Analysis of volatile compounds in Dahongpao fresh leaves showed (
Figure 1A) that 230 and 218 volatile compounds were detected in aviation mutagenic (TM) and control (CK) Dahongpao fresh leaves, respectively. There were 14 volatile compounds specific to CK, 26 volatile compounds specific to TM, and 204 identical volatile compounds. Classification analysis of volatile compounds detected in CK showed (
Figure 1B) that 218 volatile compounds could be classified into 14 categories, of which the top 5 most abundant compounds were terpenoids (19.72%), heterocyclic compounds (16.97%), ester (14.22%), heterocyclic compounds (13.76%), and alcohol (9.17%), accounting for 73.84% of total volatile compounds. Classification analysis of the volatile compounds detected in TM showed (
Figure 1C) that 230 volatile compounds could be classified into 15 categories, of which the top 5 compounds with the highest number were terpenoids (18.70%), heterocyclic compounds (16.97%), hydrocarbons (13.48%), ester (13.04%), and alcohol (10.43%), accounting for 72.62% of total volatile compounds.
In addition, this study further analyzed the changes in the number and categories of volatile compounds in gross tea after CK and TM were processed according to the Wuyi Rock tea production process. The analysis of volatile compounds in Dahongpao gross tea showed (
Figure 1D) that 369 and 360 volatile compounds were detected in gross tea made from fresh leaves of aviation mutagenic (FTM) and control (FCK) Dahongpao, respectively. Second, there were 17 volatile compounds specific to FCK, 23 volatile compounds specific to FTM, and 343 volatile compounds that were the same in both. Classification analysis of the volatile compounds detected in FCK showed (
Figure 1E) that the 360 volatile compounds could be classified into 16 categories, of which the top 5 categories with the highest number of compounds were terpenoids (25.56%), ester (16.94%), heterocyclic compounds (13.61%), ketone (9.17%), and hydrocarbons (8.89%), accounting for 74.17% of total volatile compounds. Classification analysis of the volatile compounds detected in the FTM showed (
Figure 1F) that 369 volatile compounds could be classified into 16 categories, of which, the top 5 compounds with the highest number were terpenoids (25.68%), ester (15.57%), heterocyclic compounds (14.21%), hydrocarbons (9.56%), and ketone (9.02%), accounting for 74.04% of total volatile compounds.
It can be seen that after aviation mutagenesis, the number and categories of volatile compounds in fresh leaves and gross tea of Dahongpao were more similar to those of the control, and aviation mutagenesis had a smaller effect on the composition of volatile compounds in the fresh leaves and gross tea of the tea tree (Camellia sinensis).
3.2. Analysis of Volatile Compounds in Dahongpao Fresh Leaves and Gross Tea
Plants are affected by the take-off and landing of spacecraft during aviation mutagenesis and experience the strong effects of radiation and magnetic fields in space, which may alter the normal growth and development of plants and their metabolism and thus affect the content of different compounds in plant tissues [
20]. It has been reported that
Prunella vulgaris seeds carried by the Shenzhou VIII spacecraft which underwent aviation mutagenesis had a significantly higher rosmarinic acid content in the plant after planting than the control [
24]. Secondly, it has also been found that glycyrrhizic acid and liquiritigenin contents were significantly elevated in plants of
Glycyrrhiza glabra seeds after aviation mutagenesis compared to the control [
25]. Based on the previous analysis, this study further analyzed the effect of aviation mutagenesis on the volatile compound content in fresh leaves and gross tea of Dahongpao. Analysis of the total volatile compound content of Dahongpao fresh leaves showed (
Figure 2A) that the volatile compound content of TM was significantly higher (
p < 0.05) than that of CK. PCA analysis of the relative content of volatile compounds detected in fresh leaves revealed (
Figure 2B) that there was a significant difference in the content of different volatile compounds in CK and TM, and the two principle components could effectively differentiate between CK and TM with an overall contribution of 81.06%. After the classification of volatile compounds, the results of their content analysis showed (
Figure 2C) that there were 13 categories of volatile compounds in TM with significantly higher content than CK, which were acid, aldehyde, amine, aromatics, ether, heterocyclic compounds, hydrocarbons, ketone, nitrogen compounds, phenol, sulfur compounds, terpenoids, and others. Moreover, the above 13 categories of volatile compounds were significantly associated with TM (
Figure 2D). In contrast, the content of two categories of volatile compounds, such as alcohol and ester, was significantly lower in TM than in CK, and these two categories of volatile compounds were significantly correlated with CK (
Figure 2C).
Further analysis of the effect of aviation mutagenesis on volatile compound content in Dahongpao gross tea showed (
Figure 2E) that the total volatile compound content was significantly higher in FTM than in FCK (
p < 0.05). PCA analysis with the relative content of volatile compounds detected in the gross tea revealed (
Figure 2F) that there were significant differences in the content of different volatile compounds in FCK and FTM, and the two principle components could effectively distinguish FCK from FTM with an overall contribution of 84.39%. After the volatile compounds were categorized, the results of their content analysis (
Figure 2G) showed that there were 13 categories of volatile compounds in FTM with significantly higher content than FCK, namely acid, aldehyde, amine, aromatics, ester, ether, halogenated hydrocarbons, heterocyclic compounds, hydrocarbons, ketone, nitrogen compounds, phenol, and terpenoids. Moreover, the above 13 categories of volatile compounds were significantly associated with FTM (
Figure 2H). In contrast, the content of two categories of volatile compounds, such as alcohol and sulfur compounds, was significantly lower in FTM than in FCK, and these two categories of volatile compounds were significantly correlated with FCK (
Figure 2H). There was no significant difference in the content of other volatile compounds between FTM and FCK (
Figure 2H). It can be seen that aviation mutagenesis significantly altered volatile compound content in the fresh leaves and gross tea of Dahongpao, which in turn improved the aroma quality of Dahongpao tea.
3.3. Screening and Content Analysis of Characteristic Volatile Compounds in Dahongpao Fresh Leaves and Gross Tea
Based on the previous analysis, this study further screened the characteristic volatile compounds that changed significantly in Dahongpao fresh leaves and gross tea after aviation mutagenesis. A volcano plot analysis of volatile compound content in Dahongpao fresh leaves showed (
Figure 3A) that 70 volatile compounds were significantly increased, 25 were significantly decreased, and 149 were not significantly different in TM compared to CK. The OPLS-DA model of CK and TM was further constructed to screen for key volatile compounds. The goodness-of-fit analysis of the OPLS-DA model showed (
Figure 3B) that after 200 stochastic simulations of the constructed model, the model had R
2Y = 1 and Q
2 = 0.992, both at the
p < 0.005 level. This result indicated that the OPLS-DA model constructed in this study could effectively distinguish between CK and TM, and the model fit met the requirements and could be used for further analysis. Analysis of the OPLS-DA model score plot for CK versus TM showed (
Figure 3C) that the intra-group difference between CK and TM was within 2.99%, while the inter-group difference was 96.00%. This result showed that the reproducibility of three independent replicates of CK and TM in this study was good, and there was a significant difference in volatile compound content between CK and TM. On this basis, further analysis of the S-Plot plot of the OPLS-DA model of CK versus TM revealed (
Figure 3D) that 47 volatile compounds (VIP > 1) were critically different in CK versus TM, of which 24 volatile compounds were significantly elevated in TM and 23 were significantly decreased in TM compared to CK. The classification analysis of the key differential volatile compounds showed (
Figure 3E) that the 47 volatile compounds could be classified into 12 categories, of which, compared with CK, there was a significant increase in the content of 9 volatile compounds in TM, namely amine, aromatics, phenol, aldehyde, terpenoids, ketone heterocyclic compounds, and acid and sulfur compounds, whereas three categories of volatile compounds showed a significant decrease in the content, namely, alcohol, hydrocarbons and ester.
TOPSIS was further used to analyze the impact weights of the 12 categories of volatile compounds in distinguishing CK from TM, and the results showed (
Figure 3F) that four categories of volatile compounds had impact weights greater than 10%, namely alcohol (100%), ester (62.04%), terpenoids (36.83%), and heterocyclic compounds (11.91%). A bubble characteristic plot with the above four categories of volatile compounds was performed to screen for characteristic volatile compounds that distinguish CK from TM. It was found (
Figure 3G) that the characteristic volatile compounds distinguishing CK from TM were mainly trans-3-hexenyl acetate, beta-myrcene, 3-hydroxy-4-methyl-5-ethyl-2-furanone, (
Z)-3-hexenyl butyrate, 2-p-tolylethanal, and 2-methyl-benzaldehyde. Analysis of the content of characteristic volatile compounds showed (
Figure 3H) that beta-myrcene, 3-hydroxy-4-methyl-5-ethyl-2-furanone, 2-p-tolylethanal, 2-methyl-benzaldehyde were significantly higher in TM than in CK, whereas trans-3-hexenyl acetate and (
Z)-3-hexenyl butyrate were significantly lower than in CK. It can be seen that the content of volatile compounds, especially characteristic volatile compounds, in the fresh leaves of Dahongpao changed significantly after aviation mutagenesis, and this change may affect the transformation of substances and the formation of aroma in the subsequent post-processing process of tea.
Accordingly, the volatile compound content in the gross tea of Dahongpao changed significantly after processing was analyzed in this study. Volcano plot analysis of volatile compound content in Dahongpao gross tea showed (
Figure 4A) that there were 79 volatile compounds with a significant increase, 25 with a significant decrease, and 279 with no significant difference in FTM compared to FCK. The OPLS-DA model of FCK and FTM was further constructed to screen for key volatile compounds. The goodness-of-fit analysis of the OPLS-DA model showed (
Figure 4B) that after 200 stochastic simulations of the constructed model, the model had R
2Y = 0.999 and Q
2 = 0.995, both at the
p < 0.005 level. This result showed that the OPLS-DA model constructed in this study could effectively distinguish FCK from FTM, and the model fit met the requirements and could be used for further analysis. Analysis of the OPLS-DA model score plot for FCK versus FTM showed (
Figure 4C) that the intra-group difference between FCK and FTM for the three independent replicates was within 0.96%, while the inter-group difference was 96.30%. This result showed that the reproducibility of three independent replicates of FCK and FTM in this study was better and there was a significant difference in volatile compound content between the two. On this basis, further analysis of the S-Plot plots of the OPLS-DA model of FCK versus FTM revealed (
Figure 4D) that there were 77 volatile compounds with key differences in FCK versus FTM, of which 57 volatile compounds were significantly elevated and 20 significantly decreased in FTM compared to FCK. The classification analysis of key differential volatile compounds showed (
Figure 4E) that 77 volatile compounds could be classified into 12 categories, of which 10 categories of volatile compounds were significantly increased in the FTM compared to the FCK, namely amine, aromatics, aldehyde, terpenoids, hydrocarbons, ketone, heterocyclic compound, ester, nitrogen compounds, and ether, while the content of two categories of volatile compounds, namely alcohol and phenol, decreased significantly.
TOPSIS was further used to analyze the impact weights of the 12 categories of volatile compounds in distinguishing FCK from FTM, and the results showed (
Figure 4F) that the 6 categories of volatile compounds with impact weights greater than 10% were ester (96.82%), terpenoids (73.66%), heterocyclic compounds (66.40%), hydrocarbons (22.64%), aldehyde (17.55%), and ketone (11.55%). A bubble characteristic map analysis with six categories of volatile compounds revealed (
Figure 4G) that the characteristic volatile compounds distinguishing FCK from FTM were mainly 6-pentyloxan-2-one, beta-pinene, cubebol, (
Z,
Z)-3,6-nonadienal, zingiberene, beta-phellandrene, and ethyl cinnamate. Analysis of the content of characteristic volatile compounds showed (
Figure 3H) that FTM had a significantly higher content of 6-pentyloxan-2-one, beta-pinene, cubebol, (
Z,
Z)-3,6-nonadienal, zingiberene, and beta-phellandrene than FCK, whereas ethyl cinnamate was significantly lower than FCK. It can be seen that changes in volatile compound content in Dahongpao fresh leaves after aviation mutagenesis affected the volatile compound content in gross tea, especially the characteristic volatile compound content.
3.4. Odor Characteristic and Transformation Analysis of Characteristic Volatile Compounds in Dahongpao Fresh Leaf and Gross Tea
Changes in the content of volatile compounds can directly affect the type and intensity of aroma in tea [
26]. It has been reported that beta-myrcene, 2-p-tolylethanal and 2-methyl-benzaldehyde are important compounds that make up the aroma of tea, and their main odor characteristic is floral [
27]. The main odor characteristic of (
E)-3-hexenyl acetate, 3-hydroxy-4-methyl-5-ethyl-2-furanone and (
Z)-3-hexenyl butyrate is fruity [
28,
29]. In this study, odor wheel analysis of odor characteristics of characteristic volatile compounds in fresh leaves of Dahongpao revealed (
Figure 5A) that the main odor characteristics presented by characteristic volatile compounds in CK and TM were floral and fruity, where the intensity of the floral odor characteristic in TM was significantly higher than that in CK, while the fruity odor characteristic was slightly lower than that in CK. In addition, this study identified seven characteristic compounds with significant differences in Dahongpao gross tea after aviation mutagenesis, namely ethyl cinnamate, 6-pentyloxan-2-one, (
Z,
Z)-3,6-nonadienal, beta-phellandrene, cubebol, zingiberene, and beta-pinene. The main characteristic odor of ethyl cinnamate is reported to be floral [
30], that of 6-pentyloxan-2-one is fruity [
31], that of (
Z,
Z)-3,6-nonadienal and beta-phellandrene is green [
32,
33], that of cubebol and zingiberene is spicy [
34], and that of beta-pinene is woody [
35]. In this study, the odor wheel analysis of odor characteristics of characteristic volatile compounds in Dahongpao gross tea revealed (
Figure 5B) that the main odor characteristics presented by the characteristic volatile compounds were floral, fruity, green, spicy, and woody, where the odor intensity of fruity, green, spicy, and woody notes in FTM was significantly higher than that in FCK; the intensity of the floral odor was significantly lower than that in FCK. It can be seen that the intensity of the odor characteristics of Dahongpao fresh leaves and gross tea changed significantly after aviation mutagenesis. Aviation mutagenesis altered the content of characteristic volatile compounds in fresh leaves and gross tea of Dahongpao, which in turn affected the intensity of the tea’s odor characteristics.
Based on the previous analysis, this study further analyzed the transformation of volatile compounds in Dahongpao fresh leaves and gross tea. In this study, we constructed a transformational relationship of characteristic volatile compounds in Dahongpao fresh leaves and gross tea and found that (
Figure 5C) beta-myrcene played an important role in the transformation of volatile compounds and was a precursor for the transformation or synthesis of other characteristic volatile compounds; we also found that this compound was mainly derived from fresh leaves. In Dahongpao fresh leaves, beta-myrcene content in TM was significantly higher than that in CK. Further analysis revealed that beta-myrcene could achieve the transformation of characteristic volatile compounds through three pathways. In the first pathway, beta-myrcene could be synthesized into beta-pinene; beta-pinene could be used to synthesize cubebol after carbon chain rearrangement or could be transformed by isomerization into beta-phellandrene; beta-phellandrene could be used for the synthesis of zingiberene or oxidation and ring-opening to (
Z,
Z)-3,6-nonadienal; (
Z,
Z)-3,6-nonadienal could be transformed by esterification into a ring to form 6-pentyloxan-2-one. In the second pathway, beta-myrcene could be synthesized into (
E)-3-hexenyl acetate by oxidative esterification, into (
E)-3-hexenyl acetate through hydrolytic esterification to synthesize (
Z)-3-hexenyl butyrate, and into (
Z)-3-hexenyl butyrate through hydrolytic ring opening and oxidative ring formation to synthesize 3-hydroxy-4-methyl-5-ethyl-2-furanone. Moreover, 3 -hydroxy-4-methyl-5-ethyl-2-furanone was synthesized by hydrolytic reduction of (
Z Z)-3,6-nonadienal, and (
Z,
Z)-3,6-nonadienal could be transformed by esterification into a ring to form 6-pentyloxan-2-one. In the third pathway, beta-myrcene could be oxidized and cyclized to 2-p-tolylethanal; 2-p-tolylethanal could be synthesized to ethyl cinnamate by esterification or by oxidative rearrangement to form 2-methyl-benzaldehyde, and 2-methyl-benzaldehyde could be transformed to ethyl cinnamate by esterification.
From the first pathway of beta-myrcene transformation, there were six characteristic volatile compounds synthesized by its transformation, namely beta-pinene, cubebol, beta-phellandrene, zingiberene, (Z,Z)-3,6-nonadienal, and 6-pentyloxan-2-one, which were all derived from Dahongpao gross tea and mainly contributed to the fruity, green, spicy, and woody odor characteristics. Further analysis revealed that beta-myrcene was derived from Dahongpao fresh leaves and its content in TM was significantly higher than that in CK, whereas the six transformed synthetic characteristic volatile compounds were all derived from gross tea, and their contents and odor characteristics were significantly greater in FTM than in FCK. It can be seen that via the first pathway, Dahongpao converted beta-myrcene, which has floral odor characteristics in fresh leaves, into six other characteristic volatile compounds during processing to form the fruity, green, spicy, and woody odor characteristics; this conversion was far more intense in aviation mutagenic Dahongpao than in unmutagenic Dahongpao.
From the second pathway of beta-myrcene conversion, there were five characteristic volatile compounds converted, namely (E)-3-hexenyl acetate, (Z)-3-hexenyl butyrate, 3-hydroxy-4-methyl-5-ethyl-2-furanone, (Z,Z)-3,6-nonadienal, and 6-pentyloxan-2-one. Among them, (E)-3-hexenyl acetate, (Z)-3-hexenyl butyrate, and 3-hydroxy-4-methyl-5-ethyl-2-furanone were converted from Dahongpao fresh leaves, the main odor characteristic of which was fruity, and they were slightly higher in number in CK than in TM. Meanwhile, (Z,Z)-3,6-nonadienal and 6-pentyloxan-2-one from Dahongpao gross tea, the main odor characteristics of which were fruity and green, were significantly greater in FTM than in FCK. It can be seen that from the second pathway, Dahongpao fresh leaves first converted beta-myrcene with floral odor characteristics into (E)-3-hexenyl acetate, (Z)-3-hexenyl butyrate, and 3-hydroxy-4-methyl-5-ethyl-2-furanone, which have fruity odor characteristics. Meanwhile, 3-hydroxy-4-methyl-5-ethyl-2-furanone was then converted to (Z,Z)-3,6-nonadienal and 6-pentyloxan-2-one during processing, which gave the Dahongpao gross tea green fruity odor characteristics, and the intensity of this conversion was significantly higher in aviation mutagenic Dahongpao than in unmutagenic Dahongpao.
From the third pathway of beta-myrcene conversion, there were three characteristic volatile compounds converted, namely 2-p-tolylethanal, 2-methyl-benzaldehyde, and ethyl cinnamate. Among them, 2-p-tolylethanal and 2-methyl-benzaldehyde from Dahongpao fresh leaves, which mainly had a floral odor, were in a significantly higher proportion in TM than in CK, while ethyl cinnamate from the gross tea of Dahongpao, which mainly has a floral odor, was significantly higher in FCK than in FTM. It can be seen that from the third pathway, Dahongpao fresh leaves firstly converted beta-myrcene, which had a floral odor characteristic, into 2-p-tolylethanal and 2-methyl-benzaldehyde, and these two products were then converted to ethyl cinnamate during processing, resulting in Dahongpao gross tea showing stronger floral odor characteristics. The intensity of this conversion was significantly lower in aviation mutagenic Dahongpao than in unmutagenic Dahongpao.
It can be seen that aviation mutagenesis significantly increased the content of beta-myrcene in Dahongpao fresh leaves, and the transformation and synthesis of the compounds during processing were mainly carried out through the first and second pathways of beta-myrcene transformation, which in turn increased the content of beta-pinene, cubebol, beta-phellandrene, zingiberene, (Z,Z)-3,6-nonadienal, and 6-pentyloxan-2-one and enhanced the intensity of the fruity, green, spicy and woody odor characteristics of Dahongpao gross tea; however, this decreased the intensity of floral odor characteristics. In contrast, beta-myrcene in fresh leaves of unmutagenic Dahongpao was mainly converted and synthesized by the third pathway of compounds during tea processing, which ultimately increased the ethyl cinnamate content in Dahongpao gross tea and enhanced the intensity of floral odor characteristics but decreased the intensity of fruity, green, spicy, and woody notes in Dahongpao gross tea.