Identification of Key Aroma Compounds Responsible for the Floral Ascents of Green and Black Teas from Different Tea Cultivars

Chemicals underlying the floral aroma of dry teas needs multi-dimensional investigations. Green, black, and freeze-dried tea samples were produced from five tea cultivars, and only ‘Chunyu2’ and ‘Jinguanyin’ dry teas had floral scents. ‘Chunyu2’ green tea contained the highest content of total volatiles (134.75 μg/g) among green tea samples, while ‘Jinguanyin’ black tea contained the highest content of total volatiles (1908.05 μg/g) among black tea samples. The principal component analysis study showed that ‘Chunyu2’ and ‘Jinguanyin’ green teas and ‘Chunyu2’ black tea were characterized by the abundant presence of certain alcohols with floral aroma, while ‘Jinguanyin’ black tea was discriminated due to the high levels of certain alcohols, esters, and aldehydes. A total of 27 shared volatiles were present in different tea samples, and the contents of 7 floral odorants in dry teas had correlations with those in fresh tea leaves (p < 0.05). Thus, the tea cultivar is crucial to the floral scent of dry tea, and these seven volatiles could be promising breeding indices.


Introduction
Tea, as a widely consumed non-alcoholic beverage, is associated with various health benefits, such as anti-inflammatory, anti-tumorigenic, and cardio-protective effects [1]. It is made from the fresh leaves of tea plants (Camellia sinensis. L) that contain abundant secondary metabolites. In addition to the taste sensory of tea, the aroma is also an important factor in consumer preference. There are many types of aroma characteristics of different teas, such as toasty, floral, fruity, nutty, fresh and brisk aroma, etc. [2][3][4], the formation of which are impacted by various factors, such as processing method, tea cultivar, growing conditions, and harvest season [5][6][7][8][9][10][11].
Green tea (non-oxidized tea) and black tea (fully oxidized tea) are widely consumed over the world and have different sensory properties. Even for the same type of tea, there are many different scent types. For example, green tea has scent types of floral, fruity, nutty, chestnut-like fragrances, and so on [3,8]. Generally, the aroma quality of dry teas is associated with processing techniques, considering the chemical conversions that occur during processing, resulting in the lost or postharvest synthesis of endogenous volatiles [6,12]. Many attempts have been made to improve the aroma quality of dry teas through optimization of processing methods and parameters [13][14][15]. Withering is an important step to release unwanted aroma compounds, such as green and grassy odor, and bruising is crucial to forming favorable fragrances such as floral scents [16][17][18].
Nevertheless, the chemical components of fresh tea leaves provide the material basis for forming the aroma of final dry teas. The tea cultivar is an important factor in the chemical composition of fresh tea leaves and the enzymatic activities of aroma volatile-related enzymes [11].
In China, there are hundreds of cultivars with their very own phenotypes and characteristic metabolite profiles. Some are suitable for processing green tea, and some are suitable for processing black tea or oolong tea. The scent type of tea is related to the volatile profiles of tea cultivars [11]. Moreover, the aromatic precursors and the activities of glycosidase enzymes that modulate the formation of odorants also vary with tea cultivars [11]. The floral aroma is associated with the value and quality of tea products. Under the same processing procedures, the dry tea prepared from tea cultivar 'Foshou' had a more intense floral aroma compared with 'Zhenong139' [16]. Tea cultivars suitable for processing oolong tea are usually characterized by floral fragrances, such as 'Jinguanyin', 'Tieguanyin', and 'Jinmudan' [19,20]. 'Chunyu2', a tea cultivar suitable for processing green tea, is also wellknown for its orchid-like fragrance due to its genetic background. However, the respective contributions of cultivar and manufacturing procedure to the floral scent type of dry teas are still unclear, and the key odorants responsible for floral scent need further expatiation. Understanding the role of tea cultivar in the formation of the floral aroma of dry teas and the correlations of floral volatiles between fresh tea leaves and dry teas provides important guidance for tea breeding work.
There are many factors associated with the scents of teas, and finding out the key factor is important to understanding the formation of characteristic features. The hypothesis of the present study is that the tea cultivar is the crucial factor in the formation of floral ascents of green and black teas, supplying the abundant floral odorants in the fresh leaves as raw materials. Here, green tea, black tea, and freeze-dried samples were produced from different tea cultivars, including 'Chunyu2' and 'Jinguanyin', which are associated with floral scents, as well as 'Chunyu1', 'Zhenong117', and 'Longjing43'. The aroma features of these tea samples were identified based on the result of sensory evaluation, and the volatile compositions of tea samples were analyzed by gas chromatography-mass spectrometry (GC-MS). Cluster analysis and principal component analysis (PCA) were employed to visualize the volatile composition difference of tea samples associated with aroma features. Co-expression analysis was carried out to explore the correlations of shared volatiles for the pairs of freeze-dried tea samples vs. green tea and freeze-dried tea sample vs. black tea, and the key odorants in fresh tea leaves crucial to the floral scent of dry teas were discussed. Figure 1 shows the pictures of green teas, black teas, and the corresponding freezedried tea samples prepared from five tea cultivars, namely 'Chunyu2', 'Chunyu1', 'Jinguanyin', 'Zhenong117', and 'Longjing43'. The sensory evaluation of these green and black teas was carried out by an evaluation panel, and the comprehensive evaluation results are shown in Table S1. Based on the description of aroma (Table S1, see Supplementary Materials), 'Chunyu2' green tea was identified as a long-lasting lily of valley of fragrance, and 'Jinguanyin' was identified as a long-lasting rose fragrance, which was clearly discriminated from other green teas with no floral fragrance ( Figure 1). The volatile compositions of different green teas are shown in Table S2. A total of 68 volatiles were annotated and quantified in different green teas, including 26 alcohols, 12 aldehydes, 7 ketones, 11 esters, 1 pyrrole, and 11 miscellaneous. 'Chunyu2' green tea contained the highest content of total volatiles at 134.75 µg guaiacol equivalent (GE)/g dry tea, subsequently followed by 'Jinguanyin' (82.65 µg GE/g), while 'Zhenong117', 'Longjing43', and 'Chunyu1' contained relatively low contents of total volatiles ranging from 50.57 to 65.14 µg GE/g. For all the green teas, alcohols were the major type of volatiles in green teas accounting for 44.2-66.9% of total volatiles, among which 'Chunyu2' contained the highest content of total alcohols at 90.21 µg GE/g, followed by 'Jinguanyin' (48.34 µg GE/g), while 'Zhenong117', 'Longjing43', and 'Chunyu1' contained the relatively low contents of total alcohols ranging from 22.45 to 29.97 µg GE/g. Moreover, 'Chunyu1', 'Zhenong117', and 'Longjing43' contained higher proportions of total aldehydes (14.6-23.0%) than those of 'Chunyu2' and 'Jinguanyin' (6.5% and 9.7%). The highest level of total esters (17.34 µg GE/g) was observed in 'Chunyu2', corresponding to 12.9% of total volatiles, followed by 'Zhenong117' (9.67 µg GE/g), while 'Chunyu1', 'Jinguanyin', and 'Longjing43' had relatively low levels of total esters ranging from 3.28 to 4.86 µg GE/g. Hence, the green tea samples prepared from different tea cultivars had different volatile profiles. contained relatively low contents of total volatiles ranging from 50.57 to 65.14 μg GE/g. For all the green teas, alcohols were the major type of volatiles in green teas accounting for 44.2-66.9% of total volatiles, among which 'Chunyu2' contained the highest content of total alcohols at 90.21 μg GE/g, followed by 'Jinguanyin' (48.34 μg GE/g), while 'Zhe-nong117', 'Longjing43', and 'Chunyu1' contained the relatively low contents of total alcohols ranging from 22.45 to 29.97 μg GE/g. Moreover, 'Chunyu1', 'Zhenong117', and 'Longjing43' contained higher proportions of total aldehydes (14.6-23.0%) than those of 'Chunyu2' and 'Jinguanyin' (6.5% and 9.7%). The highest level of total esters (17.34 μg GE/g) was observed in 'Chunyu2', corresponding to 12.9% of total volatiles, followed by 'Zhenong117' (9.67 μg GE/g), while 'Chunyu1', 'Jinguanyin', and 'Longjing43' had relatively low levels of total esters ranging from 3.28 to 4.86 μg GE/g. Hence, the green tea samples prepared from different tea cultivars had different volatile profiles.

Discussion
Green teas have various types of scents, which are attributed to their different volatile profiles. There were 68 volatiles annotated in all the green teas, which was comparable to the number of volatiles reported in Biluochun green tea [10]. Terpenoid volatiles, such as C6 aldehydes, alcohols, and their esters, are important contributors to the green aroma of fruits and vegetables [21], which are also regarded as the most important odorants in tea leaves due to their high contents and low thresholds [22]. In our study, alcohols were the major volatile compounds present in all the tea samples prepared from different cultivars, which is consistent with previous studies [2,23]. Alcohol compounds were the important compounds for discriminating 'Chunyu2' and Jinguanyin' green teas with floral fragrance from other green teas, while ester compounds were the key compounds for discriminating 'Chunyu2' green tea from other green teas. 3,7-Dimethylocta-1,6-dien-3-ol and the oxides [e.g., 6-ethenyl-2,2,6-trimethyloxan-3-ol, 2-[(2S,5S)-5-ethenyl-5-methyloxolan-2-yl]propan-2-ol], (2E)-3,7-dimethylocta-2,6-dien-1-ol, phenylmethanol, and 2-phenylethanol importantly contributed to the floral aroma of oolong tea and black tea [2,9,16]. Dodecan-1ol and 2-(4-methylcyclohex-3-en-1-yl)propan-2-ol have floral smell [24,25]. (E)-Hex-2-en-1-ol, (Z)-hex-3-en-1-ol are related to green and grassy odor [26], and heptan-2-ol gives a citrusy aroma [27]. The abundant presence of floral odorants in 'Chunyu2' green tea, including 3,7-dimethylocta-1,6-dien-3-ol and the oxides, dodecan-1-ol and 2-phenylethanol, Figure 6. Correlation network of the top ten volatiles in green tea and black tea correlated with the shared volatiles in three types of tea samples. Alcohol compounds (blue nodes), aldehyde compounds (red nodes), ketone compounds (light green nodes), ester compounds (orange nodes), and miscellaneous compounds (light yellow nodes). The correlation analysis of the contents of the key volatiles in different tea samples was conducted by the Cytoscape software (version 3.8.0) (https://cytoscape.org/, accessed on: 23 March 2021). A significant correlation was presented based on the statistical test with a robust cutoff (p-value < 0.05), with a black line indicating a positive correlation and a blue line indicating a negative correlation. The absolute value of the correlation coefficient increased from 0.515 to 0.984 as the line width was increased. The number of replicates is equal to 3.
The aroma characteristics of black tea are quite different from green tea, which generally has a sweet aroma. 'Jinguanyin' and 'Chunyu2' black teas with floral aroma were discriminated from other black teas based on the composition of alcohols, which was the same as green teas. Furthermore, 'Jinguanyin' black tea was distinguished from other black teas based on the compositions of aldehydes, ketones, and esters. Odorants such as (2E)-3,7dimethylocta-2,6-dien-1-ol, (1S,2R,5S,7R,8R)-2,6,6,8-tetramethyltricyclo[5.3.1.01,5]undecan-8-ol, (6E)-3,7,11-trimethyldodeca-1,6,10-trien-3-ol, and dodecan-1-ol are associated with rose-like floral aroma, which were considered as important contributors to the floral fragrance of 'Jinguanyin' black tea. Moreover, many aldehydes, ketones, and esters have unique fruity or floral aromas [10,28]. The abundant presence of esters and aldehydes might be related to the long-lasting floral and sweet fragrance of 'Jinguanyin' black tea that was distinguished from the weaker floral aroma of 'Chunyu2' black tea as well as the rose odor of 'Jinguanyin' green tea. Most of the characteristic volatiles in 'Jinguanyin' black tea could be detected in the freeze-dried tea of 'Jinguanyin' with relatively high contents. The floral fragrance of 'Chunyu2' black tea was mainly attributed to the high levels of certain alcohols, namely 2-[(1S)-4-methylcyclohex-3-en-1-yl]propan-2-ol, 3,7-dimethylocta-1,6-dien-3-ol, 2-[(2S,5S)-5-ethenyl-5-methyloxolan-2-yl]propan-2-ol, 6-ethenyl-2,2,6-trimethyloxan-3-ol, 2ethylhexan-1-ol, and heptan-2-ol, which were also abundantly present in 'Chunyu2' green tea. The similarity of alcohol composition to 'Jinguanyin' black tea ( Figure 4) and relatively high content of alcohols could be plausible explanations for the floral aroma of 'Chunyu2' black tea. Thus, it seems the scent types of green tea and black tea could be influenced by fresh tea leaves as raw materials. The significant correlations between the contents of floral odorants in black tea and fresh tea leaves indicated that the tea cultivar is the key factor in the formation of floral scents since intense enzymatic reactions such as oxidation and polymerization occur during the processing of black tea. The original contents of floral odorants in fresh tea leaves importantly affect their levels in dry teas.

Preparation of Freeze-Dried Tea Samples
Fresh tea leaves were harvested from different tea cultivars at the standard of one bud, including 'Chunyu2', 'Chunyu1', 'Jinguanyin', 'Zhenong117', and 'Longjing43'. These tea plants are grown in the Wuyi county, Zhejiang, China (28 • 893 N, 119 • 816 E). One-third of the harvested fresh tea leaves were freeze-dried immediately (SCIENTZ-12N, Ningbo SCIENTZ Biotechnology Co., Ltd., Ningbo, China), one-third was submitted to green tea processing, and the rest was submitted to black tea processing. All of the tea samples were stored at 4 • C prior to GC-MS analysis.

Preparation of Green Tea Samples
After spreading outdoors for 12 h, the fresh tea leaves of different cultivars were submitted to a continuous tea combing machine (6CLX-10A, Anji Yuanqing tea machine Co. Ltd., Anji, China) for fixation at 250 • C for 4-5 min and then combing at 200 • C for 4 min. The leaves were oven-dried to the moisture content of 5% by a two-step process: heating at 125 • C for 5 min, followed by 30 min standing at room temperature for resurgence, and then heating at 100 • C for 7 min.

Preparation of Black Tea Samples
The fresh tea leaves of different tea cultivars were withered overnight (~15 h) to reach ∼70% of fresh weight under ambient conditions. The tea leaves were packed in a piece of cloth and manually rolled for 25 min. Then, the tea leaves were spread evenly for oxidation at 30 • C and 95% relative humidity for 300 min. At last, the tea leaves were oven-dried to the moisture content of 5% by a two-step process: heating at 125 • C for 10 min, followed by 30 min standing at room temperature for resurgence, and then heating at 82 • C for another 15 min.

Identification of the Scent Types of Different Tea Samples
Considering the distinguishable cultivar features of these five tea cultivars, especially in terms of aroma, the scent types were used to distinguish different green and black teas. According to the China Nation Standard (GB/T 23776-2018, Methodology for Sensory Evaluation of Tea) that has been previously reported [25], a tea sample (3.0 g) was placed in the evaluating cup, and 150 mL of boiling water was added. After brewing for 4 min (green tea) or 5 min (black tea), the tea infusion was filtered. The dry tea appearance, color, and taste of tea infusion, as well as the aroma and tenderness of brewed leaves, were comprehensively evaluated by the evaluation panel in terms of feature description and score. The total score was the sum of 25% of dry tea appearance score, 10% of tea infusion color score, 30% of tea taste score, 25% of tea aroma score, and 10% of brewed leaf score. Seven panelists, including three senior tea sensory evaluation experts and four junior tea sensory evaluation experts (aged 24-45 years), were recruited to identify the scent type of different tea samples.

GC-MS Analysis of Volatile Compounds
The volatile compounds were analyzed by QP2010 ultra gas chromatography/mass spectrometry (Shimadzu Co., Tokyo, Japan) according to the reported method [16]. The tea samples were ground and sifted through a 0.6 mm sifter. Our preliminary experiment showed that the contents of total volatiles were quite different among freeze-dried, green, and black tea samples. To achieve comparable total peak areas of volatiles among different types of teas and high repeatability, different amounts of ground tea samples, viz. green tea 1.00 g, black tea 0.25 g, and freeze-dried tea 0.075 g, were respectively extracted with 50 mL of water at 70 • C for 10 min. After centrifugation at 2272× g and 25 • C for 10 min, the supernatants were collected. A total of 10 mL of supernatants were transferred to a 20 mL headspace bottle, followed by the addition of 20 µg/mL internal standard guaiacol (20 µL for green tea, 120 µL for black tea, and freeze-dried tea, respectively) and 2 g dried NaCl, and then the headspace bottle was sealed immediately. A 50/30 µm divinylbenzene/carboxen/poly-dimethylsiloxane (DVB/CAR/PDMS) fiber (Supelco, Bellefonte, PA, USA) was used to adsorb the volatiles at 60 • C for 1 h above the liquid surface. Then, the SPME fiber was placed in the GC-MS injection port at 250 • C for 3.5 min for desorption. GC-MS conditions were: capillary column HP-INNOWax (30 m × 0.25 mm × 0.25 µm, Agilent Technologies, Palo Alto, CA, USA), carrier gas (helium, 99.999%), flow rate 1.0 mL/min, injector temperature 250 • C, ion source temperature 200 • C, ionization style EI, MS scan range of 35-400 m/z, injection mode splitless; column temperature program: keeping at 50 • C for the first 10 min, increasing to 150 • C at the speed of 3 • C/min, keeping at 150 • C for another 1 min, increasing to 230 • C at the speed of 15 • C/min, followed by keeping at 230 • C for 3 min. The compounds were annotated based on the published retaining index as well as the National Institute of Standards and Technology (NIST) mass spectral database. Guaiacol was selected as the internal standard for GC-MS analysis because of its suitable repeatability according to the pretest of our previous work [16], with no interference to these tea samples based on our preliminary experiment. The volatile compounds were quantified by guaiacol according to the following equation: Content (µg guaiacol equivalent/g dry tea) = (Peak area of target/Peak area of IS) × Amount of guaiacol used (µg) Amount of weighted sample(g)×( 10 mL 50 mL ) (1)

Association of the Shared Volatile Compounds between Different Tea Samples
There were 27 shared volatiles screened out from the freeze-dried tea samples, green 25 and black teas prepared from different cultivars. Pearson correlation analysis was performed based on the relative contents of these shared volatiles in different types of tea samples, using cor and corPvalueStudent functions in R (version 4.0.5), and the threshold screening criterion was: p < 0.05. Positive correlation: R > 0; negative correlation: R < 0. The Cytoscape software (version 3.8.0) was used to visualize the correlation of these 27 volatiles for freeze-dried tea samples versus green teas and freeze-dried tea samples versus black teas, as well as the internal correlations of the top 10 volatiles in both green and black teas (p < 0.05).

Data Analysis
All the tests were repeated three times, and the mean value ± SD was presented. The significant difference analysis was carried out by the SAS System for Windows version 8.1 (SAS Institute Inc., Cary, NC, USA), using the Tukey test. Principal component analysis (PCA) based on a correlation matrix was conducted by Minitab 17 statistical software (Minitab. LLC, State College, PA, USA). Three-dimensional score plots were drawn by the Origin Pro 8.5.1 software (Originlab Corporation, Northampton, MA, USA). The heatmap was plotted based on the relative content of the volatile data set and drafted using TBtools [30].
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27092809/s1, Table S1: Comprehensive sensory evaluation results of green and black teas, Table S2: The volatile compositions of green teas prepared from different tea cultivars (µg guaiacol equivalent/g dry tea), Table S3: The volatile compositions of black teas from different tea cultivars (µg guaiacol equivalent/g dry tea), Table S4: The volatile compositions of freeze-dried teas from different tea cultivars (µg guaiacol equivalent/g dry tea), Table S5: The IUPAC name list for the symbols in Figure 6, Figure S1: The hierarchical heatmap of the relative contents of volatile compounds in different freeze-dried samples, Figure S2: The PCA result of freeze-dried samples of different tea cultivars based on the volatile compositions. (A) score plot; (B) loading plot. The number of replicates is equal to 3.