HS−SPME/GC−MS Reveals the Season Effects on Volatile Compounds of Green Tea in High−Latitude Region

This study investigates the volatile compounds of green tea produced with different leaves from spring, summer, and autumn in high−latitude region. A total of 95 volatile compounds were identified by gas chromatography–mass spectrometry (GC–MS). Spring, summer and autumn green tea contained 68, 72 and 82 volatile compounds, respectively. Principal component analysis (PCA), partial least squares−discrimination analysis (PLS−DA), and hierarchical cluster analysis (HCA) classified the samples and showed the difference. And 32 key characteristic components were screened out based on variable importance in the projection (VIP) values higher than 1.0. The characteristic volatile compounds of spring green tea including 18 components, such as geranylacetone, phenethyl alcohol, geraniol, β−ionone, jasmone, 1−octen−3−ol and longifolene. 13 components such as 2−methylfuran, indole, 1−octanol, D−limonene and ethanethiol were the key compounds in summer green tea. And 2,4,6−trimethylstyrene was the major differential volatile compounds in autumn green tea. The results increase our knowledge of green tea in different seasons and provide a theoretical basis for production control of green tea.


Introduction
Green tea, classified as unfermented tea, accounts for 60% of China's annual tea production, and is widely appreciated by consumers owing to its unique aroma [1,2]. Aroma is a key indicator in determining the sensory quality and economic value of the tea [3]. Tea aroma is basically related to volatile compounds. Although the volatile compounds approximately represent only 0.01% of dry weight of tea, they play an important role in the quality of tea due to their low threshold values [4][5][6][7]. At present, almost 700 volatile compounds have been detected in tea but only about 300 in green tea [8,9]. These volatile compounds can be divided into the following classes: alcohols, aldehydes, ketones, esters, hydrocarbons, sulfur compounds, and nitrogen compounds [2,7]. Due to the complexity of aroma components and the differences in aroma of different teas, identification of aroma has attracted great interest [10].

HS−SPME Method
The extraction of green tea volatile compounds was conducted by reported HS−SPME method with minor modifications [40,41]. Briefly, 6.0 g of the ground tea sample were placed in a 100 mL vial. After adding 4 g NaCl and 20 mL 100 • C distilled water, the rotor of the magnetic stirrer was put into it, then the vial was put in a water bath at 60 • C for 5 min, followed by exposure to a 75 µm CAR/PDMS coating fiber for 1 h. After the extraction was completed, the SPME fiber was inserted into the injector of the gas chromatograph at 250 • C for 5 min to desorb the analytes. Each sample was repeated three times.

GC-MS Analysis
The GC−MS analytical procedure was based on previous study [40]. Chromatographic column is Agilent DB−5MS capillary column (30 m × 0.25 mm × 0.25 µm). The temperature of GC injector was 250 • C. Helium (percentage purity > 99.999%) was used as carrier gas at a constant flow rate of 1 mL/min. The oven temperature was held at 50 • C for 5 min, increased to 180 • C at 3 • C/min (held for 2 min), then increased to 250 • C at 10 • C/min (held for 3 min), and finally increased 280 • C at 10 • C/min (held for 3 min). The mass spectrometer was operated in an electron−impact mode of 70 eV. The temperatures of the ion source, quadrupole, and interface were 230 • C, 150 • C, and 280 • C, respectively, and the acquisition mode was full scan (from 30 to 400 aum).

Data Processing
The raw data acquired by GC-MS were first deconvolved using Agilent Mass Hunter Qualitative Analysis software (Agilent Technologies Inc. Palo Alto, CA, USA). Volatile compounds were tentatively identified by comparing their mass spectra (MS) and the practical retention indices (RI, determined by n−Alkanes C 6 −C 25 ) with information from National Institute of Standards and Technology (NIST) library. RI was calculated with the retention time of each compound according to previous literature [42]. Relative contents of the identified compounds were obtained by dividing the area of a single peak by the total areas.
All identified compounds were used for statistical analysis. One−way ANOVA (Duncan's multiple range tests) was used for data analysis by SPSS 25.0 software (Demo version, Armonk, NY, USA). p < 0.05 was considered to be significantly different. According to the composition and relative content of volatiles, PCA and PLS−DA models were conducted by SIMCA−P 14.1 software (Umetrics, Umea, Sweden). The key volatile compounds responsible for each sample were screened by variable influences in projection (VIP) > 1.0 [38]. A hierarchical cluster analysis (HCA) heat map was generated using Multi Experiment Viewer (MEV) software (Oracle Corporation, Redwood Shores, CA, USA).

Sensory Quality Analysis
The sensory evaluation results of green teas are shown in Table 1. The appearance, liquor color, aroma, taste, infused leaves of green tea in different seasons have significant differences. Spring tea had the highest total score followed by autumn tea and summer tea. Interestingly, green teas produced in different seasons had different aroma characteristics. Spring tea had an obvious chestnut−like aroma and scored the highest for aroma, while summer tea and autumn tea had clean and floral aromas, respectively, both of which performed moderately. A similar trend was also observed in the score of liquor color and taste, which followed the order of spring tea > autumn tea > summer tea. The liquor color of spring tea, summer tea and autumn tea had the tender yellowish, blue dull, yellowish green, respectively. As for taste, spring tea was fresh, thick and had a sweet aftertaste; summer tea was astringent and strong; autumn tea had a bitter aftertaste and was not strong enough. In addition, the score order of appearance and infused leaves was spring tea > summer tea > autumn tea. The appearance of spring tea was tight, thin and tender green, while summer tea and autumn tea were black green and coarse, respectively. The infused leaves of spring tea, summer tea and autumn tea were tender green, yellowish green, and dull green, respectively. The results showed that different picking seasons are important factors affecting the quality of tea, not only the score of tea aroma was different, especially the type of aroma was changed. Note: Data are presented as mean value ± standard deviation (mean ± SD). Different small letters indicate significant differences (p < 0.05)

Principal Component Analysis
The PCA model was established based on the relative content of volatile components. As shown in Figure 2A, tea samples of different seasons were successfully divided into three groups, indicating that each group possessed a unique aroma profile. Spring, summer, and autumn tea was in the third, second and fourth quadrants, respectively. Principal component 1 (PC1) and principal component 2 (PC2) explained 58.8% and 29.6% of the total variation (88.4%), respectively. With the passage of seasons, samples of different seasons were distributed from left to right on PC1. with VIP values > 1.0 were identified based on the established PLS−DA model ( Figure 2D). These 32 key differential volatile compounds played a crucial role in the formation of aroma quality of green tea in different seasons. Among them, 5−methylthiazole, 2−methyl−furan, m−Anisidine, 2,6−dimethyl−6−(4−methyl−3−pentenyl)−bicyclo [3.1.1.]hept−2−ene, geraniol, indole, 3−methyl−1−butanol, 1−octanol, geranylacetone, and β−Ionone were the major differential compounds among three green teas.   Figure 2D are serial numbers of the aroma components in Table 2).

Partial Least Squares−Discrimination Analysis
Partial least squares−discrimination analysis (PLS−DA) was adopted to compare the volatile profiles of green tea in three seasons. In Figure 2B, the score plot showed that green tea samples in three seasons were completely separated. And the model parameters (R 2 Y = 0.995, Q 2 = 0.991) indicated the robustness of the model. Then, the effect of modeling was evaluated by the method of substitution test. The low intercepts (R 2 = 0.276, Q 2 = −0.259) was obtained through 200 times cross−validations, which demonstrated that there was no overfitting phenomenon, and this model was reliable ( Figure 2C).
important in the projection (VIP > 1) key volatile components (numbers in Figure 2D are serial numbers of the aroma components in Table 2).

Discussion
Differences in the content and composition of volatile compounds result in different types of tea aroma. In our study, 32 key compounds were identified based on multivariate statistical analysis. Geraniol (Sweet), β−ionone (woody, violet−like), jasmone (woody, floral), and 1−octen−3−ol (mushroom−like, earthy) had higher content in spring tea, which might be key source of the chestnut−like characteristic of spring tea [33,44,45]. Previous study demonstrated that geranylacetone, phenethyl alcohol, 1−octen−3−ol, and longifolene were the key odorants of the chestnut−like aroma [13,15,38,46], which was consistent with our findings. Additionally, the key compounds of summer tea including 1−octanol (green), D−limonene (citrus−like, fresh), 2−methylfuran (chocolate), ethanethiol (sulfurous, fruity) play an important role in the aroma profile of summer tea. And D−limonene has been reported to contribute to the clean aroma of green tea [38], which was in keeping with our results. The research showed that (Z)−methyl epijasmonate was responsible for the orchid aroma of green tea [47]. In present study, 2,4,6−trimethylstyrene was the key compounds of autumn tea. The difference in key component from those previously reported for floral aroma may be due to differences in tea cultivars and origins.
The aroma quality is affected by the harvest season, cultivar, origin, manufacturing process [48,49], of these, season is a crucial factor. In tea leaves, aroma components are mainly produced through enzyme−assisted transformation and degradation of precursors [21]. Glycosides, carotenoids, amino acids, fatty acids, and terpene derivatives are the main tea aroma precursors [50]. The synthesis of these aroma precursors is affected by seasonal climate changes such as light, temperature and humidity, which further affect the generation of volatiles. The concentration of glycoside precursors and glycosidic enzymes seasonally change in tea leaves, expressed from high to low as spring > summer > autumn [50,51]. In our study, the contents of linalool, geraniol, benzyl alcohol, and phenethyl alcohol synthesized from their corresponding glycoside precursors showed the similar trend [51,52]. β−ionone is an important contributor to the aroma of green tea due to its low odour threshold [53], which comes from the primary oxidation of β−carotene [50]. In previous studies, carotenoids are regulated by light and temperature, and had highest content in spring tea [54][55][56]. This was similar to our results that the content of β−ionone was most abundant in spring tea. Additionally, the aroma score of summer tea was the lowest in this study. Amino acids are important substances for the formation of tea aroma though the Maillard reaction [57]. However, studies have shown that strong light in summer results in less amino acids in summer tea [19,58], reducing the source of aroma in summer tea, which is consistent with our study. At present, the exact seasonal climate effects on volatile compounds in tea have not been reported, the biosynthesis pathways of key aroma components in different seasons needs further study to clarify.

Conclusions
Season is an important factor affecting the aroma of tea. In this study, according to the sensory evaluation results, spring tea, summer tea, and autumn tea showed chestnut−like, clean, and floral aroma, respectively, and the aroma score was ranked as spring tea > summer tea > autumn tea. 32 key compounds were identified. Among them, 18 volatile compounds including geranylacetone, phenethyl alcohol, geraniol, β−ionone, jasmone, 1−octen−3−ol, longifolene were the key compounds in spring tea; 13 volatile compounds including 2−methylfuran, indole, 1−octanol, D−limonene, ethanethiol were key compounds in summer tea; the key component of autumn tea was 2,4,6−trimethylstyrene. This study enriched the aroma theory of green tea from the high latitude region and provided scientific basis for quality control of green tea production.