Aroma Difference Analysis of Partridge Tea (Mallotus oblongifolius) with Different Drying Treatments Based on HS-SPME-GC-MS Technique

Partridge tea has high medicinal value due to its rich content of terpenoids, phenols, flavonoids, and other related bioactive components. In order to study the best drying method for partridge tea, four treatments, including outdoor sun drying (OD), indoor shade drying (ID), hot-air drying (HAD), and low-temperature freeze-drying (LTD), were performed. The results showed that the OD and HAD treatments favored the retention of the red color of their products, while the ID and LTD treatments were more favorable for the retention of the green color. The HS-SPME-GC-MS results showed that a total of 82 compounds were identified in the four drying treatments of partridge tea, and the most abundant compounds were terpenoids (88.34–89.92%). The HAD-treated tea had the highest terpenoid content (89.92%) and high levels of flavor compounds typical of partridge tea (52.28%). OPLS-DA and PCA showed that α-copaene, β-bourbonene, caryophyllene, α-guaiene, and δ-cadinene could be considered candidate marker compounds for judging the aroma quality of partridge tea with different drying treatments. This study will not only provide a basis for processing and flavor quality control but also for spice and seasoning product development in partridge tea.

The drying method plays a significant role in tea processing, as it directly affects the aroma, taste, color, and shelf life of tea [9]. Previously, the traditional drying methods for partridge tea were indoor shadow drying (ID) and outdoor sun drying (OD), which were time-consuming and left the tea vulnerable to environmental factors. However, nowadays, there are numerous alternative methods available for drying tea leaves. For example, two modern drying methods for tea leaves are low-temperature freeze-drying technology (LTD) and hot-air-drying technology (HAD). HAD of agricultural products is one of the most popular preservation methods because of its simplicity and low cost [10]. Moreover, previous studies had found that the polyphenol content, volatile compound content, and antioxidant capacity of hot-air-dried tea were higher than those of outdoor sun-dried tea [11]. It is well known that LTD is a drying technology that allows the product to change very little from its original state [12]. Previous research found that low-temperature freeze-dried rose tea, compared to dryer-dried, not only had improved physicochemical L* 38.95 ± 3.33 b 40.56 ± 3.83 b 47.28 ± 1.90 a 48.14 ± 3.07 a a* 11.69 ± 2.09 b 11.91 ± 1.44 b 5.55 ± 2.25 c 16.95 ± 2.00 a b* 5.72 ± 3.46 b 5.52 ± 2.41 b 13.86 ± 4.29 a 1.11 ± 0.64 c Note: L* denotes luminance; a* denotes poor red-green, b* denotes poor yellow-blue [36,37]. Data are presented as mean ± standard deviation (n = 6). Mean values with different lowercase letters in the same column indicate significant differences according to the least significant difference (LSD) test (p < 0.05).

Classification of Volatile Compounds in Partridge Tea
A total of 82 compounds, including 41 alkenes, 19 aldehydes, 14 alcohols, 3 ketones, 1 alkane, and 4 esters, were identified in the four dried treatments of partridge tea via HS-SPME-GC-MS. As shown in Figure 2, there were differences in the percentages of volatile component species in different dried samples. Terpenoids were the highest in both content and quantity among the 82 compounds. The highest content of terpenoids (89.92%) was found in the HAD-treated partridge tea, along with ID (89.23%), OD (88.34%), and LTD (88.80%), indicating that the HAD-treated partridge tea was able to retain more flavor compounds. Terpenes are natural compounds found primarily in the plant world [38]. Most terpenes have antibacterial, anticancer, anti-inflammatory, and other biological activities [39], and are widely used in the pharmaceutical, food, and cosmetic industries [40]. Cells in the leaves shrink due to uneven dehydration during slow drying, so the dried leaves curl [32], whereas the HAD-treated leaves had less leaf curling due to their high drying temperature and faster water dissipation. The reason for less curling of the leaves in the LTD-treated tea was that during the freeze-drying process, the solid water protects the basic structure and shape of the product with minimal volume change [33]. Higher temperatures during tea drying result in elevated theaflavins, theasinensins, thearubigins, and theabrownins, and most of these substances show a yellow color [34,35], so most of the partridge tea samples dried at higher temperatures showed yellow and yellow-brown colors. The lower temperature could better preserve the original color of the samples to achieve better preservation [12]. Note: L* denotes luminance; a* denotes poor red-green, b* denotes poor yellow-blue [36,37]. Data are presented as mean ± standard deviation (n = 6). Mean values with different lowercase letters in the same column indicate significant differences according to the least significant difference (LSD) test (p < 0.05).

Classification of Volatile Compounds in Partridge Tea
A total of 82 compounds, including 41 alkenes, 19 aldehydes, 14 alcohols, 3 ketones, 1 alkane, and 4 esters, were identified in the four dried treatments of partridge tea via HS-SPME-GC-MS. As shown in Figure 2, there were differences in the percentages of volatile component species in different dried samples. Terpenoids were the highest in both content and quantity among the 82 compounds. The highest content of terpenoids (89.92%) was found in the HAD-treated partridge tea, along with ID (89.23%), OD (88.34%), and LTD (88.80%), indicating that the HAD-treated partridge tea was able to retain more flavor compounds. Terpenes are natural compounds found primarily in the plant world [38]. Most terpenes have antibacterial, anticancer, anti-inflammatory, and other biological activities [39], and are widely used in the pharmaceutical, food, and cosmetic industries [40]. Therefore, the HAD treatment was favorable for improving the nutritional and medicinal value of partridge tea. Therefore, the HAD treatment was favorable for improving the nutritional and medicinal value of partridge tea.

Identification of Volatile Compounds in Partridge Tea
Among the 82 compounds identified (Table 2, Figure 3), 55 components were significantly different between the four drying treatments (p < 0.05). In this study, the content of α-copaene, β-bourbonene, caryophyllene, α-guaiene, α-caryophyllene, and δ-cadinene was found to be higher and all were terpenes, and are typical partridge tea flavor compounds according to previous studies [41]. These flavor compounds were highest in the HAD-treated (52.28%), followed by the OD-treated (49.64%), and then, the LTD-treated (45.34%) and ID-treated (43.78%) tea. All of these typical flavor compounds of partridge tea have a chemical structure of C15H24 and are sesquiterpenoids, which have excellent anti-tumor, anti-inflammatory, and antibacterial properties [42]. Particularly, caryophyllene and β-bourbonene have been confirmed to have good anticancer activity, and caryophyllene shows a selective antiproliferative effect on colorectal cancer cells [43,44]. β-bourbonene was shown to inhibit the proliferation of prostate cancer PC-0M cells [45]. Our results show that the HAD drying method was more favorable for the retention of terpenoids, and the HAD-treated partridge tea had better biological activities such as anticancer and antivirus.

Identification of Volatile Compounds in Partridge Tea
Among the 82 compounds identified (Table 2, Figure 3), 55 components were significantly different between the four drying treatments (p < 0.05). In this study, the content of α-copaene, β-bourbonene, caryophyllene, α-guaiene, α-caryophyllene, and δ-cadinene was found to be higher and all were terpenes, and are typical partridge tea flavor compounds according to previous studies [41]. These flavor compounds were highest in the HAD-treated (52.28%), followed by the OD-treated (49.64%), and then, the LTD-treated (45.34%) and ID-treated (43.78%) tea. All of these typical flavor compounds of partridge tea have a chemical structure of C 15 H 24 and are sesquiterpenoids, which have excellent antitumor, anti-inflammatory, and antibacterial properties [42]. Particularly, caryophyllene and β-bourbonene have been confirmed to have good anticancer activity, and caryophyllene shows a selective antiproliferative effect on colorectal cancer cells [43,44]. β-bourbonene was shown to inhibit the proliferation of prostate cancer PC-0M cells [45]. Our results show that the HAD drying method was more favorable for the retention of terpenoids, and the HAD-treated partridge tea had better biological activities such as anticancer and antivirus.

Orthogonal Partial Least Squares Discriminant Analysis
PCA and OPLS-DA were used to downgrade and screen the differential components of partridge tea treated with different drying methods, and then, classify the chemotypes according to the dominant compounds among the different drying treatments. PCA

Orthogonal Partial Least Squares Discriminant Analysis
PCA and OPLS-DA were used to downgrade and screen the differential components of partridge tea treated with different drying methods, and then, classify the chemotypes according to the dominant compounds among the different drying treatments. PCA showed that PC1 and PC2 accounted for 51.6% of the total variability of the model. As shown in Figure 3B (R2X = 0.691, R2Y = 0.977, Q2 = 0.904), the orthogonal partial least squares (OPLS-DA) analysis was performed to obtain the variable importance of projection (VIP) values (important variable in the projection).
Based on the results of OPLS-DA, volatile compounds with VIP values > 2 were selected, and the S-plot showed 12 compounds with significant differences among the four different drying treatments of partridge tea (data point triangle on the plot) ( Figure 4C-H), including 2-methyl-2-butenal, (e)-2-hexenal, α-copaene, β-bourbonene, β-maaliene, caryophyllene, isogermacrene d, α-guaiene, (e)-β-famesene, eremophilene, (+)-ledene, and δ-cadinene. These 12 compounds were significantly different in the four different drying treatments and could be considered candidate marker compounds for judging the aroma quality of partridge tea with different drying treatments, and provide a reference for the use of partridge tea for making fragrances and aromatics as well as for the control of partridge tea product quality.
Molecules 2023, 28, x FOR PEER REVIEW 7 of 11 aroma quality of partridge tea with different drying treatments, and provide a reference for the use of partridge tea for making fragrances and aromatics as well as for the control of partridge tea product quality.

Sample Preparation
The experimental samples were collected on 12 March 2023 at the Wild Partridge Tea Germplasm Resource Nursery of Hainan University, and we selected partridge tea plants with relatively consistent leaf color and growth conditions as the sample collection objects. The four groups of samples were collected from the third fresh leaves under the buds, with three replicates in each group, and each replicate was collected with about 50 g for the subsequent drying process (Table 3).

Volatile Extraction via HS-SPME Procedure
The samples obtained from different drying treatments were crushed and 0.5 g added to a headspace flask, and the headspace flask was placed in a water bath at 65 • C for extraction. Before extraction, the solid-phase microextraction head was aged at the gas chromatography inlet at 250 • C for 10 min. After a period of sample equilibration (5 min), the needle of the SPME device was inserted into the vial through the septum, and the fiber was exposed to the vial headspace for 40 min. After that time, the fiber was retracted into the needle assembly, removed from the vial, transferred to the injection port of the GC unit, and immediately desorbed.

Separation of Volatile Organic Compounds via GC-MS
An Agilent 7890A GC system, coupled with a Tekmar 5975C mass spectrometer (Agilent/Tekmar, Santa Clara, CA, USA), was applied in the present study. An Agilent HP-5MS capillary column (30 m × 0.25 mm, 0.25 µm) was employed to separate the volatiles. Helium (>99.99%) was used as the carrier gas at a constant flow rate of 1 mL/min. The GC oven temperature was maintained at 40 • C for 4 min, ramped at a rate of 5 • C/min to 160 • C, and ramped at a rate of 10 • C/min to 260 • C. The mass spectrometer conditions were as follows: electron ionization energy of 70 eV, ion source temperature of 230 • C, interface temperature of 280 • C, and mass scan range of 50-550 m/z.

Identification and Relative Quantification of Volatile Organic Compounds via GC-MS
The obtained chromatograms were analyzed by determining the peak areas, retention times, spectra, and base peaks on the chromatograms, and then, each peak was examined by referring to the characteristic mass spectra of the compounds listed on the National Institute of Standards and Technology (NIST14. L). Compounds were identified based on their similarity indexes (>80%) as well as retention indexes. The relative contents of each compound in the volatile compounds of tea samples were obtained via the area normalization method.

Determination of Color Differences Using a Colorimeter
A portable colorimeter (LS173B, Shenzhen Linshang Technology Co., Ltd., Shenzhen, China) was utilized to measure the color differences of partridge tea leaves subjected to four different drying treatments. Six dried leaves were selected for each treatment group. Before taking measurements, the colorimeter was calibrated using black and white references. The leaves were then placed tightly against the test holes, completely covering them, and measurements were subsequently recorded.

Statistical Analysis
Results were expressed as the mean ± SD (n = 3) for each analysis. Statistical analysis was performed via one-way analysis of variance (ANOVA) using IBM SPSS 26.0 (SPSS Inc., Chicago, IL, USA). Differences between the groups were evaluated via the Duncan test for multiple comparisons. Origin 2018c (OriginLab, Northampton, MA, USA) was used for graphic drawing. p < 0.05 was considered to indicate a statistically significant difference. SIMCA 14.1 (Sartorius AG, Göttingen, Germany) software was used for orthogonal partial least squares discriminant analysis (OPLS-DA).

Conclusions
In this study, HS-SPME-GC-MS and a colorimeter were used to identify volatile compounds and the appearance quality of partridge tea using four drying methods. It was found that the drying methods had a greater influence on the appearance of the tea leaves. Cold drying methods (ID and LTD) had a greater effect on the retention of leaf color, while the two relatively hot-air drying methods (OD and HAD) had a poorer effect on the retention of leaf color, with an overall darker brown color. In addition, a total of 82 compounds were identified in the different drying treatments for partridge tea. Among them, terpenoids were the most important class of compounds contributing to the aroma composition of partridge tea. And HAD had the highest terpenoid content among the four drying treatments. Meanwhile, we found that the five active ingredients were α-copaene, β-bourbonene, caryophyllene, α-guaiene, and δ-cadinene, and the HAD treatment had a relatively good retention effect on these compounds, which could be used as marker compounds to evaluate the quality of partridge tea. This study investigated the effect of different drying methods on the volatile compounds and the appearance quality of partridge tea, which provides a basis for the selection of processing methods for partridge tea. Finally, this study confirmed essential volatile compounds via a chemometric method in partridge tea, which provides a basis for the application of partridge tea in spices and seasonings.