Effects of Different Fixation Methods on Color, Aroma, and Chemical Composition of Lonicerae japonicae Flos Tea

Abstract

Lonicerae japonicae flos (LJF) is a natural product with medicinal, edible, and ornamental value which has been developed into LJF tea. At present, LJF tea can be processed using four main fixation methods: fixation by sun drying (SG), hot-air oven drying (HG), stir-frying drying (CZ), and steaming (ZZ). However, a comparative analysis of the effects of different fixation methods on the quality of LJF tea has not been reported. This study comprehensively investigated the effects of these fixation methods on the appearance color, volatile components, and non-volatile components of LJF tea samples. Our findings demonstrated that LJF tea in the SG group had the highest L value, causing a brighter appearance, which was mainly caused by the retention of organic acids and flavonoids. Additionally, LJF tea in the SG group had a higher content of aroma components than other groups. These results suggested that sun drying may be beneficial for improving the quality of LJF tea. This study provided a reference for the selection of fixation methods for LJF tea and offered a clue for quality improvement of LJF tea.

1. Introduction

Lonicerae japonicae flos (LJF) is a traditional Chinese medicine with properties of both medicinal and edible use, which is cultivated in numerous regions, such as China, New Zealand, Argentina, and other countries [1]. LJF has good medicinal value, ornamental value, economic value, and ecological value [2]. Modern research has demonstrated that LJF contains multiple active substances. For instance, chlorogenic acid and caffeic acid, belonging to the category of organic acids, have significant anti-inflammatory and antioxidant characteristics [3,4]. Rutin, quercetin, luteolin and other flavonoids demonstrate potential for treating diabetes, cardiovascular and cerebrovascular diseases, liver diseases and other diseases [4]. Additionally, volatile oil is one type of active substance with multiple functions, such as promoting expectoration and relieving cough, strengthening the stomach, reducing fever and relieving pain, as well as exerting antibacterial and anti-inflammatory effects [5]. Due to the presence of these functional compounds, LJF has been developed into a variety of functional foods. These include health-promoting herbal teas, herbal tea beverages, and herbal-based dietary products [6,7]. Among them, LJF tea is the most consumed healthy herbal tea, which belongs to the green tea family. The quality of LJF tea mainly depends on the relative content of non-volatile components affecting their functional properties and volatile components influencing their aroma. Notably, the relative content of these chemical components is mainly affected by different processing methods [8].

Fixation is an important and final step in the tea-making process. During this stage, the tea leaves are heat-treated to eliminate a portion of their internal moisture, inactivate enzyme activity, drive away the grassy smell, and contribute to the formation of the characteristic flavor unique to tea [9]. At present, LJF tea can be processed using four main fixation methods: fixation by sun drying (SG), fixation by hot air oven drying (HG), fixation by stir-frying drying (CZ), and fixation by steaming (ZZ). Among them, SG is a traditional processing method for LJF tea, and this method can preserve the flavor of LJF tea [10]. HG is a method to fix tea through hot air, which has the advantage of consistent temperature and disadvantage of degrading the components [11]. CZ is a commonly used folk fixation method for LJF tea. This simple method can quickly inactivate degrading enzymes [12]. ZZ is a fixation method designed to prevent LJF tea from discoloring during the fixation process. This method can rapidly inactivate degrading enzymes in LJF tea. However, a comprehensive analysis of the effects of different fixation methods on the chemical component contents in LJF tea has not been reported.

Metabolomics based on LC–MS serves as an approach applied to reveal the composition and alterations of chemical compounds. This method has the capacity to identify potential markers, including those present at low concentrations [13]. Gas chromatography–ion mobility spectrometry (GC–IMS) is a powerful technique for the analysis of volatile compounds within samples. It provides several benefits, such as high sensitivity, straightforward sample pretreatment, and high detection efficiency [14]. Thus, metabolomics and GC–IMS are conducive to comprehensively elucidating the effects of different fixation methods on the contents of chemical components in LJF tea.

The major aim of the study was to use a colorimeter to determine the color characteristics of LJF tea samples with different fixation methods, and utilize GC–IMS and ultra-performance liquid chromatography–high resolution mass spectrometry (UPLC–HRMS) to analyze the relative content of volatile and non-volatile compounds in LJF tea with different fixation methods and, concurrently, to reveal the intrinsic mechanism of color changes in LJF tea caused by different fixation methods. This study will fill the gap in the research regarding the differences in chemical composition among LJF teas with different fixation methods. Meanwhile, this study will provide a reference for the selection and processing of high-quality LJF tea.

2. Materials and Methods

2.1. Fixation of LJF Tea

Fresh LJF samples (bud stage) were purchased from Linyi City, Shandong Province, China. The authenticity of these LJF samples was identified by Professor Jia Li, Shandong University of Traditional Chinese Medicine. Subsequently, the fixation of LJF tea was performed using four fixation methods, respectively. Briefly, the fresh LJF samples were spread evenly on a dry and clean bamboo-woven winnowing basket. The thickness of the spread layer was approximately 0.3 cm. Then, they were placed in a location with sufficient sunlight for natural sun drying until the LJF tea samples reached a constant weight. Finally, the LJF tea samples of the SG group were obtained. In the CZ group, fresh LJF samples were placed in a pre-heated wok. Stir-fry the LJF tea samples for 2 min, and then use low-temperature hot air drying (40 °C) to dry until they reach a constant weight. In the ZZ group, fresh LJF samples were placed in a pre-heated steamer. Steam for two min, and then use low-temperature hot air drying (40 °C) to dry until they reach a constant weight. In the HG groups, fresh LJF samples were placed in a hot air circulation dryer (YIHENG Technical Co., Ltd., Shanghai, China). The baking temperatures were set to 40, 60, and 80 °C, respectively [15,16]. Dry the LJF tea samples to a constant weight and name them HG40, HG60, and HG80 groups.

2.2. Color Analysis of LJF Tea Samples

The appearance color of the LJF tea samples was assessed using an NH300 high-quality colorimeter (3nh Technology Co., Shenzhen, China). All samples were in triplicate and named sequentially (biological replicates, e.g., SG replicates: SG_1, SG_2, SG_3). The color properties mainly involve three parameters, including L value, a value, and b value. Among them, the L value denotes lightness, a value represents the red–green axis, and the b value represents the yellow–blue axis [17]. Color differences (ΔE) are employed to evaluate the color changes in these samples. ΔE is calculated according to the following equation:

$$\Delta \mathrm{E}=\sqrt{{\left(L^{\ast }-L\right)}^2+{\left(a^{\ast }-a\right)}^2+{\left(b^{\ast }-b\right)}^2}$$

where L*, a*, and b* signify the parameter values of the white plate, while L, a, and b stand for the parameter values of the LJF tea samples.

2.3. Analysis of Volatile Components Based on GC–IMS

Headspace gas chromatography–ion mobility spectrometry (HS-GC–IMS) was utilized to analyze the volatile compounds (VOCs) in LJF tea samples with different fixation method groups. All samples were in triplicate and named sequentially (biological replicates, e.g., SG replicates: SG_1, SG_2, SG_3). First, all samples were ground and sieved through a 40-mesh sieve. Next, precisely 0.5 g of each sample was weighed and placed into a 20 mL headspace vial. These samples were then incubated at 80 °C for 15 min. After that, a constant headspace volume of 500 μL was automatically injected into the injector by a heated syringe (maintained at 85 °C). Separation of the LJF tea samples was carried out using an MXT-WAX capillary column (30 m × 0.53 mm, 1.0 μm, RESTEK company, PA, USA) with N₂ (99.99% purity) as the carrier gas. The column separation procedure was as follows: initially, the flow rate was maintained at 2 mL/min for 2 min. Then, after 3 min, it was increased to 10 mL/min. After 20 min, the flow rate was further ramped up to 100 mL/min and was sustained at this rate for 5 min. The temperature of the capillary column remained at 60 °C during the whole separation process. After completing the separation by the capillary column, the VOCs entered the IMS detector (Hanon, Jinan, China), where further isolation occurred with 99.99% N₂ at a flow rate of 150 mL/min.

The IMS data were acquired using Standalone (G.A.S., version 1.2.0, Hanon, Jinan, China). Subsequently, they were analyzed through instrumental software, such as LAV (version 2.2.1, Hanon, Jinan, China) and GC × IMS library Search (version 1.0.3, Hanon, Jinan, China). The identification of VOCs was achieved by integrating the retention index (RI) and drift time, along with the retrieval from the NIST database and the IMS library provided by G.A.S.

Following the GC–IMS analysis, a data matrix of the form K × N containing peak heights was produced, where K corresponded to the quantity of samples, while N denoted the number of VOCs. Prior to further analysis, this matrix underwent a normalization process. The selection of featured compounds served to eliminate redundant information and enhance the generalization capacity of models. Thus, partial least squares discriminant analysis (PLS-DA) was carried out to screen for the characteristic VOCs. Meanwhile, 100 permutation tests were used to evaluate the overfitting of PLS-DA model. Afterward, principal component analysis (PCA) was employed to visually display the differentiation among these LJF tea samples.

2.4. Analysis of Non-Volatile Components Based on UPLC–HRMS

Non-volatile components in LJF tea samples were detected using UPLC–HRMS. All samples were prepared in six biological replicates and named sequentially (SG replicates: SG_1, SG_2, SG_3, SG_4, SG_5, SG_6). Approximately 300 mg of LJF tea powder was placed in a container, and then 3.0 mL of 70% methanol was added. Using an ultrasonic constant-temperature cleaning machine (Scientz, Ningbo, China), the mixture was extracted at 30 °C under 350 W conditions for 20 min. Subsequently, it was centrifuged at a speed of 10,000 r/min for 5 min. After that, the obtained supernatant was filtered through a 0.22-micrometer microporous membrane to prepare for UPLC–Q-TOF-MS/MS analysis.

Sequential injection analysis was carried out on the supernatant obtained from the LJF tea sample extract. To ensure the integrity of the analysis, quality control (QC) samples were regularly injected after every ten samples. Specifically, three QC samples were injected at the beginning and end of the analysis process. For the analysis of the chemical composition, an ultra-high performance liquid chromatography system (H-Class, Waters, Milford, MA, USA) combined with a Q-TOF mass spectrometer (Impact II, Bruker, Karlsruhe, Germany) was employed. The separation of chemical compounds took place on a Waters ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm), which was maintained at a temperature of 30 °C. Subsequently, a gradient elution method was applied. The mobile phases used were a 0.1% formic acid aqueous solution (A) and a 0.1% formic acid acetonitrile solution (B). The gradient elution protocol was as follows: 0~10 min, 5~95% B; 10~11 min, 95–95% B; 11~12 min, 95–5% B; 12~16 min, 5–5% B. The flow rate was adjusted to 0.3 mL/min, and the injection volume was set at 1 μL.

An electrospray ionization (ESI) source was used in the mass spectrometer. Specifically, for positive ionization, the capillary voltage was tuned to 3500 V, while for negative ionization, it was set at 3000 V. The drying gas flowed at a steady rate of 8 L/min, and the nebulizer gas pressure was held constant at 200 kPa. The quadrupole ion energy was fixed at 3 eV. Inside the collision pool, the collision energy was 7-Ev, the transfer time was 60 μs, and the RF voltage amplitude was 750 Vpp. The mass spectrometer scanned over a mass-to-charge ratio (m/z) range from 50 to 1500.

The data obtained from the analysis were processed with AntDAS software, which involved peak detection and peak alignment. The following parameter settings were applied: the m/z precision was also set at 0.02 Da, the m/z tolerance was also 0.02 Da, the time shift was configured to 2 min, the signal-to-noise ratio (S/N) was set to 10, and the Gaussian similarity was set at 0.6. For peak alignment, the time shift was 2 min, and for component registration, the time shift was 0.3 min. The identification of metabolites was conducted based on m/z and secondary fragments according to public databases such as PubChem and PMhub. After the processing, a K × N data matrix containing peak area was generated. Then, PLS-DA was carried out to select the characteristic compounds, and 100 permutation tests were used to evaluate the overfitting of the PLS-DA model. Afterward, PCA was employed to visually display the differentiation among these LJF tea samples.

2.5. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 10 and R-studio software (version 2024.04.2-764). One-way analysis of variance (ANOVA) was used to evaluate significant differences among multiple groups. A p value < 0.05 was considered indicative of a statistically significant difference.

3. Results and Discussions

3.1. Appearance Color of LJF Tea Samples

The appearance color is one of the important indicators of tea appearance quality. As shown in Figure 1, in the SG group, the appearance color of LJF tea samples was yellow, while the b value of LJF tea samples in the SG group (78.61 ± 0.60) was lower than that in other groups (Table S1). This may be attributed to the black color masking the yellow color. Meanwhile, LJF tea samples in the ZZ group retained their original appearance color. Further, after the stir-frying process, LJF tea samples showed a dark brown color, which may be caused by the decrease in the L value. On the contrary, the L value of the SG group was the highest, indicating that the appearance of the samples in this group was brighter. In the HG groups, as the drying temperature increased, the color of LJF tea gradually darkened, which was consistent with changes in the L value. Based on the appearance color of these LJF tea samples, ΔE was calculated, and showed results consistent with the appearance color of LJF tea samples (Figure S1).

3.2. VOCs Detected in LJF Tea Samples

Volatile organic compounds (VOCs) constitute an important component and possess various functional characteristics, including antibacterial, anticancer, and anti-inflammatory properties [1,18]. These compounds could be principally categorized into four classes: aldehydes, alcohols, ketones, and esters [19]. Consequently, GC–IMS was utilized to investigate the disparities in the content of volatile compounds among LJF tea samples processed with different fixation methods. Based on the results of GC–IMS, the 3D chromatograms of LJF tea samples with different fixation methods were established. As depicted in Figure S2, distinct variables were represented by the axes: the x-axis stood for the drift time, while the y-axis represented the retention time. The red dots on the graph signified a higher content of VOCs, and the blue dots indicated a lower content. Then, based on their actual drift times and retention indices, the VOCs were identified using the IMS database. As depicted in Table S2, a total of forty VOCs were identified across the LJF tea samples with different fixation methods. These VOCs could be categorized into eight groups: six esters, nine aldehydes, one organic acid, one ether, seven alcohols, seven ketones, eight alkanes, and one other. Table S2 provides the detailed information, including abbreviations and full names, CAS number, molecular weight (Mw), retention index (RI), retention time (Rt) and drift time (Dt), of the VOCs identified by GC–IMS. When a “D” was appended to the name of a compound, it indicated that the compound existed in a dimer form and exhibited longer drift times. This phenomenon might have been caused by its proton affinity and relatively high content.

Subsequently, a heatmap of the VOCs in LJF tea samples with different fixation methods was generated using the peak heights from the top view of 3D chromatograms. As presented in Figure 2D, LJF tea samples within the same group exhibited comparable VOC content, demonstrating the relative stability of the processing methods. Moreover, the content of certain VOCs underwent specific alterations. For example, as shown in the purple box of Figure 2D, the content of these VOCs increased with the temperature of the fixation methods, mainly involving alcohols, ketones and aldehydes. In the green box of Figure 2D, the content of these VOCs relatively increased, indicating that the LJF tea samples in the SG group had a distinct aroma.

Alcohols belong to the category of volatile compounds. They are chiefly characterized by scents reminiscent of flowers and fruits [20]. Their origin can be mainly traced back to the degradation process of fatty acids [21]. In this study, a total of seven alcohols were detected. Meanwhile, three alcohols, including (Z)-3-hexen-1-ol, 2-hexen-1-ol and 1-Hexanol, presented higher content in LJF tea samples in the SG group compared with groups such as the CZ group and the HG40 group (Figure S3A–C, p < 0.05). However, there was no significant difference between the SG group and other groups, such as the ZZ group and the HG60 group (p > 0.05). Among them, (Z)-3-hexen-1-ol is a key component that is beneficial for the increase in floral and sweet aromas [22]. 2-hexen-1-ol is an aroma component that contributes to the generation of a green odor [23]. Therefore, fixation by sun drying was beneficial for preserving the aroma of LJF tea, which may be due to the absence of wind and high temperature during the sun drying process.

Aldehydes were generated as degradation products from precursors such as fatty acids, amino acids, and carotenoids [21]. Nine aldehydes and seven ketones were annotated in the present study. As shown in Figure S3D,E, two aldehydes, namely (E, E)-2,4-hexadienal and (E)-2-hexen-1-al, were found in LJF tea samples. Among them, (E, E)-2,4-hexadienal is a volatile component, which is related to the aroma of this fruit [24]. A previous study demonstrated that (E)-2-hexen-1-al is a key aroma compound detected in flat black tea [25]. The two aldehydes showed higher content in the SG group compared with the CZ and HG60 groups (p < 0.05). The relative content of (E, E)-2,4-hexadienal showed no significant differences between the SG group and other groups, such as the CZ group and the HG80 group (p > 0.05). The relative content of (E, E)-2,4-hexadienal presented no significant difference between the SG group and the HG40 group (p > 0.05). Similarly, compared with groups such as CZ, ZZ and HG60, the SG group showed higher content of two ketones, including 2-heptanone and 2-propanone (Figure S3F,G, p < 0.05). However, there was no significant difference in these two ketone components between the SG group and the HG80 group (p > 0.05). Overall, these higher levels of aldehydes and ketones may result in the retention of aroma in LJF tea samples in the SG group.

Esters are essential aroma-forming components in tea. They are mostly formed from the degradation of amino acids [26]. These substances typically emit charming aromas, like those of fruits, sweetness, and flowers [27]. A total of six esters were annotated in the present study. Among them, ethyl 2-methy lpropionate was identified as a characteristic VOC with a sweet and rubber essence [28]. The content of Ethyl 2-methylpropanoate presented relatively high content in the SG group compared with other groups, except for the HG80 group (Figure S3H, p < 0.05), which could be attributed to the degradation of amino acids and the formation of esters within a certain temperature range. Ethyl propanoate is a key VOC contributing to the fruity aroma of red wine [29]. In this study, there was a significant difference between the SG and HG80 groups (Figure S3I, p < 0.01). However, there was no significant difference between the SG group and other groups, including the CZ, ZZ, HG40, and HG60 groups (p > 0.05). These results suggested that the SG group had a relatively high content of these esters, which contributes to the generation of aromas.

PCA, an unsupervised multivariate analysis method, was employed to examine the differences among samples within the same group or between different groups, which was accomplished by reducing data dimensionality. PCA transformed complex datasets into a more understandable format, thereby uncovering the latent patterns and variations existing among the samples [30]. As presented in Figure 2A, it was evident that LJF tea samples processed with different fixation methods could be distinctly differentiated. This indicated that there were significant changes in the content of VOCs among the LJF tea samples obtained from these various processing methods. PLS-DA is a multivariate statistical method that combines the benefits of partial least squares regression and discriminant analysis. By extracting latent variables from complex datasets, it aims to effectively classify and discriminate between different groups, thereby revealing the underlying patterns and relationships hidden within the data [31]. Thus, PLS-DA was further performed to screen featured non-volatile components that had significant changes in their relative content. The Q² and R² of the PLS-DA model were 0.908 and 0.774, respectively. Concurrently, the results of 100 permutation tests showed that the intercept of Q² on the y-axis did not exceed 0.05, indicating that the PLS-DA model had good generalization ability and no overfitting phenomenon occurred (Figure 2B). As depicted in Figure 2C, a total of six VOCs with variable importance in projection (VIP) > 1.15 and false discovery rate (FDR) < 0.05, namely 1-octen-3-one, 1-octen-3-one-D, (E)-2-heptenal, undecane, ethyl 2-methy lpropionate and 2-propanone, were screened. The changes in the relative content of these featured VOCs resulted in the specific aromas of LJF tea samples processed by different fixation methods (Figure S4).

3.3. Non-Volatile Components Detected in LJF Tea Samples

Metabolomics based on LC–MS was performed to comprehensively determine the relative content of LJF tea samples with different fixation methods, and analyze their content changes. Through qualitative matching analysis, in the positive ion mode, 1208 peak signals with relative standard deviation (RSD) < 30% were detected, while in the negative ion mode, 312 peak signals with RSD < 30% were found. The PCA score plot suggested that the clustering of the QC group samples indicated good instrument stability (Figure S5). Non-volatile components were identified based on the precursor ions and their fragment ions detected in the spectra, in combination with the PMhub, MassBank, and PubChem databases. In total, 82 metabolites were annotated, with 63 in the positive ion mode and 19 in the negative ion mode. The detailed information of these non-volatile components is presented in

Table 1. The detailed information of non-volatile components in LJF tea samples with different fixation methods.

ID	Rt/min	m/z	Compound Name	MS/MS	Class
chem1	0.84	278.1190 [M+H]+	Gly Cys Val	198.1091, 161.0652, 232.1142, 244.1149, 198.0739, 118.0847, 260.1082	Amino acids and their derivatives
chem2	0.86	210.0754 [M+H]+	3-carboxy-DL-phenylalanine	164.1039, 192.099, 118.0645, 210.1084	Amino acids and their derivatives
chem3	0.86	294.1130 [M+H]+	Met Ser Gly	230.1351, 87.0321, 132.1003, 131.9513, 115.0384	Amino acids and their derivatives
chem4	1.27	268.1011 [M+H]+	Adenosine	136.0611, 119.035	Amino acids and their derivatives
chem5	1.37	284.0946 [M+H]+	Guanosine	152.0556, 135.0295, 110.0347	Amino acids and their derivatives
chem6	1.76	249.1192 [M+H]+	Glu-Thr	120.0803, 84.0448, 102.0547	Amino acids and their derivatives
chem7	2.18	375.1215 [M+H]+	Secologanic acid	195.0628, 151.0732, 151.038, 213.0734	Organic acids and their derivatives
chem8	2.39	311.1214 [M+H]+	Glu-Tyr	146.0593, 120.0805, 294.0926, 132.0805, 249.1206, 91.0549	Amino acids and their derivatives
chem9	2.42	166.0840 [M+H]+	Phenylalanine	103.054, 120.0799, 121.0831	Amino acids and their derivatives
chem10	2.62	474.1874 [M+H]+	Glu Tyr Tyr	130.0483, 136.0768, 247.1071, 274.5686	Amino acids and their derivatives
chem11	2.65	278.0977 [M+H]+	Ser Gly Asp	117.0552, 278.0981, 145.0725, 214.1414	Amino acids and their derivatives
chem12	2.70	382.1623 [M+H]+	Asp Phe Thr	98.0232, 116.0331, 382.1765, 235.0853	Amino acids and their derivatives
chem13	2.88	492.1956 [M+H]+	Thr Glu Gln Asp	262.1044, 331.1548, 313.1432, 492.192, 116.0715	Amino acids and their derivatives
chem14	3.01	355.0961 [M+H]+	Neochlorogenic acid	163.0369, 145.0268, 135.0426, 117.0322, 164.0403	Organic acids and their derivatives
chem15	3.20	535.2023 [M+H]+	Genipin 1-O-alpha-L-rhamnopyranosyl(1->6)-beta-D-glucopyranoside	165.0534, 195.0634, 209.0788, 209.0971, 145.0488	Terpenes and their derivatives
chem16	3.28	405.1316 [M+H]+	Secoxyloganin	167.0327, 211.0579, 193.048	Terpenes and their derivatives
chem17	3.33	389.1368 [M+H]+	Vogeloside II	151.0372, 177.0527, 195.0626, 227.0883, 151.0733, 209.0786	Terpenes and their derivatives
chem18	3.41	355.0959 [M+H]+	Chlorogenic Acid	163.0378, 164.0413	Organic acids and their derivatives
chem19	3.41	554.2083 [M+H]+	Hydro-dimethyl lonijaposide C	374.1536, 392.1636	Terpenes and their derivatives
chem20	3.54	285.0921 [M+H]+	Tyrosylcysteine	147.0412, 105.0672, 164.069, 119.0477, 136.0757, 147.0746, 119.0838	Amino acids and their derivatives
chem21	3.54	552.1958 [M+H]+	Dimethyl lonijaposide C	320.1084, 288.0832, 390.1482	Terpenes and their derivatives
chem22	3.70	348.1942 [M+H]+	Leu Ala Met	154.0833, 145.0994, 111.0423, 150.0939, 83.0841, 104.0693	Amino acids and their derivatives
chem23	3.73	594.2059 [M+H]+	Lonijaposide T	362.1175	Terpenes and their derivatives
chem24	3.80	359.1272 [M+H]+	Sweroside	127.0385, 197.0789, 179.0688	Terpenes and their derivatives
chem25	3.91	391.1515 [M+H]+	Loganin II	179.0679, 167.0681, 193.0832, 197.0782	Terpenes and their derivatives
chem26	3.99	538.2179 [M+H]+	Lonijaposide B	358.1594, 211.0945, 376.1696, 344.1443	Terpenes and their derivatives
chem27	4.12	384.1583 [M+H]+	Met Ala Tyr	107.0478, 165.0519, 109.0269, 149.0574, 177.0517	Amino acids and their derivatives
chem28	4.14	389.1370 [M+H]+	Vogeloside I	151.0382, 177.0534, 195.0636, 209.0789	Terpenes and their derivatives
chem29	4.28	595.1556 [M+H]+	Kaempferol-7-neohesperidoside	287.0513, 288.0547	Flavonoids and their derivatives
chem30	4.33	575.2121 [M+H]+	5α-Carboxystrictosidine	395.1542, 413.1642, 343.1239	Alkaloids and their derivatives
chem31	4.35	303.0450 [M+H]+	Quercetin	285.0363, 153.0174	Flavonoids and their derivatives
chem32	4.35	449.0995 [M+H]+	Kaempferol-7-o-glucoside	287.0513, 288.0549	Flavonoids and their derivatives
chem33	4.35	465.0942 [M+H]+	Isoquercetin	303.0459, 304.0494	Flavonoids and their derivatives
chem34	4.49	625.1646 [M+H]+	Isorhamnetin-3-O-rutinoside	317.0596, 318.0631	Flavonoids and their derivatives
chem35	4.62	287.0499 [M+H]+	Kaempferol	287.0505, 153.0172, 135.0437, 145.0276, 171.0409, 109.0282, 258.0521, 103.0546, 213.0165, 96.5292, 127.0542, 137.0312, 165.0176	Flavonoids and their derivatives
chem36	4.62	337.0863 [M+H]+	5-Caffeoylshikimic acid	163.0376, 145.0275, 135.0433, 117.0327, 89.0389	Organic acids and their derivatives
chem37	4.62	499.1138 [M−H2O+H]+	Isochlorogenic acid A	163.0379, 319.0767, 145.0278	Organic acids and their derivatives
chem38	4.62	517.1240 [M+H]+	Isochlorogenic acid B	163.0379, 164.0414, 145.0278	Organic acids and their derivatives
chem39	4.83	463.1151 [M+H]+	Chrysoeriol 7-O-glucoside	301.0667, 302.0702	Flavonoids and their derivatives
chem40	4.90	413.1606 [M+H]+	Dihydroamorphigenin	413.1631, 339.1357, 381.1377, 219.1079	Terpenes and their derivatives
chem41	5.11	303.1750 [M+H]+	Arg Gln	147.0428, 139.9902, 156.9959, 303.0456	Amino acids and their derivatives
chem42	5.11	400.1319 [M+H]+	Cys Met Phe	149.0589, 121.0646, 93.0699, 280.0932, 91.0541	Amino acids and their derivatives
chem43	5.11	431.1505 [M+H]+	Polygalatenoside C	147.0436, 145.0628, 395.159, 195.0635	Carbohydrates and their derivatives
chem44	5.30	439.2218 [M+H]+	Tyr Glu Lys	439.2222, 147.1148, 259.1635, 136.0616	Amino acids and their derivatives
chem45	5.61	627.1583 [M+H]+	Quercetin-3,4′-O-di-beta-glucoside	465.109	Flavonoids and their derivatives
chem46	5.66	287.0503 [M+H]+	Luteolin	287.0507, 288.0539, 153.017	Flavonoids and their derivatives
chem47	5.71	1045.5322 [M+H]+	Jujubogenin 3-O-alpha-L-arabinopyranosyl-(1-2)-[3-O-(trans)-p-coumaroyl-beta-D-glucopyranosyl-(1-3)]-alpha-L-arabinopyranoside	437.3332, 455.3433, 147.0642, 309.1139, 133.0493	Terpenes and their derivatives
chem48	5.87	477.1301 [M+H]+	Flavoyadorinin B	315.0808	Flavonoids and their derivatives
chem49	6.23	271.0559 [M+H]+	Aloeemodin	271.0555, 153.0166, 119.0479	Others
chem50	6.38	301.0658 [M+H]+	Diosmetin	286.042, 258.0476	Flavonoids and their derivatives
chem51	7.08	331.0756 [M+H]+	Tricin	331.0739, 332.1815, 333.1908	Flavonoids and their derivatives
chem52	7.10	349.1919 [M+H]+	Cys Lys Val	349.1753, 121.0982, 117.5463, 229.0671, 187.1075	Amino acids and their derivatives
chem53	7.91	448.1685 [M+H]+	Asp Gln Ala Asp	187.147, 187.0366, 448.3186, 431.1786, 402.9901, 205.144	Amino acids and their derivatives
chem54	7.96	439.1822 [M+H]+	Tyr Gln Glu	439.1801, 264.235, 275.1926, 263.5572, 164.1481	Amino acids and their derivatives
chem55	8.09	433.2267 [M+H]+	Ala Thr Leu Asp	155.0076, 261.217, 173.019, 243.207, 127.0345, 59.2304	Amino acids and their derivatives
chem56	8.41	350.1691 [M+H]+	Tyr Pro Ala	261.2171, 187.1449, 119.0821, 233.2215	Amino acids and their derivatives
chem57	8.41	358.1576 [M+H]+	Cys His Val	121.1005, 358.1554, 254.8643, 278.1364, 277.6353	Amino acids and their derivatives
chem58	8.52	391.2381 [M+H]+	Met Lys Ile	149.0216, 132.0652, 391.2377, 86.0605, 328.3121, 149.1306	Amino acids and their derivatives
chem59	8.81	329.0961 [M+H]+	5-Hydroxyl-3′,4′,7-trimethoxy flavone	313.0638, 314.0694	Flavonoids and their derivatives
chem60	9.07	617.3293 [M+H]+	Lys Gln Ala Gly Asp Val	385.2329, 581.3045, 129.0549, 582.2958, 553.3069, 600.3156	Amino acids and their derivatives
chem61	9.35	299.0862 [M+H]+	Apigenin 7,4′-dimethyl ether	256.068, 299.0853, 284.0624, 167.0312	Flavonoids and their derivatives
chem62	11.54	331.2185 [M+H]+	Arg Arg	313.0643, 285.0699, 314.07, 331.2079	Amino acids and their derivatives
chem63	12.42	432.1853 [M+H]+	Glu His Phe	149.0219, 432.1844, 149.0945, 93.069, 303.042, 166.0921	Amino acids and their derivatives
chem64	0.81	191.0534 [M−H]−	Citric Acid	191.0528, 111.0436	Organic acids and their derivatives
chem65	0.81	195.0481 [M−H]−	D-Gluconic acid	85.0291, 87.0084, 99.0447, 111.0442	Organic acids and their derivatives
chem66	0.90	475.1221 [M+HCOO]−	Formononetin-7-O-glucoside	133.0119, 135.0418, 135.0057, 183.0637, 267.0455, 133.0494, 137.0229, 135.026, 209.0431, 196.9716, 251.062, 238.3721, 180.0663, 183.0239, 180.0323	Flavonoids and their derivatives
chem67	1.19	243.0584 [M−H]−	Uridine	110.0228, 111.0078, 152.8954, 152.0179, 151.8918, 122.0232, 152.035, 120.0818, 124.0383, 82.029, 152.994, 138.0611	Amino acids and their derivatives
chem68	2.93	455.1674 [M+HCOO]−	Morroniside	101.0232, 191.0522, 119.0334	Terpenes and their derivatives
chem69	3.22	375.1220 [M−H]−	7-Epi-loganic acid	151.0737, 125.0588, 169.0838	Organic acids and their derivatives
chem70	3.22	697.2042 [M−H]−	Arbutoside II	341.1034, 373.1085, 355.097	Others
chem71	3.89	729.2087 [M−H]−	Demethyl-strychoside A	453.1279, 409.1397, 505.1574, 497.116, 549.1459	Others
chem72	3.99	403.1170 [M−H]−	Kingiside	121.0271, 101.0227, 165.0522, 119.0327, 149.0213, 121.0634	Terpenes and their derivatives
chem73	4.27	423.1783 [M−H]−	Cys Arg Phe	119.0334, 345.2224, 363.1782, 147.0428, 337.0301	Amino acids and their derivatives
chem74	4.27	593.1392 [M−H]−	Kaempferol-7-O-neohesperidoside	593.1336, 285.033, 594.1366, 284.0254, 286.0364, 595.1396	Flavonoids and their derivatives
chem75	4.37	463.0804 [M−H]−	Quercetin-3-O-glucoside	300.0199, 301.0263, 271.0179	Flavonoids and their derivatives
chem76	4.66	515.1094 [M−H]−	Isochlorogenic acid C	179.0306, 191.0515, 173.0413, 353.0785, 135.042	Organic acids and their derivatives
chem77	4.75	161.0223 [M−H]−	Umbelliferone	133.0274, 105.034, 119.0494, 91.0547	Others
chem78	5.04	499.1150 [M−H]−	Coumaroyl caffeoylquinic acid isomer	191.051, 179.0303, 163.0359, 173.0412, 135.0418, 161.0205, 119.0472, 353.0784	Organic acids and their derivatives
chem79	5.13	529.1241 [M−H]−	3,4-O-Dicaffeoylquinic acid methyl ester	179.0302, 135.0417, 161.0202	Organic acids and their derivatives
chem80	5.90	579.2902 [M−H]−	Tyr Lys Asp Arg	131.0343, 533.2913, 179.0544, 173.0445, 179.0334	Amino acids and their derivatives
chem81	10.32	279.2275 [M−H]−	Linoleic acid	99.9254, 195.0529, 279.228, 235.1025	Organic acids and their derivatives
chem82	12.92	421.2183 [M−H]−	Phenylalanylthreonylarginine	361.1398, 421.2156, 218.9713, 362.1425, 421.2523, 375.2785	Amino acids and their derivatives

, including retention time (Rt), precursor ions (m/z), ion mode, and fragment ions (MS/MS). These metabolites can be mainly classified into seven categories: amino acids and their derivatives (32), organic acids and their derivatives (13), terpenes and their derivatives (14), flavonoids and their derivatives (17), alkaloids and their derivatives (1), carbohydrates and their derivatives (1), and others (4). Subsequently, the heatmap of these non-volatile components was established (Figure 3). As depicted in the purple box of Figure 3, it could be found that LJF tea samples in the SG group showed higher content of some non-volatile components, which mainly involved flavonoids and their derivatives (such as quercetin, isoquercetin, and kaempferol) and organic acids and their derivatives (such as chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid B). As illustrated in the green box of Figure 3, these non-volatile components showed relatively high content in the ZZ and SG groups, while they exhibited relatively low content in other groups, indicating that the content of these non-volatile components was easily influenced by high temperatures.

Organic acids are the main active compounds in the LJF, mainly composed of derivatives of chlorogenic acid and cinnamic acid [19]. In this study, 13 organic acids and their derivatives were detected, such as neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and 3,4-O-dicaffeoylquinic acid methyl ester. Interestingly, compared with other groups, the content of these organic acids was relatively high in the LJF tea samples from the ZZ and the SG groups (Figure S6, p < 0.05). This could be explained by enzymes and high temperatures leading to the degradation of these non-volatile components under continuous high temperature drying conditions. Compared with LJF tea samples in the SG group, LJF tea samples in the ZZ group had higher content of these organic acids (except for isochlorogenic acid A), which might be attributed to the rapid inactivation of enzymes caused by the momentary high temperature (p < 0.05). Furthermore, these organic acids and their derivatives had numerous healthy characteristics. For instance, chlorogenic acid is a phenolic compound with antioxidant, anti-inflammatory, and antispasmodic activities [32]. Isochlorogenic acid is a natural component that has three isomers, isochlorogenic acids A, B, and C, which have evident functional benefits such as being antioxidant, antibacterial, and anti-inflammatory [33]. Notably, these organic acids were the main active substances of LJF tea. Consequently, the higher content of these components shaped the health-promoting properties of LJF tea samples in the SG and ZZ groups.

Flavonoids, which were frequently present as secondary metabolites in numerous natural plants, served as the principal active ingredients in LJF tea. These compounds had a wide range of health-promoting properties, such as the ability to lower blood lipid levels, exhibit antioxidant activity, and protect the liver [5]. Among them, quercetin and its glycosides were polyphenols and their derivatives, widely present in numerous vegetables, fruits, and other natural products. Various health benefits of them were found, including antioxidative, anti-carcinogenic, and anti-inflammatory activities [34]. Isoquercetin was a secondary metabolite derived from multiple plants, which exhibited anti-inflammatory, immuno-modulatory, and anticoagulant effects [35]. Kaempferol and its derivatives were natural phytochemicals with neuroprotective, anti-inflammatory, and hepatoprotective benefits [36]. Interestingly, as illustrated in Figure S7, compared with other groups, these flavonoids, except for luteolin, showed higher content in LJF tea samples from the SG group (p < 0.01), demonstrating that fixation by sun drying might be beneficial for the retention of flavonoids in LJF tea.

Subsequently, PCA was conducted to present the similarities and differences in non-volatile components between different groups. As depicted in Figure 4A, it could be found that LJF tea samples in the same group showed close distance, while LJF tea samples from distinct groups could be evidently distinguished. These results demonstrated that different processing methods shaped the content differences in non-volatile components. Then, PLS-DA was performed to screen the featured non-volatile components with significant changes in their content between different groups. The Q² and R² of the PLS-DA model were 0.596 and 0.592, respectively. Concurrently, the results of 100 permutation tests showed that the intercept of Q² on the y-axis did not exceed 0.05, indicating that the PLS-DA model had good generalization ability and no overfitting phenomenon occurred (Figure 4B). A total of ten featured non-volatile components with VIP > 1.2 and FDR < 0.05, namely adenosine, secologanic acid, phenylalanine, Ser Gly Asp, dimethyl lonijaposide C, lonijaposide T, Met Lys Ile, vogeloside I, Met Lys Ile and linoleic acid, were screened (Figure 4C). Based on these featured non-volatile components, the heatmap was established (Figure 4D). It could be found that these components were mainly present in LJF tea samples from CZ and HG60 groups, indicating that these components were products generated under consistent high temperature conditions. Furthermore, LJF tea samples from the HG80 group also had low content of these components, which could be attributed to the degradation of these components under the consistent higher temperature (80 °C).

Based on the analysis results of non-volatile components, the correlation analysis between the color difference and non-volatile components content was performed. As shown in Figure S8, some non-volatile components showed negative correlation with color difference, including quercetin and its glycosides, isochlorogenic acid and its isomers, and kaempferol and its glycosides. These results suggested that the darker the color of the LJF tea samples is, the lower the content of these components (organic acids and flavonoids) is. Overall, the changes in appearance color were predominantly caused by the changes in organic acids and flavonoids in the LJF tea samples.

4. Conclusions

In summary, this study explored the effects of six fixation methods on LJF tea samples through comprehensive analysis of the appearance color, volatile components, and non-volatile components. Our findings illustrated that compared with other fixation methods, LJF tea samples in the SG group had a brighter appearance, which was caused by the high content of organic acids and flavonoids. Additionally, LJF tea samples in the SG group also retained higher relative content of aroma components and non-volatile components. These characteristics shaped the high quality of the LJF tea samples in the SG group. Overall, compared with other methods, sun drying may be beneficial for improving the quality of LJF tea. This study provides a reference for the selection of fixation methods for LJF tea and offers a theoretical basis for the quality improvement of LJF tea.