## 2 Materials and Methods
### Determination of the Main Components of Black Tea  
Moisture content was determined using the existing method (GB/T 8304-2013) [17]. The total soluble sugar (TSS) was measured using the anthrone-sulfuric acid method [17]. Total free amino acids were calculated by the Ninhydrin method [1]. Total polyphenol (TPP) content was determined using the Folin-Ciocalteu method [18]. The theaflavins (TFs), thearubignis (TR), and theabrownins (TBs) were measured by the previously used procedure [19]. Tea polysaccharide (TPS) was determined according to the methods described previously [20] and total flavonoid content (TFC) was measured using aluminum nitrate colorimetric methods [21]. Untargeted nonvolatile constituents in black tea were determined [22]. In short, 0.15 g of each sample was weighed and added to 7.5 mL of a 75% methanol-water solution and 0.15 mL of etophylline (250 g/mL) as an internal standard. After vortexing, samples were placed in a water bath (70%) for half an hour. UHPLC (Infinity 1290, Agilent Technologies, Santa Clara, CA, USA), combined with Q-TOF/MS Machine (Q-TOF 6520, Q-TOF 6520, Agilent Technologies In., Santa Clara, USA), was used for LC-MS analysis. The separation was conducted at a steady temperature (35 \({}^{\circ}\)C) with an Agilent ZORBAX Eclipse Plus C18 Column (100 \(\times\) 2.1 mm, 1.8 um). Inlet injection volume was 3 \(\upmu\)L with a volumetric flow rate of 0.3 mL/min. The samples were eluted with a gradient aqueous solvent prepared by 1 mL of formic acid in 1000 mL of ultra-distilled water, denoted as mobile phase A, and methanol as an organic solvent, denoted as mobile phase B. The linear gradient was recorded as follows: 10-15% of B (0-4 min); 15-25% of B (4-7 min); 25-32% of B (7-9 min); 32-40% of B (9-16 min); 40-55% of B (16-22%); 55-95% of B (22-28 min); 95% of B (28-30 min); 95-100% of B (30-31 min); and 100% of B (31-35 min). The tea metabolome was determined by positive electrospray ionization-tandem mass spectrometry (+ESI-MS) with the following characteristics: higher ESI source and stream of drying gas temperature of 300 \({}^{\circ}\)C; higher constant voltage (3.5 kv) of the capillary tube; dispersal of a fine spray pressure 3.5 psi; sample flow rate 11 L/min enhanced by higher temperature of nebulizing gas (300 \({}^{\circ}\)C); and the mass/charge (m/z) ratio ranged between 100 and 1200. The analyte molecules were analyzed with the separation capacity of three distinctive voltages (10, 20 and 30) by the automated tandem mass spectrometry (MS/MS) model. Different metabolites were identified using MS\({}^{2}\) spectra, accurate mass, and respective standards. Volatile compounds were determined by gas chromatography-mass spectrometry (GC-MS) (DSQII, Thermo Fisher Scientific, Waltham, MA, USA), using a modified method [1]. Briefly, 1.0 g of sample was weighed into a 10 mL vial, then 5 mL of 20% of sodium chloride (boiled) was added, and 1 \(\upmu\)L of capric acid (0.04 \(\upmu\)L/100 mL of ethanol) was added as an internal standard. Solid-phase microextraction (SPME) was inserted into the headspace of a vial and placed immediately into a 60% water bath for 60 min. Inserted SPME fiber (DVB-PDMS 65 \(\upmu\)m) was coated with adsorbents into the headspace of a vial based on the adsorption of flavor volatile compounds for extraction purpose. Then, SPME was moved to the GC-MS injection section for volatile compound identification, separation, and quantification. GC-MS was conditioned as follows: capillary column (DB-5 MS; 0.25 mm \(\times\) 30 mm \(\times\) 0.22 \(\upmu\)m; Agilent, CA, USA); carrier gas was helium with 1.0 mL/min flow rate; temperature started at 45 \({}^{\circ}\)C for 2 min, then increased at a rate of 5 \({}^{\circ}\)C/min, and kept increasing to 90 \({}^{\circ}\)C for 2 min; temperatures continued to increase up to 120 \({}^{\circ}\)C for 2 min at 2.0 \({}^{\circ}\)C/min, reaching a climax of 220 \({}^{\circ}\)C for 7 min at 5 \({}^{\circ}\)C/min.