An In Vitro Catalysis of Tea Polyphenols by Polyphenol Oxidase

Tea polyphenol (TPs) oxidation caused by polyphenol oxidase (PPO) in manufacturing is responsible for the sensory characteristics and health function of fermented tea, therefore, this subject is rich in scientific and commercial interests. In this work, an in vitro catalysis of TPs in liquid nitrogen grinding of sun-dried green tea leaves by PPO was developed, and the changes in metabolites were analyzed by metabolomics. A total of 441 metabolites were identified in the catalyzed tea powder and control check samples, which were classified into 11 classes, including flavonoids (125 metabolites), phenolic acids (67 metabolites), and lipids (55 metabolites). The relative levels of 28 metabolites after catalysis were decreased significantly (variable importance in projection (VIP) > 1.0, p < 0.05, and fold change (FC) < 0.5)), while the relative levels of 45 metabolites, including theaflavin, theaflavin-3′-gallate, theaflavin-3-gallate, and theaflavin 3,3′-digallate were increased significantly (VIP > 1.0, p < 0.05, and FC > 2). The increase in theaflavins was associated with the polymerization of catechins catalyzed by PPO. This work provided an in vitro method for the study of the catalysis of enzymes in tea leaves.


Introduction
Tea is manufactured from the fresh leaves of Camellia sinensis, which is the most consumed beverage in the world after water, and widely believed to be rich in flavor compounds and have positive effects on human health, especially anti-oxidation, anti-inflammatory, gut barrier protection, and bile acid metabolism regulatory effects [1][2][3]. Polyphenols in tea leaves (TPs) account for 18% to 36% of dried tea leaves [4], mainly including catechins, O-glycosylated flavonols, C-glycosylflavones, proanthocyanidins, phenolic acids, and their derivatives, and also containing the fermented oxidation products of catechins, e.g., theaflavins, thearubigins, and theabrownins in oolong, black, and dark teas [5][6][7]. Among them, O-glycosylated flavonols, tannins, and galloylated catechins are the main astringent compounds, and nongalloylated catechins enhance the tea bitterness [8,9]. Furthermore, TPs are a major class of aroma compounds giving clove-like, smoky, and phenolic characteristics to dark teas, particularly Pu-erh tea [10]. Hence, TPs are important for the healthful functions and flavors of tea beverages [2,4], which have important scientific and commercial interests in tea manufacture.
According to the manufacturing process, tea can be classified into six types: green tea, white tea, black tea, yellow tea, ooloog tea, and dark tea [11]. The oxidation of TPs caused by polyphenol oxidase (PPO) or peroxidase in the manufacturing process is critical for the
Under the action of the 500 U/mL PPO, whether the contents of TPs could be significantly (p < 0.05) reduced with the extension of reaction time needed further discussion, so different enzyme reaction times were selected to verify this. As shown in Figure 1B, the contents of all TPs (e.g., GA, GC, EGC, C, Ca, EGCG, EC, GCG, ECG, Ti, CG, Rt, EA, My, Qc, Lu, Kp) in sun-dried green tea leaves decreased significantly (p < 0.05) with the extend of enzyme reaction time from 6 h to 7 h. Furthermore, except for EGCG, ECG, and EA, the contents of most TPs did not change significantly (p > 0.05) at the PPO reaction time of 7 h and 8 h ( Figure 1B). Therefore, 500 U/mL PPO reaction for 7 h can catalyze most TPs (e.g., GA, GC, EGC, C, Ca, EGCG, EC, GCG, ECG, Ti, CG, Rt, EA, My, Qc, Lu, Kp) in sun-dried green tea leaves ( Figure 2A). The color of sun-dried green tea leaves and tea infusions became deeper after PPO catalysis ( Figure 2B). These changes in TPs were similar to those after PPO metabolism of tea leaves and are in agreement with previous reports [28][29][30]. and phenolic characteristics to dark teas, particularly Pu-erh tea [10]. Hence, TPs are important for the healthful functions and flavors of tea beverages [2,4], which have important scientific and commercial interests in tea manufacture.

Metabolomic Analysis of Catalysis of Metabolites in Sun-Dried Green Tea Leaves by PPO
Metabolomics is broadly applied in tea sciences and has tremendous potential for establishing correlations between tea metabolites and quality characteristics, and assessing the physiological changes in tea plants induced by cultivation conditions and metabolic responses to abiotic and biotic stress, and construction of metabolic pathways [31,32]. Therefore, the changes in metabolites of sun-dried green tea leaves under 500 U/mL PPO reaction for 7 h were further subjected to a metabolomic analysis. Metabolites were extracted from catalyzed tea powder (CTP) and CK, and analyzed using a non-targeted liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) based metabolomics approach. As shown in Figure 3A, PCA showed that the variance contributions of PC1 and PC2 were 56.8% and 25.9%, respectively, with a cumulative variance contribution of 82.7%, which was much larger than the confidence value of 60%, suggesting that this metabolomics analysis had good stability and reproducibility. While, a great distance was observed among the three different samples, ETP samples were clustered in the upper right area, CK samples were mainly located in the bottom right, and quality control (QC) samples were clustered in the left area. Differential clustering of CK and CTP samples indicated that metabolites in sun-dried green tea leaves were significantly changed after the catalysis of PPO.

Metabolites
Class Sub-Class
In comparison with CK, the levels of EC, EGC, ECG, and EGCG in CTP decreased from 2.94 mg/g, 9.34 mg/g, 17.60 mg/g, 31.98 mg/g to 0.68 mg/g, 2.04 mg/g, 6.05 mg/g, and 8.96 mg/g, respectively, whereas the levels of TF 1 , TF 2 A, TF 2 B, and TF 3 increased 3.82-, 5.11-, 5.92-, and 6.01-fold, respectively ( Figure 5). We suggested that TF 1 was synthesized through the polymerization of EC and EGC under the catalysis of PPO; PPO could catalyze the polymerization of EC and EGCG to form TF 2 A; ECG and EGC could be polymerized to form TF 2 B under the catalysis of PPO; and TF 3 was synthesized through the polymerization of ECG and EGCG under the catalysis of PPO. Theaflavins are the general name for a class of compounds with a benzodiazepine structure formed by the condensation of catechins under the catalytic action of PPO [35,36], and they have great potential and broad application prospects in the fields of food, health products, and natural medicine [37][38][39]. In fresh tea leaves, the phenolic hydroxyl groups on the B ring of catechins are oxidized by PPO to form theaflavin intermediates (o-quinones) [40,41], which are easily oxidized and polymerized to form theaflavins [42,43]. Therefore, it is proved that TF 1 , TF 2 A, TF 2 B, and TF 3 can be produced by enzymatic oxidation of PPO only in the presence of dihydroxy-B-cycloflavanol (e.g., EC and ECG) and trihydroxy-B-cycloflavanol (e.g., EGC and EGCG) through the structural formula and the change of the levels of metabolites ( Figure 5).

Materials and Chemical Standards
The raw material (RM) was sun-dried green tea leaves with one bud and three leaves, which were collected from Pu'er City Institute of Tea Science, Yunnan Province, China. PPO (500 U/mg) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

Optimization of the PPO Catalysis Conditions
Sun-dried green tea leaves were ground to fine powder with liquid nitrogen 30 min to obtain tea leaves with broken cell walls, and the tea powder can be passed through the 40 mesh sieve. After that, 1 g tea powder was added to 1 mL PPO at a concentration of 0 U/mL (control check, CK), 100 U/mL, 200 U/mL, 300 U/mL, 400 U/mL, and 500 U/mL, respectively, and the reaction was carried out at 35 • C for 7 h, then terminated by boiling water for 10 min. Since then, 1 mL PPO (500 U/mL) was added to the tea powder (1 g) and the reaction was terminated after 6 h, 7 h, and 8 h at 35 • C. TPs were extracted with the methanol extraction method and subjected to HPLC analysis described in our previous report [44]. Briefly, 1 g of sample was extracted with 44.00 mL of methanol:hydrochloric acid (40:4, v/v) in a flask equipped with a reflux condenser. The extraction was performed in a water bath (at 85 • C) for 90 min. The extractions were diluted to 50 mL, filtered through a 0.2 µm nylon filter, and then analyzed directly by HPLC. Samples were determined using an Agilent 1200 series HPLC system consisting of an LC-20AB solvent delivery unit, an SIL-20A autosampler, a CTO-20A column oven (35 • C), a G1314B UV variable wavelength detector, and an LC Ver1.23 workstation (Agilent Technologies, Santa Clara, CA, USA). Partitioning was performed using an Agilent Poroshell 120 EC-C18 column (4.6 × 100 mm, 2.7 µm) fitted with a C 18 guard column (Agilent Technologies). The mobile phase was a mixture of (A) 5% acetonitrile and 0.261% ortho-phosphoric acid in water and (B) 80% methanol in water. In an elution gradient, from 0-16 min, buffer B was increased from 10 to 45%; from 16-22 min, buffer B was increased to 65%; and from 22-25.9 min, buffer B was increased to 100%. Three replicates of each sample were extracted, and each extraction was analyzed twice. the standards [47,48] and comparing their m/z values, retention times and fragmentation patterns with those of the standards. The chromatographic peak area of each was calculated. Positive and negative data were combined to obtain a combined data set.

Statistical Analysis
Statistical analyses were performed using IBM SPSS Statistics 26.0 (SPSS Inc., Chicago, IL, USA). Principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) results were generated by SIMCA 14.1 (Umetrics, Umea, Sweden) to visualize the metabolic differences between the experimental groups after normalization and standardization processing. Variable importance in projection (VIP) analysis ranked the overall contribution of each variable to the OPLS-DA model, and those variables with VIP > 1.0, p < 0.05, and fold change (FC) > 2 or < 0.5 were classified as differentially changed metabolites (DCMs) [49].
Therefore, an in vitro catalysis of TPs by PPO was established and provided technical references for the study of the catalytic mechanism of PPO in tea leaves.