Effects of Tannase and Ultrasound Treatment on the Bioactive Compounds and Antioxidant Activity of Green Tea Extract

The present study investigated the effects of tannase and ultrasound treatment on the bioactive compounds and antioxidant activity of green tea extract. The single-factor experiments and the response surface methodology were conducted to study the effects of parameters on antioxidant activity of green tea extract. The highest antioxidant activity was found under the optimal condition with the buffer solution pH value of 4.62, ultrasonic temperature of 44.12 °C, ultrasonic time of 12.17 min, tannase concentration of 1 mg/mL, and ultrasonic power of 360 W. Furthermore, phenolic profiles of the extracts were identified and quantified by high-performance liquid chromatography. Overall, it was found that tannase led to an increase in gallic acid and a decrease in epigallocatechin gallate, and ultrasounds could also enhance the efficiency of enzymatic reaction.


Introduction
Reactive oxygen species (ROS) are produced in the human body, and excessive ROS could cause oxidative stress in organisms and promote the development of some chronic diseases [1,2]. The natural antioxidants from plants play an important role in human health. They have been demonstrated to have free radical scavenging activity and protect against oxidative stress [3]. Polyphenols are one of the main natural antioxidants, and have shown many bioactivities and beneficial health effects, such as antioxidant, anti-inflammatory, anticancer, anti-ageing, anti-diabetic, anti-obese, cardioprotective and neuroprotective activities [4][5][6][7][8][9][10]. Other bioactive compounds (such as carotenoids, polysaccharides, alkaloids, and saponins) could also contribute to human health benefits [7,11]. The natural antioxidants could be used for the prevention and management of several diseases, but it has caused some argument. For example, an in vitro antioxidant could exhibit pro-oxidant property in vivo under certain conditions, such as vitamin C [12]. In addition, the antioxidant is usually considered to inhibit the onset of cancers [10]. However, it is also reported that the antioxidant could accelerate lung cancer progression, and increase melanoma metastasis in mice [13,14]. On the other hand, one of the most important uses of natural antioxidants is as the food additives in the food industry [15]. The antioxidants have been defined by several different terms in the literature [16]. For example, when a substance presents at a low concentration compared with that of an oxidizable substrate,

Preparation of Green Tea Extract
Dianqing tea was purchased in the local market of Guangzhou, China, which showed the strongest antioxidant activity among 30 Chinese teas in a previous study [49]. It is a type of green tea produced in Kunming, China. Three different batches of Dianqing tea were bought from different markets, and the mixed tea samples were used for the experiments. The green tea was ground into powders by using a grinder (Royalstar Co. Ltd., Hefei, China), and then the powders were filtered through a 100 meshes sieve. The filtered powders were sealed in plastic bags and stored under 4 • C.
The green tea powders were mixed with 85 • C distilled water (50 g/L, w/v), and then the mixture was placed in a 85 • C water bath, which was kept at 85 • C for 30 min according to the literature [50,51]. With this tea/water ratio, the tea extract was reported to show stronger antioxidant activity and more abundant phenolic compounds [51]. The mixture was centrifugated at 4200× g for 30 min, and the extract was collected and stored at 4 • C for further experiments.

Treatment of Tannase and Ultrasound
Tannase was diluted to different concentrations with 0.1 M citrate-phosphate buffer. The tannase solution was added to the green tea extract. The combined treatment of ultrasound and tannase (UST) was carried out by using an ultrasonic device (Kejin Ultrasonic Equipment Factory, China). The reactions were performed at consistent ultrasonic power of 360 W and other different process parameters in the single-factor tests, including the pH value of citrate-phosphate buffer (3.0, 4.0, 5.0, 6.0, 7.0), tannase concentration (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/mL), ultrasonic temperature (25,35,45,55,65,75, 85 • C), and ultrasonic time (0, 5, 10, 15, 20, 25, 30, 35 min). After the completion of ultrasonic treatment, the reaction mixture was heated at 100 • C for 10 min to inactivate tannase. The mixture was cooled to room temperature and centrifugated at 4200× g for 10 min. The obtained supernatant was stored at −20 • C and further used for the determination of antioxidant activity and HPLC assay. Based on the results of single-factor tests, the pH value of citrate-phosphate buffer, ultrasonic temperature, and time were further optimized by RSM to study their interaction.
For comparison, the ultrasonic treatment (US) was conducted by adding 0.1 M citrate-phosphate buffer (the optimized pH value) without tannase. The tannase treatment (TAN) was carried out by placing the mixture in the water bath without ultrasound. The green tea extract (GTE) was obtained by dilution of the original extract of green tea with the same buffer solution. The other parameters (such as the pH value, ultrasonic temperature, and time) were the same as those of UST.

Design by Response Surface Methodology
RSM was used to investigate the impacts of independent variables on the antioxidant capacities (TEAC) of the extracts. Three crucial factors which could significantly affect the antioxidant capacities of the extracts were selected based on the results of single factor tests of UST. Those factors were further used to perform three-variable and five-level central composite design (CCD) via Design Expert v8.0.6 software. Three variables (X 1 , the pH value of citrate-phosphate buffer; X 2 , ultrasonic temperature; X 3 , ultrasonic time) were chosen to be optimized, while other parameters (the concentration of tannase of 1 mg/mL; ultrasonic power of 360 W) were kept constant.
There was a total of 20 runs of experiments, including 8 combinations of factorial points, 6 combinations of axial points, and 6 replicates of center points. The response values were TEAC values. Data from CCD were analyazed by multiple regressions to fit into the response surface quadratic model, as follows: where Y was the value of response, X i and X j were different independent variables, β 0 were the intercept, β i , β ii , and β ij were the linear, quadratic, and interaction coefficients, respectively. The analysis of variance (ANOVA) was conducted to analyze the adequacy of the fitted model, of which the significant level was p < 0.05. The individual and interactive effects of variables on the response value were visualized on the 3D surface plots. The actual value of the experiment under the optimal conditions was compared with the predicted value, verifying the accuracy of the model.

Determination of Antioxidant Activity
The antioxidant property could be referred to the radicals scavenging capacity, reducing capacity, metal chelating capacity, activity as antioxidative enzyme, and the inhibiting capacity against oxidative enzymes, and many methods have been reported for the evaluation of antioxidant activities [18][19][20][21]. In this study, TEAC, DPPH, and FRAP assays were chosen, because the TEAC and DPPH assays are to determine the radical scavenging capacity, while the FRAP assay is to test the reducing capacity, and they could assess the antioxidant capacities of the extract from different aspects [18]. Furthermore, these methods have been widely used because they are easy to implement and the obtained results are reproducible [52].

Trolox Equivalent Antioxidant Capacity (TEAC) Assay
The antioxidant activities of the extracts were determined by the TEAC assay based on the procedure in the literature [53]. The ABTS ·+ stock solution was prepared by mixing 7 mmol/L ABTS and 2.45 mmol/L K 2 S 2 O 8 at the volume ratio of 1:1, and was incubated in a dark place for 16 h at room temperature. The ABTS ·+ stock solution was diluted to an absorbance of 0.70 ± 0.05 at 734 nm. A 100 µL of diluted extract was mixed with 3.8 mL of ABTS ·+ working solution, and then the mixture was incubated in a dark place for 6 min at room temperature. The absorbance of the reaction mixture was tested at 734 nm, and the percent of inhibition of absorbance was calculated. Trolox was used as the standard to make the calibration curve. The values of TEAC were presented as µmol Trolox/g dry weight (DW) of green tea.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay
The assay was used to determine the DPPH radical scavenging ability of extracts based on the procedure in the literature, with minor modifications [51]. A 300 µL of diluted extract was mixed with 3 mL of DPPH solution (2 × 10 -4 M). The reaction mixture was incubated in a dark place for 30 min at room temperature. The control contained the distilled water instead of extracts. The absorbance was determined at 517 nm by using a spectrophotometer. All measurements were carried out in triplicate. The DPPH radical scavenging activity of the extract was caculated as: (1 − As/Ac) × 100%, where As is the absorbance of tested samples and Ac is the absorbance of the control.

Ferric Reducing Antioxidant Power (FRAP) Assay
The reducing activities of extracts were analyzed according to the literature [49]. The FRAP reaction solution was prepared by mixing 0.3 M acetate buffer (pH 3.6), 0.01 M TPTZ solution, and 0.02 M FeCl 3 solution at a volume ratio of 10:1:1. A 100 µL of diluted extract was mixed with 3.0 mL FRAP reagent, and the mixture was incubated at room temperature for 4 min. The absorbance was measured at 593 nm. The standard curve was made by using FeSO 4 , and the FRAP values were expressed as µmol Fe 2+ /g DW of green tea.

Phytochemical Analysis of the Extracts by HPLC
The contents of phytochemicals in the extracts were measured by HPLC system, according to the previous method [54]. The phenolic compounds were identified by retention time and UV-vis spectrum compared with those of the standards. The quantification of phenolic compounds was determined by comparing the peak area of the UV response at the maximal absorption wavelength with that observed by mean of pure standards (standard curve method), and was represented as mg/g DW.

Statistical Analysis
All the experiments were conducted in three independent tests, and data were presented as mean ± standard deviation (SD). The statistical analysis was performed with SPSS 19.0 (IBM SPSS Statistics, IBM Corp, Somers, NY, USA). One-way ANOVA and post hoc least-significant difference (LSD) test were used to determine the significance of differences for each group, with statistical significance at p < 0.05.

Effects of the pH Value of Buffer Solution on Antioxidant Capacities
The effects of buffer solution pH value on antioxidant capacity of the extracts were investigated in 0.1 M citrate-phosphate buffer. Other parameters were set as the tannase concentration of 1 mg/mL, ultrasonic temperature of 35 • C, and ultrasonic time of 30 min. Figure 1A showed that the TEAC value increased at pH value of 3.0 to 5.0, and decreased at pH value of 5.0 to 7.0. It showed that tannase had an optimal pH value of 5.0, and the result is similar to a study reported by Costa et.al, which illustrated that the free tannase was active and stable at pH 5.0 [55]. The pH value of buffer solution is one of the most important parameters affecting enzymatic activity. It was mainly related to the protonation and deprotonation of amino acids at the active site, and the alternation in conformation induced by the ionization from other amino acids [56]. Thus, using the buffer at an appropriate pH value could greatly enhance the activity of enzymes. Tannase was mainly produced by fungus, and the fungal tannase was generally acidic protein. The activity of tannase increased at the acidic solution and began to decrease when the solution became alkaline [57]. There were also other studies resulting in the pH value around 5.0 as the optimum for tannase activity [58]. Thus, the pH value of 5.0 was chosen for the following experiments.

Effects of Tannase Concentration on Antioxidant Capacities
The tannase concentrations ranged from 0.0 to 2.5 mg/mL, which were evaluated as its influence on the antioxidant capacities of the extracts, while other conditions were controlled as 0.1 M citrate-phosphate buffer (pH 5.0), ultrasonic temperature of 35 • C, and ultrasonic time of 30 min.
In addition, 50 g/L of the tea/water ratio was utilized, because the tea extract was reported to show stronger antioxidant activity and more abundant phenolic compounds with this ratio [51]. The results in Figure 1B revealed that the antioxidant capacities initially increased in the range of 0.0-1.0 mg/mL. As the concentration of tannase was subsequently increased, there was no significant change in TEAC values. When the concentration of substrate was constant and the amount of enzyme was small, substrate was not saturated by the enzyme [59]. With more enzyme added to substrate, there was a greater enzymatic reaction. However, the substrate would be saturated by a certain amount of enzyme, and the effect of enzyme was no longer significant on the substrate. The adding of tannase enhanced the biotransformation of tea polyphenols, and more antioxidant compounds were produced from green tea extracts. Taking the cost and effectiveness into consideration, 1 mg/mL was selected as the concentration of tannase in following experiments, because TEAC values had little difference when tannase concentration was beyond 1 mg/mL.

Effects of Ultrasonic Temperature on Antioxidant Capacities
To investigate the effect of temperature in ultrasound treatment on the antioxidant capacities, the temperature was varied from 25 to 85 °C , and other parameters were set as 0.1 M citratephosphate buffer (pH 5.0), tannase concentration of 1 mg/mL, and ultrasonic time of 30 min. As shown in Figure 1C

Effects of Ultrasonic Temperature on Antioxidant Capacities
To investigate the effect of temperature in ultrasound treatment on the antioxidant capacities, the temperature was varied from 25 to 85 • C, and other parameters were set as 0.1 M citrate-phosphate buffer (pH 5.0), tannase concentration of 1 mg/mL, and ultrasonic time of 30 min. As shown in Figure 1C, the TEAC value increased under the ultrasonic temperature from 25 to 45 • C. The antioxidant values at the temperature of 45 and 55 • C made no significant difference. With a further increase in temperature from 55 to 85 • C, there was a downward trend in the antioxidant capacity because the high temperature could decrease the activity of enzyme [60]. Thus, 45 • C was selected in subsequent experiments.

Effects of Ultrasonic Time on Antioxidant Capacities
The antioxidant capacity of the extracts varied greatly depending on the time in ultrasound treatment. The sample was treated under different ultrasonic time, and other procedures were 0.1 M citrate-phosphate buffer (pH 5.0), tannase concentration of 1 mg/mL, and ultrasonic temperature of 45 • C. As shown in Figure 1D, the antioxidant capacities increased significantly from 0 to 10 min, and it reached the peak at 10 min. The prolongation of ultrasonic time allowed enzymes to react with substrate completely, which led to an increase in the antioxidant capacities. However, after the ultrasonic time was longer than 10 min, the antioxidant capacities began to decline. Due to the too-long ultrasonic time, the activity of enzyme could decrease, and antioxidants could be degraded. Therefore, the time of 10 min was selected in the treatment.

Statistical Analysis and Model Fitting
Based on the results of single-factor tests, three individual factors (the pH value of buffer solution, ultrasonic temperature and ultrasonic time) were further optimized via CCD. The actual and coded levels of three individual variables and corresponding response values are listed in Table 1 where X 1 , X 2 , and X 3 are the pH value of buffer solution, ultrasonic temperature, and ultrasonic time, respectively. Y represents the TEAC values. The ANOVA of the response surface quadratic model is shown in Table 2. The p-value was used to evaluate the statistical significance of the regression model. In general, the model can be considered as significant if the p-value is smaller than 0.05. As shown in Table 2, the regression model was indicated to be significant (p < 0.05). In addition, the linear coefficients (X 1 , X 3 ) and quadratic coefficients (X 1 2 , X 2 2 , X 3 2 ) had statistical significance (p < 0.05). Furthermore, the lack-of-fit testing was non-significant (p = 0.1508), which further verified that the model was suitable in predicting the response. After the response surface regression, the determination coefficient value (R 2 ) of the predicted model was 0.9450, which suggested a good fit of the model. Also, 94.50% of the variation could be reflected by the predicted model. The adjusted R 2 was 0.8954 which was closed to R 2 , suggesting that the actual responses were highly correlated with the predicted responses. Moreover, the coefficient of variation (CV) represented the reproducibility of a model. The CV was 1.61%, which was smaller than 10%, indicating that the model was reproducible.

Response Surface Plots and Graphical Analysis
The interrelation of independent variables and response values were visually displayed via the 3D response plots in Figure 2. As is shown in Figure 2A, the TEAC values were performed as a function of pH value and ultrasonic temperature with a fixed ultrasonic time. When ultrasonic time was kept at 10 min, the TEAC values increased slowly with the increase of pH value from 4.00 to 5.00, then dropped from 5.00 to 6.00. In addition, the TEAC values increased with the increase of ultrasonic temperature from 35 • C to 45 • C, then decreased slightly from 45 • C to 55 • C. Figure 2B presents the interactions between the pH value and ultrasonic time on the TEAC values at 45 • C. The effects of the pH value on the TEAC value were similar to that shown in Figure 2A. The increase in the ultrasonic time slightly elevated the TEAC values from 5.00 to 15.00 min. Moreover, Figure 2C displays that under the condition of fixed pH value, the antioxidant capacities markedly improved as the ultrasonic temperature and time increased. The effect of ultrasonic time was more potent than that of ultrasonic temperature. According to the results of ANOVA and response surfaces plots, the pH value and ultrasonic time had stronger effects on antioxidant capacities than that of ultrasonic temperature. then dropped from 5.00 to 6.00. In addition, the TEAC values increased with the increase of ultrasonic temperature from 35 °C to 45 °C, then decreased slightly from 45 °C to 55 °C. Figure 2B presents the interactions between the pH value and ultrasonic time on the TEAC values at 45 °C. The effects of the pH value on the TEAC value were similar to that shown in Figure 2A. The increase in the ultrasonic time slightly elevated the TEAC values from 5.00 to 15.00 min. Moreover, Figure 2C

Optimal Treatment Conditions
Based on the single-factor tests and RSM study, the optimal condition for tannase and ultrasound treatment of the extracts was 0.1 M citrate-phosphate buffer with a pH value of 4.62, ultrasonic temperature of 44.11 °C , ultrasonic time of 12.17 min, tannase concentration of 1 mg/mL, and ultrasonic power of 360 W. The determination of antioxidant capacities was conducted three times under the optimal conditions, and TEAC value was 2028.66 ± 4.59 μmol Trolox/g DW, which was close to the predicted values of 2053.73 μmol Trolox/g DW.

Comparison of Different Treatments
The effects of different treatments were compared on the antioxidant capacities of the extracts ( Figure 3A). The results showed that US had higher antioxidant capacities than that of GTE, and TAN exhibited a significant increase in antioxidant capacities compared with GTE. Furthermore, UST showed higher antioxidant capacities than that of TAN. That is, the tannase could induce biotransformation in polyphenols and produced a large number of antioxidant compounds like gallic acids, and ultrasound could improve the efficiency of an enzymatic reaction. In addition, the results obtained by the DPPH and FRAP assays showed the similar trends as that by TEAC assay (Figure 3B and C).

Optimal Treatment Conditions
Based on the single-factor tests and RSM study, the optimal condition for tannase and ultrasound treatment of the extracts was 0.1 M citrate-phosphate buffer with a pH value of 4.62, ultrasonic temperature of 44.11 • C, ultrasonic time of 12.17 min, tannase concentration of 1 mg/mL, and ultrasonic power of 360 W. The determination of antioxidant capacities was conducted three times under the optimal conditions, and TEAC value was 2028.66 ± 4.59 µmol Trolox/g DW, which was close to the predicted values of 2053.73 µmol Trolox/g DW.

Comparison of Different Treatments
The effects of different treatments were compared on the antioxidant capacities of the extracts ( Figure 3A). The results showed that US had higher antioxidant capacities than that of GTE, and TAN exhibited a significant increase in antioxidant capacities compared with GTE. Furthermore, UST showed higher antioxidant capacities than that of TAN. That is, the tannase could induce biotransformation in polyphenols and produced a large number of antioxidant compounds like gallic acids, and ultrasound could improve the efficiency of an enzymatic reaction. In addition, the results obtained by the DPPH and FRAP assays showed the similar trends as that by TEAC assay (Figures 3B and 3C).
( Figure 3A). The results showed that US had higher antioxidant capacities than that of GTE, and TAN exhibited a significant increase in antioxidant capacities compared with GTE. Furthermore, UST showed higher antioxidant capacities than that of TAN. That is, the tannase could induce biotransformation in polyphenols and produced a large number of antioxidant compounds like gallic acids, and ultrasound could improve the efficiency of an enzymatic reaction. In addition, the results obtained by the DPPH and FRAP assays showed the similar trends as that by TEAC assay (Figure 3B and C).

Antioxidants of the Extracts
The antioxidants in the extracts obtained from different treatments were identified and quantified by HPLC. Ten antioxidants were identified and quantified, including gallic acid, GC,

Antioxidants of the Extracts
The antioxidants in the extracts obtained from different treatments were identified and quantified by HPLC. Ten antioxidants were identified and quantified, including gallic acid, GC, GCG, EC, EGC, ECG, EGCG, caffeine, ellagic acid, and astragalin ( Table 3). The addition of tannase in green tea extract resulted in a significant increase in gallic acid, EGC, and EC, and a significant reduction in EGCG, ECG, and GCG (p < 0.05). By using the pure standards as the reference, the results revealed that the antioxidant capacities of UST and TAN were mainly attributed to the content of gallic acid, which took the percentage of 28.70% and 26.00% in the TEAC values, respectively. On the other hand, the antioxidant capacities of US and GTE mainly depended on the contents of EGCG, with the percentage of 14.5% and 16.38% in the TEAC values, respectively ( Figure 4). It confirmed that tannase catalyzes the hydrolysis of ester and depside bonds in gallic acid tannins or hydrolyzable tannins. In addition, the combined treatment of tannase and ultrasound significantly increased the yield of gallic acid in the extracts compared with those only treated with tannase (p < 0.05). It indicated that the simultaneous treatment of tannase and ultrasound not only accelerated the release of phenolic compounds from green tea extracts, but also increased the rate of enzymatic reaction on biotransformation, resulting in the improved antioxidant activity of green tea extracts. Furthermore, other compounds like caffeine, ellagic acid, and astragalin were changed slightly with the use of ultrasound or tannase. 0.58 ± 0.02 a 0.45 ± 0.10 b 0.53 ± 0.04 a,b 0.51 ± 0.02 a,b a, b, c Data between groups were significantly different (p < 0.05).

Conclusions
The effects of tannase and ultrasound treatment have been studied on the bioactive compounds and antioxidant activity of green tea extract. The optimal conditions were 0.1 M citrate-phosphate buffer at pH value of 4.62, ultrasonic temperature of 44.12 °C , ultrasonic time of 12.17 min, tannase concentration of 1 mg/mL, and ultrasonic power of 360 W. The tannase enzymatic reaction led to bioconversion of bioactive components in green tea extracts, and resulted in the increase of the

Conclusions
The effects of tannase and ultrasound treatment have been studied on the bioactive compounds and antioxidant activity of green tea extract. The optimal conditions were 0.1 M citrate-phosphate buffer at pH value of 4.62, ultrasonic temperature of 44.12 • C, ultrasonic time of 12.17 min, tannase concentration of 1 mg/mL, and ultrasonic power of 360 W. The tannase enzymatic reaction led to bioconversion of bioactive components in green tea extracts, and resulted in the increase of the antioxidant activity. The ultrasound exerted positive effects on the tannase enzymatic reaction, and the combined treatment of tannase and ultrasound markedly increased the antioxidant activity of the green tea extract. Furthermore, the ten antioxidants in the extracts have been identified and quantified by an HPLC method. The results showed an increase in gallic acid, EC, and EGC, and a decrease in EGCG, ECG, and GCG after the treatment. Since the consumption of EGCG has been reported to have potential toxicity in liver, the reduction of EGCG content via the tannase and ultrasound treatment might improve the human benefits of green tea extracts. The simultaneous ultrasound and tannase treatment could be a potent tool in the food industry with low cost, high efficiency, and shortened processing steps. It also could promote the production of natural antioxidants which may be used as nutritional supplements or food additives in the food industry. In the future, the in vivo antioxidant activities of these extracts should be evaluated in order to compare the effects of different treatments on the in vivo and in vitro antioxidant activites. In addition, more tea samples could be tested to verify the effectivity of these methods, such as white, yellow, oolong, black, and dark teas.