Microsoft Word - 55castellanos.docx CHEMICAL ENGINEERING TRANSACTIONS VOL. 64, 2018 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Enrico Bardone, Antonio Marzocchella, Tajalli Keshavarz Copyright © 2018, AIDIC Servizi S.r.l. ISBN 978-88-95608- 56-3; ISSN 2283-9216 Effects of High Voltage Pulsed Electric Field on Antioxidant Activity and Extraction of Tea Polysaccharides for Third Grade Ripe Pu’er Tea Baijuan Wanga, Ting Chenb, Yan Zhaoa, Wen Penga, Jing Wanga, Ji-Yuan Xiaa, Mingzhong Jiang*c a Yunnan Agricultural University, Kunming 650201, China b Chong Qing Electromechanical Vocational Institute, Chongqing 402760, China c Yunnan Poice College, Yunming 650223, China jmz4269@sina.com To enhance the antioxidant activity of third grade ripe Pu’er tea, this paper adopts different high-voltage pulsed electric fields (HPEF) to extract the tea polysaccharides and determine their antioxidant activity in each tea sample. The antioxidant activity was measured by four indices, namely the hydroxyl radical (·OH) scavenging effect, the superoxide anion (O2·–) scavenging effect, the DPPH radical scavenging effect, and the total reducibility. Based on the stepwise regression on Matlab, the mathematical model was established to depict the relationship between antioxidant activity, HPEF voltage, and HPEF frequency, and 3D relationship diagrams of the model were drawn to find the best HPEF parameters. It is concluded that the proper HPEF conditions can promote the extraction rate and antioxidant activity of tea polysaccharides in ripe Pu’er tea. The research findings open up a new way to enhance the antioxidant activity of Pu’er tea polysaccharides and shed new light on the extraction and development of natural antioxidants in Pu’er tea. 1. Introduction As the geographical indication (GI) product of China’s Yunnan Province, Pu’er tea is immensely popular at home and abroad thanks to its unique health effects (Wang et al., 2015), including but not limited to the prevention of hypertension, hyperlipidaemia and coronary heart disease (Hwang et al., 2003; Hou et al., 2009). The prominent health effects are attributable to the antioxidant activity of bioactive components, namely tea polysaccharides, theanine, caffeine, tea saponins, catechins, tea pigments and minerals. These components have been extracted as raw materials for various pharmaceuticals and healthcare products. In recent years, researchers are competing to enhance the antioxidant activity of Pu’er tea. One of the most desirable tools for antioxidant enhancement is the high-voltage pulse electric field (HPEF). By this method, the target material is placed between two electrode plates, and applied with continued high- voltage pulse wave. With a short treatment time, low energy consumption, no pollution and limited heat release, the HPEF can preserve the quality of the target material to the greatest extent. Currently, the method has been extensively applied in field like agricultural product processing (Zhong et al., 2007; Liao et al., 2003; Zhong et al., 2004), food sterilization and preservation (Ganeva et al., 2003), natural product extraction (Yin et al., 2005; Eshtiaghi et al., 2002; Loginova et al., 2011), wine aging (Fang Sheng, et al, 2003), assisted extraction and food defrosting (Zhou et al., 2012), to name but a few. In view of the above, this paper adopts the HPEF to extract tea polysaccharides from ripe Pu’er tea and examines the effect of different HPEFs on the extraction rate and antioxidant activity of the tea polysaccharides. By stepwise regression on Matlab, the antioxidant activity was discussed from such four aspects as hydroxyl radical (·OH) scavenging effect, the superoxide anion (O2· –) scavenging effect, the DPPH radical scavenging effect, and the total reducibility. Then, a mathematical model was established to depict the relationship between antioxidant activity, HPEF voltage, and HPEF frequency, and 3D relationship diagrams of the model were drawn to find the optimal HPEF parameters. DOI: 10.3303/CET1864054 Please cite this article as: Baijuan Wang , Ting Chen , Yan Zhao , Wen Peng , Jing Wang , Ji-Yuan Xia , Ming-Zhong Jiang , 2018, Effects of high voltage pulsed electric field on antioxidant activity and extraction of tea polysaccharides for third grade ripe pu’er tea, Chemical Engineering Transactions, 64, 319-324 DOI: 10.3303/CET1864054 319 2. Materials and Methods 2.1 Materials Raw material: third grade ripe Pu’er tea produced in 2011 from Lincang, Yunnan Province. Apparatuses: ISO 9001-certified electronic balance (Beijing Sartorius Instrument System Co., Ltd.), DRHH-1 thermostat bath (Shanghai Shuangjie Laboratory Instrument Co., Ltd.), 1,000mL round-bottom flask, condenser pipe, RE-52AA rotary evaporators (Shanghai Yarong Biochemical Instrument Factory), SHZ-D (III) water circulating multi-purpose vacuum pump (Henan Yuhua Instrument Co., Ltd.), funnel, beaker, erlenmeyer flask, watch glass, 722S visible spectrophotometer (Shanghai Jinghua Technology Instruments Co., Ltd.), and test tubes. 2.2 HPEF device The HPEF device consists of a DMC-200 high-voltage pulsed power supply (Dalian Dingtong Technology Development Co., Ltd.) and a processing device. The main performance parameters of the high-voltage pulsed power supply are as follows: Output voltage: 0~60kV; Input voltage: AC 220V±10%; Duty ratio of output pulse: 0~70%; Output pulse frequency: 80Hz~2,000Hz; Output power: 2,000W. 2.3 Experimental Method (1) Setting of HPEF parameters The main HPEF parameters include voltage, frequency and duration. Based on the previous findings, the voltage was set to 11 levels in 12kV~22kV, the frequency to 80Hz, 99Hz, 121Hz, 139Hz and 162Hz, and the duration to 55min. The specific parameters are listed in Table 1 below. Table 1: HPEF voltage and frequency settings (Time=55min) Group Voltage (kV) Frequency (Hz) Group Voltage (kV) Frequency (Hz) Group Voltage (kV) Frequency (Hz) 1 12 80 20 20 99 39 17 139 2 13 80 21 21 99 40 18 139 3 14 80 22 22 99 41 19 139 4 15 80 23 12 121 42 20 139 5 16 80 24 13 121 43 21 139 6 17 80 25 14 121 44 22 139 7 18 80 25 15 121 45 12 162 8 19 80 27 16 121 46 13 162 9 20 80 28 17 121 47 14 162 10 21 80 29 18 121 48 15 162 11 22 80 30 19 121 49 16 162 12 12 99 31 20 121 50 17 162 13 13 99 32 21 121 51 18 162 14 14 99 33 22 121 52 19 162 15 15 99 34 12 139 53 20 162 16 16 99 35 13 139 54 21 162 17 17 99 36 14 139 55 22 162 18 18 99 37 15 139 19 19 99 38 16 139 (2) Extraction and measurement of tea polysaccharides The alcohol precipitation method was employed to extract tea polysaccharides. First, the tea was placed in rotary evaporators to remove most of its moisture. Then, the Sevage solution (trichloromethane: n-butyl alcohol = 4:1) was added to the tea, and the mixture was oscillated for 20~30min. After that, absolute ethyl alcohol was added by twice the volume percent of the supernatant. Next, the mixture was filtered and let stand for 24h for drying. In this way, the author managed to extract the tea polysaccharides. The content of tea polysaccharides was determined by phenol-sulfuric acid spectrophotometry, and the calibration curve was drawn as follows. 320 Figure 1: Polysaccharides standard curve (3) Determination of antioxidant activity of tea polysaccharides As mentioned above, the antioxidant activity of tea polysaccharides was measured by four indices: the ·OH scavenging effect, the O2· – scavenging effect, the DPPH radical scavenging effect, and the total reducibility. The scavenging effects can be obtained by the following formula: scavenging rate (%) = [1-((A1-A2 ))/A0 ]×100 The ·OH scavenging effect was determined by sodium salicylate complexometry (Xu et al., 2006). The specific steps are as follows. First, mix 2mL 20mg/mL tea polysaccharides solution with 2mL 9mmol/L FeSO4 and 2mL 9mmol/L H2O2, and let the mixture react at room temperature for 20min. Then, add 2mL 9mmol/L sodium salicylate solution, and let the mixture react at room temperature for another 20min. After that, measure the absorbance A1 of tea polysaccharides in different groups at λ=510nm. Next, replace sodium salicylate solution with 2mL distilled water, and repeat the steps above to obtain the absorbance A2. In addition, substitute the tea polysaccharides solution with 2mL distilled water, and repeat the steps above to obtain the absorbance A0. The O2·– scavenging effect was determined by pyrogallol autoxidation (Guo et al., 2007). The specific steps are as follows. First, take 4mg/mL tea polysaccharides solution as the base solution, and prepare tea polysaccharides solutions with the concentration of 0.05mg/mL, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, and 0.6mg/mL, respectively. Then, take 0.2mL tea polysaccharides solution of each concentration, add it with 5.7mL 50mmol/L Tris-HCl buffers (pH=8.2), and let the mixture react in 25°C water for 20min. After that, add 0.1mL 6mmo1/L pyrogallol solution and let the mixture react for 5min. Finally, measure the absorbance A1 at λ=360nm. Next, replace pyrogallol solution with distilled water of the same volume, and repeat the steps above to obtain the absorbance A2. In addition, substitute the tea polysaccharides solution with 2mL distilled water, and repeat the steps above to obtain the absorbance A0. The DPPH radical scavenging effect was determined by the method of Zhang et al. (2007) The specific steps are as follows. First, prepare 0.2mmol/L DPPH radical anhydrous ethanol solution in a brown bottle at 0~4 °C. Then, mix 2mL 20mg/mL tea polysaccharides solution with 2mL 0.2mmol/L DPPH radical anhydrous ethanol solution in a test tube, and let the mixture stand for 0.5h. After that, measure the absorbance A1 at λ=517nm. Next, replace the DPPH radical anhydrous ethanol solution with anhydrous ethanol solution of the same concentration, and repeat the steps above to obtain the absorbance A2. In addition, substitute the tea polysaccharides solution with 2mL 20mg/mL anhydrous ethanol solution, and repeat the steps above to obtain the absorbance A0. The total reducibility of tea polysaccharides was determined by the method of Zou et al. (2014) The specific steps are as follows. First, take 4mg/mL tea polysaccharides solution as the base solution, and prepare 20mL tea polysaccharides solutions with the concentration of 0mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL and 5mg/mL, respectively. Then, take 1mL tea polysaccharides solution of each concentration, add it with 1mL 2mol/L phosphate buffer (pH=6.6) and 1mL potassium ferricyanide solution (ω=1%), and let the mixture stand for 20min. After that, add 0.25mL ferric chloride (ω=0.1%) and 2mL distilled water, and let the mixture stand for 20min. Finally, measure the absorbance A at λ=360nm. The value of A is positively correlated with the total reducibility. 3. Results and Analysis 3.1 Effect of HPEF on the extraction of tea polysaccharides For the tea samples in the blank group and those under different HPEFs, the content of tea polysaccharides was calculated according to the standard curve equation: y=8.2200x+0.0147 (R2=0.9997). The results are listed in Table 2. 321 Table 2: The content of tea polysaccharides in its crude extraction Group Content of tea polysaccharides (mg/mL) Group Content of tea polysaccharides (mg/mL) Group Content of tea polysaccharides (mg/mL) CK 0.1590 19 0.1638 38 0.1659 1 0.1661 20 0.1649 39 0.1633 2 0.1672 21 0.1595 40 0.1629 3 0.1631 22 0.1632 41 0.1628 4 0.1644 23 0.1605 42 0.1637 5 0.1595 24 0.1646 43 0.1633 6 0.1644 25 0.1650 44 0.1646 7 0.1632 26 0.1641 45 0.1668 8 0.1560 27 0.1661 46 0.1649 9 0.1656 28 0.1606 47 0.1635 10 0.1596 29 0.1611 48 0.1640 11 0.1588 30 0.1660 49 0.1629 12 0.1654 31 0.1643 50 0.1627 13 0.1640 32 0.1629 51 0.1657 14 0.1616 33 0.1660 52 0.1649 15 0.1637 34 0.1645 53 0.1640 16 0.1628 35 0.1637 54 0.1598 17 0.1632 36 0.1634 55 0.1531 18 0.1645 37 0.1635 It is clear that the tea samples under HPEF had a higher content of tea polysaccharides than those in the blank group. For instance, the content in group 2 (13kV/80Hz) reached the peak value of 0.1672mg/mL, 5.09% higher than that of the blank group. This means proper HPEF conditions can increase the content of tea polysaccharides in its crude extraction. 3.2 Effect of HPEF on the ·OH scavenging effect of tea polysaccharides The best-fit binary cubic equation was established through multiple nonlinear fittings of the relationship between the scavenging effect (Y), HPEF voltage (X1) and frequency (X2). The regression model is as follows: Y=94.232+0.55945X1+0.061745X2-0.035053X1 2- 0.00049171X2 2+0.00027475X1X2+0.00067568X1 3+0.000001202X2 3 Since the P-value (5.34e-08) is less than 0.01 and the intercept/P-value of each coefficient is less than 0.5, the model is applicable to the prediction of the ·OH scavenging effect of tea polysaccharides in tea samples under HPEF. The 3D diagram was drawn below according to the model. As shown in Figure 2, when the HPEF voltage fell in 12~18kV, the ·OH scavenging effect of tea polysaccharides grew stronger and then became weaker with the increase in frequency; when the HPEF voltage was higher than 18kV, the scavenging effect was improved on the whole. The best scavenging effect appeared at X1=22kV and X2=162Hz. 3.3 Effect of HPEF on the O2·– scavenging effect of tea polysaccharides The best-fit binary cubic equation was established through multiple nonlinear fittings of the relationship between the scavenging effect (Y), HPEF voltage (X1) and frequency (X2). The regression model is as follows: Y=-35.784+2.2099X1+2.1266X2-0.0073939X2 2-0.028182X1X2 Since the P-value (3.75e-08) is less than 0.01 and the intercept/P-value of each coefficient is less than 0.05, the model is applicable to the prediction of the O2· – scavenging effect of tea polysaccharides in tea samples under HPEF. The 3D diagram was drawn below according to the model. Figure 2: Three-dimensional diagram of the rate of Figure 3: Three-dimensional diagram of the rate of scavenging tea polysaccharides for ·OH scavenging tea polysaccharides for O2·– 322 Figure 4: Three-dimensional diagram of the rate of Figure 5: Three-dimensional diagram of scavenging tea polysaccharides for DPPH total reducibility of tea polysaccharides As shown in Figure 3, when the HPEF voltage ranged from 12kV to 18kV, the O2· – scavenging effect of tea polysaccharides grew stronger and then became weaker with the increase in frequency; when the HPEF frequency was lower than 100Hz, the scavenging effect remained basically stable with the change in voltage; when the frequency was higher than 100Hz, the scavenging effect decreased with the increase in voltage. The best scavenging effect appeared at X1<14kV and X2 ∈ (120, 140) Hz. 3.4 Effect of HPEF on the DPPH scavenging effect of tea polysaccharides The best-fit binary cubic equation was established through multiple nonlinear fittings of the relationship between the scavenging effect (Y), HPEF voltage (X1) and frequency (X2). The regression model is as follows: Y=109.72-0.083284X1-0.173017X2+0.00059166X2 2 Since the P-value (4.54e-08) is less than 0.001 and the intercept/P-value of each coefficient is less than 0.05, the model is applicable to the prediction of the DPPH radical scavenging effect of tea polysaccharides in tea samples under HPEF. The 3D diagram was drawn below according to the model. As shown in Figure 4, when the voltage was regular, the DPPH radical scavenging effect of tea polysaccharides generally decreased with the increase in frequency; when the frequency was irregular, the scavenging effect was basically immune to voltage increase. Hence, frequency is the main influencing factor of the DPPH radical scavenging effect. The best scavenging effect appeared at X1<14kV and X2≈80Hz. 3.5 Effect of HPEF on the total reducibility of tea polysaccharides The best-fit binary cubic equation was established through multiple nonlinear fittings of the relationship between the scavenging effect (Y), HPEF voltage (X1) and frequency (X2). The regression model is as follows: Y=-44.954+5.3492X1+0.70703X2-0.14596X1 20-0.0026079X2 2- 0.081965X1X2+0.0022262X1 2X2+0.00029876X1X2 2-0.000008071X1 2X2 2 Since the P-value (9.22e-08) is less than 0.001 and the intercept/P-value of each coefficient is less than 0.05, the model is applicable to the prediction of the reducibility of tea polysaccharides in tea samples under HPEF. The 3D diagram was drawn below according to the model. As shown in Figure 5, when λ=360nm and X1>14kV, the total reducibility exhibited a decline trend with the increase in frequency; when X1<14kV, the total reducibility first increased and then decreased with the frequency growth; the strongest reducibility appeared at X1>18kV and X2≈80Hz. 4. Conclusion In this paper, the HPEF was adopted to extract the polysaccharides from third grade ripe Pu’er tea. Then, the content of tea polysaccharides in different groups of tea samples were compared one by one. The comparison shows that proper HPEF conditions can increase the extraction rate, i.e. the content of tea polysaccharides in its crude extraction. After that, the scavenging effects and total reducibility of tea polysaccharides were analysed by stepwise regression on Matlab. Based on the P-value, the regression model was proved applicable to the prediction of the relationship between the antioxidant activity of tea polysaccharides, HPEF voltage and HPEF frequency. Then, the 3D diagrams were drawn according to the model. Since the P-value is always below 0.01, the optimal regression model is significant enough for prediction of the relationship between antioxidant activity of tea polysaccharides, HPEF voltage and frequency. The model analysis reveals that the HPEF can improve the antioxidant activity of tea polysaccharides in ripe Pu’er tea to a certain extent. It is also concluded that the best ·OH scavenging effect appeared at X1=22kV and X2=162Hz; the best O2· – scavenging effect appeared at X1<14kV and X2 ∈ (120, 140) Hz; the best DPPH radical 323 scavenging effect appeared at X1<14kV and X2≈80Hz; the strongest reducibility appeared at X1>18kV and X2≈80Hz. To sum up, the proper HPEF conditions can promote the extraction rate and antioxidant activity of tea polysaccharides in ripe Pu’er tea. 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