CHEMICAL ENGINEERING TRANSACTIONS VOL. 62, 2017 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Fei Song, Haibo Wang, Fang He Copyright © 2017, AIDIC Servizi S.r.l. ISBN 978-88-95608- 60-0; ISSN 2283-9216 Effects of High Voltage Pulsed Electric Field on Antioxidant Activity of Tea Polyphenols for Yunnan Pu’er Tea Mingzhong Jianga,b, Ting Chen*c, Yan Zhaoa, Qi Wua, Bo Fenga, Shan Xionga, Baijuan Wanga* a Yunnan Agriculture University, Kunming 650201, China b Yunnan Police College, Kunming 650223, China c Chongqing Electromechanical Vocational Institute, Chongqing 402760, China wangbaijuan123@126.com In order to improve the antioxidant activity of Pu’er tea, the authors use different high voltage pulsed electric field (HPEF) to deal with the third grade Pu’er ripe tea, extract the tea polyphenols and determine antioxidant activity of tea polyphenols in each tea samples by four indexes, namely the ability of scavenging hydroxyl radical (·OH), the ability of scavenging superoxide anion (O2·–), the ability of scavenging radical DPPH and total reducibility. Then antioxidant activity of tea polyphenols is analyzed by stepwise regression analysis on Matlab, establish the mathematical model of its antioxidant activity and voltage, frequency. The results show that HPEF could decrease the content of tea polyphenols in Pu’er ripe tea, and promote it to translate into Theabrownines (TBs) and so on. The suitable conditions of HPEF can improve the antioxidant activity of Pu’er tea to a certain extent. This study provides a new physical method for improving the antioxidant activity of Pu’er tea polyphenols, and provides technical support for the extraction and development of natural antioxidants in Pu’er tea. 1. Introduction Pu’er Tea, as a geographical indication product in Yunnan Province, has been beloved by domestic and international tea lovers because of its unique quality and many health benefits (Wang et al., 2015; GB/T 30766-2014). There are many health functions of Pu’er tea like the lipid-lowering diet, prevention and treatment of coronary heart disease, falling hypertension, anti-aging and so on(Hwang et al., 2003; Hou et al., 2009; Kuo et al., 2005; Lee et al., 2013). Among of these functions, the most important mechanism is its antioxidant effect. High voltage pulsed electric field (HPEF) is the process of treating high voltage pulse waves intermittently between two parallel electrode plates. It has been widely applied in agricultural products and food processing industry for its many advantages such as short processing time, low energy consumption, no pollution and so on (Yin et al., 2007; Zhong et al., 2007; Liao et al., 2003). At present, domestic and foreign scholars have achieved certain results in food sterilization and preservation (Zhang et al., 2012), extraction of natural products (Ganeva et al., 2003; Yin et al., 2007; Yin et al., 2005; Eshtiaghi et al., 2002; Loginova et al., 2011), wine aging (Fang et al., 2003), extraction (López et al., 2008) and other fields mainly using the physical means HPEF. This paper deals with the extraction of tea polyphenols from tea samples by HPEF, and studies the effect of HPEF on the extraction rate and antioxidant activity of Pu’er tea polyphenols. Using Matlab to analyze the antioxidant activity of tea polyphenols by stepwise regression analysis, the author establishes the mathematical model between antioxidant activity and HPEF voltage and frequency, and draws the three- dimensional relationship graph to find the best HPEF treatment parameters. 2. Materials and methods 2.1 Experimental materials and equipment Experimental materials: three-grade Pu’er ripe tea produced in 2011 in Lincang. DOI: 10.3303/CET1762210 Please cite this article as: Mingzhong Jiang, Ting Chen, Yan Zhao, Qi Wu, Bo Feng, Shan Xiong, Baijuan Wang, 2017, Effects of high voltage pulsed electric field on antioxidant activity of tea polyphenols for yunnan pu’er tea, Chemical Engineering Transactions, 62, 1255- 1260 DOI:10.3303/CET1762210 1255 Experimental instruments: ISO 9001 type electronic balance (Beijing Sartorius Co. Ltd.), reflux extraction device (1000ml round bottom flask, an condensing tube, an iron stand, alcohol lamp and asbestos net), extraction device (a 250ml separatory funnel, an iron stand, 150ml beakers and 400ml beakers); drying device (a tripod, an alcohol lamp, 250ml beaker, surface vessel and asbestos net); 722S spectrophotometer (Shanghai Jinghua Science and Technology Co. Ltd.), some beaker tubes and some 50mL beakers. 2.2 High voltage pulsed electric field device The HPEF process device consists of two parts: high voltage pulse power supply and processing device. The core part of the device is a high voltage pulse power supply. The main performance parameters of the high voltage pulse power supply are as follows: Output voltage: 0~60kV; Input voltage: AC 220V ± 10%Output pulse duty ratio: 0~70%; Output pulse frequency: 80Hz~2000Hz;Output power: 2000W. 2.3 Experimental method High voltage pulsed electric field process Pu’er ripe tea Voltage, frequency and time are the main parameters affecting the high voltage pulsed electric field. According to the preliminary research results of the project team, the setting time is 55min, the frequency is 80Hz, 99Hz, 121Hz, 139Hz and 162Hz, the voltage is 12kV-22kV, and their parameters are shown in Table 1. Table 1: HPEF voltage and frequency settings (Time=55min) NO. Voltage(kV) (Hz) Frequency NO. Voltage(kV) (Hz) Frequency NO. Voltage(kV) (Hz) Frequency 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 Extraction and measurement of tea polyphenols Extract the tea polyphenol standard solution from 1.0mL, 2.0mL, 3.0mL and 4.0mL into the four 450mL flask, and add distilled water to filter and dilute to 10mL, then add 10mL ferrous tartrate solution, finally add phosphate buffer (pH=7.5) to the scale, mixing into the cuvette with blank reagent as a reference. The absorbance value (A) is measured at λ=540nm, and then draw a standard curve as shown in Figure 1. Figure 1: Polyphenol standard curve 1256 Figure 2: Scavenging·OH for polyphenols Figure 3: Scavenging O2·– for tea tea polyphenols Determination of antioxidant activity of tea polyphenols The determination of antioxidant activity of tea polyphenols contains four indexes: hydroxyl radical (·OH) scavenging ability, superoxide anion (O2·–) scavenging ability, DPPH radical scavenging ability and total reducibility. Among of these, the calculation formula of the scavenging rate is: The scavenging rate(%)=[1-(A1-A2)/A0]×100 Determination of the scavenging ability of ·OH: Sodium salicylate (XU Jianguo, et al, 2006) was used to determine the scavenging ability of tea polyphenols on ·OH. Determination of the scavenging ability of O2·–: utilize the pyrogallol autoxidation method (Guo et al., 2007) to determine scavenging effects of tea polyphenols on O2·– determination of scavenging capacity of DPPH radical: According to the method from Zhang et al. (2007), and others, we can determine the scavenging effect of tea polyphenols on DPPH radical Determination of total reducibility of tea polyphenols: According to the method from Zou et al. (2014) and others, we determine the total reducibility. 3. Results and analysis 3.1 Effect of HPEF on Extraction of tea polyphenols Table 2: The content of tea polyphenols in its crude extraction NO. Tea polyphenols content(mg/mL) NO. Tea polyphenols content(mg/mL) NO. Tea polyphenols content(mg/mL) CK 0.0118 19 0.0087 38 0.0105 1 0.0077 20 0.0136 39 0.0104 2 0.0082 21 0.0112 40 0.0100 3 0.0090 22 0.0067 41 0.0107 4 0.0089 23 0.0091 42 0.0094 5 0.0099 24 0.0093 43 0.0089 6 0.0091 25 0.0102 44 0.0087 7 0.0099 26 0.0110 45 0.0110 8 0.0096 27 0.0100 46 0.0106 9 0.0077 28 0.0089 47 0.0098 10 0.0094 29 0.0106 48 0.0100 11 0.0085 30 0.0094 49 0.0089 12 0.0098 31 0.0093 50 0.0085 13 0.0091 32 0.0104 51 0.0101 14 0.0106 33 0.0098 52 0.0091 15 0.0114 34 0.0106 53 0.0083 16 0.0098 35 0.0114 54 0.0077 17 0.0102 36 0.0112 55 0.0088 18 0.0075 37 0.0108 1257 According to the standard curve equation of polyphenols y=7.5350x+0.0123 (R2=0.9997), to calculate the content of tea polyphenols in blank group and treated by different HPEF groups in crude extraction of tea, its contents are shown in Table 2. From the table, most of crude extraction after treated by HPEF, its content of tea polyphenols in tea samples is lower than the blank group (CK). Among that, the content of tea polyphenols of the twenty-second group (22kV/99Hz) is the lowest, which is 0.0067mg/mL, and the content of tea polyphenols is 43.22% lower than that in the blank group. 3.2 The effect of HPEF on tea polyphenols to scavenge·OH The regression model is as follows: Y=-107.31+34.214X1+0.21157X2-0.00413X1X2-2.0427X 2 1 -0.000597X 2 2 +0.040711X 3 1 The optimal regression model P = 2.56e-08 (less than 0.001), this model is significant, in the model, the P values of the intercept and the coefficients are <0.05.The model can be used to forecast and analyze the tea polyphenols’ scavenging ability on ·OH after the HPEF treatment in tea samples. Draw a three-dimensional graph according to the model as Figure 2. When the HPEF voltage is 12-18kV, the removal ability of tea polyphenols on ·OH strengthens with the increase of frequency, then weaken; When the HPEF voltage is greater than 18kV, the total removal rate increases with the increase of frequency. When the HPEF condition is about 22kV/162Hz, the tea polyphenols has the best scavenging effect on ·OH. 3.3 The effect of HPEF on tea polyphenols to scavenge O2·– The regression model is as follows: Y=1703.9-169.71X1-30.035X2+3.1584X1X2+4.3041X 2 1 +0.12554X 2 2 -0.08333X 2 1 X2-0.01357X1X 2 2 +0.000367X 2 1 X 2 2 The optimal regression model P = 3.75e-08 (less than 0.001), this model is significant, in the model, the P values of the intercept and the coefficients are <0.05. The model can be used to forecast and analyze the tea polyphenols’ scavenging ability on O2·– after the HPEF treatment in tea samples. Draw a three-dimensional graph according to the model as Figure 3. When the HPEF voltage is constant, the removal ability of tea polyphenols on O2·– strengthens with the increase of frequency, then weakens. When the voltage is 22kV, the tea polyphenols has the best scavenging effect on O2·–. 3.4 The effect of HPEF on tea polyphenols to scavenge radical DPPH Stepwise regression analysis is performed the ability of tea polyphenols to radical DPPH after HPEF treatment , and use two third-order polynomial equations to have multivariate nonlinear fitting, on the scavenging capacity (Y) and voltage of HPEF (X1)and the frequency (X2), establish equation, and then get the optimal fitting equation. The regression model is as follows: Y=838.81-6.0972X1-19.195X2+0.18985X 2 1 +0.16874X 2 2 -0.000478 X 3 2 The optimal regression model P =6.52e-12 (less than 0.001), this model is significant, in the model, the P values of the intercept and the coefficients are <0.05. The model can be used to forecast and analyze the tea polyphenols’ scavenging ability on radical DPPH after the HPEF treatment in tea samples. Draw a three- dimensional graph according to the model as Figure 4. Figure 4: Scavenging DPPH for tea polyphenols Figure 5: Total reducibility of tea polyphenols 1258 When the HPEF voltage is constant, the removal ability of tea polyphenols on radical DPPH weakens with the increase of frequency, then strengthens and weakens. When the frequency is about 140Hz, the tea polyphenols has the best scavenging effect on DPPH. 3.5 The effect of HPEF on total reducibility of tea polyphenols The optimal regression model P =2.43e-12 (less than 0.001), this model is significant, in the model, the P values of the intercept and the coefficients are <0.05. The model can be used to forecast and analyze the tea polyphenols’ total reducibility after the HPEF treatment in tea samples. Draw a three-dimensional graph according to the model as Figure 5. When the wavelength is 360nm and the HPEF frequency is constant，the total reducibility of tea polyphenols strengthens with the increase of frequency, then weakens. When the voltage is 16-20kV and the frequency is about 80Hz or 160Hz, HPEF can improve the total reducibility of tea polyphenols. 4. Conclusions By using high voltage pulsed electric field to deal with the Pu'er Tea and extract the tea polysaccharides from tea samples, the contents of tea polyphenols in tea samples are compared, and the results show that the appropriate HPEF could decrease the content of tea polyphenols. Secondly, scavenging effects of tea polyphenols and its total reducibility are analyzed by stepwise regression analysis on Matlab, then establish the corresponding optimal regression model and draw the three-dimensional relationship diagram. The results show that the significance test probability of the optimal regression model P<0.01, that is the model is distinct. We can see that through the analysis of the corresponding regression model: HPEF can improve the antioxidant activity of Pu’er tea polyphenols to a certain extent, and 1) When the voltage is 22kV, the tea polyphenols have the best scavenging effect on ·OH; 2) When the frequency is less than 100Hz and the voltage is less than 16kV, tea polyphenols has the greatest scavenging ability for O2·–; 3)The main factor affecting the scavenging ability on DPPH radical is frequency; when the frequency is about 140Hz, the scavenging ability of tea polyphenols on DPPH radical is the highest; 4) When the voltage is 16kV~20kV and the frequency is about 80Hz and 160Hz, the total reducibility of tea polyphenols is the strongest. Therefore, the suitable high voltage pulsed electric field can improve the antioxidant activity of Pu’er tea polyphenols. This study provides a new physical method for improving the antioxidant activity of Pu’er tea polyphenols, and provides a new technical support for the extraction and development of natural antioxidants, and the deep processing and the development of functional food of Pu’er tea. Acknowledgments Fund Source: National Natural Science Foundation of China (61561054); Corresponding author is BaiJuan Wang, Yunnan Jianchuan (Bai nationality), Master, Associate Professor, Research direction: Biophysics. E- mail: wangbaijuan123@126.com Reference Eshtiaghi M.N., Knorr D., 2002, High electric field pulse pretreatment: potential for sugar beet processing, Journal of Food Engineering, 52(3), 265-272, DOI: 10.1016/S0260-8774(01)00114-5. Fang S., Sun X.B., Lu S.D., 2003, The study on the acceleration rule and thepringiple of the ice thawing processing by HPEF, Journal of Beijing Technology and Business University (Natural Science Edition), 21 (4), 44-45. Ganeva V., Galutzov B., Teissie J., 2003, High yield electroextraction of proteins from yeast by a flow process, Analytical Biochemistry., 315(1), 77-84, DOI: 10.1016/S0003-2697(02)00699-1. Guo Y.H., Hu S.Q., 2007, Study on Antioxidant Activities of Eleocharis Tuberosa Peel, Food and fermentation industry, 33(10), 128-130. Hou Y., Shao W.F., Xiao R., Xu K., Ma Z., 2009, Pu’er Tea Aqueous Extracts Lower Atherosclerotic Risk Factors in a Rat Hyperlipidemia Model, Experimental Gerontology, 44(6/7), 434-439, DOI: 10.1016/j.exger.2009.03.007. Hwang L.S., Lin L.C., Chen N.T., Liuchang H.C., Shiao M.S., 2003, Hypolipidemic effect and antiatherogenic potential of Pu-Erh tea, ACS Symp, Ser, (859), 87-103, DOI: 10.1021/bk-2003-0859.ch005. Kuo K.L., Weng M.S., Chiang C.T., 2005, Comparative Studies on the Hypolipidemic and Growth Suppressive Effects of Oolong, Black, Pu’er, and Green Tea Leaves in Rats, Journal of Agricultural and Food Chemistry, 53(2), 480-489, DOI: 10.1021/jf049375k. Lee L.Y., Foo K.Y., 2013, Recent Advances on the Beneficial Use and Health Implications of Pu’er Tea, Food Research International, 53(2), 619-628, DOI: 10.1016/j.foodres.2013.02.036. 1259 Liao X.J., Zhong K., Wang L.M., Shi Z.N., 2003, Inactivation of Saccharomyces cerevisiae and E.coli by Pulsed Electric Fields Technology, Food and Fermentation Industries, 29(10),19-22. Loginova K., Logino M., Vorobiev E., Lebovka N., 2011, Quality and filtration characteristics of sugar beet juice obtained by“cold”extraction assisted by pulsed electric field, Journal of Food Engineering, 106(2), 144- 151, DOI: 0.1016/j.jfoodeng.2011.04.017. López N., Puertolas E., Condon S., ÁLvarez I., Raso J., 2008, Effects of pulsed electric fields on the extraction of phenolic compounds during the fermentation of must of Tempranillo grapes, Innovative Food Science and Emerging Technologies, 9, 477-482, DOI: 10.1016/j.ifset.2007.11.001. Wang B.J., Zhang J.G., 2015, Drinking and tasting of Yunnan Pu’er tea, Kunming: Yunnan Science and Technology Press. Xu J.G., Hu Q.P., 2006, Study on Free Radical Scavenging Capacity by Cassia Seed Water Extract in vitro, Food Science, 27(6), 73-76. Yin Y.G., Hao G.D., Ding Y., 2007, The high voltage electric field (PEF) new technology, Agro Food Science And Technology, (1), 11-14. Yin Y.G., He G.Dan, Shi J., 2005, Experiment of Liquor Aging by High Voltage Pulse Electric Field, Liquor- Making Science & Technology, 1, 47-50. Yin Y.G., Jin Z.X., Wang C.L., 2007, The effect of pulsed electric field on DNA extraction from bovine spleens, Separation and Purification Technology, 56(2), 127-132, DOI: 10.1016/j.seppur.2007.01.033. Zhang J.M., Xiao X.N., Yi X., Ma Y.H., 2007, The Extraction of Soluble Dietary Fiber from Plantago asiatica and Its Free Radicals Scavenging Activity, Natural products Research and development, 19(4), 667-670. Zhang W., Han Q.G., Zhu Y.J., Zheng X.H., He J., Liu K.Z., Yang Y., 2012, Study on the application of high pulse electric field technology in preserving Myrica rubra fruits, Science and Technology of Food Industry, 33(2), 389-392. Zhao W.Q., Yin Y.G., Wang Z.D., 2001, Present Situat ion and Development of Pasteurization Technology by High-Voltage Electric Field, Transactions of the CSAE, 17(5), 139-141. Zou Z.H., Xie H.K., Deng G.Q., Peng X.M., Li H.G., Hu J.S., Li X., Zhang L.P., Tan X.H., 2014, The Research of Antioxidant Effect and Molecular Weight of Liquid Hyaluronic Acid by Electron Beam, Journal of Nuclear Agricultural Sciences, 28(11), 2010-2014. DOI: 10.11869/j.issn.100-8551.2014.11.2010. 1260