Using cellulase to improve the extraction efficiency of the antioxidant compounds from white radish (raphanus sativus l.) powder - Pham Thi Kim Ngoc

Journal of Science and Technology 54 (4A) (2016) 48-54 USING CELLULASE TO IMPROVE THE EXTRACTION EFFICIENCY OF THE ANTIOXIDANT COMPOUNDS FROM WHITE RADISH (Raphanus sativus L.) POWDER Pham Thi Kim Ngoc 1, 3, * , Nguyen Thi Minh Nguyet 2 , Dong Thi Anh Dao 3 1 Ba Ria-VungTau University, 80 Truong Cong Dinh, 3 Ward, Vung Tau City, BR-VT Province 2 Industrial University of Ho Chi Minh City, 12 Nguyen Van Bao, Go Vap, HCMCity 3 Ho Chi Minh City University of Technology, 268 Ly ThuongKiet, 10 District, HCM City * Email: kimngoc080283@gmail.com Received: 15 August 2016; Accepted for publication: 5 October 2016 ABSTRACT White radish (Raphanus sativus L.) is an edible root vegetable of the Brassicaceae family. It is cultivated and consumed all over the world. Nowadays, radishes are used and eaten by many people in their daily meals. Some studies showed that it has a high content of antioxidant compounds. In this study, we used enzyme - assisted extraction (EAE) to produce an antioxidant - rich extract. The antioxidant activity of the extract was determined by the ABTS radical cation scavenging capacity. The optimal extraction conditions of the antioxidant compounds from white radish were: enzyme concentration of 1.75 % (v/w), extraction time of 81 minutes, pH 4.56 and extraction temperature of 48 o C. As a result, the antioxidant activity of the extract was 0.1594 ± 0.033 m Moltrolox equivalent antioxidant capacity (TEAC)/g and 161 % higher than that of the control sample(CE).Furthermore, SEM (scanning electron microscopy) analysis result showed that there was a difference in the surface structure of white radish powder after being extracted by CE and EAE. Keywords: white radish, Raphanus sativus L., antioxidant compound, EAE, cellulase. 1. INTRODUCTION White radish (Raphanus sativusL., family Brassicaceae) is grown mainly for its edible and fleshy root. The root can beaten raw, cooked or preserved in salt. It is a rich source of bioactive molecules including anthocyanins, glucosinolates, isothiocyanates, flavonoids and phenolics. Some previous studies showed that white radish has a high content of antioxidant compounds. The total phenolics content in the freeze – dried juice of Thai radish root was approximately 10.09 mg/g. The scavenging effects of this juice on DPPH radical, superoxide radical and singlet oxygen were 0.64, 4.2 and 1.42 mg/ml, respectively [1]. The substances associated with the antioxidant activity are the flavonoids (quercetin, kaempferol, myricetin, apigenin, luteolin, Using cellulase to improve the extraction efficiency of antioxidant compounds from whiteradish 49 catechin, rutin) [2] and phenolic acids (caffeic, p-coumaric, ferulic, hydroxycinnamic, p- hydroxybenzoic, vanillic, syringic and sinapic acid) [3, 4]. There were many researches on enzyme-assisted extraction (EAE) that might improve the recovery of phenolics and preserve the functional properties. This method has been reported for extracting the phenolics from green tea and citrus peel [5, 6]. The mechanism of EAE is that enzymes have the ability to degrade or disrupt cell walls and membranes, thus it enables better release and more efficient extraction of bioactivities [7]. The aim of this study is to use cellulase enzyme to extract antioxidant compounds from white radish powder. The antioxidant activity of the extract was demonstrated basing on inhibition capacity of ABTS free radical. The optimal parameters of EAE were determined by RSM and compared with CE. 2. MATERIALS AND METHODS 2.1. Materials Plant material: Fresh white radish roots were harvested from Duc Trong district, Lam Dong province. Fresh roots were washed, cut into small slices and dried at 50 o C until the moisture content was 6 – 7 %. After that, they were ground to powder, sieved through a sifter (40 meshes) and stored in a plastic sealed bag and kept in the dark with a freezer at -20 o C. Chemicals: cellulase (Celulast1.5L) was derived from Trichodermareesei and manufactured by Novo Nordisk Ferment (Switzerland). It is liquid, brown and slight fermentation odor. Its activity is about 700 EGU/g with a density of 1.22 g/ml. The suitable range of pH and temperature, where active enzyme is stable - typically, is 4 - 9 and 45 – 55 oC, respectively. The product should be stored with recommended temperature from 0 o C to 25 o C. Trolox and ABTS were purchased from Sigma-Aldrich. Other chemicals were analytical grade. 2.2. Antioxidant extraction process EAE: 1 g sample was mixed with 75 mL of distilled water in a 250 mL beaker. Then, cellulase enzyme was added. The mixture was placed into a water bath for different times at the required temperature. After finishing the extraction process, the enzyme was inactivated at 90 °C for 5 min and filtered. The filtrate was collected, made up to 100 mL and used for determining the antioxidant activity. CE: Another sample was extracted in the same optimal conditions of the EAE without enzyme and used as the control sample for the comparison. Experimental design: primary trials of EAE were carried out with enzyme concentration (0.5, 1.0, 1.5, 2.0 and 2.5 % v/w dry mass - DM), temperature (40, 50, 60 and 70 o C), time (30, 60, 90, 120 and 150 minutes) and pH (4, 5, 6 and 7) to choose the suitable values for central points in optimal experiment. A four independent variables, three - level central composite face response surface design (RSM - CCF) was used to optimize these factors. A second - order polynomial model was used to describe the capacity of antioxidant activity. After evaluating the model and collecting the estimates of the optimal points, the final optimum values were determined by experiments. Pham Thi Kim Ngoc, Nguyen Thi Minh Nguyet, Dong Thi Anh Dao 50 2.3. Analytical methods The antioxidant activity of the extract which was determined by the reduction of the ABTS radical cations by antioxidants present in the extract was described by Re et al. [8]. The percentage inhibition of absorbance at 734 nm was calculated and plotted as a function of concentration of antioxidants and Trolox for the standard reference data. The overall relationship between concentration and absorbance was described by a straight line with the equation y = 32.37x + 1.1808 (R 2 = 0.998). The results were expressed as mM TEAC/g DM. All experiments were carried out in triplicate; results were expressed as mean ± SD and analyzed by Statgraphics Centurion XV.I. The optimal extraction conditions were estimated though RSM using Modde 5.0 software. 3. RESULTS AND DISCUSSION 3.1. Single - factorexperiments 3.1.1. Effectsofenzyme concentration Enzyme concentration is an important parameter that can influence the extraction efficiency. In this study, enzyme concentrations were performed at 0.5, 1.0, 1.5, 2.0 and 2.5 % v/w when the other reaction conditions were as follows: extraction time 90 minutes, extraction temperature 50 °C and pH 5.0. The results are illustrated in Fig. 1, the antioxidant activity is highest at cellulase concentration of 1.5 % (0.101 ± 0.005 mM TEAC/g DM) and significantly different from the others. The lower concentrations were not enough to extract all antioxidants; but the higher concentrations (from 2.0 to 2.5 %) did not increase the antioxidant activity of the extracts. Therefore, the enzyme concentration of 1.5 % was the most favorable. 3.1.2. Effects of different extraction temperatures Extraction temperature would influence the molecular movement because heat could accelerate dissolution of antioxidant compounds. In addition, temperature effects strongly on the activity of the enzyme. However, when the temperature rises to a certain ambit, the enzymes will be denatured and inactivated because of protein nature of enzyme. In this experiment, different temperatures (40, 50, 60, and 70 °C) were chosen to study theeffects on Figure 2. Effects of temperature on antioxidant activity of extracts from white radish powder. Figure 1. Effects of enzyme concentration on antioxidant activity of extracts from white radish powder. Using cellulase to improve the extraction efficiency of antioxidant compounds from whiteradish 51 antioxidant activity of the extract. The data was illustrated in Fig.2, the suitable temperature for extraction was 50 o C. 3.1.3. Effects of extraction time The antioxidant activity of extraction at different extraction times is shown in Fig. 3. When the extraction time increased, the yield of antioxidant compounds would also increase, hence, the antioxidant activity of the extracts is higher and reaches the maximum values between 90 and 120 minutes. After that, there is a light decrease because antioxidant compounds are sensitive to high temperature for a long time. In the other hand, this decrease might be possibly caused by the loss of enzyme activity. The difference in antioxidant activity of extract between 90 and 120 minutes is not significant. As a result, we concluded that 90 minutes were suitable time to extract antioxidant compounds from white radish powder. 3.1.4. Effects of pH During enzymatic treatment, the pH values were varied from 4 to 7 to investigate its effect on antioxidant activity of the extract. The results in Fig. 4 declare that antioxidant activity is highest at pH = 5. Therefore, we chose this value for the further experiments. 3.2. Optimization EAE parameters by RSM-CCF In our research, RSM - CCF was used to optimize 4 factors: temperature extraction (X1), time extraction (X2), pH (X3) and enzyme concentration(X4), 3 replicate runs at center points. All the response values obtained from the 27 run experiments are presented in Table 1. Multiple regression analysis was applied on the experiment data, where Y was the antioxidant activity (mM TEAC/g DM). The results had R 2 = 0.9095, Q 2 = 0.6138, and difference between R 2 and Q 2 was less than 0.3, hence the model can be considered acceptable [9]. 2 2 1 3 1 40.1304 0.0055 0.024 0.0195 0.0138Y X X X X The time extraction(X2) was removed from the model. This proved that the time of enzyme incubation in range 75 - 105 minutes did not effect on antioxidant activity of the extract. Temperature (X1), pH (X3) and enzyme concentration (X4) had dependent effects on the response. The regression equation showed the important role of pH. Each enzyme has a suitable Figure 4. Effects of pH on antioxidant activity of extracts from white radish powder. Figure 3. Effects of extraction time on antioxidant activity of extracts from white radish powder. Pham Thi Kim Ngoc, Nguyen Thi Minh Nguyet, Dong Thi Anh Dao 52 pH range. When we increase or decrease pH value, enzymatic structure is altered and can lead to irreversible denaturation. If the extraction temperature were increased, the enzyme would be inactive. Table 1. Response surface analytical plan and experimental results. No X1 X2 X3 X4 TEAC No X1 X2 X3 X4 TEAC 1 45 75 4.5 1.25 0.1486 15 45 105 5.5 1.75 0.0779 2 55 75 4.5 1.25 0.1359 16 55 105 5.5 1.75 0.0865 3 45 105 4.5 1.25 0.1166 17 45 90 5.0 1.5 0.1321 4 55 105 4.5 1.25 0.1198 18 55 90 5.0 1.5 0.1149 5 45 75 5.5 1.25 0.1536 19 50 75 5.0 1.5 0.1481 6 55 75 5.5 1.25 0.1290 20 50 105 5.0 1.5 0.1024 7 45 105 5.5 1.25 0.1028 21 50 90 4.5 1.5 0.1284 8 55 105 5.5 1.25 0.0961 22 50 90 5.5 1.5 0.0870 9 45 75 4.5 1.75 0.1359 23 50 90 5.0 1.25 0.1366 10 55 75 4.5 1.75 0.1333 24 50 90 5.0 1.75 0.1454 11 45 105 4.5 1.75 0.0910 25 50 90 5.0 1.5 0.1346 12 55 105 4.5 1.75 0.1025 26 50 90 5.0 1.5 0.1329 13 45 75 5.5 1.75 0.1386 27 50 90 5.0 1.5 0.1426 14 55 75 5.5 1.75 0.1408 Enzyme concentration: 1.75% Enzyme concentration: 1.5% Enzyme concentration: 1.25% Figure 5.Response surface graph showing interaction between extraction time, enzyme concentration, temperature and pH on antioxidant activity of extracts from white radish powder. The optimum conditions for extracting antioxidant compounds from white radish were: enzyme concentration of 1.75 % (v/w), extraction time 81 minutes, extraction temperature 48 o C and pH 4.56, the antioxidant capacity was obtained 0.1609 mM TEAC/g DM. Using these parameters in real experiments, we determined the real antioxidant capacity was 0.1594 ± 0.033 mMTEAC/g DM, the difference was less than 5 %. All of these indicated that the quadratic model was in good agreement with the experimental results. Fig. 5 illustrated the graphs of the response surface given by Optimizer tools of Modde 5.0 from the regression equation. Using cellulase to improve the extraction efficiency of antioxidant compounds from whiteradish 53 3.3. Comparison of UAE with CE The antioxidant activity of the extract from white radish in using EAE with optimal conditions was 0.1594 ± 0.033 mM TEAC/g DM. This value was 161 % higher than that of CE that was treated at similar conditions (incubation temperature 48 o C, pH 4.56 and extraction time 81 minutes) without enzyme treatment. This result affirmed efficiency of EAE to collect bioactive compounds. Our results are similar to some previously published papers, confirming the effectiveness of the EAE in improving the efficient retrieval of bioactive compounds [5, 6]. SEM analysis showed a rupture on the surfaces of CE sample compared to EAE one (Fig. 6). In EAE, cellulase had been used particularly for the treatment of sample prior to extraction. It disrupted the structural integrity of the plant cell wall and hydrolyzed cell wall components, thereby increasing cell wall permeability, which results in higher extraction yields of bioactive [7]. Figure 6. SEM images of residues in EAE (A) and CE (B) of white radish powder. 4. CONCLUSION In this study, the optimal conditions of EAE to extract antioxidant compounds from white radish powder were based on RSM - CCF design. Under optimal extraction conditions: enzyme concentration of 1.75 % (v/w), extraction time 81 minutes, extraction temperature 48 o C and pH 4.56), the highest antioxidant activity was 0.1594 ± 0.033 (mM TEAC/g). The results showed that EAE is more effective to extract the antioxidant compounds from white radish powder. In addition, the other conclusion of this study is that white radish can be considered as a good source of antioxidant compounds. Acknowledgements. The research is funded by Viet Nam National University Ho Chi Minh City (VNU- HCM) under grant number T911-KTHH-2016-06. REFERENCES 1. Jakmatakul R. et al. - Evaluation of anti tyrosinase and antioxidant activities of Raphanus sativus root: comparison between freeze-dried juice and methanolic extract, Thai journal pharmacy science 33(2009) 22–30. 2. Ghasemzadeh A. et al. - Flavonoid compounds and their antioxidant activity in extract of some tropical plants, Journal of medicinal plants research 6(13) (2012) 2639–2643. 3. Gutierrez R.M.P and PerezR.L.- Raphanus sativus (Radish): their chemistry and biology, The scientific word journal 4 (2004) 811–837. A B Pham Thi Kim Ngoc, Nguyen Thi Minh Nguyet, Dong Thi Anh Dao 54 4. Beevi S.S. et al. - Polyphenolics profile and antioxidant properties of Raphanus sativus L., Natural product res. 26(6) (2012) 557–563. 5. Li B.B., Smith B. and HossainM.M. - Extraction of phenolics from citrus peels: Enzyme- assisted extraction method, Separation and purification technology 48 (2006) 189–196. 6. Hong Y. H. et al. - Enzymatic improvement in the polyphenol extractability and antioxidant activity of green tea extract, Biosci. Biotechnol. Biochem.77 (1) (2013) 22– 29. 7. Puri M. et al. - Enzyme assisted extraction of bioactives from plants, Trends in biotechnology 30 (1) (2012) 37–44. 8. Re R. et al. - Antioxidant activity applying an improved ABTS radical cationdecolorization assay, Freeradic. Biol. Med.26 (1999) 1231–1237. 9. Gabrielsson J. et al. - Multivariate methods in pharmaceutical applications, Journal of chemometrics 16 (3) (2002)141–160. TÓM TẮT SỬ DỤNG ENZYME CELLULASE ĐỂ CẢI THIỆN HIỆU QUẢ THU NHẬN DỊCH CHIẾT GIÀU HỢP CHẤT CHỐNG OXI HÓA TỪ BỘT CỦ CẢI TRẮNG (Raphanus sativus L.) Phạm Thị Kim Ngọc1, 3, *, Nguyễn Thị Minh Nguyệt2, Đống Thị Anh Đào3 1Trường Đại học Bà Rịa – Vũng Tàu, 80 Trương Công Định, phường 3, Tp.Vũng Tàu, BR-VT 2Trường Đại học Công nghiệp, 12 đường Nguyễn Văn Bảo, Gò Vấp, Tp. Hồ Chí Minh 3Trường Đại Học Bách khoa HCM, 268 Lý Thường Kiệt, quận 10, Tp. Hồ Chí Minh * Email: kimngoc080283@gmail.com Củ cải trắng (Raphanus sativus L.) là một loại rau ăn củ thuộc họ cải. Nó được trồng và sử dụng trên khắp thế giới. Ngày nay, củ cải được dùng phổ biến trong bữa ăn hàng ngày của chúng ta. Một số nghiên cứu cho thấy củ cải trắng có chứa một lượng lớn các chất có hoạt tính chống oxi hóa. Trong nghiên cứu này, chúng tôi sử dụng phương pháp trích li có sự hỗ trợ của enzyme cellulase để thu dịch chiết giàu chất chống oxi hóa. Hoạt tính chống oxi hóa của dịch chiết được xác định thông qua khả năng khử gốc tự do ABTS. Các điều kiện tối ưu cho quá trình trích li có sự hỗ trợ của cellulase đã được xác định gồm: nồng độ enzyme cellulase sử dụng 1,75 % so với lượng chất khô trong nguyên liệu, thời gian 81 phút, pH 4,56 và nhiệt độ 48 oC. Kết quả hoạt tính chống oxi hóa của dịch chiết đạt cao nhất ở mức 0,1594 ± 0,033 mMol TEAC/g, cao hơn 161 % so với mẫu trích li với các điều kiện tương tự nhưng không sử dụng enzyme. Ngoài ra, kết quả chụp SEM cho thấy có sự khác biệt lớn về cấu trúc bề mặt sau trích li của mẫu bột có và không có xử lý với enzyme cellulase. Từ khóa: củ cải trắng, Raphanus sativus L., chất chống oxi hóa, trích li có sự hỗ trợ của enzyme, cellulase.