College of Food Science and Engineering, Beijing University of Agriculture, Beijing, , China

Transcription

1 2018 International Conference on Modeling, Simulation and Optimization (MSO 2018) ISBN: Optimization of the Ultrasound-assisted Extraction of Saponins from Germinated Quinoa and the Evaluation of the Radical-scavenging Activity Shan WANG, Xiao-meng GUO and Ting-jun MA * College of Food Science and Engineering, Beijing University of Agriculture, Beijing, , China * Corresponding author Keywords: Quinoa, Germination, Saponins, Ultrasonic extraction, Response surface analysis, Radical-scavenging activity. Abstract. This study aimed to optimize the extracting process for the content of saponins in germinated quinoa. Saponins from germinated quinoa was ultrasonically extracted. This study the effects of extractive temperature, extractive time, liquid ratio and ethanol volume fraction on the content of saponins in germinated quinoa under the condition of single factor. The extraction process of saponins in germinated quinoa was optimized by the Box-behnken center combination and response surface method (RSM). Results indicated that the highest saponins content of mg/g in germinated quinoa was obtained after the optimized conditions which was extractive temperature 54 C, extractive time 15 min, liquid ratio 1:50, ethanol volume fraction of 75%. The content of saponins in germinated quinoa was 2.6 times higher than unwashed quinoa seeds. The saponins after being optimized have strong antioxidant properties. This finding suggested that the ultrasonic method is beneficial to the extraction of saponin from germinated quinoa, which provides a theoretical basis for the enrichment of saponins in germinated quinoa. Introduction Chenopodium quinoa belongs to Chenopodiaceae family, an annual dicotyledonous plant [1]. Bolivia of South Africa and Peru are the two major producers of quinoa [2]. Quinoa is not only rich in nutrition, but also rich in multiple bioactive substances like saponins, polyphenol, flavone and many other functional components. Saponins are mainly distributed in quinoa seeds [3], and saponins is a large group of complex structure and has a biological activity of naturel organic compounds [4]. It is reported that quinoa saponins have antifungal activity, the purified monosodium saponins activity is very small, while total saponins at 50 μg/ml can effectively inhibit Candida albicans, indicating a synergistic effect between saponins [5]. On one hand, quinoa can absorb water and germinate extremely easily, and its biological activity is enhanced after the germination. On the other hand the main advantage of ultrasound is the reduction in the extraction time with a low solvent consumption and low temperature, which helps to avoid thermal damages in the extraction of heat-labile products and therefore preserve the structural and molecular properties [6-7]. And most researchers focus on the studies of quinoa seeds and bran, while there are few reports on the germinated quinoa. In order to optimize the content of saponin with high radical-scavenging activities, this study used germinated quinoas as raw materials, and utilized RSM to optimize four factors, this providing certain theoretical basis for further research on germinated quinoa. Materials and Methods Materials and Reagents The quinoas are provided by Hebei Zhangjiakou Academy of Agricultural Sciences. 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH), 2, 2-azinobs-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) were purchased from Sigma-Aldrich. Oleanolic acid was purchased from Biological 580

2 Products; all chemicals used in the experiments were of analytical grade. Methods The Determination of germination days. A certain amount of quinoas on the 1 st, 2 nd, 3 rd, 4 th, 5 th day of germination respectively was taken under the same culture condition and dried and smashed less than 60 mesh sieve. Quinoas with different germination days based on the methods of Wangjuwei et al [8] then the content of saponins corresponding to different days of germination was obtained. The maximum content was used as the material for subsequent experiments. Extraction and Determination of Saponins in Germinated Quinoa. The extraction were carried out in an ultrasonic processor by using a direct method. An ultrasound probe of 150W operating at 20 KHz was used for ultrasound treatment. The method based on Wangjuwei et al [8]. The study chose the 3 rd germination of quinoa as material, then the ethanol volume fraction were set at 50%, 60%, 70%, 80%, 90% and the ultrasonic temperature were set at 30, 40, 50, 60, 70, which correspond to the liquid ratio of 1:20, 1:30, 1:40, 1:50, 1:60, respectively. Each homogenate was sonicated for 5, 10, 15, 20 and 25 min, as the predetermined extraction time. Then the content of saponins in germinated quinoa solution was determined according to the method of Wangjuwei et al [8].. Standard Curve Preparation and Radical Scavenger Capacity (DPPH and ABTS Assays). The method was based on Wangjuwei et al [8]. The method of Zielinska et al. [9], Karamac [10] was used to calculate the free radical-scavenging capacity of DPPH and ABTS assay. The Design of Optimized Box-Behnken RSM. According to the design principle of Box-Behnken, the test level variables respectively by -1, 0, 1 encoding. The Test factors and levels are shown in Table 1. Horizontal Table 1. Factors and their coded levels used in experimental design for RSM. Ultrasonic Time/min Ultrasonic Temperature / Factor Ethanol Volume Fraction/% Liquid Ratio(g/mL) : : :60 Results and Analysis Results of Single Factor Experiment Effects of Ultrasonic Time on Saponins Content of Germinated Quinoa. It can be seen from Figure. 1 that the content of saponin increased first and then decreased with the increase of ultrasonic time. At 15 min the content of saponin in germinated quinoa is the highest, which is mg/g. Figure 1. Effects of ultrasonic time on saponins content of germinated quinoa. Effects of Ultrasonic Temperature on Saponins Content of Germinated Quinoa. Figure. 2 shows, in the range of temperature of 20~50 C, the content of saponin increased with the temperature rises. At 50 C, the content of saponin in germinated reaches the maximum which is mg/g. When the temperature range from 50 C to 70 C, the content of saponin decreased. 581

3 Figure 2. Effects of ultrasonic temperature on saponins content of germinated quinoa. Effects of Ethanol Volume Fraction on Saponins Content of Germinated Quinoa. As we can see from Figure. 3, under the condition of ethanol volume fraction being 70%, saponin content reaches the maximum, which is mg/g. And high concentrations of ethanol can suppress the dissolution of saponin by influencing osmotic pressure and alcohol soluble impurities dissolve out. Considering the cost, 70% concentration of ethanol was adopted. Figure 3. Effects of ethanol volume fraction on saponins content of germinated quinoa. Effects of Liquid Ratio on Saponins Content of Germinated Quinoa. Figure. 4 shows that under the circumstance that the liquid ratio is 1:50, saponin content reaches the maximum, which is mg/g. The reason may be that excessive ethanol has a negative influence on osmotic pressure, thus suppressing the dissolution of saponins. Therefore, 1:50 is the best liquid ratio. Figure 4. Effects of liquid ratio on saponins content of germinated quinoa. The Design and Experimental Results of Response Surface The experiment was designed using Design-Expert software with ultrasonic time(a), ultrasonic temperature(b), ethanol volume fraction(c) and liquid ratio(d) being arguments and four-factor and three-level experiment was operated. The design and results are shown in Table

4 Run Table 2. The experimental design and results response surface analysis. A.Ultrasonic Time/min B.Ultrasonic Temperature/ C.Ethanol Volume Fraction/% D.Liquid Ratio/g/mL Saponins Content (mg/g) Regression Model Analysis Multiple regression fitting was carried out according to the test design of Table 3, and the regression model was set up among the saponins content in germinated quinoa (Y) and ultrasonic time(a), ultrasonic temperature(b), ethanol volume fraction(c) and liquid ratio(d). The equation of fitting quadratic multivariate regression model is Y= A+1.74B+2.44C+0.56D-4.8AB-1.44C-1.04AD+.96BC+1.18BD-1.48CD-5.65A B C D 2 As is shown in Table 3, the model P<0.001 and there is significant difference in it. The lack-of-fit item P=0.1586>0.05 with no significant difference, suggesting that the model fitted well. The coefficient of determination of this model R 2 =0.8856>0.8, indicating that the quadratic equation can fit the real response surface better. As we can see from Table 3, the effect of four factors on the saponin content in germinated quinoa is C>B>A>D. Linear term C, exchange term AB and quadratic terms A 2, B 2, C 2, D 2 have significant effect on saponin content (P< 0.01). Linear term B 583

5 and quadratic terms BC have significant effect on the response value of saponin content (P< 0.05), the effect of the rest terms on saponin content in germinated quinoa is not significant. Therefore, we can draw a conclusion that the effect of various influence factors on saponin content in germinated quinoa is not simply linear. Remove the non-significant items and the fitting equation is revised as: Y= B+2.44C-4.8AB+3.96BC-5.65A2-5.19B2-5.14C2-5.21D2 Table 3. Analysis of variance for the regression model. Sum of Mean Source Squares DF Square F Value P Value Model A-Time B-Temperature C-Ethanol Volume Fraction D-Liquid Ratio AB AC AD BC BD CD A < B C D Residual Lack of Fit Pure Error Cor Total R R 2 Adj Ultrasonic time 14.5 min, ultrasonic temperature C, ethanol volume fraction 73.85%, liquid ratio 1: Under these conditions, the prediction of maximum saponin content is mg/g. Considering the reality, the original theoretical model was revised as follows: ultrasonic time 15 min, ultrasonic temperature 54 C, ethanol volume fraction 75%, liquid ratio 1:50. The verification test was operated on such conditions and was repeated 3 times. The average saponin content was mg/g. The relative error was 0.19%, indicating that the results are basically consistent with the predicted values of the model. The DPPH radical scavenging rate of saponin is 96.07% ± 0.18%, the ABTS free radical scavenging rate is 86.28% ± 0.20%, indicating DPPH was a better indicator of radical-scavenging activity of saponins than ABTS. Conclusions Saponins have proven to have significant antioxidant activity, which is directly correlated with the removal of free radicals in the DPPH assay. Under the theoretical model, the saponin content was measured as mg/g, which is 2.6 times as much as that in quinoa seeds. This experiment is easy to operate with short extraction time, which fully indicates that ultrasonic extraction is 584

6 beneficial to enrichment and accumulation of saponin in quinoa. The research above can provide some theoretical basis for the development of quinoa products, and provide help for human production and life. Acknowledgement Public sector (agriculture) research- activity of rice, grains and other food processing key technology and equipment research and demonstration ( ). References [1] Wang JC; Zhao X.W., et al. Research Progress on characteristics and development and utilization of quinoa [J]. Journal of Zhejiang agriculture and Forestry University, 2014, 31(2): [2] Vicente Gianna, Juan Manuel Montes, Edgardo Luis Calandri, et al. Impact of several variables on the microwave extraction of Chenopodium quinoa wild on the microwave extraction of Chenopodium quinoa wild saponins [J]. Food Science & Technology, 2012, 47: [3] Woldemichael G.M. Wink M. Identification and biological activities of triterpenoid saponins from Chenopodium quinoa [J]. Agric Food Chem, 2001, 49(5): [4] Osbourn A., Goss R.J., Field R.A. The saponins: polar isoprenoids with important and diverse biological activities. [J]. Natural Product Reports, 2011, 28(7): [5] Woldemichael G.M., Wink M. Identification and biological activities of triterpenoid saponins from Chenopodium quinoa. [J]. Journal of Agricultural & Food Chemistry, 2001, 49(5): [6] Vilkhu K., Mawson R., et al. Applications and opportunities for ultrasound assisted extraction in the food industry A review [J]. Innovative Food Science & Emerging Technologies, 2008, 9(2): [7] Tian Y., Xu Z., Zheng B., et al. Optimization of ultrasonic-assisted extraction of pomegranate (Punica granatum L.) seed oil. [J]. Ultrasonics Sonochemistry, 2013, 20(1): 202. [8] Wang J.W., Ma T.J., et al. Process optimization and kinetic model for extraction of soybean saponins by ultrahigh pressure [J]. Journal of Chinese Institute of Food Science and Technology, 2012, 12(4): [9] Zielinska D., Szawaranowak D., Zielinski H. Comparison of spectrophotometric and electrochemical methods for the evaluation of the antioxidant capacity of buckwheat products after hydrothermal treatment. [J]. Journal of Agricultural & Food Chemistry, 2007, 55(15): [10] Karamać M. Antioxidant Activity of Tannin Fractions Isolated from Buckwheat Seeds and Groats [J]. Journal of the American Oil Chemists Society, 2010, 87(5):