An economical and efficient technology for the extraction of resveratrol from peanut (Arachis hypogaea) sprouts by multi-stage countercurrent extraction

Food Chemistry 179 (2015) 15–25

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

An economical and efficient technology for the extraction of resveratrol from peanut (Arachis hypogaea) sprouts by multi-stage countercurrent extraction Qianghua Zhang a,1, Yanhong Bian b,1, Yingying Shi a, Shangyong Zheng e,1, Xu Gu a, Danyan Zhang d, Xiufang Zhu a, Xiaoli Wang a, Dingyun Jiang a, Qingping Xiong a,c,⇑ a

College of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huaian 223003, Jiangsu, PR China Affiliated Huaian Hospital of Xuzhou Medical College, Huaian 223002, Jiangsu, PR China c Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huaiyin Institute of Technology, Huaian 223003, Jiangsu, PR China d School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong, PR China e School of Medicine, Yunnan University, Kunming 650091, Yunnan, PR China b

a r t i c l e

i n f o

Article history: Received 4 September 2014 Received in revised form 10 December 2014 Accepted 20 January 2015 Available online 2 February 2015 Keywords: Resveratrol Extraction Peanut sprouts Economical and efficient technology Multi-stage countercurrent extraction

a b s t r a c t In this paper, an economical and efficient technology for the extraction of resveratrol from peanut sprouts by multi-stage countercurrent extraction (MSCE) was investigated based on the alkaline extraction and acid precipitation method (AEAP). Firstly, the MSCE equipment and operation procedures were designed. Then, the optimal parameters of MSCE were obtained by using single-factor experiments and Box– Behnken design (BBD) as follows: extraction temperature of 46.6 °C, CaO to raw material ratio of 6:100, water to raw material ratio of 8.8:1 and extraction time of 51.7 min. Finally, the performance of MSCE was compared against the single pot extraction (SPE) under optimal conditions. The results demonstrated that MSCE was a time-saving, energy-saving, and cost-saving extraction technology for manufacturing resveratrol from peanut sprouts. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Resveratrol (3,5,40 -trihydroxy stilbene) belongs to the large group of polyphenols. It is a low molecular weight secondary metabolites produced by plants as a defensive response to situations of stress, such as microbial infection, UV irradiation or mechanical damage (Iwuchukwu & Nagar, 2008). Over the past three decades, the plant polyphenols have gained significant worldwide interest based on a number of associated health benefits (Frémont, 2000; Petrovski, Gurusamy, & Das, 2011; Smoliga, Baur, & Hausenblas, 2011), including anti-oxidant, anti-cancer, anti-viral, anti-inflammatory, cardioprotective and neuroprotective properties. Recent studies have shown that resveratrol is emerging as a very promising functional ingredient, which might be be incorporated into a number of pharmaceutical and functional food products (Gülçin, 2010; Santos, Carvaho-Gustavo, Oliveira,

⇑ Corresponding author at: College of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huaian 223003, Jiangsu, PR China. Fax: +86 517 83591165. E-mail address: [email protected] (Q. Xiong). 1 These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.foodchem.2015.01.113 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

Raposo-Nadia, & Silva, 2013). Peanut sprouts prepared from the germination of peanut kernels have been used in the diet as health food for several centuries. Peanut sprouts are rich in resveratrol and have been regarded as one of the most important and economical food sources of resveratrol (Sobolev & Cole, 1999; Xiong, Zhang, et al., 2014). At present, the isolation of resveratrol from peanut sprouts is an interesting alternative in terms of cost because isolation from other plants is very expensive (Rudolf & Resurreccion, 2007). Therefore, the extraction of resveratrol from peanut sprouts is relevant to scientific and medical, but also to economic interests. Resveratrol is a polyphenol of the stilbene kind compounds, which has two phenolic benzene rings linked by an ethane bridge (Rabesiaka, Rakotondramasy-Rabesiaka, Mabille, Porte, & Havet, 2011). As a polyphenol with three hydroxyl groups, resveratrol is acidic and has a weak polarity. Hence, resveratrol is difficult to dissolve in water, but easy soluble in many organic solvents, such as ethanol, ethyl acetate and chloroform. Based on the physical properties, the conventional method for extracting resveratrol from herbal samples commonly employs solid-organic solvent extraction method (OSEM) (Malovaná, Garcıa-Montelongo, Pérez, & Rodrıguez-Delgado, 2001). Nevertheless, resveratrol extracted by

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OSEM is not suitable for many applications in the food and pharmaceutical sectors because of high levels of toxic organic solvent residual. In addition, OSEM has many other disadvantages, including high toxic solvent consumption, long extraction time, low extraction efficiency and selectivity (Pascual-Martl, Salvador, Chafer, & Berna, 2001). Therefore, more and more research organizations and companies are exploring new generation of extraction techniques. In a previous study, for the first time, we found that the alkaline extraction and acid precipitation method (AEAP) is a faster and less expensive method that also produces better yields of resveratrol (Zhang, Xiong, & Shi, 2009). More importantly, AEAP is a cleaner and green production techniques due to using pure water as extracting solvent (Egüés, Sanchez, Mondragon, & Labidi, 2012), making the quality of the products meet the standards of food and pharmaceutical applications. Unfortunately, this study employed the single pot extraction (SPE) in batch mode, requiring excess consumption of solvent, which can lead to higher energy costs and longer time consumption for the subsequent concentration. These facts result in the limited commercial application of AEAP in the industrialization resveratrol. Multi-stage countercurrent extraction (MSCE) combines circulatory dynamic extraction and continuous countercurrent extraction technology (Wang, Ma, Fu, Lee, & Wang, 2004). The key theory of the MSCE is the exchange of extracts between different extraction stages by creating and maintaining a steady concentration gradient between the solvent and the herbal matrix (Xiong, Zhang, & Shi, 2010). The steady concentration gradient and the repeated use of solvent in different extraction stages not only enable a significant increase in extraction efficiency but also substantially decrease the dosage of extraction solvent. Recently, MSCE has been employed to the extraction of several bioactive components from plant materials with promising results (Veloso, Krioukov, & Vielmo, 2005; Xie, Liu, Chen, & Wang, 2009; Yu et al., 2012). On the basis of AEAP, we report here an economical and efficient technology for the extraction of resveratrol from peanut sprouts by MSCE. In order to obtain further understanding of the effects of major process variables on extraction efficiency, extraction temperature, CaO to raw material ratio, water to raw material ratio and extraction time were studied by single-factor experiments and Box–Behnken design (BBD). The performance of MSCE was also compared against SPE under comparable conditions. 2. Materials and methods 2.1. Materials and reagents The fresh peanut sprouts used in the experiment were purchased from Huaiyin Vegetable Product Market (Huaiyin, China). Standard substance of resveratrol was purchased from Sigma– Aldrich (St. Louis, MO, USA). Methanol of chromatographic grade was purchased from J&K Chemical Co., Ltd. (Beijing, China). Analytical grade calcium oxide (CaO) was obtained from Shanghai Chemical Reagents Co. (Shanghai, China). Chromatographic grade acetic acid was purchased from Dima Technology Inc. (USA). Deionized water was purified by a Milli-Q Water Purification system (Millipore, MA, USA). 2.2. Pretreatment of peanut sprouts Briefly, the fresh peanut sprouts were collected and washed carefully with deionized water. After removing the impurities, the peanut sprouts were crushed by a high speed disintegrator (HX-200A, Yongkang Hardware and Medical Instrument Co. Ltd., China) and the homogenate was soaked in petroleum ether for

24 h. Then, the solutions were centrifuged at 3000 rpm for 10 min (22R, Heraeus Sepatech, Germany). The collected peanut sprouts powder was dried at 50 °C in air dryer for 48 h, and then sifted through a 60 mesh sieve. 2.3. Extraction methods of resveratrol 2.3.1. MSCE extraction MSCE was carried out on the 50 L pilot-scale MSCE equipment, which was designed and manufactured by our laboratory. An extraction flow diagram and equipment schematic diagram were shown in Fig. 1A and B, respectively. The instrument has four extraction units labeled respectively as I, II, III and IV in Fig. 1B. They were linked by common discharge pipe of extracting solution, countercurrent pipe and 50 L storage pot of cycle solvent. Each unit had the same configuration and dimensions, consisting of a 50 L extraction pot with mechanical stirrer and heat-exchanger, a flow meter, a pump, and three valves. In MSCE process, all extraction units were operated in a closed loop configuration. The cycle solvent was pumped from the storage pot to the extraction pot through countercurrent pipe, mixed with the herbal samples slurry and then flowed out from the extraction pot through the bottom valve and back to the discharge pipe of extracting solution. Finally, the extracting solution was pumped respectively to the storage pot of cycle solvent and target extracting solution which finished all operation programs. The operation procedure of MSCE was referred to the instructions illustrated in Fig. 1C. The transfer directions of cycle solvent, target extracting solution, herbal residue and fresh solvent were expressed as arrow-pointing symbol , , and , respectively. The subscript number (from 1 to 4) of I, II, III and IV represents the first to fourth extracting solution, respectively. In MSCE extraction process, each unit filled with herbal samples was extracted four times using respectively fresh solvent and cycle solvents according to the pre-schemed operation procedure. All first extraction solution were regarded as target extracting solution and discharged to the storage pot. Meanwhile, all units applied fresh solvent in the fourth extraction. The whole MSCE extraction process could be divided into two stages: the first, a conditioning stage, followed by the second, a cycle extraction stage. Each extraction pot in the sample conditioning stag was loaded with an appropriate amount of pretreated dry powder of peanut sprouts and CaO, respectively. Under the process given in Fig. 1C, the extraction solvent was pre-designed in order to produce a systematic concentration gradient along the pot sequence. For example, the samples in unit I were continuously extracted four times with an appropriate amount of fresh solvent. The concentration of resveratrol was different from I1 to I4 (I1 > I2 > I3 > I4). In the sample conditioning stage, I1 was regarded as an approximate target extracting solution and discharged to the storage pot containing target extracting solution. I2  I4 were regarded as cycle solvents and transferred to unit II as the first, second and third extraction solvent, respectively. In a similar manner, II2  II4 were transferred to unit III. III2  III4 were added to unit IV. After III2 arrived at unit IV, fresh solvent was run for a cycle as follows: fresh solvent ? I4 ? II3 ? III2 ? IV1. The concentration gradient for the four-stage cycle solvent (fresh solvent, IV2, IV3 and IV4) was obtained by extracting the sample in the respective pots for a cycle. They indicated that the conditioning stage operation had completed. After the completion of the above conditioning operation, the cycle extraction stage of MSCE extraction started according to the sequence of steps illustrated by the flow diagram given in Fig. 1C. In the cycle extraction stage, resveratrol was continuously transferred between the solvent and the herbal sample in each of the sequential steps. The sequence of operations from unit I to IV was repeated. The transfer directions of solvent and operation procedure were similar to the conditioning

Q. Zhang et al. / Food Chemistry 179 (2015) 15–25

17

Fig. 1. Extraction flow diagram of resveratrol (A), equipment (B) and operation procedure (C) of MSCE process.

stage operation. The only difference being type of solvent, the fourstage cycle solvents of different concentration gradient were employed to extract resveratrol in the cycle extraction stage. 2.3.2. SPE extraction In the SPE extraction process, the flow diagram, configuration and dimensions of equipment were same to MSCE. However, each

unit was separated and operated independently. Fresh solvent and CaO were applied to each extraction process. Under the above SPE process, the herbal sample was extracted with the reported optimum parameters as follows: extraction temperature was 60 °C, CaO to raw material ratio was 5:100, water to raw material ratio was 8:1, extraction time was 86 min and the extraction was performed thrice (Zhang et al., 2009).

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Fig. 1 (continued)

2.4. Determining content and calculating extraction ratio of resveratrol

total volume of extraction solution (L), and W is the weight of dry powder samples (mg).

A Waters liquid chromatography system (Waters company, USA), which consist of a Model 600 controller liquid chromatograph, a Model Delta 515 pump, Millennium 32 system software, and a Model 2487 ultraviolet detector (UVD) was adopted to determine the contents of resveratrol (Nevado, Salcedo, & Peñalvo, 1999). Chromatographic separation was carried out by HIQ Sil C18V reversed-phase column (250 mm  4.6 mm i.d., KYA TECH Corporation, Japan) packed with 5 lm diameter particles. The mobile phase was methanol–water–acetic acid (27:70:3, V:V:V) and was filtered through a 0.45 lm membrane filter (Millipore, USA) prior to use (Xiong, Zhang, et al., 2014). Resveratrol was quantified at a wavelength of 303 nm following RP-HPLC separation (Piñeiro, Palma, & Barroso, 2006). The flow rate was 1.0 mL/ min, the injection volume was 10.0 lL, and the column temperature was set at 30 °C. The chromatographic peaks of resveratrol were confirmed by comparing their retention time and UV spectrum with those of the reference compounds. Six experimental points were employed for establishing a calibration curve. A good linear relationship was obtained over the range of 10–240 mg/L and the regression lines for resveratrol was as follows:

2.5. Optimization of extraction process

Y ¼ 51274:4X þ 316:7 ðR2 ¼ 0:9994; n ¼ 6Þ

ð1Þ

where Y is the peak area of resveratrol, and X is the concentration of resveratrol (mg/L). The extraction ratio (%) of resveratrol was then calculated using the following equation:

Extraction ratioð%Þ ¼

CNV  100 W

The preliminary range of the extraction parameters for the resveratrol extraction were determined by a single-factor experiment, then, a three-level, four-variable BBD was applied to systematically optimize the extraction of resveratrol from peanut sprouts. Based on the results of preliminary experiments, the extraction equipment was kept four units in the experimental design to avoid a waste of resources and decreased extraction process. Four independent variables considered were extraction temperature (X1), CaO to raw material ratio (X2), water to raw material ratio (X3) and extraction time (X4) while the response variable was the extraction ratio (%) of resveratrol. For statistical calculation, the variables were coded according to the following equation (Qiao et al., 2009):

xi ¼

ðX i  X 0 Þ DX i

where xi is the coded value of an independent variable, Xi is the actual value of an independent variable, X0 is the actual value of an independent variable at center point, and DXi is the step change value of an independent variable. The range of independent variables and their levels were presented in Table 1. Whole design consisted of 29 experimental points carried out in random order, and the experimental data (Table 1) were fitted to the following second-order polynomial model (Hsu, 1995):

ð2Þ

where C is the concentration of resveratrol calculated from the calibrated regression equation (mg/L); N is the dilution factor, V is the

ð3Þ

Y ¼ a0 þ

3 X i¼1

ai X i þ

3 X

2 X 3 X

i¼1

i¼1 j¼iþ1

aii X 2i þ

aij X i X j

ð4Þ

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Q. Zhang et al. / Food Chemistry 179 (2015) 15–25 Table 1 Box–Behnken design matrix and the response values for the extraction rate of resveratrol. No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Extraction temperature (°C)

CaO to raw material ratio (g/g, m:m)

Water to raw material ratio (ml/g, V:m)

Extraction time (min)

X1

Code x1

X2

Code x2

X3

Code x3

X4

Code x4

40 50 40 50 45 45 45 45 40 50 40 50 45 45 45 45 40 50 40 50 45 45 45 45 45 45 45 45 45

1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0

5:100 5:100 7:100 7:100 6:100 6:100 6:100 6:100 6:100 6:100 6:100 6:100 5:100 7:100 5:100 7:100 6:100 6:100 6:100 6:100 5:100 7:100 5:100 7:100 6:100 6:100 6:100 6:100 6:100

1 1 1 1 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 0 0

9:1 9:1 9:1 9:1 7:1 11:1 7:1 11:1 9:1 9:1 9:1 9:1 7:1 7:1 11:1 11:1 7:1 7:1 11:1 11:1 9:1 9:1 9:1 9:1 9:1 9:1 9:1 9:1 9:1

0 0 0 0 1 1 1 1 0 0 0 0 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0

50 50 50 50 40 40 60 60 40 40 60 60 50 50 50 50 50 50 50 50 40 40 60 60 50 50 50 50 50

0 0 0 0 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0

where Y is the predicted response extraction ratio (%) of resveratrol,

a0 is a constant, ai, aii and aij are the regression coefficients for intercept, linear, quadratic and interaction terms, respectively. Xi and Xj represent the level of the independent variables (i – j). 2.6. Statistical analysis The statistical analysis was performed by using the reported procedure (Jiang, Wang, Liu, Gan, & Zeng, 2011). Analysis of the experimental design and data was carried out using Design Expert software of version 7.0 (Stat-Ease Inc., Minneapolis, USA). Analysis of variance (ANOVA) was carried out and the fitness of the polynomial model equation was expressed as the coefficient of determination R2. The significances of the regression coefficients were tested by the F-test. One-way ANOVA was performed using the SPSS 13.0 for windows (SPSS, Chicago, IL, USA). Multiple comparisons of means were done by the least significance difference (LSD) test. P-values of less than 0.05 were regarded as significant.

Extraction rate (%)

0.577 0.762 0.607 0.768 0.732 0.672 0.762 0.760 0.567 0.723 0.623 0.757 0.750 0.762 0.747 0.682 0.622 0.803 0.565 0.768 0.668 0.675 0.768 0.762 0.882 0.877 0.878 0.888 0.902

In a previous study, it was found that the reaction rate of the extraction temperature is generally increased by 3–4 times as the temperature increased 10 °C (Xiong, Zhang, et al., 2014). And in a certain temperature range, the movement-active force of molecular matter was strengthened due to the temperature rise. The temperature increase may change the physical properties of the material, while reducing its absorption intensity and making resveratrol easier to be extracted (Alexandre & Dubois, 2000). Therefore, high temperatures enhance the extraction efficiency. However, resveratrol was affiliated with polyphenols. Excessively high temperature will result in the oxygenolysis of resveratrol, change the molecular structure of resveratrol and adversely affect the extraction efficiency (Liu & Liu, 2004; Yang, Ou-Yang, Wu, & Hu, 2008). Therefore, 45 °C was selected as the center point of extracting temperature in the BBD experiments.

3.2. Effect of CaO to raw material ratio on extraction ratio of resveratrol

3. Results and discussion 3.1. Effect of extraction temperature on extraction ratio of resveratrol To investigate the effect of extraction temperature on the extraction ratio of resveratrol, the extraction process was carried out using different extraction temperatures of 25, 30, 35, 40, 45, 50, 55, 60 or 65 °C, while other extracting parameters were fitted as following: CaO to raw material ratio of 5:100, water to raw material ratio of 7:1, and extraction time of 40 min. As shown in Fig. 2A, the resveratrol extraction ratio was rapidly increased as the temperature increased, but when the temperature was increased more than 45 °C, the resveratrol extraction ratio was slowly decreased. These results can be explained as extraction at the lower temperatures is not conducive to dissolution and mass-transfer of resveratrol between the solvent and the sample.

Resveratrol is a hydrophobic organic compound. In order to be extracted effectively by water, resveratrol must be transformed into salts to improve its solubility and polarity by a neutralization reaction with CaO. Therefore, CaO dosage is the most critical factor affecting the extraction ratio of resveratrol. In the present study, the effect of CaO dosage on the extraction rate of resveratrol was investigated using different CaO to raw material ratios (2:100, 3:100, 4:100, 5:100, 6:100, 7:100, 8:100 and 9:100) while other extraction variables were set as follows: extraction temperature of 45 °C, water to raw material ratio of 7:1, extraction time of 40 min. As shown in Fig. 2B, the extraction ratio improved as the ratio of CaO to raw material increased from 2:100 to 6:100, but there were no significant differences between 6:100 and 9:100. Therefore, CaO to raw material ratio of 6:100 was used in the following experiments as an excess of CaO dosage would not only

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Fig. 2. Effect of extraction temperature (A), CaO to raw material ratio (B), water to raw material ratio (C) and extraction time (D) on the yield of resveratrol.

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Q. Zhang et al. / Food Chemistry 179 (2015) 15–25 Table 2 Estimate parameter of regression model. Variables

Sum of squares

DOF

Mean square

F-value

P-value Prob. > F

Model X1 X2 X3 X4 X1X2 X1X3 X1X4 X2X3 X2X4 X3X4 X21 X22 X23 X24 Residual Lack of fit Pure error Correlation total

0.260647 0.0867 0.0000213 0.004681 0.013002 0.000144 0.000121 0.000121 0.001482 0.000042 0.000841 0.106884 0.041635 0.029986 0.049653 0.003962 0.003543 0.000419 0.264609

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 10 4 28

0.018618 0.0867 0.0000213 0.004681 0.013002 0.000144 0.000121 0.000121 0.001482 0.000042 0.000841 0.106884 0.041635 0.029986 0.049653 0.000283 0.000354 0.000105

65.7847 306.3514 0.075381 16.53927 45.9424 0.508819 0.427549 0.427549 5.237478 0.149289 2.971644 377.672 147.1147 105.9551 175.4461

<0.0001 <0.0001 0.7877 0.0012 <0.0001 0.4874 0.5238 0.5238 0.0382 0.7050 0.1067 <0.0001 <0.0001 <0.0001 <0.0001

3.380646

0.1259

R2 = 0.9854, adjusted R2 = 0.9711.

Fig. 3. Response surface plots (A, C, E, G, I and K) and contour plots (B, D, F, H, J and L) showing the effects of extraction temperature, CaO to raw material ratio, water to raw material ratio and extraction time and their mutual effects on extraction yield of resveratrol.

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Fig. 3 (continued)

cost more, but also produce more hydrochloric acid (subsequent acid precipitation process) waste. 3.3. Effect of water to raw material ratio on extraction ratio of resveratrol

tion ratio of resveratrol as the water to raw material ratio was increased from 3:1 to 9:1, but insignificant difference between 9:1 and 15:1 (Fig. 2C). Thus, water to raw material ratio of 9:1 was adopted in the following BBD experiment. 3.4. Effect of extraction time on extraction ratio of resveratrol

Ratios of water to raw material significantly affects the extraction rate (Bezerra, Santelli, Oliveira, Villar, & Escaleira, 2008). Resveratrol can be diffused more quickly by a larger ratio of water to raw material, which makes a greater concentration difference between the interior plant cells and the exterior solvent. In addition, more resveratrol molecules can dissolve in water, resulting in an enhancement of the extraction ratio. Nevertheless, if the ratio of solvent to raw material was too high, the concentration of resveratrol dissolved in water would become low, leading to greater energy consumption for solvent volatilization. Therefore, a suitable ratio of water to raw material should be selected for extraction of resveratrol in MSCE. To study the effect of different ratios of water to raw material on extraction rate of resveratrol, water to raw material ratios were set at 3:1, 5:1, 7:1, 9:1, 11:1, 13:1 and 15:1. Other extraction conditions were set as follows: extraction temperature of 45 °C, CaO to raw material ratio of 6:100, extraction time of 40 min. The results showed that there was a significant increasing trend in the extrac-

Extraction time is another important factor that affects the extraction efficiency and selectivity of MSCE. It had been reported that a long extraction time favored the production of bioactive compounds (Soria & Villamiel, 2010). However, excessive extension of extraction time may also cause the changes in molecular structure of resveratrol. Therefore, the effects of different extraction times on the extraction ratio of resveratrol were investigated. Extraction times of 20, 30, 40, 50, 60, 70 or 80 min were tested with other extraction parameters were set as followings: extraction temperature of 45 °C, CaO to raw material ratio of 6:100 and water to raw material ratio of 9:1. As shown in Fig. 2D, the extraction ratio of resveratrol increased as extraction time increased from 20 to 50 min, reaching a maximum at 50 min. After this point, the extraction ratio of resveratrol started to decrease with increasing extraction time. The changes of the extraction ratio might be due to the different chemical stability of resveratrol. This tendency was in agreement with other reports (He, Gongke, & Zhan, 2003).

Q. Zhang et al. / Food Chemistry 179 (2015) 15–25

23

Fig. 3 (continued)

Hence, extraction time of 50 min was selected as the center point for further experiment. 3.5. Model fitting and optimization for extraction of resveratrol 3.5.1. Model fitting The experimental data was analyzed according to previous reported methods (Guo, Zou, & Sun, 2010; Jiang et al., 2011). The correlation between response variables and test variables (the extraction ratio of resveratrol) associated with the following second-order polynomial equation:

Y ¼ 17:43308 þ 0:48687X 1 þ 111:69417X 2 þ 0:29284X 3 þ 0:091158X 4  0:12X 1 X 2 þ 0:00055X 1 X 3  0:00011X 1 X 4  0:96250X 2 X 3  0:0325X 2 X 4 þ 0:0000725X 3 X 4  0:0051346X 21  801:16667X 22  0:016998X 23  0:000874917X 24

ð5Þ

where Y represents the extraction ratio of resveratrol, and X1, X2, X3 and X4 represent extraction temperature, CaO to raw material ratio, water to raw material ratio and extraction time, respectively. The results of ANOVA, lack-of-fit and the adequacy of the model are summarized in Table 2. The model F-value of 65.7847 implied

that the model was highly significant. There was only a 0.01% chance that a model F-value could occur due to noise. The determination coefficient R2 of the model was 0.9854, indicating that 98.54% of the variability in the response could be explained by the model. In addition, the P-value of P = 0.1259 for lack-of-fit implied the lack-of-fit was not significant relative to the pure error, indicating the model equation was adequate to predict the extraction ratio of resveratrol within the range of experimental variables. The significance of the regression coefficients was tested by F-test, and the corresponding P-values for the model terms are also listed in Table 2. The P-value is used as a tool to check the significance of each coefficient, which in turn may indicate the pattern of the interaction between the variables. The smaller the P-value is, the more significant the corresponding coefficient is. Accordingly, X1, X3, X4, X2X3, X21, X22, X23 and X24 were significant (P < 0.05), while X2, X1X2, X1X3, X1X4, X2X4, X3X4 were not significant (P > 0.05).

3.5.2. Optimization for extraction of resveratrol Process variables and experimental data are shown in Table 1. In order to better understand the interactions of the variables, the response surface plots and contour plots for the model were produced by the Stat-Ease Design-Expert software. The shapes of the contour plots, circular or elliptical, indicate whether the

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Table 3 The comparison of MSCE and SPE under the optimal conditions. Method

MSCE SPE

Evaluation indexes Amount of pot

Amount of samples (kg)

Extraction temperature (°C)

Time consumption (min/kg)

Solvent consumption (L/kg)

CaO consumption (kg/kg)

Extraction ratio (%)

4 4

20 20

47b 60

41.6a 51.5

8.8b 24.0

0.06b 0.15

0.897 ± 0.035 0.908 ± 0.041

a,b

Designate a significant differences. P < 0.05 compared with SPE. P < 0.01 compared with the SPE.

a

b

mutual interactions between variables are significant or not (Xiong, Li, et al., 2014; Xiong, Zhang, et al., 2014). A circular contour plot indicates that the interaction between related variables is negligible, while an elliptical contour plot indicates that the interaction between related variables is significant (Muralidhar, Chirumamila, Marchant, & Nigam, 2001; Zhang, Li, Xiong, Jiang, & Lai, 2013). The response surface plots and contour plots as shown in Fig. 3 were generated using Design-Expert, which depicted the interactions between two variables by keeping the other variables at their zero levels for extraction ratio of resveratrol. It was evident that these three-dimensional plots and their corresponding contour plots provided a visual interpretation of the interaction for two variables and facilitated the location of optimum experimental conditions. By using the software Design-Expert, the solved optimum values of the tested variables for the extraction of resveratrol were extraction temperature of 46.6 °C, CaO to raw material ratio of 6:100, water to raw material ratio of 8.8:1 and extraction time of 51.7 min. Under the optimal conditions, the maximum predicted extraction ratio of resveratrol was 0.903%, which corresponded fairly well to that of real extraction (0.897 ± 0.035%, n = 3). The result suggested that the regression model was accurate and adequate for the prediction of resveratrol extraction.

3.6. Comparison of MSCE and SPE As described above, MSCE was carried out by creating a concentration difference between the solvent and solid samples. In the MSCE process, the concentration of resveratrol was dynamic gradient course and enhanced continuously in four extraction units by stages. It has been reported that mass transfer from the sample surface to the liquid phase is resulted mainly from the diffusion from one region to another region of different concentrations (Geller & Hunt, 1993; Jönsson, Lövkvist, Audunsson, & Nilvé, 1993). On the basis of Fick’s first law for steady-state condition states, the mass transfer flux by ordinary molecular diffusion is equal to the product of the diffusion coefficient (diffusivity) and the negative diffusion with the concentration gradient increased (Docˇekalová & Diviš, 2005; Straub, Graue, Heitmeir, Nebendahl, & Wurst, 1987; Ujihara, Fujiwara, Sazaki, Usami, & Nakajima, 2002). Therefore, MSCE could greatly facilitate mass transfer between immiscible phases through the increase of the driving force, result in higher extraction efficiency. In order to evaluate the advantages and disadvantages of MSCE, a comparison of MSCE and SPE was performed based on the difference of extraction temperature, extraction ratio, consumption of time, and consumption of solvent and CaO, with same samples, solvent and CaO. The experimental result was shown in Table 3. Although the extraction ratio of resveratrol from MSCE was slightly lower than that of SPE operation with insignificant difference (P > 0.05), MSCE offered overwhelming advantages with respect to extraction temperature, extraction time, and consumption of solvent and CaO. For the same weight of samples, MSCE was complete in about four-fifth of extraction time, using half the amount of CaO and one third of

the amount of solvent relative to SPE. Moreover, the SPE technique had the extraction temperature of 60 °C higher than MSCE process. This may due to afterheat and redundant CaO of previous units were reused along with countercurrent operation of the solvent in the MSCE process. These facts indicated that SPE needs longer extraction time, higher extraction temperature, and more extraction solvents and CaO to obtain the equivalent amount of resveratrol relative to MSCE. Higher consumption of solvent and CaO, meaning lower resveratrol concentration and more CaO residues in extraction solution, would inevitably result in more energy and acid cost for the subsequent concentration and acidic precipitation process. Therefore, MSCE is a time-saving, energy-saving, and cost-saving extraction technology for manufacturing extraction of resveratrol from peanut sprouts.

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