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Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

1 Optimization of enzyme-assisted extraction of polysaccharides from

2 alfalfa and its antioxidant activity
3 Q1 Shaopu Wang, Xiaofang Dong ∗ , Jianming Tong
4 Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
5

6 a r t i c l e i n f o a b s t r a c t
7
8 Article history: In this present study, an efficient complex enzyme-assisted extraction technology was developed and
9 Received 13 August 2013 optimized to extract polysaccharides from alfalfa using four factors at five levels central composite
10 Received in revised form rotatable response surface design (CCRD). The experimental data was fitted to a second order polyno-
11 16 September 2013
mial equation with high coefficient of determination values (R2 > 0.95). The results of statistical analysis
12 Accepted 20 September 2013
Available online xxx
showed that the linear and quadratic terms of these four variables had significant effects (P < 0.05) on the
yield of polysaccharides from alfalfa. The optimum conditions were as follows: enzyme concentration
13
of 2.5%, 2.0%, 3.0% (weight of alfalfa) of cellulase, papain and pectase, extraction temperature 52.7 ◦ C,
14 Keywords:
15 Alfalfa
extraction pH 3.87, ratio of water to raw material 78.92 mL/g and extraction time 2.73 h. Under the
16 Polysaccharides optimal conditions, the experimental extraction yield of alfalfa polysaccharides was 5.05 ± 0.02%, which
17 Extraction was well matched with the value (5.09%) predicted by the CCRD model. Moreover, evaluation of the
18 Response surface methodology antioxidant activity of polysaccharides from alfalfa in vitro suggested that the polysaccharides had good
19 Antioxidant antioxidant effect, especially scavenging activity for hydroxyl radical and DPPH radical, which indicated
that the polysaccharides from alfalfa may be explored as a novel natural antioxidant.
© 2013 Published by Elsevier B.V.

20 1. Introduction APS have been explored to be a kind of potential natural antioxidant 41

or feed additives in the future. 42

21 Alfalfa (Medicago sativa Linn) is perennial flowering plant of The conventional extraction methods of polysaccharides from 43

22 the family Fabaceae widely grown as one of the important forage alfalfa are hot-water extraction [12] and alcohol abstraction [13] 44

23 crops because of the highest feeding value and yield throughout the which not only depend largely on maceration, mechanical rab- 45

24 world and is also utilized as a typical medicine herb for hundreds bling and continuous energy consumption, but also require higher 46

25 of years in China [1]. So far, several beneficial physiological effects temperature, longer extraction time with lower extraction yield. 47

26 [2], immunostimulatory activity [3–5], and pharmacological activ- Polysaccharides structures responsible for bioactivities may be 48

27 ities, such as anti-tumor [6], lipid-lowing [7,8] and antioxidation destroyed to some extent suffering from a high temperature for 49

28 [2,9] of polysaccharides from alfalfa (APS) have been proved and long extraction time, leading to a drop in the effects of pharmacol- 50

29 published. Besides, APS have been found to promote the growth of ogy [14]. Thus, it would be desirable to devise an extraction means 51

30 livestock and poultry with a trait of dosage effect [2,7]. Oxidation is operated under moderate conditions which facilitates the polysac- 52

31 an essential biological process to many living organisms for energy charides to overcome cell wall limitations into the solvent. Based on 53

32 production. However, excessive reactive oxygen species produced the above considerations, enzyme-assisted extraction technology 54

33 in vivo during some oxidative reactions are strongly associated has been widely applied in the extraction of target compounds from 55

34 with some chronic diseases [10]. Recent researches suggested that different plants owing to its moderate, efficient and energy-saving 56

35 synthetic antioxidants were restricted due to their potential car- advantages which have been widely investigated compared to the 57

36 cinogenicity [11]. Therefore, there has been increasing interest in conventional extraction methods [14–20]. However, an important 58

37 developing and utilizing natural, effective and safe antioxidants, point of this method is that the effectiveness of enzyme-assisted 59

38 such as plant polysaccharides, to protect the human body from var- extraction could vary greatly from one material to another [21]. 60

39 ious diseases, such as cancer, arthritis and the aging process [10]. Up to the best of our knowledge, there were no reports describ- 61

40 Because of the special biological functions and abundant source, ing about optimization extraction of polysaccharides from alfalfa 62

by enzyme-assisted extraction technology. 63

Response surface methodology (RSM) is an effective collection 64

∗ Corresponding author: Tel.: +86 10 62819257; fax: +86 10 62819257. of statistical and mathematical technique useful for developing 65

E-mail addresses: shaopu.1988@163.com (S. Wang), xiaofangd1124@sina.com and optimizing complex processes. The main advantage of RSM 66

(X. Dong), tjm606@263.net (J. Tong). is to reduce the number of experimental trials needed to evaluate 67

0141-8130/$ – see front matter © 2013 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.ijbiomac.2013.09.029

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Table 1
Orthogonal test and results of proportion of complex enzymes.

No. (A) Cellulase concentration (B) Papain concentration (C) Pectase concentration Extraction
(w% of alfalfa) (w% of alfalfa) (w% of alfalfa) yield (%)

1 1(1.5) 1(1.0) 1(1.0) 4.23

2 1(1.5) 2(2.0) 2(2.0) 4.38
3 1(1.5) 3(3.0) 3(3.0) 4.98
4 2(2.0) 1(1.0) 2(2.0) 4.35
5 2(2.0) 2(2.0) 3(3.0) 5.00
6 2(2.0) 3(3.0) 1(1.0) 4.33
7 3(2.5) 1(1.0) 3(3.0) 5.09
8 3(2.5) 2(2.0) 1(1.0) 4.44
9 3(2.5) 3(3.0) 2(2.0) 4.51
k̄1 4.531 4.556 4.336
k̄2 4.562 4.611 4.413
k̄3 4.680 4.605 5.023
R 0.149 0.055 0.687

68 multiple parameters and their interactions and generate a mathe- of 80% (v/v) and kept overnight at 4 ◦ C. The precipitate was col- 110

69 matical model to find out optimal values [22]. lected at 2800 × g for 10 min, washed successively with anhydrous 111

70 The present study was designed to optimize the complex ethanol, acetone respectively and lyophilized at −40 ◦ C to get the 112

71 enzyme-assisted extraction parameters of the alfalfa polysaccha- crude APS. Finally, the content of the polysaccharides was deter- 113

72 rides using central composite rotatable response surface design mined by the phenol–sulfuric acid method [23,24], and d-glucose 114

73 (CCRD), an independent quadratic design belonging to RSM. was used to construct a standard curve. The polysaccharides yield 115

74 Besides, the antioxidative activities of APS including reducing (%) is calculated as follows: 116

75 power, scavenging activities of hydroxyl radical and DPPH radical

polysaccharides yield(%) 117
76 were evaluated as a novel potential antioxidant.
the polysaccharides content of extraction (g)
= × 100 (1) 118
weight of alfalfa powder (g)
77 2. Materials and methods
119

78 2.1. Materials and chemicals

2.2.2. Orthogonal test design of complex enzymes concentrations 120

An orthogonal L9 (34 ) test design was applied to optimize the 121

79 The fresh whole alfalfa, collected from a local farm at full bloom,
concentrations of cellulase, papain and pectase with disodium 122
80 was ground in a sample grinder and sieved through a 0.63 mm sifter
hydrogen phosphate–citric acid buffer. As seen from Table 1, nine
(40 mesh) after oven drying for 24 h at 65 ◦ C to the moisture con-
123
81
experiments were conducted with 3 factors and 3 level on the basis 124
82 tent less than 15%. The obtained powder (moisture content 6.31%
of preliminary single-factor extractions. The other extraction con- 125
83 in dry basis) was stored in plastic sealed bags and kept in dry envi-
ditions were as followed: pH 4.0, extraction temperature 50 ◦ C, 126
84 ronment prior to conduct the experiments. Cellulase (15,000 U/g),
ratio of water to raw material 70 mL/g and extraction time 2.5 h. 127
85 papain (6000 USP-U/mg), and pectase (50 U/g) were obtained from
The extraction process was as described in Section 2.2.1, and the 128
86 Sinopharm Chemical Reagent Co., Ltd. d-Glucose, 1,1-diphenyl-2-
yield of APS was the only dependent variable and also calculated 129
87 picrylhydrazyl (DPPH) and ascorbic acid was obtained from Sigma
according to Eq. (1). 130
88 Chemical Company Co. (St. Louis, MO, USA). All other chemicals
89 used in the experiment were of analytical grade and purchased
2.2.3. CCRD and statistical analysis 131
90 from Beijing Chemicals Co. (Beijing, China).
On the basis of orthogonal test of complex enzymes concentra- 132

tions, CCRD (Design Expert Software, Version 8.0.5, Stat-Ease Inc., 133
91 2.2. Optimization of complex enzyme-assisted extraction of Minneapolis, MN, USA) with four independent variables at five lev- 134
92 polysaccharides from alfalfa (APS) els was employed for the optimization of the extraction variables 135

for the best yields of alfalfa polysaccharides. Extraction tempera- 136

93 2.2.1. Extraction procedure ture (X1 : 30–70 ◦ C), extraction pH (X2 : 2.0–7.0), ratio of water to raw 137
94 The powder of dried alfalfa was defatted in a Soxhlet appara- material (X3 : 50–90 mL/g) and extraction time (X4 : 1.5–3.5 h) were 138
95 tus with petroleum ether (boiling point: 30–60 ◦ C) for 24 h and the selected independent variables to be optimized in this design, 139
96 pretreated with 80% (v/v) ethanol twice to remove some colored and the coded and uncoded (actual) levels of the four variables were 140
97 materials, some small molecule materials, such as monosaccha- presented in Table 2, which were based on the preliminary range of 141
98 rides, oligosaccharides. The organic solvent was volatilized at 40 ◦ C extraction variables through single-factor tests. The variables were 142
99 to obtain the pretreated material. The defatted powder (3 g) was coded according to the equation [25]: 143
100 extracted with complex enzymes solution at the given concentra-
Xi − X0
101 tion in a scheduled extraction temperature, extraction pH, ratio xi = (2) 144
Xi
102 of water to raw material and extraction time during the entire
103 extraction process. After extraction, the extracted slurry was fil- where xi is the (dimensionless) coded value of an independent vari- 145

104 tered through a filter paper under vacuum and inactivated the able, Xi is the real value of an independent variable, X0 is the real 146

105 enzyme activity in boiling water for 6 min. The obtained filtrate value of Xi at the center point, and Xi is the step change of the real 147

106 was concentrated to one-fifth of the initial volume using a rotary value of the variable i. Table 2 listed the whole design consisted of 30 148

107 evaporator (RE-5298A, Shanghai Yarong Biochemistry Instrument experimental points with six center points in order to estimate the 149

108 Factory, Shanghai, China) at 50 ◦ C under vacuum, and then precip- pure error, and the experiment was carried out at random in order 150

109 itated by adding dehydrated ethanol to give a final concentration to minimize the effect of unexplained variability in the observed 151

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Table 2
Central composite experimental design with results for extraction of polysaccharides yield.

Run X1 (extraction temperature, ◦ C) X2 (extraction pH) X3 (ratio of water to material, mL/g) X4 (extraction time, h) Extraction yield (%)

1 60 (1) 5.0 (1) 60 (−1) 2.0 (−1) 3.29

2 50 (0) 4.0 (0) 70 (0) 2.5 (0) 4.97
3 50 (0) 4.0 (0) 70 (0) 2.5 (0) 4.83
4 60 (1) 5.0 (1) 60 (−1) 3.0 (1) 3.51
5 60 (1) 3.0 (−1) 80 (1) 2.0 −1) 4.22
6 40 (−1) 3.0 −1) 60 (−1) 2.0 −1) 3.64
7 70 (2) 4.0 (0) 70 (0) 2.5 (0) 3.63
8 50 (0) 2.0 (−2) 70 (0) 2.5 (0) 3.41
9 30 (−2) 4.0 (0) 70 (0) 2.5 (0) 2.99
10 40 (−1) 3.0 (−1) 80 (1) 2.0 (−1) 4.37
11 50 (0) 4.0 (0) 90 (2) 2.5 (0) 5.01
12 40 (−1) 3.0 (−1) 60 (−1) 3.0 (1) 3.60
13 50 (0) 4.0 (0) 70 (0) 2.5 (0) 5.03
14 50 (0) 4.0 (0) 70 (0) 2.5 (0) 4.73
15 60 (1) 3.0 (−1) 80 (1) 3.0 (1) 4.58
16 50 (0) 4.0 (0) 50 (−2) 2.5 (0) 3.48
17 40 (−1) 5.0 (1) 60 (−1) 2.0 (−1) 3.53
18 60 (1) 5.0 (1) 80 (1) 2.0 (−1) 4.03
19 50 (0) 4.0 (0) 70 (0) 2.5 (0) 5.03
20 60 (1) 3.0 (−1) 60 (−1) 3.0 (1) 3.91
21 40 (−1) 5.0 (1) 80 (1) 3.0 (1) 3.89
22 60 (1) 5.0 (1) 80 (1) 3.0 (1) 4.17
23 60 (1) 3.0 (−1) 60 (−1) 2.0 (−1) 3.56
24 50 (0) 6.0 (2) 70 (0) 2.5 (0) 3.36
25 40 (−1) 3.0 (−1) 80 (1) 3.0 (1) 3.78
26 50 (0) 4.0 (0) 70 (0) 2.5 (0) 4.90
27 50 (0) 4.0 (0) 70 (0) 3.5 (2) 4.96
28 40 (−1) 5.0 (1) 80 (1) 2.0 (−1) 3.73
29 40 (−1) 5.0 (1) 60 (−1) 3.0 (1) 3.52
30 50 (0) 4.0 (0) 70 (0) 1.5 (−2) 4.21

152 responses due to systematic errors. Besides, the triplicates were ascorbic acid as a positive control. A higher spectrophotometrical 181

153 performed at all design points and the average yield obtained was absorbance indicated a higher reducing power activity. 182

154 taken as the response. Data from CCRD were analyzed by multiple
155 regressions to fit the following quadratic polynomial model: 2.3.2. Hydroxyl radical scavenging assay 183

Hydroxyl radical scavenging activity was measured according 184


4 
4 
4
to Smirnoff’s work with a certain modification [28]. The reaction 185
156 Y = ˇ0 + ˇj xj + ˇjj xj2 + ˇij xi xj + ei (3) mixture contained 0.3 mL FeSO4 (7.5 mM), 0.3 mL H2 O2 (7.5 mM), 186
j=1 j=1 i <j=2 0.3 mL salicylic acid (7.5 mM) in ethanol solution and 1 mL sam- 187

ple with different concentration, and then incubated at 37 ◦ C for 188

157 where Y is the response; xi and xj are variables (i and j range 30 min. The absorbance of the hydroxylated salicylic acid was mea- 189
158 from 1 to 4); ˇ0 is the model intercept coefficient; ˇj , ˇjj and sured at 510 nm. Ascorbic acid was used as the positive control. 190
159 ˇij are regression coefficients of linear, quadratic and the interac- The antioxidant activity of APS was calculated according to the 191
160 tive terms, respectively; and ei is the error [26]. Analysis of the following equation: 192
161 experimental design and calculation of predicted data were car-  A1 − A2

162 ried out by using Design Expert Software (Version 8.0.5, Stat-Ease Scavenging effect (%) = 1 − × 100 (4) 193
163 Inc., Minneapolis, MN, USA) to estimate the response of the inde- A0
164 pendent variables. Subsequently, three additional confirmation where A1 was the absorbance of the sample or ascorbic acid, and 194
165 experiments were conducted to verify the validity of the statistical A0 was the absorbance of the solvent control, whereas A2 was the 195
166 experimental strategies. P-values of less than 0.05 were considered absorbance of the reagent blank without H2 O2 . 196
167 to be statistically significant.
2.3.3. DPPH free radical scavenging assay 197

168 2.3. Assay for antioxidant activities of APS The scavenging of DPPH radical was measured according to the 198

method of Chen et al. [14]. A 0.1 mM solution of DPPH in ethanol 199

169 2.3.1. Reducing power solution (0.75 mL) was added to the sample solution (1.5 mL) with 200

170 The reducing power of APS was evaluated according to the various concentrations. The mixture was shaken and left to stand 201

171 method of Yen and Chen with slight modifications [27]. An aliquot for 30 min in the dark, and the absorbance was measured at 517 nm 202

172 of each sample (0.5 mL) at different concentrations (0–1.6 mg/mL) against ethanol as a blank. Ascorbic acid was used as the posi- 203

173 was mixed with 0.5 mL of phosphate buffer (pH 6.6, 0.2 M), fol- tive control. The percentage scavenging effect was calculated as 204

174 lowed by 0.5 mL of potassium ferricyanide [K3 Fe(CN)6 ] (1%, w/v). following: 205

175 The mixture was incubated at 50 ◦ C for 20 min in a water bath. A  A1 − A2


176 portion (0.5 mL) of trichloroacetic acid (10%, w/v) was added to Scavenging effect (%) = 1 − × 100 (5) 206
A0
177 the mixture for terminating the reaction, and then the mixture
178 was centrifuged at 1500 × g for 10 min. The supernatant (0.5 mL) where A1 was the absorbance of the sample or ascorbic acid, A0 was 207

179 was mixed with 1.5 mL of distilled water and 0.1 mL of FeCl3 the absorbance of the solvent control, and A2 was the absorbance 208

180 (0.1%, w/v), then the absorbance was measured at 700 nm using of the reagent blank without DPPH. 209

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210 3. Results and discussion under some temperature and long extraction time [34]. Therefore, 270

extraction time ranging from 1.5 to 3.5 h was adopted in the present 271

211 3.1. Effect of different temperature on extraction yield of APS work. 272

212 To investigate the effect of extracting temperature on the yield 3.4. Effect of different ratio of water to raw material on extraction 273

213 of APS, extraction process was set at 30, 40, 50, 60 and 70 ◦ C, yield of APS 274

214 while other extraction variables (extraction pH, ratio of water to

215 raw material, extraction time, cellulase amount, papain amount, Different ratio of water to raw material is another factor that 275

216 and pectase amount) were fixed at 5.0, 30 mL/g, 1.5 h, 2.0%, 2.0%, could significantly alter the extraction yield of polysaccharides 276

217 2.0%, respectively. The result show that the increase of APS extrac- [35,36]. Therefore, optimal ratio of water to raw material should 277

218 tion production was sharp when the temperature was increased be selected for extraction of targeted polysaccharides (APS). In the 278

219 from 30 to 50 ◦ C, beyond which the production declined gradually present study, ratio of water to raw material was performed at 279

220 with further increasing temperatures. The maximum production 30, 40, 50,60, 70, 80, 90 mL/g when other parameters were as fol- 280

221 (3.39%) of APS was observed at 50 ◦ C, as shown in Fig. 1(a). The lows: extraction temperature 50 ◦ C, extraction pH 4.0, extraction 281

222 reasons for this result may be due to the increase of APS sol- time 2.5 h, cellulase amount 2.0%, papain amount 2.0% and pec- 282

223 ubility, extraction speed at higher temperature, and the better tase amount 2.0%, respectively. The yield continued to increase 283

224 enzyme activities at the suitable temperature. Because different significantly from 3.24% to 4.79% as the ratio of water to raw 284

225 enzymes have the best hydrolyzing effect under their own optimal material increased from 30 to 70 mL/g, and thereafter it still main- 285

226 effect temperatures, which significantly affect the enzyme activi- tained a mild slope with the ratio of water to raw material rising 286

227 ties. Furthermore, extremely high temperatures can cause thermo from 70 to 90 mL/g (Fig. 1d). This was due to the reason that the 287

228 degradation of polysaccharides, increased energy cost, acceleration increase of ratio of water to raw material could increase diffusiv- 288

229 of solvent volatilization, and enhancement of impurity of extrac- ity of the enzyme and solvent into cells and enhance desorption of 289

230 tion. Therefore, extraction temperature ranging from 30 ◦ C to 70 ◦ C the polysaccharides from the cell [36]. However, in order to reduce 290

231 was adopted in the present work. the process cost, ratio of water to raw material 50–90 mL/g was 291

considered to be optimal in this experiment. 292

232 3.2. Effect of different pH on extraction yield of APS

3.5. Effect of different doses and proportion of complex enzyme 293

233 Optimal pH value that different enzymes own differently is a on extraction yield of APS 294

234 crucial factor ensuring the best enzyme activity. One reason might
235 be the change of enzyme spatial structure leading to the weaken- Different enzymes with various doses (cellulase, papain and 295

236 ing or even lose of enzyme activities when the enzyme is subjected pectase) exert different effects on the yield of APS, which are 296

237 to different pH. In this study, different pH levels (3.0, 4.0, 5.0, shown in Fig. 1e–g. Other extraction parameters were fixed at 50 ◦ C 297

238 6.0 and 7.0) were selected to evaluate the influence on APS yield. extraction temperature, pH 4.0, ratio of water to raw material of 298

239 Other extraction variables were fitted as following: extraction tem- 70 mL/g, 2.5 h extraction time, respectively. Fig. 1e–g indicated that 299

240 perature 50 ◦ C, ratio of water to raw material 30 mL/g, extraction all of the APS productions reached the peak value at 2.0% assisted 300

241 time 1.5 h, cellulase amount 2.0%, papain amount 2.0% and pectase extraction with each enzyme, and then there is no increase when 301

242 amount 2.0%, respectively. The effect of different pH on the extrac- the amount of enzymes continued to rise which demonstrated 302

243 tion yield of APS is shown in Fig. 1(b). The yield of APS continued to that each enzyme amount of 2.0% was sufficient to obtain perfect 303

244 increase with the increase of pH value (3.0–4.0) and was enhanced polysaccharides yield. A possible explanation is that higher enzyme 304

245 to a critical value (3.59%) at pH 4.0 which was in agreement with doses leading to faster total hydrolysis, and end-product inhibi- 305

246 Chen et al. and Yin et al. [14,16], while the yield of APS exhibits a tion may have happened. Consequently, 2.0% of each enzyme was 306

247 negative relationship with pH (4.0–7.0) which may be caused by selected as an optimal dose in further research. 307

248 the decrease of enzyme activities at inappropriate pH value [29]. In addition to consideration of the dose of enzyme that should 308

249 Besides, the result of orthogonal test in Table 1 showed that the be as low as possible to guarantee the total APS degradation and 309

250 pectase played the most important role in the yield of APS and the economic costs, the proportion of compound enzymes (cellulase, 310

251 optimum pH for pectase was 4.0 [30,31]. Therefore, the pH value of papain and pectase) might also affect the yields of APS. Orthogonal 311

252 4.0 was favorable for the next extraction of APS. experiments L9 (34 ) were carried out to obtain the suitable com- 312

pound enzymes proportion suing the fixed extraction temperature 313

253 3.3. Effect of different time on extraction yield of APS (50 ◦ C), pH (4.0), extraction time (2.5 h) and ratio of water to raw 314

material (70 mL/g), and the results of orthogonal test are listed in 315

254 Extraction time is an important parameter that can influence Table 1. The orthogonal analysis shows that the pectase plays the 316

255 the extraction efficiency. Numerous studies have demonstrated most important role in the yield of APS (R = 0.687), probably due to 317

256 that a long extraction time contributed to the yield of polysaccha- its better performance in degradation of plant cell wall with its 318

257 rides [16,32]. At the meantime, excessive lengthening of extraction comprised activities from pectinesterase, polygalacturonase and 319

258 time may induce the change of polysaccharides molecule structure pectinlyase [37,38]. The optimal combination of parameters was 320

259 [33]. The extraction yield of APS affected by different extraction A3 B2 C3 , namely, cellulase concentration (2.5%), papain concentra- 321

260 time is showed in Fig. 1(c) when other extraction conditions were tion (2.0%) and pectase concentration (3.0%). 322

261 as follows: extraction temperature 50 ◦ C, extraction pH 4.0, ratio

262 of water to raw material 30 mL/g, cellulase amount 2.0%, papain 3.6. Optimization of the extraction parameters of APS 323

263 amount 2.0% and pectase amount 2.0%, respectively. The results
264 show that the extraction yield of APS was increased as the extrac- 3.6.1. Statistical analysis and the model fitting 324

265 tion time prolonged from 1.0 h to 2.5 h, and the maximum yield The total number of 30 statistically designed experiments was 325

266 (3.91%) was obtained at the time of 2.5 h. After this point, there carried out for different combinations of the physical parameters 326

267 was a slight decrease and then maintained a dynamic equilibrium in order to optimize the combined effect of independent variables 327

268 with increasing the extraction time. This phenomenon maybe due (extraction temperature, extraction pH, ratio of water to raw mate- 328

269 to partial of the polysaccharide hydrolysis with the exist of enzymes rial and extraction time) on the yield of APS. Table 2 shows the 329

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Fig. 1. Effects of different (a) extraction temperature; (b) extraction pH; (c) extraction time; (d) ratio of water to raw material; (e) cellulase concentration; (f) papain
concentration; and (g) pectase concentration on the extraction yield of polysaccharides. Each value is the mean ± SD of triplicate measurements.

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Table 3
Analysis of variance and regression coefficients of the predicted quadratic polynomial model.

Source Coefficient estimate Sum of squares Degree of freedom Mean square F-value P-Value

Model 4.910 10.950 14 0.780 21.49 <0.0001

X1 0.100 0.250 1 0.250 6.97 0.0185
X2 −0.088 0.180 1 0.180 5.06 0.0400
X3 0.300 2.200 1 2.200 60.47 <0.0001
X4 0.087 0.180 1 0.180 5.00 0.0409
X1 X2 −0.034 0.019 1 0.019 0.51 0.4842
X1 X3 0.080 0.100 1 0.100 2.79 0.1153
X1 X4 0.096 0.150 1 0.150 4.06 0.0623
X2 X3 −0.017 0.004 1 0.004 0.12 0.7319
X2 X4 0.027 0.012 1 0.012 0.33 0.5765
X3 X4 −0.029 0.014 1 0.014 0.38 0.5483
X1 2 −0.410 4.580 1 4.580 125.80 <0.0001
X2 2 −0.390 4.170 1 4.170 114.65 <0.0001
X3 2 −0.180 0.850 1 0.850 23.22 0.0002
X4 2
−0.091 0.230 1 0.230 6.24 0.0246
Residual 0.550 15 0.036
Lack of fit 0.480 10 0.048 3.36 0.0965
Pure error 0.071 5 0.014
Cor total 11.500 29
R2 = 0.9525; Adj R2 = 0.9082; Pred R2 = 0.7530; Adeq Precision = 13.6390; C.V.% = 4.70

330 experimental conditions and the result of yield of APS according to experimental values. Adequate precision is greater than 4 of this 371

331 the factorial design. The maximum production of APS (5.03%) was value is desirable and the ratio in this study was found to be > 13, 372

332 obtained under the experimental conditions of extraction tempera- which showed an adequate signal and confirm that, the model is 373

333 ture 50 ◦ C, extraction pH 4.0, ratio of water to raw material 70 mL/g significant for this extraction method [42]. Besides, Table 3 showed 374

334 and extraction time 2.5 h. By applying multiple regression analysis that the linear coefficients (X1 , X2 , X3 , X4 ), quadratic term coeffi- 375

335 on the experimental data, Design Expert 8.0.5 expresses the empir- cient (X1 2 , X2 2 , X3 2 , X4 2 ) were significant, with very small P-values 376

336 ical relationship between response variable and the test variables (P < 0.05). The other term coefficients were not significant (P > 0.05). 377

337 by a second-order polynomial equation in terms of coded factors:

338 Yield(%) = 4.91 + 0.10X1 − 0.088X2 + 0.30X3 + 0.087X4 3.6.2. Optimization of extraction conditions of APS 378

339 − 0.034X1 X2 + 0.080X1 X3 + 0.096X1 X4 − 0.017X2 X3 The three dimensional (3D) response surface and contour plots 379

drawn by Design Expert Software (Version 8.0.5) were constructed 380

340 + 0.027X2 X4 − 0.029X3 X4 − 0.041X12 − 0.39X22 over the independent variables (extraction temperature, extraction 381

pH, ratio of water to raw material and extraction time) to illustrate 382
341 − 0.18X32 − 0.091X42 (6)
the main and interactive effects on the yield of APS and obtain the 383

342 where Yield (%) is the APS yield; X1 , X2 , X3 and X4 are the coded optimum as presented in Figs. 2 and 3. In the 3D response surface 384

343 values of extraction temperature, extraction pH, ratio of water to plot and contour plot, the yield of APS was obtained along with two 385

344 raw material and extraction time, respectively. constant variables (in turn at its central level), whereas the other 386

345 The statistical significance of the regression model was checked two variables were continuous variables varying within the experi- 387

346 by corresponding F-test and P-value, and the analysis of variance mental range under investigation. In the two figures, the maximum 388

347 (ANOVA) for the response surface quadratic model is shown in predicted value indicated by the surface was confined in the small- 389

348 Table 3. The model F-value of 21.49 indicated that the model was est ellipse in the contour diagram. Circular contour plots indicate 390

349 highly statistically significant at P < 0.0001. The lack of fit F-value negligible interactions between the corresponding variables, while 391

350 and P-value was found to be 3.36 and 0.0965, which indicated the elliptical contours are obtained when there is a perfect interaction 392

351 suitability of the model to predict the variations. The determina- between the independent variables [43]. The independent variables 393

352 tion coefficient (R2 ), predicted determination coefficient (R2 pred ), and maximum predicted values from the figures corresponded with 394

353 adjusted determination coefficient (R2 adj ) and coefficient of vari- the optimum values of the dependent variables obtained by the 395

354 ance (C.V.) are usually used to evaluated the goodness of the equations. 396

355 fit of the model and it was listed in Table 3. The value of the As expected, extraction temperature (X1 ) and extraction pH (X2 ) 397

356 determination coefficient (R2 = 0.9525) of the quadratic regression exerted a quadratic effect on APS production. Exactly, a greater 398

357 model indicated that only 4.75% of the total variations could not increase in APS yield resulted when the extraction temperature 399

358 be explained by the model. The adjusted determination coeffi- (X1 ) was rising from 40.0 to 52.7 ◦ C, but beyond 52.7 ◦ C, the extrac- 400

359 cient (R2 adj = 0.9082) was also very high, showing the high degree tion yield of APS decreased gradually as the temperature ascended. 401

360 of correlation between the experimental and predicted values. In Likewise, the extraction pH (X2 ) had a similar effect on extraction 402

361 addition, the R2 adj was very close to the R2 value, which exhibited yield as extraction temperature (X1 ) and the maximum yield was 403

362 the large enough of the sample size [39,40]. The values of R2 adj achieved at pH 3.87 (Figs. 2a and 3a). In Figs. 2b and 3b, it can be seen 404

363 and R2 pred should be approximately within 0.20 of each other to be that the ratio of water to raw material (X3 ) curve started to level off 405

364 in reasonable agreement. Otherwise, there may be a problem with at 79 mL/g indicating that a ratio of water to raw material (X3 ) of 406

365 either the data or the model [41]. Thus, the values of R2 pred (0.7530) 79 mL/g is required to obtain maximum yield. From Figs. 2c and 3c 407

366 was in reasonable agreement with the R2 adj (0.9082), showing that showed that the yield of APS was increased with extraction tem- 408

367 the form of the model chosen to explain the relationship between perature (X1 ) and extraction time (X4 ). Increased extraction time 409

368 the factors and the response is well-correlated. At the same time, (X4 ) up to a threshold level of 2.7 h led to increased polysaccharides 410

369 a low value 4.70 of the coefficient of the variation (C.V.) clearly yield. Beyond this level, APS yield slight decreased. In Figs. 2d and 411

370 stated a very high degree of precision and good reliability of the 3d, there was a sharply increase in the yield of APS with increase 412

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Fig. 2. Response surface plots (3D) showing the effects of variables on the response yield of APS.

413 in the ratio of water to raw material (X3 ) before the ratio value the APS from alfalfa are as follows: extraction temperature (X1 ) 427

414 of 79 mL/g. With the increasing extraction pH (X2 ), the APS yield 52.7 ◦ C, extraction pH (X2 ) 3.87, ratio of water to raw material 428

415 reached the peak value at pH 3.87, then dropped from 3.87 to 5.0. (X3 ) 78.92 mL/g and extraction time (X4 ) 2.73 h. Under these con- 429

416 Figs. 2e and 3e showed that the maximum yield of APS was achieved ditions, the model predicted a maximum response of 5.09%. For 430

417 when extraction pH (X2 ) and extraction time (X4 ) were 3.87 and their validation of the suitability of the model equations, tripli- 431

418 2.7 h, respectively. In Figs. 2f and 3f, it was obvious that there was cate confirmatory experiments were performed under the optimal 432

419 an upsurge in extraction yield with increase in ratio of water to conditions, and the average yield of APS was 5.05 ± 0.02%, which 433

420 raw material (X3 ), but extraction yield showed a slight escalating indicated that the model was appropriate for the extraction process. 434

421 trend with increase in extraction time (X4 ), which demonstrated

422 that ratio of water to raw material (X3 ) was more important than 3.7. Antioxidant activity of APS 435
423 extraction time (X4 ).
3.7.1. Reducing power assay 436

424 3.6.3. Verification of predictive model The reducing capacity of polysaccharides always serves as a sig- 437

425 According to Figs. 2 and 3, and the above single factor study, nificant indicator of its potential antioxidant activity. The higher 438

426 it could be concluded that the optimized extraction parameters of the absorbance at 700 nm is, the stronger the reducing power is 439

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Fig. 3. Contour plots showing the effects of variables on the response yield of APS.

440 [27]. In Fig. 4a, the absorbance at 700 nm increased with the con- correlation between the concentration of the polysaccharide and 453

441 centration of polysaccharides. Compared with that of ascorbic acid, the hydroxyl radical scavenging activity was observed from 0 to 454

442 the reducing power of APS was much lower at all tested concen- 1.6 mg/mL. At a concentration of 1.6 mg/mL, the scavenging activ- 455

443 trations. At 1.6 mg/mL, the reducing power of APS was only 0.239, ities were 70.3% and 99.7% for APS and ascorbic acid, respectively. 456

444 while the ascorbic acid showed an excellent reducing power with The IC50 value of APS and ascorbic acid for eliminating hydroxyl 457

445 1.252 at the same concentration. radicals was 0.43 and 0.16 mg/mL, which indicated that the scav- 458

enging activity of APS against hydroxyl radical was lower than that 459

of ascorbic acid. 460

446 3.7.2. Scavenging effects on hydroxyl radicals
447 Hydroxyl radical as one of the most reactive oxygen radicals can
448 easily induce oxidative damage by crossing cell membranes and 3.7.3. Scavenging effects on DPPH radicals 461

449 reacting with most biomolecules including such as carbohydrates, DPPH, a stable free radical discovered by Goldschmidt and Renn 462

450 proteins, lipids, and DNA in cells, as well as inflict tissue damage [45], would be scavenged and the absorbance would be reduced 463

451 or cause cell death [44]. The results of hydroxyl radical scavenging at 517 nm when encountering an electron or hydrogen donating 464

452 activity of APS and ascorbic acid were given in Fig. 4b. A positive substrate such as antioxidant. Thus, the decrease in absorbance is 465

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Fig. 4. Antioxidant activity assay of APS and ascorbic acid. (a) Reducing power. (b) Scavenging effects on hydroxyl radicals. (c) Scavenging effects on DPPH. Each value is the
mean ± SD of triplicate measurements.

466 always utilized as a tool to evaluate the radical-scavenging activity further works on purification, structure, functions evaluation and 495

467 of natural compounds [46]. The DPPH radical-scavenging activity application studies are in progress to make it an effective product. 496

468 of APS was investigated at different concentrations (0–1.6 mg/mL)

469 and the result was presented in Fig. 4c. The results indicated that
Acknowledgement 497
470 APS and ascorbic acid showed obvious scavenging activity on DPPH
471 radical in a concentration-dependent manner. The DPPH scaveng-
The authors appreciate the financial support provided by the 498
472 ing activity increased from 40.0% to 87.9% when the concentration
Grant (2013BAD10B04) from the National key Technology R&D 499
473 of APS increased from 0.1 to 1.6 mg/mL, and exhibited an interesting
Program for the 12th Five-year Plan of China. 500
474 radical scavenging activity with an IC50 value of 0.19 mg/mL. The
475 ability to scavenge DPPH radical was lower than that of ascorbic
476 acid (IC50 = 0.05 mg/mL), which was similar to other plant polysac- References 501

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