Enzymatic Preparation and Characterization of Soybean Oligosaccharides from Okara

Available online at www.sciencedirect.com

Procedia Engineering 37 (2012) 186 – 191

The Second SREE Conference on Engineering Modelling and Simulation (CEMS 2012)

Enzymatic Preparation and Characterization of Soybean Oligosaccharides from Okara Jinhong Wua, Yan Wua, Chunmei Yanga, Zhengwu Wanga, a* a

Department of Food Science and Technology, Bor S. Luh Food Safety Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China

Abstract The objective of this study was to investigate the enzymatic preparation method of soybean oligosaccharides (S0S) from okara by using Viscozyme L, characterize the physicochemical properties as well as antioxidant activity of S0S. The effect of Viscozyme L treatment on the preparation of S0S from okara was researched by orthogonal test. Under the fixed extraction time of 2.5 h and the ratio of okara to water of 1:15 (w/v), the optimal enzymatic preparation conditions of S0S by using Viscozyme L were identified as pH of 3.5, temperature of 45 ºC, enzyme dosage of 3% (w/v) with a maximum yield of 10% (w/w). S0S were found to be crude oligosaccharides containing 56.5% neutral sugar, having a low molecular weight distribution range between 860 and 9380 Da, composing of galactose, xylose, rhamnose, arabinose, mannose and glucose. Furthermore, S0S showed the free radical scavenging activity with IC50 value of 1.81 mg/mL. These results suggest that S0S are novel oligosaccharides derived from okara, and could be improve value-added utilization of okara as antioxidants in functional foods and nutraceuticals.

© 2012 Published by Elsevier Ltd. Selection Keywords: Okara, Enzymatic preparation, Soybean oligosaccharides, Antioxidant, Properties

* Corresponding author. Tel.:+86-021-34205748; fax: +86-021-34205748. E-mail address: [email protected].

1877-7058 © 2012 Published by Elsevier Ltd. doi:10.1016/j.proeng.2012.04.224

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Jinhong Wu et al. / Procedia Engineering 37 (2012) 186 – 191

1. Introduction The residue left from ground soybeans after extraction of the water extractable fraction used to produce soy milk and tofu, is usually called okara. In China about 20,000,000 tons of moist okara were produced annually from the soybean production industry. However, until now it has been consumed only as feed stuff, or even incinerated as factory waste in China, and the reuse of okara, especially for food application, is very difficult. Several physiological functions of okara have been found, such as antioxidation [1], anticholesterol action [2], and antiobesity action [3]. Thus, okara is recognized as an important functional food material with potential use in the food industry. So it is of great significance to develop useful food products from this byproduct and reduce environmental consequences of its disposal. Viscozyme L is a multi-component carbohydrase, which could effectively hydrolyze plant cell wall polysaccharide and cleave the linkages within the polysaccharide matrix to liberate more intercellular constituents [4]. Furthermore, the hydrolytic breakdown of high molecular weight (HMW) polysaccharides may contribute to enhanced antioxidant activities [5]. Thus, we firstly tried to hydrolyze plant cell wall polysaccharide from okara by using Viscozyme L to obtain new soybean oligosaccharides (SOS), and their characteristics as well as antioxidant biological function of SOS were investigated in order to evaluate their possible applications in the food industry. 2. Methods 2.1. Enzymatic preparation of SOS from okara by orthogonal experiment The raw fresh okara supplied from Yi Xing Food Co., Ltd.(shanghai company, China) was dried at 80 °C for 5 h, ground using an electric grinder and sieved through 80 mesh to obtain dried okara powder. The enzymatic hydrolysis experiments were carried out in a jacketed glass reactor connected to a thermostatically controlled water heater. 10 g of dried okara powder and 150 mL of water were placed in the glass vessel, pretreatment at 90 °C for 10 min and cooled to the hydrolysis temperature. Viscozyme L (100 units/g) was added, mixed, and reacted during stirring with a stirrer bar. The hydrolysis reaction was stopped by boiling at 100 °C for 10 min, and cooled to 60 °C. To removal of protein, proteolysis of the above hydrolysis product was carried out by using 1% ([E/S],w/w) Alcalase at 60 °C, pH 8.0 for 2 h, then stopped by 100 °C for 10 min. The hydrolysate was centrifuged at 5000 rpm for 15 min, then the supernatant were precipitated by the addition of ethanol to a final concentration of 75% (v/v), and the precipitates were collected by centrifugation, dissolved in deionized water and finally lyophilized. The yield of SOS from okara by means of enzymatic hydrolysis was calculated as the following Eq.(1). Yield (%)= (weight of lyophilized SOS / weight of dried okara)h100

(1)

The main affecting factors of enzymatic hydrolysis of okara are pH, temperature and enzyme dosage for a given okara concentration of 1:15(w/v) and hydrolysis time of 2.5 h. In order to optimize the hydrolysis conditions, the orthogonal experiment (L9(34)), including three-factors and three-levels (as shown in Table 1) was used to get the best reaction conditions.

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Jinhong Wu et al. / Procedia Engineering 37 (2012) 186 – 191 Table. 1 Factors and levels of the orthogonal experiment Factors Level

pH

Temperature (°C)

Enzyme dosage (%) ([E/S],w/w)

1

2.5

45

1

2

3.0

50

2

3

3.5

55

3

2.2. Characterization of physicochemical properties of SOS The chemical compositions of SOS, including total neutral sugar, protein, fat and moisture were analyzed. Total neutral sugar content was determined by the reaction with phenol in the presence of sulfuric acid using glucose as a standard [6]. Content of protein, fat and moisture was determined by the AOAC official method (976.06, 996.06, 934.01), respectively. The molecular weight distribution of SOS was determined by high-performance gel-permeation chromatography (HPGPC) with a Waters HPLC system according to the method of Jia et al. [7]. The molecular weight was estimated by reference to the calibration curve made from Dextran standards of known molecular weight (2500, 4600, 7200, 13380, 21400 Da). The monosaccharide composition of SOS was determined by gas chromatography according to the procedure as previously described by Li et al. [8].

2.3. DPPH Radical Scavenging Activity of SOS DPPH was dissolved in methanol to give a 0.2 mM solution. 100 ȝL of DPPH methanol solution mixed with 100 ȝL of SOS water solution at five indicated concentrations (or water itself as control) for 10 s, then transferred to the cavity of the EMX-8 ESR spectrometer (Bruker Biospin corp, Germany). ESR spectra were recorded after the mixture was reacted for 60 s. The conditions of ESR measurement were as follows: microwave frequency 9.8 GHz, modulation frequency 100 kHz, microwave power 20 mW, modulation amplitude 3 G, scan width 100 G, time constant 1.28 ms, temperature 298 K. The scavenging effect of SOS on the DPPH radical was calculated by Eqs. (2). E(%) = (h0-hx)/h0 h100

(2)

Here h0 and hx were the largest ESR signal intensity obtained from the highest peak of samples with and without SOS, respectively. 3. Results and discussion 3.1. Optimization of the enzymatic hydrolysis conditions of okara In order to obtain the best okara enzymatic hydrolysis conditions by using Viscozyme L according to the yield of S0S, an orthogonal design test (L9(3)4) was carried out and the results and statistical analysis were shown in Table 2. The K and R values in Table 2 were calculated following the procedures described by Ling et al. [9]. The R value showed that enzyme dosage had the most significant effect on

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yield, and the order of importance that influenced yield was found to be as follows: enzyme dosage > pH > temperature. In addition, the K value for pH, temperature and enzyme dosage had the highest value at level 3, level 1 and level 3, respectively. It indicated that the optimal enzymatic hydrolysis conditions were pH 3.5, temperature 45 °C and enzyme dosage 3% (E/S, w/w), and a maximum 10.0% yield was obtained under the optimal conditions. Table. 2 Statistical analysis of the results from orthogonal experiment No.

pH

Temperature ˄°C˅

Yield ˄%˅

1

1

1

1

6.4

2

1

2

2

6.2

3

1

3

3

7.6

4

2

1

2

7.4

5

2

2

3

7.8

6

2

3

1

6.6

7

3

1

3

10.0

8

3

2

1

8.4

9

3

3

2

6.6

a

K1

6.733

7.933

7.133

K2

7.267

7.467

6.733

K3

8.333

6.933

8.467

1.600

1.000

1.734

b a

Enzyme dosage (%) (E/S,w/w)

R

Ki represents the average yield at level i; b R represents the value of range.

3.2. Proximate analysis of SOS Results of proximate analysis of SOS and okara were present in Table 3. Compared with okara, the protein and fat contents of SOS decreased, while the total neutral sugar content increased. Results indicated that SOS have a higher level of carbohydrate compared to okara, which implied that some insoluble plant cell polysaccharides of okara might be transformed into soluble polysaccharides by treatment with Viscozyme L. Table. 3 Proximate composition of SOS and okara Sample

Total neutral Sugar (%)

Protein (%)

Fat (%)

Moisture(%)

Others (%)

SOS

56.5 ± 1.2

2.1 ± 0.2

4.5 ± 0.4

15.8 ± 0.9

21.1

Okara

5.1 ± 0.6

24.6 ± 1.0

10.5± 0.5

10.5±0.7

49.4

The chromatogram from the high-performance gel-permeation chromatography (HPGPC) analysis of SOS was shown in Fig.1. In Fig.1, it was revealed that SOS has six fractions with significantly difference in molecular weight distribution. The percentage of each fraction was calculated according to the linear regression equation obtained by the standard samples and shown in Table 4. The result showed that the

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Jinhong Wu et al. / Procedia Engineering 37 (2012) 186 – 191

3088 2031

molecular weight fractions of SOS mostly distributed between 860 and 9380 Da, and reached about 65% of total. Result indicated that SOS obtained after hydrolyzing okara with Viscozyme L were mostly low molecular weight oligosaccharides. The monosaccharide composition of SOS was tabulated in Table 5, and the relative concentration of each corresponding identified monosaccharide was evaluated by the internal standard method and also presented in Table 5. Results demonstrated that major monosaccharides of SOS were galactose, xylose and rhamnose based on the relative content. 10.00 8.00

2.00 0.00

730 330

13351

4.00

248498

MV

6.00

-2.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 Minutes

28.00 30.00

Fig.1. The molecular weight distribution profile of SOS as determined by HPGPC. Table. 4 molecular weight (MS) distribution of SOS Fraction

MS distribution (Da)

Table. 5 Monosaccharide analysis of SOS

Percentage˄%˅

Monosaccharide

Content˄%˅

1

74224~751638

4.57

Rhamnose(Rha)

18.24

2

9379~74224

12.96

Arabinose(Ara)

12.47

3

2586~9379

30.56

Xylose(Xyl)

22.26

4

861~2586

34.30

Mannose(Man)

11.08

5

424~861

8.97

Glucose(Glu)

10.92

6

99~424

8.63

Galactose(Gala)

25.03

3.3. Antioxidant activity of SOS DPPH is one of the free radicals widely used for testing preliminary radical scavenging activity of a compound or a plant extract, so antioxidant activity of SOS was tested by the inhibition of the stable free radical of DPPH. Electron spin resonance (ESR) spectra of DPPH radical with or without SOS were shown in Fig.2. The scavenging effect of SOS increased with the increase in their concentrations from 1.18 mg/mL to 9.00 mg/mL as shown in Fig.3, which implied that SOS was a dose-dependent radical scavenger. According to the regression equation deduced from regression curve shown in Fig.3, the IC50 value of SOS was calculated as 1.81 mg/mL.

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90 80

E˄%˅

70 60 50

y = 20.35 Ln(x) + 37.97

40

2

R = 0.9934

30 20 10 0 0

1

2

3

4

5

6

7

8

9

10

Concentration of SOS˄mg/mL˅

Fig.2. ESR spectra of DPPH with or without SOS. The arrow showed the highest peak of ESR spectrum.

Fig.3. Regression curve to illustrate the relation between the concentration of SOS and E value on the DPPH scavenging activity.

4. Conclusion The enzymatic hydrolysis of okara by treated with Viscozyme L could produce soybean oligosaccharides (S0S) with a maximum yield of 10%. SOS contained 56.5% of total neutral sugar, had a low molecular weight distribution range between 860 and 9380Da, whose content reached about 65% of total. Moreover, the major monosaccharides of SOS were galactose, xylose and rhamnose. SOS was found to be effective on DPPH radical scavenging activity, which implied that SOS could be applied in food industry as a natural antioxidant. However, for application in food as natural antioxidants, the antioxidant activities of SOS for scavenging of other radical species and its application properties in relevant food matrices needs to be studied in the future. Acknowledgements This work was supported by the National Natural Science Foundations of China (No.31000814 and No.30900998), the projects from Science and Technology Commission of Shanghai Municipality (No. 11495801600). References [1] Mateos-Aparicio I, Mateos-Peinado C, Jiménez-Escrig A, Rupérez P. Multifunctional antioxidant activity of polysaccharide fractions from the soybean byproduct okara. Carbohyd Polym. 2010; 82:245-250. [2] Matuso M., Hitomi E.. Suppression of plasma cholesterol elevation by okara tempe in rats. Biosci Biotechnol Biochem 1993; 57:1188-1190. [3] Préstamo G., Rupérez P., Espinosa-Martos I., Villanueva M.J., Lasunción M.A.. The effects of okara on rat growth, cecal fermentation, and serum lipids. Eur Food Res Technol. 2007; 225 (5-6) 925-928. [4] Xiao G., Huiyuan Y.. Optimization of Viscozyme L-assisted extraction of oat bran protein using response surface methodology. Food Chemistry 2008; 106 (1):345-351. [5] Siriwardhana N., Kim K.N., Lee K.W., Kim S.H., Ha J.H., et al., Optimisation of hydrophilic antioxidant extraction from Hizikia fusiformis by integrating treatments of enzymes, heat and pH control. Int J Food Sci Tech. 2008; 43(4):587-596. [6] Dubois M., Gilles K.A., Hamilton J.K., Rebers P.A., Smith F.. Colorimetric method for determination of sugars and related substances. Analytical Chemistry1956;. 28 (3):350-356. [7] Song J., Ma Y., Zhang C., Zhao X.Y., Qian H.F.. Separation and properties of polysaccharides from asparagus (Officinalis L) old stalks. Advanced Materials Research. 2012; 361-363:789-793. [8] Wang L., Zhang H.B., Zhang X.Y., Chen Z.X.. Purification and identification of a novel heteropolysaccharide RBPS2a with anti-complementary activity from defatted rice bran. Food Chemistry. 2008; 110:150-155. [9] Ling J.Y., Zhang G.Y., Cui Z.J., Zhang C.K.. Supercritical fluid extraction of quinolizidine alkaloids from Sophora flavescens Ait. and purification by high-speed counter-current chromatography. J Chromatogr A. 2007; 1145:123-127.