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Preparation and antioxidant activities of oligosaccharides from Crassostrea gigas
Food Chemistry 216 (2017) 243–246
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Preparation and antioxidant activities of oligosaccharides from Crassostrea gigas Shengjun Wu a,b,c,d,⇑, Xiaolian Huang a a
Jiangsu Marine Resources Development Research Institute, Lianyungang, Jiangsu 222005, China School of Marine Science and Technology, Huaihai Institute of Technology, 59 Cangwu Road, Xinpu 222005, China c Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang 222005, China d Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang 222005, China b
a r t i c l e
i n f o
Article history: Received 3 July 2016 Received in revised form 14 August 2016 Accepted 16 August 2016 Available online 17 August 2016 Keywords: Crassostrea gigas Oligosaccharides Antioxidant activity
a b s t r a c t Oligosaccharides were prepared from Crassostrea gigas by hydrolysis of polysaccharide in C. gigas with peroxide oxygen (H2O2). The hydrolysates were cleared of protein, filtered, ultrafiltered and precipitated with absolute ethanol to give C. gigas oligosaccharides (CGOs). Factors affecting CGO yields, i.e., reaction time, temperature, and H2O2 concentration, were optimised as follows: 2.96 h reaction time, 84.71 °C reaction temperature, and 2.46% H2O2 concentration. Under these conditions, the maximum yield of CGOs reached 10.61%. The CGOs were then partially characterised by Fourier transform infrared spectroscopy, UV spectroscopy, monosaccharide composition, and antioxidant activities. Results indicate that CGOs possessed strong hydroxyl radical activity, 2,2-diphenyl-b-picrylhydrazyl-radical-scavenging activity and reducing capacity at a concentration of 100 lg/mL. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Free radicals are produced by many factors, such as physical effects, chemical reactions, and metabolic processes. Free radicals exert many pathological effects on living organisms, such as DNA damage, thereby causing carcinogenesis and inducing agingrelated cellular degeneration (Liu, Ooi, & Chang, 1997). A variety of antioxidants can scavenge strong radicals (Liu et al., 1997). These antioxidants can be divided into synthetic chemicals, such as phenolic compounds and various naturally occurring substances, such as saccharides. Moreover, natural antioxidants from organism extracts have attracted increasing interest because of consumer concern about the safety of synthetic antioxidants in food (Yao, Cao, & Wu, 2013). Crassostrea gigas, one major cultured shellfish in China, is nutritious and delicious and is thus considered a valuable food resource (Hou et al., 2014). Recent research found that C. gigas is rich in polysaccharides [4–8]. Gao et al. found that the water-soluble polysaccharide from C. gigas comprised mainly of ?4)-a-DGlc-(1? with few ?3,4)-b-D-Glc-(1? and ?2,4)-b-D-Glc-(1? branched units) (Gao, Zhao, Wang, & Luan, 2014). C. gigas polysaccharides have been demonstrated to contain many functional ⇑ Corresponding author at: Jiangsu Marine Resources Development Research Institute, Lianyungang, Jiangsu 222005, China. E-mail address: [email protected] (S. Wu). http://dx.doi.org/10.1016/j.foodchem.2016.08.043 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
activities, such as immunostimulatory (Jiang, Zhang, & Zhao, 2013), antimicrobial (Tian et al., 2013), antitumour (Chen et al., 2010), hepatoprotective (Shi et al., 2015) and antihypertensive (Wang et al., 2016) activities. However, data on the C. gigas oligosaccharides (CGOs) are limited. At present, a number of methods for preparation of oligosaccharides from different organisms have been developed, e.g. peroxide oxygen (H2O2)-assisted preparation (Wu & Yu, 2015), microwaveassisted preparation (Li, Zhao, Lv, Li, & Yu, 2015) and enzymaticassisted preparation (Tao et al., 2016). Nevertheless, the data regarding peroxide oxygen (H2O2)-assisted preparation of CGOs are limited. In the present study, we prepared water-soluble CGOs from C. gigas by hydrolysis with H2O2, partially characterised the product and evaluated their antioxidant activities.
2. Materials and methods 2.1. Materials Live C. gigas were purchased from a local farmers’ market (Xinpu, China)H2O2 (30%, v/v) was purchased from the Laiyang Kant Chemical Co., Ltd. (Laiyang, China). All other reagents used were of analytical grade.
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S. Wu, X. Huang / Food Chemistry 216 (2017) 243–246
2.2. Preparation of CGOs
2.5. Statistical analysis
C. gigas meat, including the adductor muscle and mantle was stripped from the shells, shredded, dried in a hot air oven (JKOOI-240A, China) at 60 °C to a constant weight, pulverised and sifted through a 40-mesh sieve. The lipids in the dried powder were removed by the Soxhlet extraction method using light petroleum as the solvent. The lipid-removed powder was suspended in distilled water to give a suspension with a concentration of 2% (w/v)H2O2 (2%, 2.5% and 3%, respectively) were added to the reactor containing 100 mL of the suspension, and the reactor was incubated in a thermostatic water bath at different temperatures (80, 85, and 90 °C, respectively) for designated time periods (2, 2.5 and 3 h, respectively). The hydrolysates were filtered, proteins were removed using the Sevag method, concentrated (approximately 10%), precipitated using six volumes of absolute ethanol, filtered again and freezedried. The percentage yield of CGOs was calculated using Eq. (1) as follows:
All data are presented as mean ± standard deviation. Statistical analysis was performed using Statgraphics Centurion XV version 15.1.02. A multifactor ANOVA with posterior multiple range test was used for determining statistical significance.
Yield ¼ 100 W 2 =W 1
3. Results and discussion 3.1. Effect of reaction time, temperature and H2O2 concentration on CGO yield The reaction conditions, i.e. time, temperature and H2O2 concentration, were optimised using a central composite design (CCD), which is shown along with the results in Table 1. The regression model was obtained by analysing the results of the experiment using a multifactor ANOVA as follows:
Y ¼ 895:96474 þ 31:08210 X 1 þ 18:61347 X 2 þ 58:88526 X 3 0:021100 X 1 X 2 0:50000 X 1 X 3
ð1Þ
0:23400 X 2 X 3 2:02601 X 21 0:10278 X 22
where W1 and W2 represent the weights of the recovered CGOs and the original C. gigas powder, respectively. 2.3. CGO characterisation Total sugar, protein and reducing sugar contents were determined using the phenol–sulphuric acid colorimetric method, the Kjeldahl method and a method proposed by Hou (2004). Meanwhile, monosaccharide composition was analysed using the procedure reported by Sheng et al. (2007). The Fourier transform infrared (FTIR) spectra of the resulting CGO sample was recorded in KBr pellets by a Nicolet Nexus FTIR 470 spectrophotometer (Nicolet, USA) over a wavelength range of 400 cm1 to 4000 cm1. The UV spectra were recorded on a UV spectrometer (Spectra Test, German). 2.4. Antioxidant activity assays The hydroxyl radical (HO) scavenging activity (HRSA) of the CGO sample was measured in accordance with the method of Qu, Li, Zhang, Zeng, and Fu (2016). The hydroxyl-radical-scavenging activity was calculated as follows:
HRSAð%Þ ¼
A1 A2 100 A 1 A0
ð2Þ
where A0 is the absorbance of the reagent blank absorbance, A1 is the positive control absorbance and A2 is the absorbance of the sample. 2-Diphenyl-b-picrylhydrazyl-radical-scavenging activity (DRSA) was determined in accordance with the method of Carmona-Jiménez, García-Moreno, Igartuburu, and Garcia Barroso (2014). The DPPH-free-radical-scavenging percentage was calculated by the following equation:
DRSAð%Þ ¼
½A0 ðA1 A2 Þ 100 A0
ð3Þ
where A0 is the absorbance of the control (water instead of CGOs solution), A1 is the absorbance of the sample and A2 is the absorbance of the sample under identical conditions as A1 with water instead of DPPH solution. Reducing capacity was measured in accordance with the method of Qiao et al. (2009). A higher absorbance indicates a better reducing capacity.
7:63336 X 23
ð4Þ
where Y is the CGO yield (%), X1 is the time (h), X2 is the temperature (°C) and X3 is the H2O2 concentration. ANOVA for the response surface quadratic model verified the statistical significance of Eq. (4). The results in Table 2 showed that the model obtained was highly significant, which was demonstrated by the values of F and P ((P > F) < 0.0001). The fit accuracy was verified by the high value of multiple correlation coefficient (R2 = 97.75%), which indicated that the response model explained the total variations by 97.75%. A regression model with an R2 value > 0.9 is generally considered to reflect a high correlation (Haaland, 1989). Moreover, the value of the adjusted multiple correlation coefficient (R2Adj = 95.73%) was sufficiently high to indicate the statistical significance of the model. The interaction between reaction time and temperature and that between temperature and H2O2 concentration were significant (p < 0.05). However, the interaction between reaction time and H2O2 concentration was not significant (p > 0.05) (Table 2). According to the model, the optimum reaction conditions of time, temperature and H2O2 concentration maximum CGO yield were Table 1 The central-composite design for optimizing extraction conditions. Run
X1
X2
X3
Yield (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
2.50 2.50 2.50 3.00 3.00 2.50 3.34 2.00 2.00 2.00 2.50 3.00 2.50 2.50 3.00 2.50 2.50 2.00 1.66 2.50
85.00 85.00 76.59 90.00 80.00 85.00 85.00 90.00 90.00 80.00 85.00 90.00 85.00 93.41 80.00 85.00 85.00 80.00 85.00 85.00
2.50 2.50 2.50 2.00 2.00 2.50 2.50 2.00 3.00 2.00 2.50 3.00 2.50 2.50 3.00 1.66 2.50 3.00 2.50 3.34
10.61 10.72 3.12 6.71 6.21 10.16 10.29 5.68 4.94 2.91 9.85 5.63 10.35 3.17 7.31 6.18 11.08 4.67 7.67 3.85
X1 = time (min), X2 = temperature (°C), X3 = H2O2 concentration (%).
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S. Wu, X. Huang / Food Chemistry 216 (2017) 243–246 Table 2 Analysis of variance for the experimental results of the central-composite design. Factora
Sum of square
Degree of freedom
F value
P>F
Significance
X1 X2 X3 X21 X22 X23 X1 X2 X1 X3 X2 X3 Model Lack of Fit Pure Error
10.66 0.28 0.61 3.70 95.15 52.48 2.23 0.13 2.74 151.71 2.54 0.95
1 1 1 1 1 1 1 1 1 9 5 5
30.56 0.79 1.74 10.60 272.74 150.44 6.38 0.36 7.85 48.32 2.68
0.0003 0.3940 0.2166 0.0269 <0.0001 <0.0001 0.0301 * 0.5628 0.0187 <0.0001 0.1518
*
* ** **
* **
X1 = time (min), X2 = temperature (°C), X3 = H2O2 concentration (%). * Statistically significant at 95% of probability level. ** Statistically significant at 99% of probability level.
Fig. 2. Ultraviolet visible spectrum of Crassostrea gigas oligosaccharides.
calculated as 2.96 h, 84.71 °C and 2.46%, respectively. The maximum predictable CGO yield was determined by substituting values of the factors into the regression equation. The maximum CGO yield obtained experimentally under the optimised conditions was 10.61%, which was comparable to the predicted value of 10.87% obtained using CCD regression analysis. 3.2. CGO characterisation The CGO sample appeared as a white-yellow powder and contained 1.97% ash, 2.41% moisture, 12.37% protein and 82.09% total sugar. The CGOs were composed of glucose, as indicated by sugar composition analysis by high-performance liquid chromatography. This result was consistent with previous reports (Gao et al., 2014; Wang et al., 2016). The dextrose equivalent value of the resulting products was 7, indicating that the average degree of polymerisation was 14. The FTIR spectra of the CGO sample showed peaks at 2930–3390 cm1 (OAH, NAH), 1412 cm1 (symmetrical deformation of –CH3 and –CH2) and 1152 (special absorption peaks of the a (1 ? 4) glucoside bond) (Fig. 1). The UV spectra of the CYOs displayed peaks at 200–220 nm (Fig. 2), indicating that the CYOs contained protein, which could be in the form of glycoprotein.
3.3. Antioxidant activities of CGO HO holds the highest activity among reactive oxygen species; induces severe damage to biomolecules and causes oxidative injury to biomolecules, such as lipids, proteins, carbohydrate and DNA. DPPH is a relatively stable free radical that can accept an electron or hydrogen atom to form a stable diamagnetic molecule. Therefore, both HO and DPPH have been widely accepted as tools for evaluating free-radical-scavenging antioxidant activity (Qiao et al., 2009). The DRSA of PGDO is presented in Fig. 3. At the concentration of 100 mg/mL, the HRSA and DRSA were 82.31% and 89.17%, respectively. These results indicate that CGOs hold the ability to scavenge HO and DPPH. The reducing capacity of a material is positively correlated with its antioxidant activity and therefore is important in evaluating its antioxidant activity (Fan, Li, Deng, & Ai, 2012). The CGO’s reducing capacity was positively correlated with their concentrations (Fig. 4). The CGOs exhibited a high reducing capacity (absorbance value of 0.792) at a maximum dose of 100 mg/mL. The antioxidant activities of CGO were comparable with those of the oligosaccharides from Chinese yam (Chen, Zhu, & Wu, 2015), Lycium barbarum (Jiang, 2014) and Flammulina velutipes (Xia, 2015). However, the antioxidant activity mechanisms of CGO require further investigation. 100
HRSA
90
DRSA
HRSA, DRSA (%) aa
80 70 60 50 40 30 20 10 0 3.12
6.25
12.5
25
50
100
Conce ntration (mg/mL)
Fig. 1. Fourier transform infrared spectrum of Crassostrea gigas oligosaccharides.
Fig. 3. Hydroxyl radical scavenging activity (HRSA) and 2-Diphenyl-b-picrylhydrazyl-radical-scavenging activity (DRSA) of Crassostrea gigas oligosaccharides. Data are shown as mean ± SD (n = 3). Bars represent the standard deviation (n = 3).
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S. Wu, X. Huang / Food Chemistry 216 (2017) 243–246
0.9
Absorbance (700 nm) aa
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 3.12
6.25
12.5
25
50
100
Conce ntration (mg/mL) Fig. 4. Reducing capacity of Crassostrea gigas oligosaccharides. Data are shown as mean ± SD (n = 3). Bars represent the standard deviation (n = 3).
4. Conclusions In this study, water-soluble CGOs derived from C. gigas were prepared by hydrolysing C. gigas polysaccharides with H2O2. The optimum hydrolytic conditions were investigated by CCD. Preliminary structural characterisations were conducted using chemical analysis, FTIR and UV spectra. The CGOs exhibited high antioxidant activities and may be used as functional food ingredients. Acknowledgements This research was supported by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Carmona-Jiménez, Y., García-Moreno, M. V., Igartuburu, J. M., & Garcia Barroso, C. (2014). Simplification of the DPPH assay for estimating the antioxidant activityof wine and wine by-products. Food Chemistry, 165, 198–204. Chen, Y. F., Zhu, Q., & Wu, S. J. (2015). Preparation of oligosaccharides from Chinese yam and their antioxidant activity. Food Chemistry, 173, 1107–1110. Chen, Y. H., Li, C. Z., Wu, L., Chen, Y. H., Hou, H. X., Zhang, Y. J., & Li, D. R. (2010). Separating and purifying ploysaccharides from oyster of guangxi province and a preliminary study of its antineoplastic activity. China Journal of Modern Medicine, 20, 1004–1007.
Fan, L. P., Li, J. W., Deng, K. Q., & Ai, L. Z. (2012). Effects of drying methods on the antioxidant activities of polysaccharides extracted from Ganoderma lucidum. Carbohydrate Polymers, 87, 1849–1854. Gao, M. M., Zhao, X., Wang, Q. C., & Luan, X. H. (2014). Extraction, separation and structural characterization of polysaccharides from Crassostrea gigas. Chinese Journal of Marine Drugs, 33, 8–14 (in Chinese). Hou, L., Wang, Q. K., He, Y. H., Ren, D. D., Song, Y. F., Jiang, X. D., & Li, L. D. (2014). The extraction of oyster polysaccharide and its hepatoprotective effect against alcohol-induced hepatic injury in mice. Science and Technology of Food Industry, 35, 356–359 (in Chinese). Hou, M. L. (2004). Food analysis. Beijing: Chemical Industry Press (in Chinese). Haaland, P. D. (1989). Separating signals from the noise. In P. D. Haaland (Ed.), Experimental design in biotechnology (pp. 61–83). New York: Marcel Dekker. Jiang, L. F. (2014). Preparation and antioxidant activity of Lycium barbarum oligosaccharides. Carbohydrate Polymers, 99, 646–648. Jiang, C. Y., Zhang, J., & Zhao, J. H. (2013). Study on the effect of oyster polysaccharide on immune function in mice. Chinese Traditional patent Medicine, 35, 1062–1065 (in Chinese). Li, G., Zhao, X., Lv, Y., Li, M., & Yu, G. (2015). Preparation of j-carra-oligosaccharides with microwave assisted acid hydrolysis method. Journal of Ocean University of China, 14, 345–349. Liu, F., Ooi, V. E. C., & Chang, S. T. (1997). Free radical scavenging activities of mushroom polysaccharide extracts. Life Sciences, 60, 763–771. Qu, Y., Li, C. X., Zhang, C., Zeng, R., & Fu, C. M. (2016). Optimization of infraredassisted extraction of Bletilla striata polysaccharides based on response surface methodology and their antioxidant activities. Carbohydrate Polymers, 148, 345–353. Qiao, D. L., Ke, C. L., Hu, B., Luo, J. G., Ye, H., Sun, Y., Yan, X. Y., & Zeng, X. X. (2009). Antioxidant activities of polysaccharides from Hyriopsis cumingii. Carbohydrate Polymers, 78, 199–204. Shi, X., Ma, H. H., Tong, C. Q., Qu, M., Jin, Q., & Li, W. (2015). Hepatoprotective effect of a polysaccharide from Crassostrea gigas on acute and chronic models of liver injury. International Journal of Biological Macromolecules, 78, 142–148. Sheng, J., Yu, F., Xin, Z., Zhao, L., Zhu, X., & Hu, Q. (2007). Preparation, identification and their antitumor activities in vitro of polysaccharides from Chlorella pyrenoidosa. Food Chemistry, 105, 533–539. Tao, Z. P., Sun, L. C., Qiu, X. J., Cai, Q. F., Liu, G. M., Su, W. J., & Cao, M. J. (2016). Preparation, characterisation and use for antioxidant oligosaccharides of a cellulase from abalone (Haliotis discus hannai) viscera. Journal of the Science of Food and Agriculture, 96, 3088–3097. Tian, Z. H., Li, Y. B., Li, Y., Chu, H. H., Hao, L. L., Su, T. T., & Zhang, L. (2013). Optimization of preparation technology and antimicrobial activity of polysaccharide extracts from Rushan Bay Oyster (Crassostrea gigas) in China. Food Research and Development, 34, 23–26 (in Chinese). Wang, T., Ding, J., Li, H. B., Xiang, J. J., Wen, P., Zhang, Q., Yin, L. L., Jiang, W., & Shen, C. E. (2016). Antihypertensive activity of polysaccharide from Crassostrea gigas. International Journal of Biological Macromolecules, 83, 195–197. Wu, S. J., & Yu, L. (2015). Preparation and characterisation of the oligosaccharides derived from Chinese water chestnut polysaccharides. Food Chemistry, 181, 15–18. Xia, Z. Q. (2015). Preparation of the oligosaccharides derived from Flammulina velutipes and their antioxidant activities. Carbohydrate Polymers, 118, 41–43. Yao, X. C., Cao, Y., & Wu, S. J. (2013). Antioxidant activity and antibacterial activity of peach gum derived oligosaccharides. International Journal of Biological Macromolecules, 62, 1–3.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Preparation and antioxidant activities of oligosaccharides from Crassostrea gigas Shengjun Wu a,b,c,d,⇑, Xiaolian Huang a a
Jiangsu Marine Resources Development Research Institute, Lianyungang, Jiangsu 222005, China School of Marine Science and Technology, Huaihai Institute of Technology, 59 Cangwu Road, Xinpu 222005, China c Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang 222005, China d Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang 222005, China b
a r t i c l e
i n f o
Article history: Received 3 July 2016 Received in revised form 14 August 2016 Accepted 16 August 2016 Available online 17 August 2016 Keywords: Crassostrea gigas Oligosaccharides Antioxidant activity
a b s t r a c t Oligosaccharides were prepared from Crassostrea gigas by hydrolysis of polysaccharide in C. gigas with peroxide oxygen (H2O2). The hydrolysates were cleared of protein, filtered, ultrafiltered and precipitated with absolute ethanol to give C. gigas oligosaccharides (CGOs). Factors affecting CGO yields, i.e., reaction time, temperature, and H2O2 concentration, were optimised as follows: 2.96 h reaction time, 84.71 °C reaction temperature, and 2.46% H2O2 concentration. Under these conditions, the maximum yield of CGOs reached 10.61%. The CGOs were then partially characterised by Fourier transform infrared spectroscopy, UV spectroscopy, monosaccharide composition, and antioxidant activities. Results indicate that CGOs possessed strong hydroxyl radical activity, 2,2-diphenyl-b-picrylhydrazyl-radical-scavenging activity and reducing capacity at a concentration of 100 lg/mL. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Free radicals are produced by many factors, such as physical effects, chemical reactions, and metabolic processes. Free radicals exert many pathological effects on living organisms, such as DNA damage, thereby causing carcinogenesis and inducing agingrelated cellular degeneration (Liu, Ooi, & Chang, 1997). A variety of antioxidants can scavenge strong radicals (Liu et al., 1997). These antioxidants can be divided into synthetic chemicals, such as phenolic compounds and various naturally occurring substances, such as saccharides. Moreover, natural antioxidants from organism extracts have attracted increasing interest because of consumer concern about the safety of synthetic antioxidants in food (Yao, Cao, & Wu, 2013). Crassostrea gigas, one major cultured shellfish in China, is nutritious and delicious and is thus considered a valuable food resource (Hou et al., 2014). Recent research found that C. gigas is rich in polysaccharides [4–8]. Gao et al. found that the water-soluble polysaccharide from C. gigas comprised mainly of ?4)-a-DGlc-(1? with few ?3,4)-b-D-Glc-(1? and ?2,4)-b-D-Glc-(1? branched units) (Gao, Zhao, Wang, & Luan, 2014). C. gigas polysaccharides have been demonstrated to contain many functional ⇑ Corresponding author at: Jiangsu Marine Resources Development Research Institute, Lianyungang, Jiangsu 222005, China. E-mail address: [email protected] (S. Wu). http://dx.doi.org/10.1016/j.foodchem.2016.08.043 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
activities, such as immunostimulatory (Jiang, Zhang, & Zhao, 2013), antimicrobial (Tian et al., 2013), antitumour (Chen et al., 2010), hepatoprotective (Shi et al., 2015) and antihypertensive (Wang et al., 2016) activities. However, data on the C. gigas oligosaccharides (CGOs) are limited. At present, a number of methods for preparation of oligosaccharides from different organisms have been developed, e.g. peroxide oxygen (H2O2)-assisted preparation (Wu & Yu, 2015), microwaveassisted preparation (Li, Zhao, Lv, Li, & Yu, 2015) and enzymaticassisted preparation (Tao et al., 2016). Nevertheless, the data regarding peroxide oxygen (H2O2)-assisted preparation of CGOs are limited. In the present study, we prepared water-soluble CGOs from C. gigas by hydrolysis with H2O2, partially characterised the product and evaluated their antioxidant activities.
2. Materials and methods 2.1. Materials Live C. gigas were purchased from a local farmers’ market (Xinpu, China)H2O2 (30%, v/v) was purchased from the Laiyang Kant Chemical Co., Ltd. (Laiyang, China). All other reagents used were of analytical grade.
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S. Wu, X. Huang / Food Chemistry 216 (2017) 243–246
2.2. Preparation of CGOs
2.5. Statistical analysis
C. gigas meat, including the adductor muscle and mantle was stripped from the shells, shredded, dried in a hot air oven (JKOOI-240A, China) at 60 °C to a constant weight, pulverised and sifted through a 40-mesh sieve. The lipids in the dried powder were removed by the Soxhlet extraction method using light petroleum as the solvent. The lipid-removed powder was suspended in distilled water to give a suspension with a concentration of 2% (w/v)H2O2 (2%, 2.5% and 3%, respectively) were added to the reactor containing 100 mL of the suspension, and the reactor was incubated in a thermostatic water bath at different temperatures (80, 85, and 90 °C, respectively) for designated time periods (2, 2.5 and 3 h, respectively). The hydrolysates were filtered, proteins were removed using the Sevag method, concentrated (approximately 10%), precipitated using six volumes of absolute ethanol, filtered again and freezedried. The percentage yield of CGOs was calculated using Eq. (1) as follows:
All data are presented as mean ± standard deviation. Statistical analysis was performed using Statgraphics Centurion XV version 15.1.02. A multifactor ANOVA with posterior multiple range test was used for determining statistical significance.
Yield ¼ 100 W 2 =W 1
3. Results and discussion 3.1. Effect of reaction time, temperature and H2O2 concentration on CGO yield The reaction conditions, i.e. time, temperature and H2O2 concentration, were optimised using a central composite design (CCD), which is shown along with the results in Table 1. The regression model was obtained by analysing the results of the experiment using a multifactor ANOVA as follows:
Y ¼ 895:96474 þ 31:08210 X 1 þ 18:61347 X 2 þ 58:88526 X 3 0:021100 X 1 X 2 0:50000 X 1 X 3
ð1Þ
0:23400 X 2 X 3 2:02601 X 21 0:10278 X 22
where W1 and W2 represent the weights of the recovered CGOs and the original C. gigas powder, respectively. 2.3. CGO characterisation Total sugar, protein and reducing sugar contents were determined using the phenol–sulphuric acid colorimetric method, the Kjeldahl method and a method proposed by Hou (2004). Meanwhile, monosaccharide composition was analysed using the procedure reported by Sheng et al. (2007). The Fourier transform infrared (FTIR) spectra of the resulting CGO sample was recorded in KBr pellets by a Nicolet Nexus FTIR 470 spectrophotometer (Nicolet, USA) over a wavelength range of 400 cm1 to 4000 cm1. The UV spectra were recorded on a UV spectrometer (Spectra Test, German). 2.4. Antioxidant activity assays The hydroxyl radical (HO) scavenging activity (HRSA) of the CGO sample was measured in accordance with the method of Qu, Li, Zhang, Zeng, and Fu (2016). The hydroxyl-radical-scavenging activity was calculated as follows:
HRSAð%Þ ¼
A1 A2 100 A 1 A0
ð2Þ
where A0 is the absorbance of the reagent blank absorbance, A1 is the positive control absorbance and A2 is the absorbance of the sample. 2-Diphenyl-b-picrylhydrazyl-radical-scavenging activity (DRSA) was determined in accordance with the method of Carmona-Jiménez, García-Moreno, Igartuburu, and Garcia Barroso (2014). The DPPH-free-radical-scavenging percentage was calculated by the following equation:
DRSAð%Þ ¼
½A0 ðA1 A2 Þ 100 A0
ð3Þ
where A0 is the absorbance of the control (water instead of CGOs solution), A1 is the absorbance of the sample and A2 is the absorbance of the sample under identical conditions as A1 with water instead of DPPH solution. Reducing capacity was measured in accordance with the method of Qiao et al. (2009). A higher absorbance indicates a better reducing capacity.
7:63336 X 23
ð4Þ
where Y is the CGO yield (%), X1 is the time (h), X2 is the temperature (°C) and X3 is the H2O2 concentration. ANOVA for the response surface quadratic model verified the statistical significance of Eq. (4). The results in Table 2 showed that the model obtained was highly significant, which was demonstrated by the values of F and P ((P > F) < 0.0001). The fit accuracy was verified by the high value of multiple correlation coefficient (R2 = 97.75%), which indicated that the response model explained the total variations by 97.75%. A regression model with an R2 value > 0.9 is generally considered to reflect a high correlation (Haaland, 1989). Moreover, the value of the adjusted multiple correlation coefficient (R2Adj = 95.73%) was sufficiently high to indicate the statistical significance of the model. The interaction between reaction time and temperature and that between temperature and H2O2 concentration were significant (p < 0.05). However, the interaction between reaction time and H2O2 concentration was not significant (p > 0.05) (Table 2). According to the model, the optimum reaction conditions of time, temperature and H2O2 concentration maximum CGO yield were Table 1 The central-composite design for optimizing extraction conditions. Run
X1
X2
X3
Yield (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
2.50 2.50 2.50 3.00 3.00 2.50 3.34 2.00 2.00 2.00 2.50 3.00 2.50 2.50 3.00 2.50 2.50 2.00 1.66 2.50
85.00 85.00 76.59 90.00 80.00 85.00 85.00 90.00 90.00 80.00 85.00 90.00 85.00 93.41 80.00 85.00 85.00 80.00 85.00 85.00
2.50 2.50 2.50 2.00 2.00 2.50 2.50 2.00 3.00 2.00 2.50 3.00 2.50 2.50 3.00 1.66 2.50 3.00 2.50 3.34
10.61 10.72 3.12 6.71 6.21 10.16 10.29 5.68 4.94 2.91 9.85 5.63 10.35 3.17 7.31 6.18 11.08 4.67 7.67 3.85
X1 = time (min), X2 = temperature (°C), X3 = H2O2 concentration (%).
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S. Wu, X. Huang / Food Chemistry 216 (2017) 243–246 Table 2 Analysis of variance for the experimental results of the central-composite design. Factora
Sum of square
Degree of freedom
F value
P>F
Significance
X1 X2 X3 X21 X22 X23 X1 X2 X1 X3 X2 X3 Model Lack of Fit Pure Error
10.66 0.28 0.61 3.70 95.15 52.48 2.23 0.13 2.74 151.71 2.54 0.95
1 1 1 1 1 1 1 1 1 9 5 5
30.56 0.79 1.74 10.60 272.74 150.44 6.38 0.36 7.85 48.32 2.68
0.0003 0.3940 0.2166 0.0269 <0.0001 <0.0001 0.0301 * 0.5628 0.0187 <0.0001 0.1518
*
* ** **
* **
X1 = time (min), X2 = temperature (°C), X3 = H2O2 concentration (%). * Statistically significant at 95% of probability level. ** Statistically significant at 99% of probability level.
Fig. 2. Ultraviolet visible spectrum of Crassostrea gigas oligosaccharides.
calculated as 2.96 h, 84.71 °C and 2.46%, respectively. The maximum predictable CGO yield was determined by substituting values of the factors into the regression equation. The maximum CGO yield obtained experimentally under the optimised conditions was 10.61%, which was comparable to the predicted value of 10.87% obtained using CCD regression analysis. 3.2. CGO characterisation The CGO sample appeared as a white-yellow powder and contained 1.97% ash, 2.41% moisture, 12.37% protein and 82.09% total sugar. The CGOs were composed of glucose, as indicated by sugar composition analysis by high-performance liquid chromatography. This result was consistent with previous reports (Gao et al., 2014; Wang et al., 2016). The dextrose equivalent value of the resulting products was 7, indicating that the average degree of polymerisation was 14. The FTIR spectra of the CGO sample showed peaks at 2930–3390 cm1 (OAH, NAH), 1412 cm1 (symmetrical deformation of –CH3 and –CH2) and 1152 (special absorption peaks of the a (1 ? 4) glucoside bond) (Fig. 1). The UV spectra of the CYOs displayed peaks at 200–220 nm (Fig. 2), indicating that the CYOs contained protein, which could be in the form of glycoprotein.
3.3. Antioxidant activities of CGO HO holds the highest activity among reactive oxygen species; induces severe damage to biomolecules and causes oxidative injury to biomolecules, such as lipids, proteins, carbohydrate and DNA. DPPH is a relatively stable free radical that can accept an electron or hydrogen atom to form a stable diamagnetic molecule. Therefore, both HO and DPPH have been widely accepted as tools for evaluating free-radical-scavenging antioxidant activity (Qiao et al., 2009). The DRSA of PGDO is presented in Fig. 3. At the concentration of 100 mg/mL, the HRSA and DRSA were 82.31% and 89.17%, respectively. These results indicate that CGOs hold the ability to scavenge HO and DPPH. The reducing capacity of a material is positively correlated with its antioxidant activity and therefore is important in evaluating its antioxidant activity (Fan, Li, Deng, & Ai, 2012). The CGO’s reducing capacity was positively correlated with their concentrations (Fig. 4). The CGOs exhibited a high reducing capacity (absorbance value of 0.792) at a maximum dose of 100 mg/mL. The antioxidant activities of CGO were comparable with those of the oligosaccharides from Chinese yam (Chen, Zhu, & Wu, 2015), Lycium barbarum (Jiang, 2014) and Flammulina velutipes (Xia, 2015). However, the antioxidant activity mechanisms of CGO require further investigation. 100
HRSA
90
DRSA
HRSA, DRSA (%) aa
80 70 60 50 40 30 20 10 0 3.12
6.25
12.5
25
50
100
Conce ntration (mg/mL)
Fig. 1. Fourier transform infrared spectrum of Crassostrea gigas oligosaccharides.
Fig. 3. Hydroxyl radical scavenging activity (HRSA) and 2-Diphenyl-b-picrylhydrazyl-radical-scavenging activity (DRSA) of Crassostrea gigas oligosaccharides. Data are shown as mean ± SD (n = 3). Bars represent the standard deviation (n = 3).
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0.9
Absorbance (700 nm) aa
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 3.12
6.25
12.5
25
50
100
Conce ntration (mg/mL) Fig. 4. Reducing capacity of Crassostrea gigas oligosaccharides. Data are shown as mean ± SD (n = 3). Bars represent the standard deviation (n = 3).
4. Conclusions In this study, water-soluble CGOs derived from C. gigas were prepared by hydrolysing C. gigas polysaccharides with H2O2. The optimum hydrolytic conditions were investigated by CCD. Preliminary structural characterisations were conducted using chemical analysis, FTIR and UV spectra. The CGOs exhibited high antioxidant activities and may be used as functional food ingredients. Acknowledgements This research was supported by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Carmona-Jiménez, Y., García-Moreno, M. V., Igartuburu, J. M., & Garcia Barroso, C. (2014). Simplification of the DPPH assay for estimating the antioxidant activityof wine and wine by-products. Food Chemistry, 165, 198–204. Chen, Y. F., Zhu, Q., & Wu, S. J. (2015). Preparation of oligosaccharides from Chinese yam and their antioxidant activity. Food Chemistry, 173, 1107–1110. Chen, Y. H., Li, C. Z., Wu, L., Chen, Y. H., Hou, H. X., Zhang, Y. J., & Li, D. R. (2010). Separating and purifying ploysaccharides from oyster of guangxi province and a preliminary study of its antineoplastic activity. China Journal of Modern Medicine, 20, 1004–1007.
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