Optimization, Characterization and Antioxidant Activities of Selenized Polysaccharides from Hypsizygus marmoreus

Corresponding Author: Bing Xu School of Biology and Food Engineering, Changshu Institute of Technology, Changshu 215500, PR China Email: Xubing@cslg.cn Abstract: In this study, hypsizygus marmoreus was used as raw material to extract polysaccharides by hot water extraction method. Preparation technology of Selenized Polysaccharides from Hypsizygus marmoreus (SPHM) and its antioxidant activities were investigated. Chelation rate was selected as the index, effects of initial concentration of sodium selenite (mg/mL), the mass ratio of polysaccharide to sodium selenite, chelation time and pH on the chelation rate was investigated by single factor test. Response surface method was used to optimize the preparation technology of SPHM. The preliminary structure characterization of Polysaccharides from Hypsizygus marmoreus (PHM) and SPHM was performed by infrared spectroscopy and scanning electron microscopy. The antioxidant activities of SPHM were studied by the scavenging effect of 1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1(2,4,6-trinitrophenyl) Hydrazyl (DPPH) and hydroxyl radical. The results showed that the optimum preparation technology of SPHM was as follows: the initial concentration of sodium selenite was 7.95 (mg/mL), the mass ratio of polysaccharide to sodium selenite was 6.11:1 (mg/mg), chelation time was 6.89h and pH was 5.11. Under the optimum conditions, chelate rate was 10.36%. Infrared spectroscopy and scanning electron microscopy analysis showed that selenium ions successfully combined with PHM and the surface structure of PHM and SPHM was different. Compared with HPM, the antioxidant activities of SPHM enhanced.


Introduction
Se is one of the essential trace elements in human life activities. Se exhibits antitumor activity, improves immunity and has antiaging properties (Rayman, 2000). The main two forms of Se generally present in nature are inorganic Se and organic Se. Malgorzata et al. (2012) reported that organic Se compounds have the advantage of mild toxicity, easy absorption and high bioavailability compared with inorganic Se. To reduce the original toxicity of Se, we combined the Se and polysaccharide. Chelate has the Se's advantages and also has the advantages of polysaccharide. Therefore, chelate has antiviral and antitumor properties and is easily absorbed and utilized by the bodies. The chelate's biological activity is generally higher than that of polysaccharide and inorganic Se (Zhu et al., 2016). On the basis of the present condition, synthetic Se polysaccharides, such as Enteromorpha selenide (Haitao et al., 2018), Tussilago farfara L. Se (Gaiyan et al., 2016), Cucurbita moschata (Duch. ex Lam.) Duch. ex Poiret Se (Selvi et al., 2012), Punica granatum L. bark Se (Gharzouli et al., 1999), Taraxacum mongolicum Hand-Mazz selenide (Sui et al., 2004) and Lilium brownii var. viridulum Se polysaccharide (Li et al., 2010), showed that the bioactivities of Se polysaccharides, such as bacteriostatic, antioxidant and antitumor, were improved to some extent compared with unselenized polysaccharides.

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This study, according to the modification of selenized polysaccharide, it showed improved biological activity and bioavailability by utilizing the response surface analysis method to optimize the chelating process of SPHM. We also studied the ant oxidative activity of PHM before and after chelation. The effect of selenization on the antioxidant activity of PHM was also discussed. Considering the added value of H. marmoreus, this study also aims to serve as a theoretical reference for the development of the emerging antioxidant food of SPHM.

Materials and Instruments
H. marmoreus was provided by the Kunshan Zhengxing Edible Fungus Co., Ltd. DPPH was also obtained from the Shenzhen Xinbosheng Biotechnology Co., Ltd. The reagents that were used in this experiment, such as sodium selenite, hydrogen peroxide and potassium sodium tartrate were all of analytical grade.
A small-pulverizer high-speed engine was from Shandong Weinuo Medical Equipment Manufacturing Co., Ltd. HH-2 intelligent digital display constant temperature water bath was provided by the Changzhou Guohua Electric Co., Ltd. The 722-type digital visible spectrophotometer was provided by Shanghai Optical Instrument Factory Co., Ltd. IRTracer-100 Fu Li Leaftransformed infrared spectrometer was provided by Japan Shimadzu Corporation. RE-52AA rotary evaporator was from the Beijing Chengmeng Weiye Technology Co., Ltd. SHB-B95 circulating water type multipurpose vacuum pump was provided by Zhengzhou Century Shuangke Experimental Instrument Co., Ltd. CR22GII high-speed refrigerated centrifuge was provided by the Hitachi Company, Japan. Alpha 1-2 LD plus vacuum freeze dryer was provided by CHRIST Company, Germany. ZEISS SIGMA thermal field emission scanning electron microscope was provided by Carl Zeiss Company, Germany.

PHM Preparation
H. marmoreus passed through 60 meshes to remove the impurities, drying (30°C, 24 h), crushing and sieving through 60 meshes. Then, the H. marmoreus powder was obtained. Afterward, 100 g of H, marmoreus powder was mixed with 2000 mL of distilled water and extracted at 90°C for 5 h. The precipitate was removed by centrifugation (4000 r/min, 15 min) and the clear supernatant extract was concentrated at 70°C for 3 h (Zhang et al., 2015b). Then, the protein was removed by sevag method (Kumar and Satheesan, 2010) and the supernate was collected after centrifugation, followed by addition of three times the volume of 85% ethanol. Then, the supernate was centrifuged (4000 r/min, 20 min) after ethanol precipitation for 24 h at 4°C. Cryodesiccation technique was also applied to the precipitate to obtain the PHM.

SPHM Preparation
We took 0.1-0.3 g PHM, added 5-15 mL of HNO 3 solution (volume fraction of 0.5%), heated and stirred until the polysaccharide was completely dissolved. A certain mass ratio of sodium selenite was also added, stirred at 70°C for 6-8 h under a certain pH condition, cooled and centrifuged at 3000 r/min for 5 min. Then, the supernatant was placed in a bag filter (interception molecular weight of 3500 Da). Dialysis was performed using flowing water for 24 h. Then, we took a small amount of dialysate and checked for residual sodium selenite by adding ascorbic acid until the dialysate color changed from blood red. The dialysate was cryodesiccated to obtain a SPHM.

Chelating Rate Calculation
The standard mother liquor of sodium selenite should be accurately prepared at concentration of 1 mg/mL. Then, the standard liquor concentrations of 0, 4, 8, 12, 16 and 20 mL were measured separately and added with 10 mL of ultrapure water. The mass concentrations of sodium selenite were 0, 0.4, 0.8, 1.2, 1.6 and 20 mg/mL and that of Se was measured by the flame atomic absorption method. The abscissa and ordinate were the mass concentration and absorbance of Se, respectively. We also drew a standard curve. We obtained the regression equation Y = 0.2569X+0.0375, R2 = 0.9826, where Y is the absorbance; and X is the mass Se concentration (mg/mL) (Xue et al., 2009). The chelation rate was calculated using the following equation: where, C 0 is the mass Se concentration in the solution before chelation (mg/mL) and C is the mass Se concentration in the solution after chelation (mg/mL).

Effect of Initial Mass Sodium Selenite Concentration on Chelating Rate
We formulated sodium selenite solution with the initial mass concentrations of 3, 4, 5, 6, 7 and 8 mg/mL with PHM (dissolved in 50 mL of 0.5% HNO 3 solution) and the sodium selenite mass ratio (mg/mg) was 4: 1. Then, we added PHM and the reaction was stirred at 70°C for 7 h at pH of 5 to determine the optimum initial sodium selenite concentration.

Effect of Chelating Time on Chelating Rate
We removed the sodium selenite solution at a concentration of 4 mg/mL and added 4 to 1 by mass ratio (mg/mg) of the PHM (dissolved in 50 mL of 0.5% HNO 3 solution) to sodium selenite. PHM was chelated for 4, 5, 6, 7, 8 and 9 h at the pH of 5 and 70°C to determine the optimal chelating time.

Effect of Mass Ratio of PHM to Sodium Selenite Referred to as Mass Ratio on Chelating Rate
We removed the sodium selenite solution at a concentration of 4 mg/mL at the mass ratios (mg/mg) of PHM (dissolved in a volume fraction of 0.5% HNO 3 solution 50 mL) and sodium selenite of 3: 1, 4: 1, 5: 1, 6: 1, 7: 1 and 8: 1 We added the PHM and stirred at 70°C for 7 h at pH 5 while the reaction progressed. Consequently, the proper mass ratio of H. marmoreus and sodium selenite was determined.

Effect of pH on Chelating Rate
We removed the sodium selenite solution at a concentration of 4 mg/mL and added PHM 4 to 1 by mass ratio (mg/mg) of the PHM (dissolved in 50 mL of a 0.5% HNO 3 solution) and sodium selenite and agitated at the pH levels of 3, 4, 5, 6, 7 and 8 at 70°C for 7 h to determine the optimum pH.

Response Surface Test
According to the results of the single factor experiment, we used the design principle of Box-Benhnken's Central Combination Experiment with chelating rate as the index to design the regression equation of the initial mass concentration, mass ratio, chelation time and pH of the sodium selenite and fit the relationship between each factor and the response values. We also used the Design-Expert 8.0.6 software to conduct response surface analysis and obtain the optimal preparation process of SPHM.

Infrared Characterization of PHM and SPHM
We took appropriate amount of dried PHM and SPHM powder and mixed with appropriate amount of KBr. The infrared spectrum was scanned at the wave number (WN) range of 4000-400 cm −1 . The infrared spectra of the polysaccharide and chelate were also determined.

Scanning Electron Microscopy Analysis of PHM and Se Chelate
We took a certain amount of dried PHM and SPHM that adhered to the specimen holder, blew the floating sample out and then sprayed gold. The appearance and morphology of the specimen were observed at the magnification of 2000× and a high pressure of 20.00 kV by using the ZEISS thermal field emission electron microscope.

Determination of Antioxidant Activity of PHM and Se Chelate
Determination of DPPH Free Radical Scavenging Ability (Suja et al., 2004) We accurately weighed 0.0128 g free radicals of DPPH, added 70% ethanol and then constant volume in a 50 mL volumetric flask, shook and stored in the dark after it is completely dissolved. We transferred 2 mL of DPPH solution into the comparison tubes, added 1 mL of different mass concentrations (0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL) of PHM and SPHM solution and reacted at room temperature in the dark for 30 min. Distilled water was also added for blank zero correction. The absorption was measured at the wavelength of 517 nm and the clearance rate of DPPH free radical was calculated as follows: where, A i consists of 2 mL of DPPH+1 mL of PHM solution (SPHM solution); A j comprises 2 mL PHM solution (SPHM solution) +1 mL ethanol solution with the volume fraction of 70%; A 0 is composed of 2 mL of DPPH+1 of mL ethanol solution and the volume fraction was 70%.
OH Radical Scavenging Ability Determination (Hirayama and Yida, 1997) We added ED 1 mL of FeSO 4 solution (9 mmol/L), 2 mL of salicylic acid-ethanol solution (9 mmol/L), 2 mL of different mass concentrations (0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL) of polysaccharide/SPHM solution and 2 mL of H 2 O 2 solution (8.8 mmol/L) to the colorimetric tube. The solution was subjected to water bath at 37°C for 30 min. The sample absorbance was measured at the wavelength of 510 nm with distilled water used for zero correction. The clearance rate of the OH radicals was calculated as follows: where, A 0 is the absorbance of the blank control solution and A j is the absorbance of the sample group.

Results and Discussion
Effects of Single Factor on Chelating Rate

Effect of Initial Mass Sodium Selenite Concentration on Chelating Rate
The effects of the initial mass sodium selenite concentration on the chelating rate are shown in Fig. 1. Figure 1 shows the increasing chelating rates of PHM and Se along with the mass sodium selenite concentration. When the mass sodium selenite concentration was 7 mg/mL, the chelating rate reached the maximum. Then, the chelating rate began to decrease possibly because the chelation is incomplete because of the extremely low initial Se concentration. However, when the concentration was exceedingly high, the reaction mixture was saturated. By contrast, sodium selenite addition hindered the chelation reaction. Therefore, the optimum initial mass sodium selenite concentration was 6 mg/mL.

Effects of Chelation Time on Chelating Rate
The effects of chelation time on chelating rate are shown in Fig. 2. Figure 2 shows that when the chelation time was extended from 4 h to 9 h, the chelating rate tended to increase first and then slowly decrease and the latter tended to be stable. When the chelation time was 6 h, the chelating rate reached the maximum. Thus, the optimal chelation time is 6 h.

Effects of Mass Ratio on Chelating Rate
The effects of the mass ratio of PHM to sodium selenite on the chelating rate are shown in Fig. 3. As shown in Fig. 3, along with the PHM, the mass ratio (mg/mg) to sodium selenite increased first and then decreased. When the mass ratio was 6:1, the chelating rate reached the maximum. Therefore, the most appropriate mass ratio of the PHM to sodium selenite was 6:1. On the basis of this condition, we combined PHM with other optimization conditions and obtained the final chelating rate by taking the average value through many experiments.

Effects of pH on Chelating Rate
The effect of pH on the chelating rate is shown in Fig. 4. Figure 4 shows the chelating rate of PHM and sodium selenite with increased pH, thereby showing a trend that increased first and then decreased. When the pH was 5, the chelating rate reached the maximum. This result may be due to the fact that the reaction liquid pH has a certain influence on the strength of Se ions.  Chelating rate (%) If the pH is extremely high, then the anion in the solution will compete with the active group of the PHM, thereby hindering chelate formation. If the pH is extremely small, then the H ions generated in the reaction solution increase, thereby leading to incompletely chelated Se ions, thereby resulting in reduced chelation rate (Schmidbaur and Mitschke, 1971). Therefore, the optimum pH for chelation is 5.

Response Model Establishment and Analysis
According to the results of single factor experiments, the chelation rate was the index. The experimental design factors and the horizontal values are shown in Table 1. The consequence of the experimental design of Box-Behnken and the chelating rate response values are shown in Table 2. The results of variance analysis are shown in Table 3. We used Design-Expert 8.0.6 to analyze the data in Table 2. The chelating rate (Y) to the initial mass concentration (A), mass ratio (B), chelation time (C) and pH (D) of sodium selenite were obtained after regression analysis of each factor.
The regression equation:   According to variance analysis results in Table 3, the model selected in this experiment was extremely significant (P<0.001) and the equations' lack-of-fit was insignificant (P = 0.4379>0.05). This finding indicated that the unknown factors had low influence on the test. This model was suitable for this experiment, with the R 2 =0.9347, thereby indicating that the model had good goodness of fit and can be used for preliminary analysis and prediction of the Se preparation in PHM. In addition to D, the linear effects of the three factors on the chelation degree was significant (P<0.05 or P<0.001). The interaction items AC and AD and the quadratic terms A 2 , B 2 , C 2 and D 2 had significant effects on response chelating rate (P<0.05 or P<0.001).

Response Surface Analysis
The response surface reflects the effects of the interaction of the other factors on the H. marmoreus and Se chelation when the initial selenite concentration, chelation time, mass ratio and pH are 0. The degree of steepness of the surface indicates whether the influence of different factors on the chelating rate was significant. Meanwhile, the strength of the interaction between the two factors depends on the shape of the isotypes. If the contour is elliptical, then the interaction between the two factors is strong and the impact is significant. If the contour is round, then the result is reversed. The contour sparseness reflects the strength of the influence, its closeness indicates a considerable impact on the chelating rate and the sparsity indicates minimal impact (Bonis, 1964).
The effects of the interaction of the two factors on the chelating rate are shown in Fig. 5. As shown in the Figure, the interaction between the initial mass sodium selenite concentration and pH (AD) had the most significant effect on the chelating rate, followed by the effect on the chelation rate of the initial mass concentration and chelation time interaction (AC). The interaction effects of mass ratio and chelation time (BC), mass ratio and pH (BD) and chelation time and pH (CD) on the chelation rate were insignificant.

Optimum Process Condition Determination and Model Verification
Design-Expert software was used to obtain the optimal process for chelating Se from PHM by analyzing the response surface. The initial mass sodium selenite concentration was 7.95 mg/mL, the mass ratio was 6.11: 1 (mg/mg), the chelation time was 6.89 h and the pH was 5.11. Under the optimized conditions, the Se of SPHM rate can reach 10.36%. In verifying the accuracy of the response surface optimization model, according to convenient operation, the adjustment process parameters are as follows: The initial mass sodium selenite concentration was 8 mg/mL, the ratio was 6:1 (mg/mg), the chelation time was 7 h, the pH was 5 and the chelating rate was 10.07%. The difference between the number and our prevalue was insignificant, thereby verifying the prediction experiment reliability.

Infrared Characterization of PHM and Se Chelate
The infrared spectrum of the PHM/SPHM is shown in Fig. 6. Figure 6 reveals that the PHM infrared spectroscopy had characteristic absorption peaks of polysaccharides. The absorption peaks that appeared at the

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The peaks at 1078 and 1025 cm −1 were the characteristic peaks caused by the stretching vibration of the furan nucleus and the pyran ring of the sugar, respectively. Compared with PHM, the peak shape of SPHM infrared spectrum showed insignificant change. Only the absorption wavelengths of few functional peaks showed certain bathochromic and shift. A characteristic absorption peak was observed near 923 cm −1 , which was a stretching vibration of Se = O and a peak near 717 cm −1 was a stretching vibration peak of Se-C (Franco et al., 1985). This finding indicated that sodium selenite participated in Se polysaccharide synthesis in the PHM, thereby causing the polysaccharide structure to change to some extent.

Scanning Electron Microscopic Analysis of PHM and Se Chelate
The results of scanning electron microscopy analysis of PHM and its Se chelate are shown in Fig. 7 and 8.
The PHM sample was in the form of flakes or debrislike materials. The surface structure of the polysaccharide was tight and the intermolecular force was high. The Se chelate had an irregular shape with a convex and concave surface possessing many holes. This condition indicated that a repulsive force was present between the complex molecules and the intermolecular attraction was weak (Liu et al., 2008). Further analysis was required regarding the PHM and SPHM structures.

Scavenging Ability of DPPH Free Radicals
The scavenging ability of DPPH free radicals before and after PHM chelating is shown in Fig. 9. PHM had a certain scavenging ability on the scavenged DPPH free radicals. With the increase in polysaccharide concentration, the scavenging rate gradually rose and tended to be stable (the ranges of up and down changes were small). Compared with the PHM, the ability of scavenged free radical of DPPH of the SPHM was enhanced. The ability of the T. farfara L. Se polysaccharide-removed DPPH free radicals was significantly enhanced after selenide modification. The reason may be related to the interaction between the chelating site of Se ions and reactive groups of polysaccharides and the specific reasons need further study.

Scavenging Ability of OH Radicals
The scavenging ability of the OH radicals before and after the PHM chelation is shown in Fig. 10. The PHM had certain scavenging ability to OH radicals before and after chelation and the scavenging ability had a certain linear relationship with the polysaccharide concentration.
The scavenging ability of the SPHM to OH radicals was higher than that of the PHM. This result indicated that selenization modification increased the degree of the anti-oxidant activity of PHM to some extent. The specific mechanism remains to be further studied. Shang Longchen et al. found that after selenization of the C. moschata (Duch. ex Lam.) Duch. Ex Poiret polysaccharide, the scavenging effect of C. moschata (Duch. ex Lam.) Duch. Ex Poiret Se polysaccharide on OH radicals was significantly higher than that of C. moschata (Duch. ex Lam.) Duch. Ex Poiret polysaccharides. Clearance rate %

Before chelation
After chelation

■■ Conclusion
We used H. marmoreus as the raw material and the PHM was extracted by water extraction and alcohol sedimentation. The optimal chelation process conditions, that is, the initial mass sodium selenite concentration, of PHM, were determined by the response surface method. The ratio was 7.95 mg/mL, the mass ratio was 6.11:1 (mg/mg), the chelation time was 6.89 h and the pH was 5.11. Under optimized conditions, the chelating rate of the H. marmoreus polysaccharide and Se reached 10.36%. Compared with PHM, the antioxidative activity of the SPHM was further enhanced. In this study, the PHM was chelated with Se and the PHM was successfully selenized. The antioxidant activity of the SPHM was considerably improved to some extent and the mechanism of activity enhancement still needs further research. The research results provided a theoretical basis for the SPHM as a new type of functional food antioxidant, thereby laying a foundation for the promotion of the added value of H. marmoreus resources.

Author's Contributions
Ting Liu: Paricipated in all experiments and article writing; also responsible for data analysis.
Xue Jiang: Paricipated in some experiments and data analysis.
Wei Wang: Paricipated in some experiments and data proceessing.
Bing Xu: Paricipated in project design and article writing; responsibilites of contacts for research materials.

Ethics
This article is original and contains unpublished material. The corresponding author confirms that all of the other authors have read and approved the manuscript and no ethical issues involved.