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Effect of solution plasma process with hydrogen peroxide on the degradation of water-soluble polysaccharide from Auricularia auricula. II: Solution conformation and antioxidant activities in vitro
Carbohydrate Polymers 198 (2018) 575–580
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Effect of solution plasma process with hydrogen peroxide on the degradation of water-soluble polysaccharide from Auricularia auricula. II: Solution conformation and antioxidant activities in vitro Fengming Maa, Jingwei Wua, Pu Lib, Dongbing Taoa, Haitian Zhaoc, Baiqing Zhanga, Bin Lia,
T
⁎
a
College of Food Science, Shenyang Agricultural University, Shenyang 110866, China College of art design and architecture, Liaoning University of Technology, Jinzhou 121001, China c School of Chemistry and Chemical Engineering, Harbin Institute of Technology, 8 Harbin, 150090, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Plasma Pulsed discharge Degradation Auricularia auricula Polysaccharide
Synergistic degradation of water-soluble Auricularia auricula polysaccharide (AAP) by solution plasma process (SPP) in the presence of hydrogen peroxide (H2O2) was investigated. The effects of H2O2 concentration, AAP concentration and the distance between the electrodes on the degradation of AAP were evaluated. The results showed that higher H2O2 concentration, lower AAP concentration and narrower distance between the electrodes were favorable for the degradation effect. Particle size, congo red (CR), scanning electron micrographs (SEM) and atomic force microscopy (AFM) results confirmed that SPP irradiation with H2O2 improved significantly the flexibility of the conformation. The degraded AAPs exhibited greater metal chelating effects and DPPH radical scavenging effect than the original AAP. It concluded that the combined SPP/ H2O2 method could be used for preparation of low-molecular-weight AAP.
1. Introduction Auricularia auricula is a precious colloid fungus widely used as a medicine and food supplement in China, Korea and Viet Nam. A. auricula polysaccharide (AAP) is one of the main active ingredients of A. auricula, which is considered to be responsible for the biological activities of this organism (Nguyen, Wang et al., 2012; Nguyen, Chen et al., 2012; Zhao et al., 2015). AAP were found to have a large variety of biological functions, including antioxidant, anticoagulant, hypoglycemic activity, hypolipidemic activity, antitumor, enhancing immunity, antiaging, antiviral and so on (Fan, Zhang, Yu, & Ma, 2006; Nguyen, Chen et al., 2012; Wu et al., 2010; Yang et al., 2011; Zeng, Zhang, Gao, Jia, & Chen, 2012; Zhang, Yang, Ding, & Chen, 1995; Zhang, Wang, Zhang, & Wang, 2011). However, many studies have demonstrated that many properties of polysaccharides depend on their molecular weights. Lower molecular weight polysaccharide had better bioactive functions, which are more favorable for its application (Hou, Wang, Jin, Zhang, & Zhang, 2011; Li, Li, Geng, Song, & Wu, 2017). Therefore, in order to obtain polysaccharide with low molecular weight, various degradation methods have been considered (Ren, Zeng, Tang, Wang, Wan, Feng
et al., 2018; Ren, Zeng, Tang, Wang, Wan, Liu et al., 2018; Villay et al., 2012; Zhang, Wang, Liu, & Li, 2016). The traditional methods have mainly chemical or enzymatic hydrolysis (Yue, Yao, & Wei, 2009). The chemical methods such as acid hydrolysis have disadvantages mainly due to low production yields and a higher risk of environmental pollution. The enzymatic methods are very complicate and expensive cost for the industrial application (Hien, Phu, Duy, & Lan, 2012; Luo, Han, Zeng, Yu, & Kennedy, 2010; Prasertsung, Damrongsakkul, Terashimad, Saito, & Takai, 2012). Therefore, the development of an emerging technology for degradation of AAP, which has the advantages of low cost, no pollution, energy saving and high efficiency, etc., is of great interest. Solution plasma process (SPP) as one of advanced oxidation technologies has attracted much attention for its advantages, such as low energy consumption, moderate cost, no secondary pollution, no any chemical reagents and so on (Ma et al., 2017; Prasertsung et al., 2012; Preis, Klauson, & Gregor, 2013). This has led to a growing interest in SPP as tools in the degradation of polymer compounds, such as chitosan (Ma et al., 2017; Tantiplapol et al., 2015). However, this method is in developmental stage. To improve degradation efficiency, SPP is often
Abbreviations: SPP, solution plasma process; H2O2, hydrogen peroxide; AAP, Auricularia auricula polysaccharide; CR, Congo red; SEM, scanning electron micrographs; AFM, atomic force microscopy ⁎ Corresponding author. E-mail addresses: [email protected] (F. Ma), [email protected] (J. Wu), [email protected] (P. Li), [email protected] (D. Tao), [email protected] (H. Zhao), [email protected] (B. Zhang), [email protected] (B. Li). https://doi.org/10.1016/j.carbpol.2018.06.113 Received 18 January 2018; Received in revised form 16 June 2018; Accepted 26 June 2018 Available online 30 June 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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(Diao et al., 2017; Solomon & Ciut, 1962). The relative viscosities of the original AAP and its degraded samples were determined at 25 ± 0.5 ℃ using an Ubbelohde capillary viscometer (Shanghai Longtuo Instrument Co., Ltd.,China). The intrinsic viscosity [η] values can be calculated based on the following experience equations:
used with hydrogen peroxide (H2O2). Some reports indicated that the synergistic effect with SPP and H2O2 could significantly improve the degradation effect compared to the individual SPP (Son, Kim, Lee, & Lee, 2016). In our previous studies (Ma, Wang, Zhao, & Tian, 2012), we have also found that the combination of SPP and H2O2 had a significant synergistic effect on the degradation of chitosan. The intrinsic viscosity reduction rate of chitosan by SPP irradiation with and without H2O2 were 82.19% and 70.04% at treatment time of 30 min, respectively. The study indicated that SPP irradiation in presence of H2O2 is an efficient oxidation process. However, the degradation of AAP by SPP with H2O2 has not yet been studied. In the present study, the degradation of AAP was investigated by SPP with H2O2. The effects of H2O2 concentration, AAP concentration and the distance between the electrodes are discussed. The chain conformation in solution and molecular morphology of the APP obtained after SPP irradiation were investigated by particle size, congo red (CR), scanning electron micrographs (SEM) and atomic force microscopy (AFM). Antioxidant properties are evaluated by testing the metal chelating effects and the scavenging abilities on DPPH.
nr =
ts , nsp = nr − 1 to
(1)
[η] =
2ηsp − ln ηr c
(2)
where ts and t0 is the efflux times of the AAP solution and the solvent, respectively, ηr is the relative viscosity, ηsp is the incremental viscosity, and c is the concentration of APP (g/mL). In order to check the reproducibility of the obtained data, all these experiments have been repeated 3 times. The intrinsic viscosity reduction rate (η%) was calculated according to the following experience equation:
η% =
[η]0 −[η]t × 100(%) [η]0
(3)
where [η]0 and [η]t are the intrinsic viscosities of the initial time and reaction time, respectively.
2. Materials and methods 2.1. Materials
2.4.2. Effect of H2O2 concentration The AAP powder (0.135 g) was dissolved in 42 ml distilled water. Then 3 ml of H2O2 (0, 7.5, 15, 30% (v/v)) was added respectively to obtain a solution containing AAP 0.3% (w/v) and H2O2 (0, 0.5, 1, 2% (v/v)). The reaction was carried out at the distance between the electrodes of 2 mm in order to evaluate the effect of H2O2 concentration.
Dried fruiting bodies of Auricularia auricula were purchased from a local market in Shenyang City, Liaoning Province, China. Hydrogen peroxide (H2O2), 3-(2-pyridyl)-5,6-bis(4-phenylsulfonicacid)-1,2,4triazine (ferrozine), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and acetic acid were obtained from Sinopharm Chemical Reagent Co. Ltd., China. All other chemicals and reagents were of analytical reagent grade and were used without further purification.
2.4.3. Effect of AAP concentration The AAP powder (0.135, 0.27, 0.405, 0.54 g) was dissolved respectively in 42 ml distilled water. Then 3 ml of H2O2 (30% (v/v)) was added to obtain a solution containing AAP (0.3, 0.6, 0.9, 1.2% (w/v)) and H2O2 (2% (v/v)). The reaction was carried out at the distance between the electrodes of 2 mm in order to evaluate the effect of AAP concentration.
2.2. Preparation of AAP The AAP was prepared according to the method reported by Ma et al. (2015) with slight modifications. In brief, dried fruit-bodies of Auricularia auricula were crushed into powder (40 mesh). The powder was extracted with distilled water (1:60, W/V) at 95℃ for 2 h for three cycles. Then, the extraction mixture was centrifuged at 4000 rpm for 20 min. The supernatant was collected, and precipitated by the addition of 4 volumes of absolute ethanol. The precipitate was collected by centrifugation (4000 rpm, 10 min), redissolved in distilled water and deproteinated with Sevag method (n-butanol:chloroform = 1:4, V/V). After removing the Sevag reagent, precipitate was washed several times with absolute ethanol. The resulting precipitate was dissolved in distilled water, and dialyzed using a dialysis tube (molecular weight cutoff 8000 Da) against purified water to remove the components of low molecular weight. Finally the solution was concentrated and lyophilized, obtained the AAP powder.
2.4.4. Effect of the distance between the electrodes The AAP powder (0.135 g) was dissolved respectively in 42 ml distilled water. Then 3 ml of H2O2 (30% (v/v)) was added to obtain a solution containing AAP (0.3% (w/v)) and H2O2 (2% (v/v)). The reaction was carried out at different distances between the electrodes (2, 4, 6, 8 mm) in order to evaluate the effect of the distance between the electrodes. 2.5. Characterization 2.5.1. Particle size analysis Particle size distribution was measured by Zetasizer Nano-ZS90 (Malvern, Britain). The polysaccharide solution were prepared at a concentration of 0.02 mg/mL. Analyses were carried out at a scattering angle of 90°, the constant temperature of 25℃ and the wavelength of 633 nm.
2.3. Experimental setup The setup of SPP system was from our previous study (Ma et al., 2017). It works at the peak pulse voltage of 60 kV, the power of 350 W, the pulse width of 40 ns, the frequency of 4.67 kHz, the distance between the electrodes of 2 mm. Air was bubbled into the bottom of the reactor through a bubble diffuser. The experiment was operated at atmospheric pressure and room temperature. After the SPP treatment, various amounts of NaHSO3 were added to remove the rest of H2O2. Then the AAP sample solution was freeze dried to yield powdered products.
2.5.2. Molecular morphology observation SEM (TM-3000, Hitachi, Japan) was employed to observe the morphologies of the AAP samples. The samples coated with a thin gold film. the SEM images were observed at a voltage of 2.0 kV under high vacuum condition. AFM (XE-70, Park Systems, Korea) was used to obtain the topographies of the samples. The sample was dissolved in deionized water, and then diluted to 10 μg/mL. Then, 5 μL of the sample solution was dropped onto a freshly cleaved mica substrate and dried at room temperature for 2 h. AFM was operated in tapping mode under ambient condition using commercial silicon nitride cantilevers.
2.4. Degradation experiments 2.4.1. Measurement of intrinsic viscosity reduction rate The intrinsic viscosity was determined by a viscometric method 576
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2.5.3. Congo red binding studies The polysaccharide (5 mg) was dissolved in distilled water with 80 mol/L Congo red (CR) mixture by stirring, followed by the drop-wise addition of 4 M NaOH to obtain a final NaOH concentration of 00.5 mol/L. The mixture alkaline solution without polysaccharide was prepared as the control. After equilibrating for 10 min, Maximum absorption wavelength (λmax) was measured in the range of 400–600 nm at room temperature (Cary50, Varian, U.S.A.). 2.6. Measurements of antioxidant activity 2.6.1. Metal chelating effect The ferrous ion chelating effect of the AAP was measured according to the method described by Tang et al. (2014) with some modifications. 1 mL of the AAP solutions with different concentrations were added to 0.1 mL of FeCl2 (2 mM)), 0.2 mL of ferrozine (5 mM) and 3.7 mL of distilled water. The mixture was incubated for 20 min at room temperature. The absorbance of the mixture was measured at 562 nm. The ferrous ion chelating effect was calculated according to the following equation:
A − A2 ⎞ Chelating effect(%) = ⎛1 − 1 × 100 A0 ⎠ ⎝ ⎜
Fig. 1. Effect of H2O2 concentration on the degradation of AAP. Experiment conditions: AAP concentration of 0.3%, the distance between the electrodes of 2 mm.
87.83, 89.24 and 89.95%, respectively. The results may be due to the production of more %OH under SPP irradiation with H2O2. The oxidative degradation mechanism of SPP treatment is that the radiolysis of H2O molecules by the discharge of SPP in the solution can produce %OH which was responsible for the degradation of AAP (Ge, Zhang, Yan, Mi, & Zhu, 2011; Ma et al., 2017; Wen, Shen, Ni, Tong, & Yu, 2012). When H2O2 was added into the AAP solution, H2O2 molecules were dissociated to form %OH, lead to the increasing of the %OH concentration (Liu & Wang, 2013). Therefore, the SPP treatment in combination with H2O2 showed the higher degradation effect compared with SPP alone process, and the intrinsic viscosity reduction rate of AAP solution strongly depends on the H2O2 concentration.
⎟
(4)
where A0, A1, and A2 refer to the absorbance of control solution (without sample), sample solution and background solution (without FeCl2), respectively. 2.6.2. DPPH radical scavenging effect DPPH radical scavenging effect of the AAP was performed in accordance with the reported method of Sun, Wang, Li, and Liu (2014) with a modification. 2 mL of the AAP solutions with different concentrations were mixed with 1 mL of methanolic solution of DPPH (200 μM). The mixture was allowed to react for 30 min at ambient temperature. Then, the absorbance was measured at 517 nm. Ascorbic acid was used for comparison. The DPPH radical scavenging was calculated from the following equation:
A⎞ Scavenging effect(%) = ⎛1 − × 100 A0 ⎠ ⎝ ⎜
3.1.2. Effect of AAP concentration The effect of AAP concentration on the degradation of AAP was studied over different AAP concentration. The experiments were performed with H2O2 concentration of 2%, the distance between the electrodes of 2 mm. The results were presented in Fig. 2. It was observed that the degradation was related to the AAP concentration. The intrinsic viscosity reduction rate of AAP decreased with the AAP concentration increasing. For irradiation time of 0, 30, 60, 90, 120, 150 and 180 min, the intrinsic viscosity reduction rate of AAP at AAP concentration of 0.6% were 0, 32.31, 56.42, 79.67, 84.41, 85.95 and 88.1%, respectively, and at AAP concentration of 1.2% were 0, 25.19, 47.27, 65.94, 77.12, 81.54 and 84.52%, respectively. The reason maybe is that the viscosity of the solution is increased with increasing AAP concentration. The higher viscosity retards the transportation of •OH. It may also be due to the fact that at the same level of energy input, the fraction of energy reacting to a single molecule is decreased because of
⎟
(5)
where A and A0 refer to the absorbance of sample solution and control solution (without sample), respectively. 2.7. Statistical analysis All results were expressed as means ± standard deviations (SD) of three replicates. Data were analyzed by SPSS version 17.0 software. 3. Results and discussion 3.1. Effects on the degradation of AAP 3.1.1. Effect of H2O2 concentration The effect of H2O2 concentration on the degradation of AAP was investigated by changing H2O2 concentration, and keeping other conditions constant with AAP concentration of 0.3%, the distance between the electrodes of 2 mm. The results were shown in Fig. 1. It was found that high concentration of H2O2 promoted the degradation of AAP, and the intrinsic viscosity reduction rate of AAP decreased with increasing of H2O2 concentration. Additionally, the intrinsic viscosity reduction rate decreased due to the irradiation time extended. For irradiation time of 0, 30, 60, 90, 120, 150 and 180 min, the intrinsic viscosity reduction rate of AAP at H2O2 concentration of 0% were 0, 11.84, 34.73, 55.53, 65.93, 72.48 and 75.67%, respectively, and at H2O2 concentration of 0.5% were 0, 24.27, 51.18, 71.98, 83.15, 85.5 and 87.2, respectively, and at H2O2 concentration of 2% were 0, 39.2, 72.76, 85.1,
Fig. 2. Effect of AAP concentration on the degradation of AAP. Experiment conditions: H2O2 concentration of 2%, the distance between the electrodes of 2 mm. 577
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Fig. 4. The maximum absorption wavelengths of AAP-Congo red complexes at various NaOH concentrations. Experiment conditions: H2O2 concentration of 2%, AAP concentration of 0.3%, the distance between the electrodes of 2 mm.
Fig. 3. Effect of the distance between the electrodes on the degradation of AAP. Experiment conditions: H2O2 concentration of 2%, AAP concentration of 0.3%.
the increase of AAP concentration (Chen, Chang, & Shyur, 1997; Huang, Wu, Huang, Yang, & Ren, 2013; Tsaih, Tseng, & Chen, 2004).
3.3. Congo red test The λmax of the AAP-Congo red complexes at the NaOH concentration range of 0–0.5 mol/L is shown in Fig. 4. Compared to CR, the initial λmax of AAP and its degraded samples did not show any obvious shift. It was indicated that AAP did not showed a triple-helical conformation (Liu et al., 2016). However, The descending behavior of the λmax for the original and degraded AAPs-CR complex was shown in low NaOH concentration (lower than 0.3 mol/L). It could be concluded that AAP showed a rigid conformation (Wang et al., 2016). Moreover, the degraded AAP with longer irradiation time exhibited a more flexible conformation.
3.1.3. Effect of the distance between the electrodes To investigate the effect of the distance between the electrodes on the degradation of AAP, the tests were carried out with different the distance between the electrodes at H2O2 concentration of 2%, AAP concentration of 0.3%. As shown in Fig. 3, the intrinsic viscosity reduction rate decreased with increasing the distance between the electrodes. For irradiation time of 0, 30, 60, 90, 120, 150 and 180 min, the intrinsic viscosity reduction rate of AAP at the distance between the electrodes of 4 mm were 0, 31.91, 65.11, 75.67, 83.77, 86.79 and 87.83%, respectively, and at the distance between the electrodes of 8 mm were 0, 17.12, 41.89, 60.88, 73.71, 77.63 and 80.5%, respectively. This may be due to the fact that the smaller the distance between the electrodes caused a stronger electric field, and lead to more discharge current, which would resulted in increasing of the active %OH (Sun et al., 2012; Yoshida, Johnson, & Go, 2017).
3.4. Molecular morphology Fig. 5 (top) shows the SEM images of the original and degraded AAPs. The original AAP exhibited a smooth and flat surface. By contrast, the degraded AAPs showed a rough surface morphology, and the cracks extent generally increased exponentially with increasing irradiation time. The results implied that the SPP treatment might lead to the depolymerization of the molecular chains of AAP (Shen, Hu, Wang, & Qu, 2011). Fig. 5 (bottom) shows the plane AFM image of the original and degraded AAPs in distilled water at the concentrations of 10 μg/mL. For irradiation time of 0, 60, 120 and 180 min, the particle diameters were in the range of 40–60, 20–60, 20–50 and 10–20 nm, respectively. The average height were 10, 9, 7, and 4 nm, respectively. The results indicated that the original AAP exhibited more compact conformation. The molecular aggregations of the degraded AAPs were weaker compared with that of the original AAP, and weaked with increasing irradiation time. A possible explanation is that AAP with high molecular weight may have provided the stronger intra- and inter-molecular hydrogen bonds (Li, Li, Geng, Song, Wu, 2017; Li, Wang et al., 2017). The results of SEM and AFM were in accordance with each other. Therefore, we could conclude that there was remarkable effect on the molecule conformation for the AAP after degradation.
3.2. Particle size analysis Table 1 shows the particle size analysis of the degraded AAPs as a function of irradiation time. The particle size distribution of the samples tended to be narrower and moved towards smaller diameters. For irradiation time of 0, 60, 120 and 180 min, the mean particle diameters of the samples were 243.1, 161.7, 66.19 and 49.02 nm, respectively. The particle dispersity index were 0.576, 0.486, 0.262 and 0.217. This result was in agreement with the report of Shen, Du, Wang, Guan, and Yao (2014). This phenomenon may be attributed to the molecular weight of AAP. A lower molecular weight leads to a lower viscosity and surface tension, which causing increased particle mobility (higher fluctuations). Therefore, the smaller particle size and the narrower particle size distribution were formed. Another possibility is that a lower viscosity results in the stronger atomization of AAP solution, which may cause a smaller particle size (Hosseini et al., 2013; Shen et al., 2014).
3.5. Evaluation of antioxidant activity Table 1 Particle size analysis of the degraded AAPs. Irradiation time (min)
Average particle size (nm)
Particle dispersity index
0 60 120 180
243.1 161.7 66.19 49.02
0.576 0.486 0.262 0.217
3.5.1. Metal chelating effects on ferrous ions Ferrous ions are known as the most effective prooxidants in the food system due to its high reactivity. Therefore, the high ferrous ion-chelating effects of AAP would influence its antioxidant activity (Abd ElRehim, El-Sawy, Hegazy, Soliman, & Elbarbary, 2012; Li et al., 2013). Fig. 6 shows metal chelating effects of the original and degraded AAPs. The chelating effect of AAPs increased with increasing concentration. Also, the chelating effect was enhanced with increasing irradiation 578
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Fig. 5. SEM (1000-fold)) and AFM images (2D image) of (a) the original AAP, and its degraded samples at irradiation times of (b) 60 min, (c) 120 min, and (d) 180 min. Experiment conditions: H2O2 concentration of 2%, AAP concentration of 0.3%, the distance between the electrodes of 2 mm.
Fig. 6. Metal chelating effects of the original and degraded AAPs. Experiment conditions: H2O2 concentration of 2%, AAP concentration of 0.3%, the distance between the electrodes of 2 mm.
Fig. 7. DPPH radical scavenging effect of the original and degraded AAPs. Experiment conditions: H2O2 concentration of 2%, AAP concentration of 0.3%, the distance between the electrodes of 2 mm.
time. The chelating effect of the original and degraded AAPs were 7, 64, 89 and 96% at polysaccharide concentration 12 mg/mL, respectively. The results proved that the degraded AAPs exhibited higher chelating effect compared to the original AAP. This observation is in good agreement with the report of Li et al. (2013). This result could be explained by the fact that the degradation treatment of AAP destroyed its intra-molecular hydrogen bonds, which improved the reactivity toward ferrous ions of the polysaccharide. The enhancing degree of reactivity was irradiation time-dependent (Abd El-Rehim et al., 2012).
degraded AAPs showed higher scavenging activity than the original AAP. This is because, the degradation of AAP may expose more active groups, which could present more hydrogen to scavenge DPPH radicals (Chen et al., 2016). 4. Conclusion In this study, the degradation of AAP by SPP with H2O2 was investigated. The results showed that AAP can be degraded effectively. The effect of degradation of AAP increased with increasing H2O2 concentration, decreased with increasing AAP concentration and the distance between the electrodes. Particle size, CR, AFM and SEM analysis confirmed that the degraded AAP exhibited a rigid conformation, and the flexibility of the conformation increased as irradiation time increased. Furthermore, antioxidant experiments revealed that the degraded AAP exerted greater metal chelating effects and DPPH radical scavenging effect. In summary, this work would give an insight that SPP irradiation could reduce the intrinsic viscosity of AAP and enhanced the bioactivity. The degradation method of AAP is feasible, which will extend the applications of the natural AAP. Further investigation still need to be carried out to increase the effect of degradation and improve
3.5.2. DPPH radical scavenging effect The DPPH free radical scavenging is based on the hydrogen-donating ability of antioxidants. Therefore, it was widely used to assess the radical scavenging ability of antioxidants (Chen et al., 2016; You, Yin, Zhang, & Jiang, 2014). Fig. 7 shows the DPPH radical scavenging activity of the original and degraded AAPs. The scavenging effects of AAPs on DPPH radical were as function of concentration. Besides, the scavenging effect increased with an increase in irradiation time. The scavenging effects of the original and degraded AAPs were 20, 46, 48 and 54% at polysaccharide concentration 1.2 mg/mL, respectively. The 579
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the enhancing effect on the bioactivity of AAP.
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Acknowledgements This work was supported by National Natural Science Foundation of China [31772011]; the 2014-2015 Research Funds for the Introduced Talents of Shenyang Agricultural University [20153033]. References Li, J., Li, B., Geng, P., Song, A. X., & Wu, J. Y. (2017a). Ultrasonic degradation kinetics and rheological profiles of a food polysaccharide (konjac glucomannan) in water. Food Hydrocolloids, 70, 14–19. Abd El-Rehim, H. A., El-Sawy, N. M., Hegazy, E. A., Soliman, E. A., & Elbarbary, A. M. (2012). Improvement of antioxidant activity of chitosan by chemical treatment and ionizing radiation. International Journal of Biological Macromolecules, 50, 403–413. Li, Q., Wang, W., Zhu, Y., Chen, Y., Zhang, W. J., Yu, P., et al. (2017b). Structural elucidation and antioxidant activity a novelSe-polysaccharide from Se-enriched Grifola frondosa. Carbohydrate Polymers, 161, 42–52. Chen, B. J., Shi, M. J., Cui, S., Hao, S. X., Hider, R. C., & Zhou, T. (2016). Improved antioxidant and anti-tyrosinase activity of polysaccharidefrom Sargassum fusiforme by degradation. International Journal of Biological Macromolecules, 92, 715–722. Chen, R. H., Chang, J. R., & Shyur, J. S. (1997). Effects of ultrasonic conditions and storage in acidic solutions on changes in molecular weight and polydispersity of treated chitosan. Carbohydrate research, 299, 287–294. Diao, Y. F., Song, M. W., Zhang, Y. L., Shi, L. Y., Lv, Y. S., & Ran, R. (2017). Enzymic degradation of hydroxyethyl cellulose and analysis of thesubstitution pattern along the polysaccharide chain. Carbohydrate Polymers, 169, 92–100. Fan, L. S., Zhang, S. H., Yu, L., & Ma, L. (2006). Evaluation of antioxidant property and quality of breads containing Auricularia auricula polysaccharide flour. Food Chemistry, 101, 1158–1163. Ge, H., Zhang, L. L., Yan, L., Mi, D., & Zhu, Y. M. (2011). Parameter optimization of excited OH radical in multi-needle to plate negative DC corona discharge. Journal of electrostatics, 69, 529–532. Hien, N. Q., Phu, D. V., Duy, N. N., & Lan, N. T. K. (2012). Degradation of chitosan in solution by gamma irradiation in the presence of hydrogen peroxide. Carbohydrate Polymers, 87, 935–938. Hosseini, S. M. H., Emam-Djomeh, Z., Razavi, S. H., Moosavi-Movahed, A. A., Saboury, A. A., Atri, M. S., et al. (2013). β-Lactoglobulinesodium alginate interaction as affected by polysaccharide depolymerization using high intensity ultrasound. Food Hydrocolloids, 32, 235–244. Hou, Y., Wang, J., Jin, W., Zhang, H., & Zhang, Q. (2011). Degradation of Laminaria japonica fucoidan by hydrogen peroxide and antioxidant activities of the degradation products of different molecular weights. Carbohydrate Polymers, 87(1), 153–159. Huang, Y., Wu, Y., Huang, W., Yang, F., & Ren, X. E. (2013). Degradation of chitosan by hydrodynamic cavitation. Polymer degradation and stability, 98, 37–43. Li, B., Liu, S., Xing, R. E., Li, K. C., Li, R. F., Qin, Y. K., et al. (2013). Degradation of sulfated polysaccharides from Enteromorpha prolifera and their antioxidant activities. Carbohydrate Polymers, 92, 1991–1996. Liu, Y. K., & Wang, J. L. (2013). Degradation of sulfamethazine by gamma irradiation in the presence of hydrogen peroxide. Journal of Hazardous Materials, 250-251, 99–105. Liu, W., Wang, H., Yu, J. P., Liu, Y. M., Lu, W. S., Chai, Y., et al. (2016). Structure, chain conformation, and immunomodulatory activity of thepolysaccharide purified from Bacillus Calmette Guerin formulation. Carbohydrate Polymers, 150, 149–158. Luo, W. B., Han, Z., Zeng, X. A., Yu, S. J., & Kennedy, J. F. (2010). Study on the degradation of chitosan by pulsed electric fields treatment. Innovative Food Science and Emerging Technologies, 11, 587–591. Ma, F. M., Wang, Z. Y., Zhao, H. T., & Tian, S. Q. (2012). Plasma depolymerization of chitosanin the presence of hydrogen peroxide. International Journal of Molecular Sciences, 13, 7788–7797. Ma, F. M., Li, P., Zhang, B. Q., Zhao, X., Fu, Q., Wang, Z. Y., et al. (2017). Effect of solution plasma process with bubbling gas onphysicochemical properties of chitosan. International Journal of Biological Macromolecules, 98, 201–207. Ma, Z., Zhang, C., Gao, X., Cui, F. Y., Zhang, J. J., Jia, M. S., et al. (2015). Enzymatic and acidic degradation effect on intracellularpolysaccharide of Flammulina velutipes SF08. International Journal of Biological Macromolecules, 73, 236–244. Nguyen, T. L., Chen, J., Hu, Y. L., Wang, D. Y., Fan, Y. P., Wang, J. M., et al. (2012). In vitro antiviral activity of sulfated Auricularia auricula polysaccharides. Carbohydrate Polymers, 90, 1254–1258. Nguyen, T. L., Wang, D. Y., Hu, Y. L., Fan, Y. P., Wang, J. M., Abula, S., et al. (2012). Immuno-enhancing activity of sulfated Auricularia auricula polysaccharides. Carbohydrate Polymers, 89, 1117–1122. Prasertsung, I., Damrongsakkul, S., Terashimad, C., Saito, N., & Takai, O. (2012).
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Effect of solution plasma process with hydrogen peroxide on the degradation of water-soluble polysaccharide from Auricularia auricula. II: Solution conformation and antioxidant activities in vitro Fengming Maa, Jingwei Wua, Pu Lib, Dongbing Taoa, Haitian Zhaoc, Baiqing Zhanga, Bin Lia,
T
⁎
a
College of Food Science, Shenyang Agricultural University, Shenyang 110866, China College of art design and architecture, Liaoning University of Technology, Jinzhou 121001, China c School of Chemistry and Chemical Engineering, Harbin Institute of Technology, 8 Harbin, 150090, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Plasma Pulsed discharge Degradation Auricularia auricula Polysaccharide
Synergistic degradation of water-soluble Auricularia auricula polysaccharide (AAP) by solution plasma process (SPP) in the presence of hydrogen peroxide (H2O2) was investigated. The effects of H2O2 concentration, AAP concentration and the distance between the electrodes on the degradation of AAP were evaluated. The results showed that higher H2O2 concentration, lower AAP concentration and narrower distance between the electrodes were favorable for the degradation effect. Particle size, congo red (CR), scanning electron micrographs (SEM) and atomic force microscopy (AFM) results confirmed that SPP irradiation with H2O2 improved significantly the flexibility of the conformation. The degraded AAPs exhibited greater metal chelating effects and DPPH radical scavenging effect than the original AAP. It concluded that the combined SPP/ H2O2 method could be used for preparation of low-molecular-weight AAP.
1. Introduction Auricularia auricula is a precious colloid fungus widely used as a medicine and food supplement in China, Korea and Viet Nam. A. auricula polysaccharide (AAP) is one of the main active ingredients of A. auricula, which is considered to be responsible for the biological activities of this organism (Nguyen, Wang et al., 2012; Nguyen, Chen et al., 2012; Zhao et al., 2015). AAP were found to have a large variety of biological functions, including antioxidant, anticoagulant, hypoglycemic activity, hypolipidemic activity, antitumor, enhancing immunity, antiaging, antiviral and so on (Fan, Zhang, Yu, & Ma, 2006; Nguyen, Chen et al., 2012; Wu et al., 2010; Yang et al., 2011; Zeng, Zhang, Gao, Jia, & Chen, 2012; Zhang, Yang, Ding, & Chen, 1995; Zhang, Wang, Zhang, & Wang, 2011). However, many studies have demonstrated that many properties of polysaccharides depend on their molecular weights. Lower molecular weight polysaccharide had better bioactive functions, which are more favorable for its application (Hou, Wang, Jin, Zhang, & Zhang, 2011; Li, Li, Geng, Song, & Wu, 2017). Therefore, in order to obtain polysaccharide with low molecular weight, various degradation methods have been considered (Ren, Zeng, Tang, Wang, Wan, Feng
et al., 2018; Ren, Zeng, Tang, Wang, Wan, Liu et al., 2018; Villay et al., 2012; Zhang, Wang, Liu, & Li, 2016). The traditional methods have mainly chemical or enzymatic hydrolysis (Yue, Yao, & Wei, 2009). The chemical methods such as acid hydrolysis have disadvantages mainly due to low production yields and a higher risk of environmental pollution. The enzymatic methods are very complicate and expensive cost for the industrial application (Hien, Phu, Duy, & Lan, 2012; Luo, Han, Zeng, Yu, & Kennedy, 2010; Prasertsung, Damrongsakkul, Terashimad, Saito, & Takai, 2012). Therefore, the development of an emerging technology for degradation of AAP, which has the advantages of low cost, no pollution, energy saving and high efficiency, etc., is of great interest. Solution plasma process (SPP) as one of advanced oxidation technologies has attracted much attention for its advantages, such as low energy consumption, moderate cost, no secondary pollution, no any chemical reagents and so on (Ma et al., 2017; Prasertsung et al., 2012; Preis, Klauson, & Gregor, 2013). This has led to a growing interest in SPP as tools in the degradation of polymer compounds, such as chitosan (Ma et al., 2017; Tantiplapol et al., 2015). However, this method is in developmental stage. To improve degradation efficiency, SPP is often
Abbreviations: SPP, solution plasma process; H2O2, hydrogen peroxide; AAP, Auricularia auricula polysaccharide; CR, Congo red; SEM, scanning electron micrographs; AFM, atomic force microscopy ⁎ Corresponding author. E-mail addresses: [email protected] (F. Ma), [email protected] (J. Wu), [email protected] (P. Li), [email protected] (D. Tao), [email protected] (H. Zhao), [email protected] (B. Zhang), [email protected] (B. Li). https://doi.org/10.1016/j.carbpol.2018.06.113 Received 18 January 2018; Received in revised form 16 June 2018; Accepted 26 June 2018 Available online 30 June 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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(Diao et al., 2017; Solomon & Ciut, 1962). The relative viscosities of the original AAP and its degraded samples were determined at 25 ± 0.5 ℃ using an Ubbelohde capillary viscometer (Shanghai Longtuo Instrument Co., Ltd.,China). The intrinsic viscosity [η] values can be calculated based on the following experience equations:
used with hydrogen peroxide (H2O2). Some reports indicated that the synergistic effect with SPP and H2O2 could significantly improve the degradation effect compared to the individual SPP (Son, Kim, Lee, & Lee, 2016). In our previous studies (Ma, Wang, Zhao, & Tian, 2012), we have also found that the combination of SPP and H2O2 had a significant synergistic effect on the degradation of chitosan. The intrinsic viscosity reduction rate of chitosan by SPP irradiation with and without H2O2 were 82.19% and 70.04% at treatment time of 30 min, respectively. The study indicated that SPP irradiation in presence of H2O2 is an efficient oxidation process. However, the degradation of AAP by SPP with H2O2 has not yet been studied. In the present study, the degradation of AAP was investigated by SPP with H2O2. The effects of H2O2 concentration, AAP concentration and the distance between the electrodes are discussed. The chain conformation in solution and molecular morphology of the APP obtained after SPP irradiation were investigated by particle size, congo red (CR), scanning electron micrographs (SEM) and atomic force microscopy (AFM). Antioxidant properties are evaluated by testing the metal chelating effects and the scavenging abilities on DPPH.
nr =
ts , nsp = nr − 1 to
(1)
[η] =
2ηsp − ln ηr c
(2)
where ts and t0 is the efflux times of the AAP solution and the solvent, respectively, ηr is the relative viscosity, ηsp is the incremental viscosity, and c is the concentration of APP (g/mL). In order to check the reproducibility of the obtained data, all these experiments have been repeated 3 times. The intrinsic viscosity reduction rate (η%) was calculated according to the following experience equation:
η% =
[η]0 −[η]t × 100(%) [η]0
(3)
where [η]0 and [η]t are the intrinsic viscosities of the initial time and reaction time, respectively.
2. Materials and methods 2.1. Materials
2.4.2. Effect of H2O2 concentration The AAP powder (0.135 g) was dissolved in 42 ml distilled water. Then 3 ml of H2O2 (0, 7.5, 15, 30% (v/v)) was added respectively to obtain a solution containing AAP 0.3% (w/v) and H2O2 (0, 0.5, 1, 2% (v/v)). The reaction was carried out at the distance between the electrodes of 2 mm in order to evaluate the effect of H2O2 concentration.
Dried fruiting bodies of Auricularia auricula were purchased from a local market in Shenyang City, Liaoning Province, China. Hydrogen peroxide (H2O2), 3-(2-pyridyl)-5,6-bis(4-phenylsulfonicacid)-1,2,4triazine (ferrozine), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and acetic acid were obtained from Sinopharm Chemical Reagent Co. Ltd., China. All other chemicals and reagents were of analytical reagent grade and were used without further purification.
2.4.3. Effect of AAP concentration The AAP powder (0.135, 0.27, 0.405, 0.54 g) was dissolved respectively in 42 ml distilled water. Then 3 ml of H2O2 (30% (v/v)) was added to obtain a solution containing AAP (0.3, 0.6, 0.9, 1.2% (w/v)) and H2O2 (2% (v/v)). The reaction was carried out at the distance between the electrodes of 2 mm in order to evaluate the effect of AAP concentration.
2.2. Preparation of AAP The AAP was prepared according to the method reported by Ma et al. (2015) with slight modifications. In brief, dried fruit-bodies of Auricularia auricula were crushed into powder (40 mesh). The powder was extracted with distilled water (1:60, W/V) at 95℃ for 2 h for three cycles. Then, the extraction mixture was centrifuged at 4000 rpm for 20 min. The supernatant was collected, and precipitated by the addition of 4 volumes of absolute ethanol. The precipitate was collected by centrifugation (4000 rpm, 10 min), redissolved in distilled water and deproteinated with Sevag method (n-butanol:chloroform = 1:4, V/V). After removing the Sevag reagent, precipitate was washed several times with absolute ethanol. The resulting precipitate was dissolved in distilled water, and dialyzed using a dialysis tube (molecular weight cutoff 8000 Da) against purified water to remove the components of low molecular weight. Finally the solution was concentrated and lyophilized, obtained the AAP powder.
2.4.4. Effect of the distance between the electrodes The AAP powder (0.135 g) was dissolved respectively in 42 ml distilled water. Then 3 ml of H2O2 (30% (v/v)) was added to obtain a solution containing AAP (0.3% (w/v)) and H2O2 (2% (v/v)). The reaction was carried out at different distances between the electrodes (2, 4, 6, 8 mm) in order to evaluate the effect of the distance between the electrodes. 2.5. Characterization 2.5.1. Particle size analysis Particle size distribution was measured by Zetasizer Nano-ZS90 (Malvern, Britain). The polysaccharide solution were prepared at a concentration of 0.02 mg/mL. Analyses were carried out at a scattering angle of 90°, the constant temperature of 25℃ and the wavelength of 633 nm.
2.3. Experimental setup The setup of SPP system was from our previous study (Ma et al., 2017). It works at the peak pulse voltage of 60 kV, the power of 350 W, the pulse width of 40 ns, the frequency of 4.67 kHz, the distance between the electrodes of 2 mm. Air was bubbled into the bottom of the reactor through a bubble diffuser. The experiment was operated at atmospheric pressure and room temperature. After the SPP treatment, various amounts of NaHSO3 were added to remove the rest of H2O2. Then the AAP sample solution was freeze dried to yield powdered products.
2.5.2. Molecular morphology observation SEM (TM-3000, Hitachi, Japan) was employed to observe the morphologies of the AAP samples. The samples coated with a thin gold film. the SEM images were observed at a voltage of 2.0 kV under high vacuum condition. AFM (XE-70, Park Systems, Korea) was used to obtain the topographies of the samples. The sample was dissolved in deionized water, and then diluted to 10 μg/mL. Then, 5 μL of the sample solution was dropped onto a freshly cleaved mica substrate and dried at room temperature for 2 h. AFM was operated in tapping mode under ambient condition using commercial silicon nitride cantilevers.
2.4. Degradation experiments 2.4.1. Measurement of intrinsic viscosity reduction rate The intrinsic viscosity was determined by a viscometric method 576
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2.5.3. Congo red binding studies The polysaccharide (5 mg) was dissolved in distilled water with 80 mol/L Congo red (CR) mixture by stirring, followed by the drop-wise addition of 4 M NaOH to obtain a final NaOH concentration of 00.5 mol/L. The mixture alkaline solution without polysaccharide was prepared as the control. After equilibrating for 10 min, Maximum absorption wavelength (λmax) was measured in the range of 400–600 nm at room temperature (Cary50, Varian, U.S.A.). 2.6. Measurements of antioxidant activity 2.6.1. Metal chelating effect The ferrous ion chelating effect of the AAP was measured according to the method described by Tang et al. (2014) with some modifications. 1 mL of the AAP solutions with different concentrations were added to 0.1 mL of FeCl2 (2 mM)), 0.2 mL of ferrozine (5 mM) and 3.7 mL of distilled water. The mixture was incubated for 20 min at room temperature. The absorbance of the mixture was measured at 562 nm. The ferrous ion chelating effect was calculated according to the following equation:
A − A2 ⎞ Chelating effect(%) = ⎛1 − 1 × 100 A0 ⎠ ⎝ ⎜
Fig. 1. Effect of H2O2 concentration on the degradation of AAP. Experiment conditions: AAP concentration of 0.3%, the distance between the electrodes of 2 mm.
87.83, 89.24 and 89.95%, respectively. The results may be due to the production of more %OH under SPP irradiation with H2O2. The oxidative degradation mechanism of SPP treatment is that the radiolysis of H2O molecules by the discharge of SPP in the solution can produce %OH which was responsible for the degradation of AAP (Ge, Zhang, Yan, Mi, & Zhu, 2011; Ma et al., 2017; Wen, Shen, Ni, Tong, & Yu, 2012). When H2O2 was added into the AAP solution, H2O2 molecules were dissociated to form %OH, lead to the increasing of the %OH concentration (Liu & Wang, 2013). Therefore, the SPP treatment in combination with H2O2 showed the higher degradation effect compared with SPP alone process, and the intrinsic viscosity reduction rate of AAP solution strongly depends on the H2O2 concentration.
⎟
(4)
where A0, A1, and A2 refer to the absorbance of control solution (without sample), sample solution and background solution (without FeCl2), respectively. 2.6.2. DPPH radical scavenging effect DPPH radical scavenging effect of the AAP was performed in accordance with the reported method of Sun, Wang, Li, and Liu (2014) with a modification. 2 mL of the AAP solutions with different concentrations were mixed with 1 mL of methanolic solution of DPPH (200 μM). The mixture was allowed to react for 30 min at ambient temperature. Then, the absorbance was measured at 517 nm. Ascorbic acid was used for comparison. The DPPH radical scavenging was calculated from the following equation:
A⎞ Scavenging effect(%) = ⎛1 − × 100 A0 ⎠ ⎝ ⎜
3.1.2. Effect of AAP concentration The effect of AAP concentration on the degradation of AAP was studied over different AAP concentration. The experiments were performed with H2O2 concentration of 2%, the distance between the electrodes of 2 mm. The results were presented in Fig. 2. It was observed that the degradation was related to the AAP concentration. The intrinsic viscosity reduction rate of AAP decreased with the AAP concentration increasing. For irradiation time of 0, 30, 60, 90, 120, 150 and 180 min, the intrinsic viscosity reduction rate of AAP at AAP concentration of 0.6% were 0, 32.31, 56.42, 79.67, 84.41, 85.95 and 88.1%, respectively, and at AAP concentration of 1.2% were 0, 25.19, 47.27, 65.94, 77.12, 81.54 and 84.52%, respectively. The reason maybe is that the viscosity of the solution is increased with increasing AAP concentration. The higher viscosity retards the transportation of •OH. It may also be due to the fact that at the same level of energy input, the fraction of energy reacting to a single molecule is decreased because of
⎟
(5)
where A and A0 refer to the absorbance of sample solution and control solution (without sample), respectively. 2.7. Statistical analysis All results were expressed as means ± standard deviations (SD) of three replicates. Data were analyzed by SPSS version 17.0 software. 3. Results and discussion 3.1. Effects on the degradation of AAP 3.1.1. Effect of H2O2 concentration The effect of H2O2 concentration on the degradation of AAP was investigated by changing H2O2 concentration, and keeping other conditions constant with AAP concentration of 0.3%, the distance between the electrodes of 2 mm. The results were shown in Fig. 1. It was found that high concentration of H2O2 promoted the degradation of AAP, and the intrinsic viscosity reduction rate of AAP decreased with increasing of H2O2 concentration. Additionally, the intrinsic viscosity reduction rate decreased due to the irradiation time extended. For irradiation time of 0, 30, 60, 90, 120, 150 and 180 min, the intrinsic viscosity reduction rate of AAP at H2O2 concentration of 0% were 0, 11.84, 34.73, 55.53, 65.93, 72.48 and 75.67%, respectively, and at H2O2 concentration of 0.5% were 0, 24.27, 51.18, 71.98, 83.15, 85.5 and 87.2, respectively, and at H2O2 concentration of 2% were 0, 39.2, 72.76, 85.1,
Fig. 2. Effect of AAP concentration on the degradation of AAP. Experiment conditions: H2O2 concentration of 2%, the distance between the electrodes of 2 mm. 577
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Fig. 4. The maximum absorption wavelengths of AAP-Congo red complexes at various NaOH concentrations. Experiment conditions: H2O2 concentration of 2%, AAP concentration of 0.3%, the distance between the electrodes of 2 mm.
Fig. 3. Effect of the distance between the electrodes on the degradation of AAP. Experiment conditions: H2O2 concentration of 2%, AAP concentration of 0.3%.
the increase of AAP concentration (Chen, Chang, & Shyur, 1997; Huang, Wu, Huang, Yang, & Ren, 2013; Tsaih, Tseng, & Chen, 2004).
3.3. Congo red test The λmax of the AAP-Congo red complexes at the NaOH concentration range of 0–0.5 mol/L is shown in Fig. 4. Compared to CR, the initial λmax of AAP and its degraded samples did not show any obvious shift. It was indicated that AAP did not showed a triple-helical conformation (Liu et al., 2016). However, The descending behavior of the λmax for the original and degraded AAPs-CR complex was shown in low NaOH concentration (lower than 0.3 mol/L). It could be concluded that AAP showed a rigid conformation (Wang et al., 2016). Moreover, the degraded AAP with longer irradiation time exhibited a more flexible conformation.
3.1.3. Effect of the distance between the electrodes To investigate the effect of the distance between the electrodes on the degradation of AAP, the tests were carried out with different the distance between the electrodes at H2O2 concentration of 2%, AAP concentration of 0.3%. As shown in Fig. 3, the intrinsic viscosity reduction rate decreased with increasing the distance between the electrodes. For irradiation time of 0, 30, 60, 90, 120, 150 and 180 min, the intrinsic viscosity reduction rate of AAP at the distance between the electrodes of 4 mm were 0, 31.91, 65.11, 75.67, 83.77, 86.79 and 87.83%, respectively, and at the distance between the electrodes of 8 mm were 0, 17.12, 41.89, 60.88, 73.71, 77.63 and 80.5%, respectively. This may be due to the fact that the smaller the distance between the electrodes caused a stronger electric field, and lead to more discharge current, which would resulted in increasing of the active %OH (Sun et al., 2012; Yoshida, Johnson, & Go, 2017).
3.4. Molecular morphology Fig. 5 (top) shows the SEM images of the original and degraded AAPs. The original AAP exhibited a smooth and flat surface. By contrast, the degraded AAPs showed a rough surface morphology, and the cracks extent generally increased exponentially with increasing irradiation time. The results implied that the SPP treatment might lead to the depolymerization of the molecular chains of AAP (Shen, Hu, Wang, & Qu, 2011). Fig. 5 (bottom) shows the plane AFM image of the original and degraded AAPs in distilled water at the concentrations of 10 μg/mL. For irradiation time of 0, 60, 120 and 180 min, the particle diameters were in the range of 40–60, 20–60, 20–50 and 10–20 nm, respectively. The average height were 10, 9, 7, and 4 nm, respectively. The results indicated that the original AAP exhibited more compact conformation. The molecular aggregations of the degraded AAPs were weaker compared with that of the original AAP, and weaked with increasing irradiation time. A possible explanation is that AAP with high molecular weight may have provided the stronger intra- and inter-molecular hydrogen bonds (Li, Li, Geng, Song, Wu, 2017; Li, Wang et al., 2017). The results of SEM and AFM were in accordance with each other. Therefore, we could conclude that there was remarkable effect on the molecule conformation for the AAP after degradation.
3.2. Particle size analysis Table 1 shows the particle size analysis of the degraded AAPs as a function of irradiation time. The particle size distribution of the samples tended to be narrower and moved towards smaller diameters. For irradiation time of 0, 60, 120 and 180 min, the mean particle diameters of the samples were 243.1, 161.7, 66.19 and 49.02 nm, respectively. The particle dispersity index were 0.576, 0.486, 0.262 and 0.217. This result was in agreement with the report of Shen, Du, Wang, Guan, and Yao (2014). This phenomenon may be attributed to the molecular weight of AAP. A lower molecular weight leads to a lower viscosity and surface tension, which causing increased particle mobility (higher fluctuations). Therefore, the smaller particle size and the narrower particle size distribution were formed. Another possibility is that a lower viscosity results in the stronger atomization of AAP solution, which may cause a smaller particle size (Hosseini et al., 2013; Shen et al., 2014).
3.5. Evaluation of antioxidant activity Table 1 Particle size analysis of the degraded AAPs. Irradiation time (min)
Average particle size (nm)
Particle dispersity index
0 60 120 180
243.1 161.7 66.19 49.02
0.576 0.486 0.262 0.217
3.5.1. Metal chelating effects on ferrous ions Ferrous ions are known as the most effective prooxidants in the food system due to its high reactivity. Therefore, the high ferrous ion-chelating effects of AAP would influence its antioxidant activity (Abd ElRehim, El-Sawy, Hegazy, Soliman, & Elbarbary, 2012; Li et al., 2013). Fig. 6 shows metal chelating effects of the original and degraded AAPs. The chelating effect of AAPs increased with increasing concentration. Also, the chelating effect was enhanced with increasing irradiation 578
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Fig. 5. SEM (1000-fold)) and AFM images (2D image) of (a) the original AAP, and its degraded samples at irradiation times of (b) 60 min, (c) 120 min, and (d) 180 min. Experiment conditions: H2O2 concentration of 2%, AAP concentration of 0.3%, the distance between the electrodes of 2 mm.
Fig. 6. Metal chelating effects of the original and degraded AAPs. Experiment conditions: H2O2 concentration of 2%, AAP concentration of 0.3%, the distance between the electrodes of 2 mm.
Fig. 7. DPPH radical scavenging effect of the original and degraded AAPs. Experiment conditions: H2O2 concentration of 2%, AAP concentration of 0.3%, the distance between the electrodes of 2 mm.
time. The chelating effect of the original and degraded AAPs were 7, 64, 89 and 96% at polysaccharide concentration 12 mg/mL, respectively. The results proved that the degraded AAPs exhibited higher chelating effect compared to the original AAP. This observation is in good agreement with the report of Li et al. (2013). This result could be explained by the fact that the degradation treatment of AAP destroyed its intra-molecular hydrogen bonds, which improved the reactivity toward ferrous ions of the polysaccharide. The enhancing degree of reactivity was irradiation time-dependent (Abd El-Rehim et al., 2012).
degraded AAPs showed higher scavenging activity than the original AAP. This is because, the degradation of AAP may expose more active groups, which could present more hydrogen to scavenge DPPH radicals (Chen et al., 2016). 4. Conclusion In this study, the degradation of AAP by SPP with H2O2 was investigated. The results showed that AAP can be degraded effectively. The effect of degradation of AAP increased with increasing H2O2 concentration, decreased with increasing AAP concentration and the distance between the electrodes. Particle size, CR, AFM and SEM analysis confirmed that the degraded AAP exhibited a rigid conformation, and the flexibility of the conformation increased as irradiation time increased. Furthermore, antioxidant experiments revealed that the degraded AAP exerted greater metal chelating effects and DPPH radical scavenging effect. In summary, this work would give an insight that SPP irradiation could reduce the intrinsic viscosity of AAP and enhanced the bioactivity. The degradation method of AAP is feasible, which will extend the applications of the natural AAP. Further investigation still need to be carried out to increase the effect of degradation and improve
3.5.2. DPPH radical scavenging effect The DPPH free radical scavenging is based on the hydrogen-donating ability of antioxidants. Therefore, it was widely used to assess the radical scavenging ability of antioxidants (Chen et al., 2016; You, Yin, Zhang, & Jiang, 2014). Fig. 7 shows the DPPH radical scavenging activity of the original and degraded AAPs. The scavenging effects of AAPs on DPPH radical were as function of concentration. Besides, the scavenging effect increased with an increase in irradiation time. The scavenging effects of the original and degraded AAPs were 20, 46, 48 and 54% at polysaccharide concentration 1.2 mg/mL, respectively. The 579
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the enhancing effect on the bioactivity of AAP.
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