Ultrasonic degradation ofPolysaccharides from Auricularia auricula and the antioxidant activity of their degradation products

LWT - Food Science and Technology 113 (2019) 108266

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Ultrasonic degradation ofPolysaccharides from Auricularia auricula and the antioxidant activity of their degradation products

T

Junqiang Qiua,c,∗∗, Hua Zhangb, Zhenyu Wangb,∗ a

Department of Inorganic Chemistry and Analytical Chemitry, School of Pharmacy, Hainan Medical University, Haikou, China Department of Food, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, China c Hainan Provincial Key Laboratory of R & D on Tropical Herbs, Haikou, 571199, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: AAP Polysaccharides Ultrasound Degradation Structure

Auricularia auricula polysaccharide (AAP) were purified and degraded using ultrasonic wave with the degradation kinetics model fitted to 1/Mt – 1/M0 = k • t. Followed that the morphology, intrinsic viscosity, viscosity-average molecular weight, antioxidant activity of AAP and their degradation products were in vitro investigated. Results showed the intrinsic viscosity and viscosity-average molecular weight of AAP decreased significantly (p < 0.05) with increasing ultrasonic time and their microscopic sizes were reduced after degradation. In addition, ultrasonic wave destroyed the helix structure and changed the monosaccharide proportion of AAP. SEM analysis displayed some morphological difference existing in polysaccharides before and after degradation. More importantly, the antioxidant activities in vitro of AAP after degradation were significantly increased (p < 0.05) and AAPUD12h displayed the strongest antioxidant activity. These results indicate that ultrasonic degradation may be a suitable way to improve the antioxidant activity of natural polysaccharides.

1. Introduction The mushroom Auricularia auricula is the important functional food (Zeng, Zhang, Luo, & Zhu, 2011), widely distributed in East Asia such as China, Japan, and Korea, showing some bioactivities including antioxidation (Yang et al., 2011; Zeng et al., 2011; Zhang, Wang, Zhang, & Wang, 2011), anticarcinogen (Ma, Wang, Zhang, Zhang, & Ding, 2010; Song & Du, 2012), and anticoagulant activities (Yoon, Yu, & Pyun, 2003), and can be used to prevent cardio-cerebrovascular diseases and improve immune function (Wu, Tan, Liu, Gao, & Wu, 2010). Researchers indicate polysaccharides are the main bioactive ingredients in Auricularia auricula. However, Auricularia auricula polysaccharides (AAP) usually show high molecular weight (Wu et al., 2010). However, Auricularia auricula polysaccharides (AAP) usually show high molecular weight (Mw: ∼4.1 × 105 Da) and intrinsic viscosity (93 dL/g), limiting their absorption and utilization in vivo and pharmaceutical applications. Considering the potential applications of AAP as functional foods and medicine, the degradation of AAP to low-Mw fractions with superior solubility for specific uses could be potentially beneficial. Various method such as ultrasonic degradation, chemical modification, enzymatic hydrolysis are employed to obtain low molecular weight polysaccharides (Wang, Gao, Tao, Wu, & Cui, 2017; Wang et al.,

∗

2017). Specially, ultrasonic treatment has received more and more attention due to ultrasound has the advantages of being rapid, mild and environmentally-friendly, and does not use toxic reagents or produce any waste (Pu, Zou, & Hou, 2017). In addition, ultrasonic treatments could enhance the antimicrobial activity, immunoregulatory activity, antioxidant activity, and antitumor activity of various polysaccharides (Liu, Bao, Du, Zhou, & Kennedy, 2006; Yan, Wang, Ma, & Wang, 2016; Yao, Zhu, Gao, & Ren, 2015; Zhou, Yu, Zhang, He, & Ma, 2012; Zhu, Pang, & Li, 2014), which was attributed to decreased Mw of polysaccharides. The effect of ultrasound on biological macromolecules has been widely studied. Ultrasound has been shown to affect a number of physicochemical properties such as: Mw, viscosity, anomeric configuration, monosaccharide composition, degree of branching, and spacial structure resulting in changes in their biological activities (Li & Feke, 2015; Taghizadeh & Bahadori, 2014; Savitri, Juliastuti, & Handaratri, 2014). However, few studies about the anti-oxidative activity of ultrasound treated Auricularia auricula polysaccharides (AAP) have been studied. In this study, the effects of ultrasonic treatment on intrinsic viscosity, chemical structures, microscopic morphology, monosaccharide components, and antioxidant activity of AAP were investigated.

Corresponding author. Corresponding author. Department of Inorganic chemistry and analytical chemitry, School of Pharmacy, Hainan Medical University, Haikou, China. E-mail address: [email protected] (Z. Wang).

∗∗

https://doi.org/10.1016/j.lwt.2019.108266 Received 13 March 2018; Received in revised form 10 June 2019; Accepted 13 June 2019 Available online 14 June 2019 0023-6438/ © 2019 Published by Elsevier Ltd.

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2. Materials and methods

were dialyzed [dialysis tubing was boiled in distilled water before being used] (nominal molecular weight cut-off of 3, 500 Da, Spectrum Medical Industries, Inc., Los Angeles, CA, USA) against distilled water for 48 h at 25 °C, and then all samples were lyophilized in vacuo. The treatment codes were: AAPUD0h, AAPUD2h, AAPUD4h, AAPUD6h, AAPUD8h, AAPUD10h, and AAPUD12h, respectively.

2.1. Materials and chemicals The fruit bodies of Auricularia auricula (L. exHook.) Underw were cultivated in the mountainous regions of Daxinganling in Yichun City, Heilongjiang Province, China, were collected in October 2013, and was initially identified by the morphological features. Standard monosaccharides (rhamnose, fucose, xylose, mannose, galactose, and GalA) were purchased from Sigma (St. Louis, MO, USA). 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azinobis-3-ethylbenzthiazoline-6-sulfonate (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DEAE-52 celluose was obtained from General Electric Company (Fairfield, CT, USA). All other chemicals and solvents were of analytical grade or better purchased from local suppliers.

2.4. Intrinsic viscosity Intrinsic viscosities of degraded and natural AAP were determined using an Ubbelohde-type viscometer (type Ф, 0.5∼0.6 mm bore, constant: 1.187 × 10−2 mm2 s-2, Ubbelohde, Hangzhou Zhuoxiang Technology Co., Ltd., Hangzhou, China)) at 25 °C using a serial dilution method (Behrouzian, Razavi, & Karazhiyan, 2014; Flory, 1953; Han, Jeon, Hong, & Lee, 2013; Tanford, 1961). Briefly, the viscometer was kept in a temperature controlled water bath (25 °C). AAP solution were prepared kept at 25 °C. The concentration of AAP solution was set as 1, 1.25, 1.5, 2, and 2.5 mg/mL (the solution was centrifuged at 5000 rpm and filtered through the 0.45 μm membrane before measurement), the elapsed time that different samples going through the Ubbelohde viscometer was recorded. The intrinsic viscosity [η] value was obtained by plotting [ηspecific viscosity/c] against c using the Huggins' equation (R2 > 0.98). The curves were extrapolated to zero concentration (Li & Feke, 2015).

2.2. Extraction and purification of the AAP The extraction and purification of AAP were done using the method reported by Zhang et al. with minor modifications (Zhang et al., 2011). Briefly, the mushrooms were washed, dried under vacuum (TC88, Nanjing Sail Machinery Co., Ltd., Nanjing, Jiangsu, China) at 60 °C for 24 h and broken into small particles using a crusher (FW135, Century Mao Yuan Machinery Co., Ltd., Tianjin, China). The samples (20 g/mL) were defatted using cold (Petroleum ether, purity > 98%, 4 °C) soaking method with 10 times the volume of petroleum ether for 24 h. The crude AAP was extracted using a 0.1 mol/L NaOH solution with 10 times the volume for 8 h. Then the obtained crude AAP solution was concentrated to 50 mg/mL, some of the coloured substances and phenolic compounds in the crude AAP solution were removed using cold (95% ethanol 4 °C) soaking and extraction three times overnight, the volume of 95% ethanol were three times as the AAP solution. Then the precipitate collected by centrifugation and washed successively with acetone, the precipitates were dialyzed [dialysis tubing was boiled in distilled water before being used] (nominal molecular weight cut-off of 3, 500 Da, Spectrum Medical Industries, Inc., Los Angeles, CA, USA) against distilled water for 48 h at 25 °C, and finally dried under reduced pressure (≥0.095 MPa) at 40 °C. The AAP were re-dissolved in distilled water (50 mg/mL) and lyophilized (GOT2000, Goodwill Technology Co., Ltd., Hong Kong, China). DEAE-52 celluose (General Electric Co., Fairfield, CT, USA) columns (2.6 × 60 cm) were used to purify the AAP. A 200 mg sample of AAP solution (20 mg/mL) was put onto the columns and washed with distilled water (200 mL) at room temperature. An automatic collector (BSZ-100, Zhenghong Industrial Co., Ltd., Shanghai, China) equipped with a constant flow pump (HL-2, Jiapeng Technology Co., Ltd., Shanghai, China) was used for the purification of AAP. The AAP were eluted and obtained using a NaCl gradient (0, 0.05, 0.1, 0.15, and 0.2 mol/L) at a flow rate of 0.5 mL/min, using colorimetric method (The wavelength of detection was 490 nm) and the content of total carbohydrate was determined using the phenol-sulfuric acid method (Dubois, Gillis, Hamilton, Rebers, & Smith, 1956). The yield of AAP was 4.95 ± 0.21%.

ηspecific

viscosity/c =

[η] + K[η]2 c

(1)

Where c is the concentration of the sample (g/mL), K is the Huggins constant, ηspecific viscosity is the specific viscosity of the sample. Then: [η] = K • Mwηα, gave the viscosity average Mw where K and α are constants that are independent of Mw. K and α values used in this study were 1.05 × 10−3 cm3/g and 0.63, respectively (Burkus & Temelli, 2003; Harding, Varum, Stokke, & Smidsrod, 1991; Ma & Pawlik, 2007; Zeng, Sun, Xue, Yin, & Zhu, 2010). 2.5. Degradation kinetics model The degradation kinetic models were developed using the viscosity average Mw of AAP with different ultrasound processing times from the Mw obtained above (2.2.3). The rate constant (k) was calculated as (Münzberg, Rau, & Wagner, 1995): ln (Mwt/Mw0) = k . t

(2)

1 / Mwt–1/Mw0 = k . t

(3)

Where k is the rate constant (mol/g/min) of the viscosity average Mw for each treatment; t is the treatment time (min); Mwt and Mw0 are the viscosity average Mw at time t and 0 h (g/mol), respectively (Yan et al., 2016). 2.6. Fourier transform infrared (FTIR) The FTIR spectra were obtained using a TENSOR27 FI-IR spectrometer (Bruker, Pittcon, Germany) (Tang et al., 2014; Zhang et al., 2013). The samples were mixed and ground with spectroscopic grade potassium bromide (KBr) powder (1: 100). Then the mixtures were pressed into KBr pellets, and FTIR determinations were done in the frequency range of 4000∼500 cm−1 with a band resolution of 4 cm−1, cumulative scans for 10 times, and the baseline was adjusted by the computer program that came with the instrument. The FTIR profile was plotted using Origin 8 Software.

2.3. Ultrasonic degradation Ultrasonic power is a major factor in fragmentation, whereas temperature is a minor factor (Yan et al., 2016). The ultrasonic treatments under 2 × 104 Hz were done using a ONK-1500W ultrasound generator (Jiangsu Bode Ultrasound Equipment Co., Suzhou, Jiangsu, China), the ultrasonic processor probe has a constant power of 500 W. A 100 mL (1.0%, w/v) solution of AAP was placed into a 200 mL glass beaker. The AAP solution was kept at a constant temperature of 25 ± 0.5 °C using an ice bath (Yan et al., 2016; Zhu et al., 2014) and processed for 2, 4, 6, 8, 10, and 12 h. To reduce the experimental errors, the samples were located just under the ultrasound source and dipped into the solution about 15 mm. The operation was repeated three times, the products

2.7. Congo red binding Congo red binding was used to determine the maximum absorption wavelength of the natural and degraded AAP at various alkaline concentrations, and interpreted in terms of the changes of the intra- and 2

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water), and the absorbance was measured at 700 nm against a blank that contained all reagents except the AAP.

inter-molecular interactions (Ogawa & Hatano, 1978). The AAP samples (2.0 mg) were dissolved in 2.0 mL NaOH solution of different concentrations: 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol/L. Congo red was added at 91 μmol/L final concentration and the mixture kept at room temperature for 1 h. The absorbance was measured from 400 to 700 nm using a UV-2550 type UV–Vis spectrophotometer (Shimadzu Corp., Kyoto, Japan).

Reducing power = Asample – Acontrol

(7)

Where Asample is the absorbance in the presence of AAP and Acontrol is the absorbance of the negative control. 2.11. Statistical analysis

2.8. Microstructure All treatments and tests were done in triplicate and data were expressed as mean ± SD. Statistical analyses of the single-factor data was done using SPSS13.0 software (IBM, Armonk, NY, USA), Origin Pro 9.0 (OriginLab Corp., Northampton, MA, USA). A one-way analysis of variance (ANOVA) was used to determine the significance of differences between groups. Significant differences were determined using a Fischer's F-test (p = 0.01 or 0.05) or an independent sample T-test (p = 0.05).

Scanning electron microscopy (SEM) observations were done on a FEI Sirion (Philips, Amsterdam, The Netherlands) (Yan et al., 2016). The SEM under high vacuum conditions was operated at an acceleration voltage of 20 kV and image magnifications of 1000 × . 2.9. Monosaccharide composition 5 mg of the AAP were hydrolysed with 3 mol/L trifluoroacetic acid at 110 °C for 8 h. The excessive trifluoroacetic acid was removed under the reduced pressure (≥0.095 MPa) at 40 °C. The hydrolysates were converted into the acetylate aldononitrile derivatives according to the converted procedure and derivatives were analyzed using GC (Agilent Company, Santa Clara, CA, USA) with an capillary column (30 m × 0.32 mm). The resultant monosaccharides were converted into alditol acetates according to Jiang et al. with minor modifications (Jiang et al., 2014) and then analysed using an Agilent 6890-5973 gas chromatograph (GC).

3. Results and discussion 3.1. Effect of ultrasound on intrinsic viscosity A majority of the polysaccharides have high viscosity including the AAP isolates. The elution curve of the AAPUD0h was shown in Fig. 1. The refined polysaccharides were subjected to a DEAE-Sephadex A-25 column to yield a distinct peak. No absorption at 280 or 260 nm in the UV-spectra of AAP was found. The purified polysaccharides were used in further experiments. The intrinsic viscosity of the degraded AAP was taken as an index of the degree of degradation. It decreased exponentially with treatment time. Fig. 2 showed the intrinsic viscosity of the AAP solutions with different ultrasonic time. The function obtained from the curve fitting was:

2.10. Antioxidant activity assays ABTS radical cation (ABTS·+) scavenging was determined using the method of Zeng et al. with minor modifications (Zeng et al., 2011). An aliquot of 200 μL of the diluted ABTS·+ solutions was mixed with 50 μL of AAP at various concentrations or a negative control (distilled water). After 15 min at 25 °C, the mixtures were measured at 734 nm. The radical-scavenging activity was calculated as: Scavenging activity (%) = (1 – Asample/Acontrol) × 100

[η] = 92e−1.45t, R2 = 0.965 During early treatment time periods, the intrinsic viscosity decreased rapidly, as the treatment time increased, the decrease in intrinsic viscosity became slow and reached a limiting value. The explanation for this phenomenon lied in the production of much more cavitation bubbles on and near the acoustic source to dampen the efficiency of energy transmission in the reaction medium with the increasing of degradation treat time (Zhang et al., 2013). The effects of low-frequency ultrasound irradiation (2–10 × 104 kHz) on the degradation of AAP may be mainly attribute to cavitation effects (mechanical effects) (Yan et al., 2016). The high temperatures and high

(4)

Where Asample is the absorbance in the presence of AAP, and Acontrol is the absorbance of the control. The hydroxyl radical-scavenging activity was determined using the method described by Huang et al. with minor modifications (Huang, Ding, & Fan, 2012). Briefly, an aliquot of 100 μL of 9 mmol/L FeSO4 and 9 mmol/L alcohol-salicylic acid were successively added into colorimetric tubes; after being uniformly mixed, 50 μL of AAP samples at various concentrations or water as a negative control were added into the above solutions, respectively. Finally, 100 μL of 8.8 mmol/L hydrogen peroxide were added and shaken. The mixtures were incubated at 37 °C for 15 min. The absorbance of the reaction mixtures was measured at 510 nm. The hydroxyl radical-scavenging was: Scavenging activity (%) = 1 – (Asample – Ablank)/Acontrol × 100

(5)

Where Asample is the absorbance in the presence of AAP, Acontrol is the absorbance of the control, and Ablank is the absorbance of the control without hydrogen peroxide. The reducing power of AAP was determined using the method of Klaus et al. (Klaus et al., 2011). Briefly, an aliquot of 0.2 mL of AAP at various concentrations was mixed with PBS solution (0.5 mL, 0.2 mol/ L, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (0.5 mL, 1% w/v in water). The mixtures were incubated at 50 °C for 20 min, and the reactions were terminated by adding 2.5 mL of trichloroacetic acid (10% w/v in water), followed by centrifugation at 3, 000 rpm (TLXJ-IIC, Anting Scientific Instruments, Shanghai, China) for 10 min at room temperature (25 °C). The upper layer of the solutions (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1% w/v in

Fig. 1. Elution curve of AAP using DEAE-Sephadex chromatography (NaCl on right y axis: 0.2 mol/L). 3

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Fig. 2. Intrinsic viscosity vs ultrasonic time for AAP.

Fig. 3. Degradation kinetics curves of AAP with ultrasonic treatments at fixed ultrasonic intensities.

pressures generated by the cavitation effect of low-frequency ultrasound were strong enough to destroy the chemical bonds with a strong binding force (about 3.77–4.18 × 105 J/mol) (Zhou et al., 2012). Ultrasound can induce a recombination of radicals formed in both saccharide and aromatic components (Klaus et al., 2011; Yan et al., 2016). 3.2. Ultrasonic degradation kinetics of AAP To determine the degradation efficiency of ultrasound with AAP, the determination coefficient (R2 > 0.848) between ln (Mwt/Mw0), (1/ Mwt–1/Mw0), and treatment times gave a 1st order k value of 0.388. The Mw used in the equation was the viscosity average Mw (Table 1). A smooth linear relationship was found with the second-order kinetics equation: 1/Mwt–1/Mw0 = k·t, where k = 0.0408. It had a higher determination coefficient (R2 > 0.916). Fig. 3 showed the plots of 1/Mwt–1/Mw0 of each sample versus ultrasonic treatment time (t). Similar results have been found in many natural and synthetic polysaccharides, such as fucoidan (Guo et al., 2014), Phellinus linteus mycelia polysaccharides (Yan, Pei, Ma, & Wang, 2015; Yan et al., 2016), xanthan gum (Li & Feke, 2015), apple pectin (Zhang et al., 2013), and carboxylic curdlan (Yan et al., 2015). This suggests that the second-order kinetics equation is a more workable kinetics model for AAP.

Fig. 4. Infrared spectra of AAP with different ultrasonic treatment times.

glucopyranosyl, and the signal at 818 cm−1 was attributed to the absorption of sulfate groups linked at the C–6 position of galactose (Zhang et al., 2013). The results showed that there were no remarkable changes taking place in the IR signals of AAP with ultrasonic degradation. As the degradation time extended from 2 to 12 h, the absorption peaks between 1064 and 3430 cm−1 became stronger. These results confirmed that the hydrogen bonds of AAP had been broken during the ultrasonic process. More hydrogen bonding groups had been exposed and the hydrogen bonded networks of AAP was destroyed (Tang et al., 2014). It was noteworthy that several weak absorption at 1100∼1360 cm−1 was observed after ultrasonic treatments. The changes may be caused by the degradation of glycosidic bonds, which generated more C–O and O–H groups in the AAP structure (Czechowska-Biskup, Rokita, Lotfy, Ulanski, & Rosiak, 2005; Tang et al., 2014). The results suggested that the partial chemical structure of the AAP were changed after different ultrasonic treatment time.

3.3. Fourier transform infrared (FTIR) spectrometry Infrared spectra were recorded in Fig. 4. All seven samples had the characteristic peaks of polysaccharides, including peaks at 3429, 2938, 1634, 1424, 1065, and 818 cm−1. However, the majority of the AAP except AAPUD0h had a wide and strong absorption peak at 3430 cm−1 which was believed to be related to the stretching vibration of O–H and a relatively weak peak at 2938 cm−1 that was believed to be due to the C–H stretching of CH2 groups. The two absorption at 1634 and 1380 cm−1 indicated that the AAP contained carboxylic groups (Yan et al., 2016). Notable absorption between 1000 and 2000 cm−1 indicated that the samples contained C–O–C, C–O–H, C–C, and ring vibrations. The signal at 914 cm−1 was attributed to the absorption of DTable 1 Constant parameters of viscosity-average molecular weight equation of AAP solution with specific ultrasonic intensities. Ultrasonic power (W)

500

1st order

3.4. Chain conformation of AAP In general, the conformation of polysaccharides is closely related to the chain structure, intra-molecular forces, and inter-molecular forces as well as the solvent system (Yu, Ming, & Fang, 2010). Some polysaccharides usually have an ordered three-dimensional structure, especially a triple helical conformation; these can form a complex with

2nd order

k

R2

k

R2

0.388

0.848

0.408 × 10−4

0.916

4

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shapes of AAP could be observed. AAPUD0h showed rough surface-like metal sheets or cloud in aggregated micro-structures. With increasing ultrasonic treatment time small fragments and pores appeared in the surface of AAPUD2h and AAPUD4h, but the overall shape was consistent with AAPUD0h. As ultrasonic time continued to increase, the microstructure was broken and more debris were found in AAPUD6h, AAPUD8h, and AAPUD10h. In addition, the shapes of the debris were like fungal hyphae or branches. Simultaneously, aggregates were reduced. Furthermore, AAPUD12h looked more like synaptic-like filaments or branches. Fewer aggregates or network interconnections were observed. This could be induced by cavitation, turbulence shear, and/or high pressure drops during the ultrasonic treatments. The degree of branching and side chains of AAP were gradually reduced with increasing treatment time. 3.6. Monosaccharide composition The sugar residues in the AAP were identified by comparing the relative retention times with those of standard monosaccharide samples. The results showed that the AAP consisted of rhamnose, fucose, xylose, mannose, galactose, and galacturonic acid. The galactose was the principal component, the percentage increased progressively with increasing ultrasonic-treatment times (Table 2). However, the percentage of rhamnose and mannose decreased gradually with increasing ultrasonic treatment time, and the percentage of galacturonic acid remained basically unchanged. Since the chemical compositions of the polysaccharides are important for understanding their bioactivities (Yan et al., 2016), it can be suggested that the bioactivities of polysaccharides may be directly related to their main monosaccharide components.

Fig. 5. Absorption spectra of Congo red (control) and Congo red with the AAP with different ultrasonic processing times. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Congo red at different concentrations of NaOH (Yu et al., 2010). Some linear and branched β-D-glucans seemed to have a triple-helical conformation in aqueous or weak alkaline solution (< 0.15 mol/L NaOH), which became single chains in stronger alkaline solutions (> 0.25 mol/ L NaOH) (Yu et al., 2010). Thus using Congo red is a simple and convenient way to study the morphology and size of the polysaccharide chains. The results of the Congo red tests were shown in Fig. 5. Compared with Congo red, all Congo red-AAP complexes showed different degrees of bathochromic shifts at 0.1 mol/L NaOH, which was presumably due to the existence of a triple helix. The Congo red-AAPUD0h complexes showed the largest bathochromic shifts from 490 to 501 nm. This suggested that the increased ultrasound time may break up the triple helix (Mao, Hsu, & Hwang, 2007; Villares, 2013). The important effect of ultrasound is its polymers degradation, the depolymerization process takes place by the cavitation effect involving two mechanisms: mechanical degradation of the polysaccharide from collapsed cavitation bubble and chemical degradation because of the chemical reaction between the polysaccharide and high-energy molecules, for example, the hydroxyl radicals produced during cavitation.

3.7. Antioxidant activity The antioxidant activities in vitro of AAPUD0h ∼ AAPUD12h were studied with ABTS·+, hydroxyl radial, and reducing power experiments. The data were shown in Table 3∼5. ABTS·+-scavenging assays are regularly used to determine antioxidant capacity. ABTS·+ could react with K2(SO4)2 to generate the stable glaucous cation radicals, and the product had a peak absorbance at 734 nm (Yan et al., 2016). As seen in Table 3, the scavenging activities of the AAP increased with a doseresponse pattern. The AAP showed scavenging activity against ABTS·+ at all concentrations. Compared with AAPUD0h, AAPUD2h ∼ AAPUD12h showed ever more scavenging activity. The EC50 of AAPUD0h ∼ AAPUD12h were 12.6 ± 1.2, 7.7 ± 1.0, 5.6 ± 0.7, 4.7 ± 0.2, 4.3 ± 0.5, 3.4 ± 0.3, 2.3 ± 0.2 mg/mL, respectively. As the most reactive radical, hydroxyl radicals are considered to be destructive of almost all bio-macromolecules in living cells, including carbohydrates, lipids, proteins, and DNA (Yan et al., 2016). As shown in Table 4, the EC50 of AAPUD0h ∼ AAPUD12h were 10.7 ± 0.7, 9.1 ± 0.6, 5.5 ± 0.7, 4.3 ± 0.6, 4.3 ± 0.4, 3.5 ± 0.3, 2.6 ± 0.3 mg/mL, respectively. The iron reducing power is another indicator of antioxidant

3.5. Surface appearance of ultrasonic treated AAP SEM has been used to observe and analyse the surface morphology of many polysaccharides (Yan et al., 2016). The effects of rupture and mechanically damages with ultrasonic treatment can be intuitively and qualitatively evaluated. Fig. S1 showed the differences in the surface topographies between the samples. Significant changes in sizes and

Table 2 Monosaccharide composition of the AAP with different ultrasonic treatment times. Values with different letters in the same column are significantly different according to T-test (p < 0.05). Samples

Rha (%)

Fuc (%)

AAPUD0h AAPUD2h AAPUD4h AAPUD6h AAPUD8h AAPUD10h AAPUD12h

12.0 ± 0.21a 11.6 ± 0.12a 5.7 ± 0.06b 3.8 ± 0.03c 3.1 ± 0.06d 3.1 ± 0.05d 2.0 ± 0.05e

13.7 12.8 12.4 21.0 17.1 17.6 16.8

± ± ± ± ± ± ±

0.14c 0.11d 0.02d 0.15a 0.20b 0.11b 0.14c

Xyl (%)

Man (%)

Gal (%)

10.0 ± 0.23a 9.7 ± 0.07b 7.7 ± 0.15c 2.3 ± 0.10e 3.1 ± 0.05d 3.1 ± 0.06d 2.0 ± 0.05f

8.7 7.8 4.4 1.5 3.1 2.1 1.0

46.5 46.5 61.7 62.2 63.2 65.8 68.7

Values are means ± SD, n = 3. a-g Different letters in the same column indicate a significant difference (p < 0.05). 5

± ± ± ± ± ± ±

0.21a 0.17b 0.06c 0.10f 0.11d 0.05e 0.04g

± ± ± ± ± ± ±

GalUA (%) 0.21a 0.07a 0.21b 0.29c 0.20d 0.26e 0.21f

9.1 ± 0.19b 11.6 ± 0.15e 7.9 ± 0.15a 9.4 ± 0.13c 10.4 ± 0.15d 8.3 ± 0.16a 9.4 ± 0.05c

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Table 3 Antioxidant activities in vitro of AAP with different ultrasound treatment times using scavenging ability for ABTS radical cation. Samples

AAPUD0h AAPUD2h AAPUD4h AAPUD6h AAPUD8h AAPUD10h AAPUD12h

Scavenging ability (%) 1 (mg/mL)

4 (mg/mL)

8 (mg/mL)

12 (mg/mL)

16 (mg/mL)

20 (mg/mL)

5.9 ± 0.13a 16.9 ± 0.38b 23.6 ± 0.54c 24.4 ± 0.50c 26.6 ± 1.57d 28.9 ± 1.29d 32.4 ± 1.13e

26.7 35.1 39.9 44.3 45.6 48.7 56.8

36.8 51.0 54.1 58.9 61.0 61.7 71.7

51.8 62.4 63.8 68.2 71.2 72.1 75.0

56.3 62.9 69.5 76.6 77.6 77.9 79.2

58.0 66.0 73.0 81.4 82.9 84.1 86.5

± ± ± ± ± ± ±

0.39a 0.30b 2.78c 2.58d 2.81d 1.37e 0.55f

± ± ± ± ± ± ±

0.25a 0.23b 2.12c 1.90d 1.85e 1.82e 1.31f

± ± ± ± ± ± ±

0.22a 1.74b 1.68b 1.47c 1.36c 1.43c 0.96d

± ± ± ± ± ± ±

0.02a 0.72b 1.41c 1.08d 1.15d 1.02d 0.96e

± ± ± ± ± ± ±

0.19a 1.45b 1.25c 1.82d 1.44d 0.74d 0.45e

Values are means ± SD, n = 3. a-e Different letters in the same column indicate a significant difference (p < 0.05).

phenols (Gullon et al., 2018). Further research about the relationships among the hydroxymethylfurfural, phenols, and antioxidant activity of AAP was still needed.

activity. The conversion of iron (III) to iron (II) with AAP was shown in Table 5. All of the AAP showed some reducing power with a dose-response pattern. It was obvious that the AAPUD12h had significantly higher ABTS·+-scavenging and Hydroxyl radicals-scavenging activity than other samples. At 9 mg/mL AAPUD12h had the highest reducing power, which was 0.305 ± 0.003. In order to obtain polysaccharides with specific structure for better bioactivities, the natural polysaccharide solution usually requires using some processes, such as hydrolysis, chemical derivatization, or physical treatments (Cui et al., 2016). These results showed that the AAPUD0h ∼ AAPUD12h had different degrees of antioxidant activity, and the experimental results of three assays were relatively consistent. The ultrasonically degraded AAP had better antioxidant activity than the natural one (AAPUD0h). Compared with other samples, the antioxidant capabilities of AAPUD12h were highest. Longer exposure to ultrasound led to more reductions in the Mw and viscosity, chain length, and to more available O–H groups (Venegas-Sanchez, Motohiro, & Takaomi, 2013; Zhang et al., 2014; Fleita, EI-Sayed, & Rifaat, 2015; Liao et al., 2015). These results suggested that Mw and solution behavior may one of the key factors in the antioxidant activities of polysaccharides. In addition, the monosaccharide composition, chemical structure, and chain conformation of polysaccharide may be also closely related to the antioxidant activity of AAP (Meng et al., 2015; Yan et al., 2016). The results were in agreement with that of Zhou et al. (Zhou et al., 2012). On the one hand low molecular weight is favorable for the antioxidant activity of AAP, but on the other hand, it was reported that high content of neutral monosaccharide seemed to have negative effect on antioxidant activity of polysaccharides, while high acidic monosaccharide content would promote the antioxidant activity (Cui et al., 2016). The monosaccharide in AAP might play a significant role in the antioxidant activity of degraded AAP. Moreover, it had been also confirmed that the antioxidant properties of polysaccharides may be correlated with hydroxymethylfurfural content, which was one of the degradation products from polysaccharides (Kowalski, 2013; Chaikham, Kemsawasd, & Apichartsrangkoon, 2016), sulfate groups (Li et al., 2018), as well as

4. Conclusions This study evaluated the effect of the ultrasonic treatment on the physical properties and antioxidant activity of AAP. The intrinsic viscosity data showed that the modified AAP after the ultrasonic treatment had a lower intrinsic viscosity. The ultrasonic degradation kinetics model was fitted to 1/Mt – 1/M0 = k • t. The results confirmed that the ultrasonic degraded AAP had better antioxidant activity, and the antioxidant activity of AAP were closely related to the intrinsic viscosity, molecular weight, degree of hydroxyl exposure, and monosaccharide composition. Thus, ultrasonic treatment can improve the rheological properties of AAP, and improve the antioxidant activity of polysaccharides. Therefore, further studies are still needed to confirm the changes of physicochemical properties of AAP under more conditions, sucn as ultrasonic power, AAP concentration, pH of AAP solution, and reaction temperature, and so on. In conclusion, ultrasonic sound was a simple, effective, environmental, and controllable means for partial degradation of AAP solution, these revealed that ultrasonic degradation could be helpful way for improving the intrinsic viscosity and antioxidant activity of polysaccharides. Conflicts of interest The authors of the paper have no financial or personal relationships with other people or organizations that would create a conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (31401483) and the Post-Doctoral Fund of

Table 4 Antioxidant activities in vitro of AAP with different ultrasound treatment times using scavenging ability for hydroxyl radicals. Samples

AAPUD0h AAPUD2h AAPUD4h AAPUD6h AAPUD8h AAPUD10h AAPUD12h

Scavenging ability (%) 1 (mg/mL)

4 (mg/mL)

8 (mg/mL)

12 (mg/mL)

16 (mg/mL)

20 (mg/mL)

10.2 12.9 21.6 24.2 28.5 28.9 33.5

32.0 32.2 38.6 41.8 47.5 48.7 58.8

44.3 49.5 55.1 61.6 62.2 63.7 67.4

55.9 58.6 62.3 72.1 73.2 74.1 75.0

58.2 61.9 69.1 75.6 77.5 77.9 79.5

59.4 63.9 72.1 79.4 80.0 80.9 81.3

± ± ± ± ± ± ±

0.07a 0.68a 2.23b 0.89c 3.29d 1.20d 2.19e

± ± ± ± ± ± ±

1.51a 3.65a 1.75b 1.68c 1.81d 2.37d 0.81e

± ± ± ± ± ± ±

2.09a 0.39b 1.48c 1.88d 1.27d 1.82d 2.15e

Values are means ± SD, n = 3. a-e Different letters in the same column indicate a significant difference (p < 0.05). 6

± ± ± ± ± ± ±

3.15a 2.49b 3.28c 0.53d 1.36d 1.43e 0.96f

± ± ± ± ± ± ±

0.48a 1.30a 1.41b 1.35c 0.48d 1.02d 0.62e

± ± ± ± ± ± ±

1.14a 0.71b 1.92c 1.85d 0.60e 1.00e 1.80e

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Table 5 Antioxidant activities in vitro of AAP with different ultrasound treatment times using reducing power. Samples

AAPUD0h AAPUD2h AAPUD4h AAPUD6h AAPUD8h AAPUD10h AAPUD12h

Reducing power 4 (mg/mL)

5 (mg/mL)

6 (mg/mL)

7 (mg/mL)

8 (mg/mL)

9 (mg/mL)

0.110 0.112 0.114 0.131 0.132 0.147 0.148

0.120 0.121 0.124 0.143 0.148 0.162 0.168

0.121 0.120 0.135 0.154 0.156 0.171 0.170

0.126 0.127 0.140 0.161 0.169 0.172 0.175

0.127 0.128 0.147 0.166 0.176 0.184 0.188

0.136 0.136 0.163 0.215 0.225 0.268 0.305

± ± ± ± ± ± ±

0.005a 0.002a 0.002a 0.001b 0.004b 0.002c 0.003c

± ± ± ± ± ± ±

0.004a 0.002a 0.003a 0.002b 0.003b 0.002c 0.002d

± ± ± ± ± ± ±

0.005a 0.002a 0.002b 0.000c 0.001b 0.003c 0.002c

± ± ± ± ± ± ±

0.006a 0.001a 0.003b 0.002c 0.003d 0.003d 0.000d

± ± ± ± ± ± ±

0.000a 0.002a 0.002b 0.000c 0.003d 0.000e 0.003e

± ± ± ± ± ± ±

0.001a 0.000a 0.004b 0.001c 0.006c 0.002d 0.003e

Values are means ± SD, n = 3. a-e Different letters in the same column indicate a significant difference (p < 0.05).

Heilongjiang Province (LBH-Z14098). The authors also are thankful for the Innovation Funds of Harbin Institute of Technology (HIT. NSRIF. 2017025).

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