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Interactions between ascorbic acid and water soluble polysaccharide from the seeds of Plantago asiatica L.: Effects on polysaccharide physicochemical properties and stability
Food Hydrocolloids 99 (2020) 105351
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Interactions between ascorbic acid and water soluble polysaccharide from the seeds of Plantago asiatica L.: Effects on polysaccharide physicochemical properties and stability
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Ou-Ye Lia, Li Wanga, Xiao-Ying Liua, Jun-Yi Yina,b,∗∗, Shao-Ping Niea,∗ a State Key Laboratory of Food Science and Technology, China-Canada Joint Lab of Food Science and Technology (Nanchang), Nanchang University, Nanchang, Jiangxi Province, 330047, China b Department of Applied Biology and Chemical Technology, The HongKong Polytechnic University, Kowloon, Hong Kong, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Plantago asiatica L. Polysaccharide Ascorbic acid Apparent viscosity Molecular weight Hydroxyl radical
Viscous polysaccharide is one kind of main bioactive components in Plantago asiatica L. seeds. This study aims to investigate effects of various monosaccharides, disaccharides, especially ascorbic acid (VC), on apparent viscosity and physicochemical properties of crude water soluble polysaccharide from the seeds of P. asiatica L. (PLWEP-C), as well as its stability and degradation mechanism. Results showed that small molecules such as glucose, sucrose, maltose, and lactose had little effects on apparent viscosity of PLWEP-C, while VC could significantly reduce the viscosity. There was no obvious change in FT-IR spectra and monosaccharide composition of PLWEP-C after VC addition. Hydroxyl radicals were probably involved in the degradation of polysaccharide induced by VC, which was confirmed by glucose inhibition test. SEM observation showed that PLWEP-C was mainly in lamellar and filamentous appearance, and no great change was generated by VC. The solution stability of PLWEP-C can only be kept well, when VC was at low concentration and low temperature. In conclusion, VC could significantly reduce the viscosity of polysaccharide from the seeds of P. asiatica L., which was probably attributed to the reduction of molecular weight. Hydroxyl radicals participation were probably one of main reasons in the degradation process.
1. Introduction Polysaccharide is a class of biopolymer widely existed in natural resources, including terrestrial and marine plants, as well as some exogenous metabolites of bacteria. It is a very important material, widely applied in food, cosmetic, pharmaceutical, biomedicine and other industries. Structure of the polysaccharide is complicated, because it is linked by the attachment to different monosaccharides through diverse glycosidic bonds (Caffall & Mohnen, 2009; Yang & Zhang, 2009). Usually, polysaccharide solution exhibits viscous property and forms gel, which is influenced by polymer concentration, molecular weight, degree of branching, functional groups, and other non-polysaccharide chemicals (Wang & Cui, 2005). Meanwhile, the viscosity of polysaccharide has a great influence on its physicochemical properties and functions. In food industry, the texture and processing conditions of the product are closely related to the viscosity of the polymer.
Both macromolecules and small molecules exist in the food system. The interaction between them is an interesting topic (Lin et al., 2017; Takemasa & Nishinari, 2016). Polysaccharide can also interact with small molecular compounds, such as vitamins (Alyafeai & Böhm, 2018; Rodrigues & de Oliveira, 2012), polyphenols (Wang, Liu, Chen, & Chen, 2017) and other chemical compounds (Menchicchi et al., 2015; Watrelot, Schulz, & Kennedy, 2017), thereby affects the quality and function of polysaccharides. Vitamin C (ascorbic acid, VC) exists in many vegetables and fruits, and is widely used as an antioxidant. However, it can also act as a pro-oxidant at low concentration (Buettner & Jurkiewicz, 1996; Kivelä, Nyström, Salovaara, & Sontagstrohm, 2009; Yen, Duh, & Tsai, 2002). Hydroxyl radicals can be produced by the decomposition of hydrogen peroxide in Fenton reaction against the presence of VC (Guo, Yuan, Wu, Xie, & Yao, 2002), which is catalyzed by transition metals (Arts, Mombarg, Bekkum, & Sheldon, 1997; Duarte & Lunec, 2005; Haber & Weiss, 1934). Therefore, polysaccharide is prone to degradation when they coexist. Recent studies have shown
∗
Corresponding author. Corresponding author. State Key Laboratory of Food Science and Technology, China-Canada Joint Lab of Food Science and Technology (Nanchang), Nanchang University, Nanchang, Jiangxi Province, 330047, China. E-mail addresses: [email protected] (J.-Y. Yin), [email protected] (S.-P. Nie). ∗∗
https://doi.org/10.1016/j.foodhyd.2019.105351 Received 13 July 2018; Received in revised form 29 August 2019; Accepted 29 August 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Effects of fructose (a), glucose (b), lactose (c), sucrose (d) and maltose (e) on apparent viscosity of 0.5% PLWEP-C.
that the viscosity reduction of β-glucan solutions containing VC was due to degradation of β-glucan (Faure, Andersen, & Nyström, 2012; Mäkelä, Sontag-Strohm, & Maina, 2015; Reetta, Fred, & Tuula, 2009). Polysaccharide from the seeds of Plantago is usually composed of arabinose (Ara) and xylose (Xyl), whose backbone is linked by β-1,4Xylp residues (Fischer et al., 2004; Gong et al., 2015; Yin, Lin, et al., 2012). It is highly branched, where most of the Ara residues are distributed in its side chains. Health benefits of this polysaccharide are widely accepted, including relieving constipation (Ashraf, Pfeiffer, Park, Lof, & Quigley, 1997), reducing abdominal pain in children with irritable bowel syndrome (Shulman et al., 2016), lowering plasma lipids (Rodriguez-Moran, 1998; Solà et al., 2010), improving immunity (Huang, Nie, Jiang, & Xie, 2014) and promoting short chain fatty acid
production (Hu, Nie, Min, & Xie, 2012; Yadav, Sharma, Kapila, Malik, & Arora, 2016). The polysaccharide is a kind of hydrophilic colloid, which has strong water swelling ability leading to the formation of the viscous suspension or a gel (Qian, Cui, Wang, Goff, & Smith, 2009; Yin, Nie, et al., 2012; Yu et al., 2017). The pH value of its solution was another important condition (Farahnaky, Askari, Majzoobi, & Mesbahi, 2010). Kale et al. (Kale, Yadav, & Hanah, 2016) have reported that high molecular weight and low viscosity soluble polysaccharides, including maltodextrin, gum arabic, locust bean gum, and corn bio-fiber gum, could produce significant decreases in the viscosity of psyllium husk suspensions. However, investigation on the effect of non-polysaccharides on viscosity and physicochemical properties of psyllium polysaccharide is rare. According to our previous studies on the 2
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defatted residue (200 g) was extracted twice with boiling water (2 L) for 3 h. Combined aqueous extracts were concentrated to 1 L, and ethanol (95%, v/v) was added until the final concentration was 80% (v/v). It was kept at 4 °C overnight, and then centrifuged (4800 rpm, 15 min). The precipitate was collected and lyophilized to get crude polysaccharide. The crude polysaccharide was dissolved at a concentration of 5.0 mg/mL and centrifuged (4800 rpm, 15 min) again to remove the precipitate. Finally, the supernatant was lyophilized to get P. asiatica L. water extracted polysaccharide, named as PLWEP-C. PLWEP-C was determined to contain 55.1% neutral sugar, 20.3% uronic acid and 4.0% protein. 2.3. Apparent viscosity and gelling property of PLWEP-C Apparent viscosity of polysaccharide solutions were measured by an ARES G-2 Rheometer (TA Instruments, New Castle, DE, USA) using cone-and-plate (40 mm diameter with a gap of 0.046 mm). Temperature of 25.0 °C was controlled by a SR5 Peltier Circulator (TA Instruments, New Castle, DE, USA).
Fig. 2. Effect of VC on apparent viscosity of 0.5% PLWEP-C.
2.3.1. Apparent viscosity PLWEP-C or commercial arabinoxylan solution was dissolved in ultra-pure water at 60 °C. Then, VC was added into the polysaccharide solution at concentrations of 0.025, 0.05, 0.1 and 0.2% (w/v), successively. The final concentration of polysaccharide was kept at 0.5% (w/ v). At the same time, glucose was introduced to the study as a competitive hydroxyl radical scavenger (Buxton, Greenstock, Helman, & Ross, 1988), whose concentration was selected to be 1 M (appr. 20%, w/w). The effects of other small molecules including glucose, fructose, lactose, sucrose, and maltose on apparent viscosity of PLWEP-C were evaluated in the similar way. The concentrations of these small molecules were chosen as 0.025, 0.05, 1.0, 2.0 and 4.0%, successively. 2.3.2. Gelling property To determine the gelling properties of PLWEP-C before and after VC addition, PLWEP-C was completely dissolved in water. VC was added into the polysaccharide solution at final concentrations of 0.1, 0.2, 0.4 and 0.8% (w/v), successively. The final concentration of PLWEP-C was kept at 2.0% (w/v). The mixture was kept at 20 °C for more than 12 h. The range of frequency was 0.1-20 Hz at strain of 2% in the linear viscoelastic region.
Fig. 3. Effect of VC on apparent viscosity of 1% commercial arabinoxylan.
polysaccharide from seeds of P. asiatica L., we have found that some small molecular compounds have a unique influence on its viscosity. Therefore, this study aims to report different effects of monosaccharides, disaccharides, and ascorbic acid on physicochemical properties of water soluble polysaccharide from the seeds of P. asiatica L., which would guide the application.
2.4. Stability observation The effects of VC on stability of PLWEP-C at room temperature (23 °C) and low temperature (4 °C) were compared. The concentrations of VC were chosen as 0.025, 0.05, 0.1 and 0.2% (w/v), successively. Final concentration of PLWEP-C was kept at 0.5% (w/v), which contained 0.02% of NaN3 for bacterial inhibition. Photos of each sample were collected at different times during the storage period. Meanwhile, samples of PLWEP-C stored at 23 °C for 20 days was chosen to determine protein content (Bradford, 1976) and monosaccharide composition (Liu et al., 2017) of the precipitate, which was collected by centrifugation (4800 rpm, 15 min, twice).
2. Materials and methods 2.1. Materials and chemicals Seeds of P. asiatica L. were collected in November 2013 from Ji'an, Jiangxi Province, China. They were kept under dry conditions in dark until June 2015 for polysaccharide preparation. VC (≥99.0%), arabinoxylan (95%), and glucose (≥99.5%) were obtained from SigmaAldrich Shanghai Trading Co. Ltd (Shanghai, China). Other chemicals and reagents were of analytical grade.
2.5. Morphology observation by SEM The solid morphology of polysaccharide was analyzed by scanning electron microscopy (SEM). At first, polysaccharide solutions were prepared at the concentration of 0.5% which contained 0.025, 0.05, 0.1 and 0.2% of VC, successively. Then, each of them was diluted by 10 times and placed in liquid nitrogen for pre-freezing for 30 min. Finally, they were transferred to a freezer and lyophilized before test. The dried samples were placed on a sample stage, and coated with a thin layer of platinum. A JSM-6701F SEM (Jeol Company, Tokyo, Japan) was used
2.2. Polysaccharide preparation The preparation process of polysaccharide from the seeds of P. asiatica L. was conducted according to earlier report (Yin, Nie, Chao, Yin, & Xie, 2010) with some modifications. Primarily, the seeds of P. asiatica L. were defatted with ethanol (95%, v/v) and dried. Then, the 3
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Fig. 4. Inhibition effects of glucose addition on apparent viscosity of arabinoxylan added with different concentrations of VC.
Fig. 5. Effect of VC on storage modulus (G′) and loss modulus (G″) of PLWEP-C at 25.0 °C. Strain used in all the experiments was within the linear viscoelastic region where the gel structure was not damaged.
Fig. 7. Effect of VC on molecular weight of PLWEP-C determined by HPGPC. All the samples after the treatment with VC were dialyzed against distilled water, and freeze-dried. Polysaccharide concentration was chosen to be 0.5 mg/mL before membrane filtration for HPGPC analysis. Fig. 6. Effect of VC on FT-IR spectra of PLWEP-C. All the samples after the treatment with VC were dialyzed against distilled water, and freeze dried.
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chromatographic system. 2.6.3. Monosaccharide composition analysis Monosaccharide composition analysis was carried out according to the method reported by Liu et al. (Liu et al., 2017) with some modifications. Polysaccharide sample (5.0 mg) was hydrolyzed with 0.5 mL of 12 M H2SO4 in the iced water bath for 30 min, then diluted by water (2.5 mL) and hydrolyzed for another 2 h in a silicone oil bath (100 °C). The hydrolysate was diluted and analyzed by high performance anionexchange chromatography (Dionex ICS-5000, Thermo Fisher Scientific Massachusetts, USA) coupled with pulsed amperometric detection (PAD). A CarboPac™ PA20 guard column (4 mm × 50 mm) and a CarboPac™ PA20 analytical column (4 mm × 250 mm) were used. A Chromeleon 6.8 (Thermo Fisher Scientific, Massachusetts, USA) software was used for data collection and processing.
Fig. 8. SEM images of PLWEP-C (0.5%) added with 0.025, 0.05, 0.1 and 0.2% of VC. All the samples were completely mixed with VC at certain concentrations, freeze-dried and observed at magnification of 1000. The final concentration of polysaccharide in each group was kept at 0.05%.
3. Results and discussions 3.1. Effects of small molecules on apparent viscosity of PLWEP-C
for images collection at an accelerating voltage of 5 kV.
Fructose, glucose, lactose, sucrose and maltose were commonly used carbohydrates in food industry. Fig. 1 shows the effects of monosaccharides and disaccharides at concentrations from 0.025 to 4.0% (w/v) on apparent viscosity of PLWEP-C. PLWEP-C at the concentration of 0.5% displayed shear-thinning behavior (pseudoplastic) behavior. It can be found that fructose, glucose, lactose, sucrose and maltose had little effects on apparent viscosity of PLWEP-C at tested concentrations. VC is widely applied as antioxidant. Fig. 2 shows the effect of VC on apparent viscosity of PLWE-C. VC could reduce the apparent viscosity of PLWEP-C. When the concentration of added VC was 0.05%, PLWEP-C had the lowest viscosity. As the concentration of VC was over 0.05%, the viscosity of PLWEP-C increased with increasing of concentration of VC. When VC was removed from the polysaccharide after dialysis, the viscosity could not be recovered (data not shown). The viscosity of commercial arabinoxylan was much lower than PLWEP-C, while effect of VC on apparent viscosity of commercial arabinoxylan (Fig. 3) was similar to that of PLWEP-C. The greatest reductions in viscosity of polysaccharide (Figs. 2 and 3) were observed at the low concentrations of VC (0.025 and 0.05%), with progressively smaller reductions as the concentration was increased, until ultimately, reductions as the concentration of VC was increased to the highest value used (0.4%). VC has a multiplicity of antioxidant properties, but it can exert pro-oxidant effects at low concentration
2.6. Effect of VC on physicochemical properties of PLWEP-C after dialysis Polysaccharide solutions (0.5%, w/v) treated by VC at concentrations of 0.025, 0.05, 0.1 and 0.2% (w/v) were kept under stirring for 24 h, then dialyzed against ultra-pure water for three days to remove the excess VC in the mixture. After that, the samples were freeze-dried and applied for apparent viscosity, FT-IR spectrum, morphology, molecular weight, and monosaccharide composition analyses. 2.6.1. FT-IR observation FT-IR spectra of PLWEP-C and VC treated samples were recorded from 4000 cm−1 to 500 cm−1 by a Thermo Nicolet 5700 infrared spectrograph. Samples were dried prior to tableting with KBr powder. 2.6.2. Molecular weight determination by HPGPC Molecular weights of PLWEP-C and VC treated samples were detected by the high performance gel permeation chromatography (HPGPC) according to the previous reports (Feng, Yin, Nie, Wan, & Xie, 2016; Wang et al., 2017). Ultra-pure water containing 0.02% (w/v) NaN3 was used as mobile phase in the current study. Samples and dextran standards (1.0 mg/mL) dissolved in mobile phase were passed through a 0.22 μm membrane filter and then injected into the
Fig. 9. SEM images of PLWEP-C (0.5%) added with 0.025, 0.05, 0.1 and 0.2% of VC. All the samples after the treatment with VC were dialyzed against distilled water, freeze-dried and observed at magnification of 1000. The final concentration of polysaccharide in each group was kept at 0.05%. 5
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Fig. 10. Stability of 0.5% PLWEP-C added with different concentrations of VC under the temperature of 4 °C. All the solutions contain 0.02% NaN3 for bacterial inhibition. Number of 1, 2, 3, and 4 shown in the figure in black font represents the concentration of added VC was 0.025, 0.05, 0.1and 0.2%, successively.
Fig. 11. Stability of 0.5% PLWEP-C added with different concentrations of VC under the temperature of 23 °C. All the solutions contain 0.02% NaN3 for bacterial inhibition. Number of 1, 2, 3 and 4 shown in the figure in black font represents the concentration of added VC was 0.025, 0.05, 0.1 and 0.2%, successively. Table 1 Chemical component and monosaccharide composition of the precipitation from PLWEP-C and ascorbic acid treated samples (n = 3, mean ± SD). Precipitation from the solution
Protein (%,w/w)
Monosaccharide composition (%, w/w) Ara
PLWEP-C PLWEP-C+0.025%VC PLWEP-C+0.05%VC PLWEP-C+0.1%VC PLWEP-C+0.2%VC
n.d. n.d. n.d. n.d. n.d.
6.07 8.98 7.25 6.62 6.14
Gal ± ± ± ± ±
0.05 0.27 0.10 0.08 0.03
0.02 0.72 0.32 0.11 0.09
Xyl ± ± ± ± ±
0.01 0.01 0.02 0.02 0.01
28.01 32.05 28.67 27.08 28.96
Gala ± ± ± ± ±
1.35 1.01 1.26 0.40 0.19
3.76 4.17 4.65 3.75 3.73
Ara/Xyl ± ± ± ± ±
0.21 0.40 0.24 0.12 0.28
0.22 0.28 0.25 0.24 0.21
n.d., not determined.
Glucose was considered as a competitive hydroxyl radicals scavenger (Buxton et al., 1988; Reetta et al., 2009). Therefore, it was added into the solution combining with VC and arabinoxylan. Different concentrations of VC and glucose (1 M, 20 w/w %) were sequentially added to the arabinoxylan. The results (Fig. 4) showed that glucose could effectively inhibit the viscosity decrease caused by the addition of VC. It indicated that the hydroxyl radicals induced degradation of polysaccharide was inhibited by the addition of glucose. It was similar to viscosity change for PLWEP-C when glucose was added into the solution containing VC.
(Buettner & Jurkiewicz et al., 1996; Kivelä et al., 2009; Yen et al., 2002). It was probably because of the antioxidant property of VC at higher concentration, and pro-oxidant effect at low concentration. We have taken another test to reverse verify it. Hydroxyl radicals, one specific species of reactive oxygen species, have been established to scission polysaccharides (Fry, 1998). Fenton reaction, which is typically present in the aqueous phases of food, can produce aggressive hydroxyl radicals (Arts et al., 1997; Duarte & Lunec, 2005; Haber & Weiss, 1934; Slavin, Martini, Jacobs, & Marquart, 1999; Wardman & Candeias, 1996). In certain conditions, VC behaves as a pro-oxidant initiating the Fenton reaction due to the reducing capacity (Reactions 1 and 2), then produces aggressive hydroxyl radicals (Reaction 3). AH2 + 2Cu2+/Fe3+→ A + 2H+ + 2Cu+/Fe2+
(1)
AH2 + O2 → A + H2O2
(2)
Cu+/Fe2+ + H2O2 →˙OH + OH− + Cu2+/Fe3+
(3)
3.2. Effect of VC on viscoelastic properties of PLWEP-C Previous study (Hesarinejad et al., 2018; Yin, Nie, et al., 2012) showed that water extracted polysaccharide from the seeds of P. asiatica L. had weak-gel like structure at high concentration, as values of storage modulus (G′) were higher than those of loss modulus (G″) during 6
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observed. At the same time, PLWEP-C was firstly added with different concentrations of VC, then dialyzed against distilled water to remove those small molecules. Their morphological appearance was similar to each other. Much more flaky and curly aggregates appeared for the polysaccharide treated higher concentration of VC, but all samples contained linear morphology.
the whole experimental range. Effect of VC on viscoelastic properties of PLWEP-C was carried out at high polysaccharide concentration (2.0%), with the concentration ratios of VC to PLWEP-C fixed at 20, 10, 5 and 2.5, successively. Mechanical spectra of all samples (Fig. 5) showed that G′ was greater than G″ throughout the tested frequency range and they were both frequency dependent, suggesting the tested samples exhibited weak-gel structure. VC could reduce G′ values of polysaccharide, which was much lower than the original one in most cases except the additional concentration of VC reaching 0.8%. The gel strength was the lowest when VC concentration was 0.1%. With increasing concentration of VC, the gel strength increased.
3.6. Effect of VC on stability of PLWEP-C solution Stability of polysaccharide solution added with other compounds is an important issue, which affects its application. Therefore, effects of VC on stability of PLWEP-C solution at different storage conditions were investigated (Figs. 10 and 11). The original polysaccharide solution was in gray and a little yellow color. It was stable during the tested period both at 4 °C and 23 °C. The polysaccharide containing VC was much more stable at 4 °C. There was some precipitation observed when PLWEP-C containing VC was kept at 23 °C for 10 days, even when the concentration of VC was only 0.025%. We have determined the basic chemical composition of the precipitation from PLWEP-C and VC interaction, for 20 days at temperature of 23 °C. The results of the chemical component and monosaccharide composition are shown in Table 1. No protein was determined. We also determined the monosaccharide composition of precipitation for PLWEP-C and VC treated samples. The ratio of Ara/Xyl was similar to that of the untreated sample.
3.3. FT-IR observation Fig. 6 shows the effect of VC treatment on the structure of PLWEP-C by FT-IR. The strong and broad peak at around 3415 cm−1 can be assigned to stretching vibrations of hydroxyl groups. The weak peak at around 2926 cm−1 belonged to the stretching vibration of methyl group (Gong et al., 2015). Absorption band at 1730 cm−1 indicated the presence of uronic acid, while absorption at 1611 cm−1 and 1424 cm−1 arose from asymmetric and symmetric stretching vibrations of carboxylate were generated by carboxylic acid (Yin, Nie, Zhou, Wan, & Xie, 2010). A strong absorption at 1043 cm−1 was due to stretching vibration of the pyranose ring. An IR band at 899 cm−1 was the characteristic absorption of β-anomeric configuration (Yang & Zhang, 2009). It was obvious that all the above mentioned absorption peaks could be found in the polysaccharide before and after VC treatment, indicating the reservation of structural characteristics.
4. Conclusions Our study showed that the apparent viscosity of PLWEP-C could be changed by adding VC. When it was added at lower concentration, the apparent viscosity decreased significantly, then increased with the increase of VC concentration. Structural analysis by FT-IR and monosaccharide composition analysis indicated there was no significant difference in structural features between PLWEP-C and VC treated samples. SEM showed that the morphology also did not change a lot. However, great reduction was found in molecular weight of PLWEP-C after the treatment with VC. Change in viscosity of PLWEP-C might be attributed to the reduction of molecular weight. Stability of PLWEP-C solution decreased when VC was added. Macromolecules are susceptible to degradation induced by oxygen species, especially hydroxyl radicals, which belong to reactive oxygen species and can oxidize bio-macromolecules. In recent years, many researches on hydroxyl radicals degraded carbohydrates have been reported (Majzoobi, Radi, Farahnaky, & Tongdang, 2012; Wu et al., 2016). It was found that VC had degradation effects on wheat starch molecules especially after gelatinization (Majzoobi et al., 2012). Reetta et al. (Reetta et al., 2009) found that polysaccharides were sensitive to hydroxyl radicals induced depolymerization, and these radicals can be produced in cereal food systems. Reetta et al. (Reetta, Henniges, Sontag-Strohm, & Potthast, 2012) have further studied the β-glucan solution added with VC, which demonstrated that the β-glucan chain was oxidized during the addition of VC and posed a risk to the functionality of β-glucan in solution. Mäkelä et al. (Mäkelä et al., 2015) have proved that the oxidation of barley β-glucan resulted in significant degradation via glycosidic bond or cross-ring cleavage, which decreased both molecular weight and viscosity (Mäkelä et al., 2015). In this study, glucose was used as a competitive hydroxyl radical scavenger. The results showed that glucose could effectively inhibit the viscosity decrease of PLWEP-C caused by the addition of VC. It indicated that the hydroxyl radicals induced degradation of arabinoxylan by VC was inhibited by the addition of glucose. However, whether there are any changes of glycosidic bonds and which kinds of free radicals involved in the reaction should be further explored. In addition, functional properties of the polysaccharides before and after VC treatment should also be investigated. It would be helpful in
3.4. Molecular weight and monosaccharide composition Molecular weight distributions of PLWEP-C and VC treated samples are shown in Fig. 7. The weight-average molecular weight of PLWEP-C was 6.4 × 106, while those of PLWEP-C (0.5%) treated with 0.025, 0.05, 0.1 and 0.2% VC were 3.1 × 106, 3.2 × 106, 2.7 × 106, and 3.9 × 106, respectively. VC could reduce the weight-average molecular weight. This result was inconsistent with the change of apparent viscosity, as the molecular weight did not decrease with the decrease of VC concentration. Since HPGPC required membrane filtration before injection, we speculated that some samples were trapped during the filtration process, resulting in the loss of some polysaccharide fractions. Therefore, xylose was used as a standard to determine the polysaccharide content in the solution after filtration by colorimetric method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Only 26.2% of polysaccharide in PLWEP-C was retained after filtration, which were 82.6%, 81.1%, 73.3%, and 63.1% for PLWEP-C treated with 0.025, 0.05, 0.1 and 0.2% VC, successively. It indicated that the lowest molecular weight of PLWEP-C treated with 0.1% VC was probably because of its lower mass retention rate than 0.025% and 0.05% VC treated samples. According to monosaccharide composition analysis results, PLWEPC was confirmed to contain Rha (0.66%), Ara (9.40%), Gal (1.96%), Glc (0.58%), Xyl (32.64%), GalA (2.07%) and GlcA (6.00%), which was similar to our previous reports (Liu et al., 2017). After VC treatment, there was no significant difference among the samples. 3.5. Morphology observation by SEM SEM images of PLWEP-C treated with VC before and after dialysis are shown in Figs. 8 and 9, respectively. The particles of PLWEP-C were irregular in shape. Some of them were in linear style, while others were flaky curly aggregates. When VC was added into PLWEP-C, the polysaccharide morphology changed a little, but not too much. A little more linear appearance was found in the polysaccharide when it was added with 0.025, 0.05 or 0.1% of VC. The appearance of PLWEP-C containing 0.2% VC was condensed, although some linear style could still be 7
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understanding the interactions between the polysaccharide from P. asiatica L. and VC, and promoting the development of polysaccharides in food and pharmaceutical industries.
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American Journal of Clinical Nutrition, 70, 459–463. Solà, R., Bruckert, E., Valls, R. M., Narejos, S., Luque, X., Castro-Cabezas, M., et al. (2010). Soluble fibre (Plantago ovata husk) reduces plasma low-density lipoprotein (LDL) cholesterol, triglycerides, insulin, oxidised LDL and systolic blood pressure in hypercholesterolaemic patients: A randomised trial. Atherosclerosis, 211(2), 630–637. Takemasa, M., & Nishinari, K. (2016). Solution structure of molecular associations investigated using NMR for polysaccharides: Xanthan/galactomannan mixtures. Journal of Physical Chemistry B, 120(12), 3027–3037. Wang, Q., & Cui, S. W. (2005). Food carbohydrates: Chemistry, physical properties, and applications. Crc Pr I Llc. Wang, J., Liu, W., Chen, Z., & Chen, H. (2017). Physicochemical characterization of the oolong tea polysaccharides with high molecular weight and their synergistic effects in combination with polyphenols on hepatocellular carcinoma. 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Antioxidant and pro-oxidant properties of ascorbic acid and gallic acid. Food Chemistry, 79, 307–313. Yin, J. Y., Lin, H., Li, J., Wang, Y., Cui, S. W., Nie, S., et al. (2012). Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L. Carbohydrate Polymers, 87(4), 2416–2424. Yin, J. Y., Nie, S. P., Chao, Z., Yin, W., & Xie, M. Y. (2010). Chemical characteristics and antioxidant activities of polysaccharide purified from the seeds of Plantago asiatica L. Journal of the Science of Food and Agriculture, 90(2), 210–217. Yin, J. Y., Nie, S. P., Li, J., Li, C., Cui, S. W., & Xie, M. Y. (2012). Mechanism of interactions between calcium and viscous polysaccharide from the seeds of Plantago asiatica L. Journal of Agricultural and Food Chemistry, 60(32), 7981–7987. Yu, L., Yakubov, G. E., Zeng, W., Xing, X., Stenson, J., Bulone, V., et al. (2017). Multilayer mucilage of Plantago ovata seeds: Rheological differences arise from variations in arabinoxylan side chains. Carbohydrate Polymers, 165, 132–141.
Conflicts of interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work. Acknowledgments The authors are grateful to the financial supports from National Natural Science Foundation of China (21564007, 31871755), Scientific and Technological Innovation Foundation for Distinguished Young Scholars of Jiangxi Province (20192BCB23005), Outstanding Science and Technology Innovation Team Project of Jiangxi Province (20165BCB19001), and Hong Kong Scholars Program (XJ2016058). References Alyafeai, A., & Böhm, V. (2018). In vitro bioaccessibility of carotenoids and vitamin E in rosehip products and tomato paste as affected by pectin contents and food processing. Journal of Agricultural and Food Chemistry, 66(15), 3801–3809. Arts, S. J. H. F., Mombarg, E. J. M., Bekkum, H. V., & Sheldon, R. A. (1997). Hydrogen peroxide and oxygen in catalytic oxidation of carbohydrates and related compounds. Synthesis, (06), 597–613 1997. Ashraf, W., Pfeiffer, R. F., Park, F., Lof, J., & Quigley, E. M. (1997). Constipation in Parkinson's disease: Objective assessment and response to psyllium. Movement Disorders Official Journal of the Movement Disorder Society, 12(6), 946–951. Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizingthe principle of protein-dye binding. Analytical Biochemistry, 72(1–2), 248–254. Buettner, G. R., & Jurkiewicz, B. A. (1996). Catalytic metals, ascorbate and free radicals: Combinations to avoid. Radiation Research, 145(5), 532–541. Buxton, G. V., Greenstock, C. L., Helman, W. P., & Ross, A. B. (1988). Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (˙OH/˙O-) in aqueous solution. Journal of Physical and Chemical Reference Data, 17, 513–769. Caffall, K. H., & Mohnen, D. (2009). The structure, function, and biosynthesis of plant cell wall pectic polysaccharides. Carbohydrate Research, 344(14), 1879–1900. Duarte, T. L., & Lunec, J. (2005). Review: When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C. Free Radical Research Communications, 39(7), 671–686. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350–356. Farahnaky, A., Askari, H., Majzoobi, M., & Mesbahi, G. (2010). The impact of concentration, temperature and pH on dynamic rheology of psyllium gels. Journal of Food Engineering, 100(2), 294–301. Faure, A. M., Andersen, M. L., & Nyström, L. (2012). Ascorbic acid induced degradation of beta-glucan: Hydroxyl radicals as intermediates studied by spin trapping and electron spin resonance spectroscopy. Carbohydrate Polymers, 87(3), 2160–2168. Feng, L., Yin, J., Nie, S., Wan, Y., & Xie, M. (2016). Fractionation, physicochemical property and immunological activity of polysaccharides from Cassia obtusifolia. International Journal of Biological Macromolecules, 91, 946–953. Fischer, M. H., Yu, N., Gray, G. R., Ralph, J., Anderson, L., & Marlett, J. A. (2004). The gel-forming polysaccharide of psyllium husk (Plantago ovata Forsk). Carbohydrate Research, 339(11), 2009–2017. Fry, S. C. (1998). Oxidative scission of plant cell wall polysaccharides by ascorbateinduced hydroxyl radicals. Biochemical Journal, 332, 507–515. Gong, L., Zhang, H., Niu, Y., Chen, L., Liu, J., Alaxi, S., et al. (2015). A novel alkali extractable polysaccharide from Plantago asiatic L. Seeds and its radical-scavenging and bile acid-binding activities. Journal of Agricultural and Food Chemistry, 63(2), 569–577. Guo, B., Yuan, Y., Wu, Y., Xie, Q., & Yao, S. (2002). Assay and analysis for anti- and prooxidative effects of ascorbic acid on DNA with the bulk acoustic wave impedance technique. Analytical Biochemistry, 305(2), 139–148. Haber, F., & Weiss, J. (1934). The catalytic decomposition of hydrogen peroxide by iron salts. Proceedings of the Royal Society of London, 147(147), 332–351. Hesarinejad, M. A., Sami Jokandan, M., Mohammadifar, M. A., Koocheki, A., Razavi, S. M. A., Ale, M. T., et al. (2018). The effects of concentration and heating-cooling rate on rheological properties of Plantago lanceolata seed mucilage. International Journal of Biological Macromolecules, 115, 1260–1266. Huang, D., Nie, S., Jiang, L., & Xie, M. (2014). A novel polysaccharide from the seeds of Plantago asiatica L. induces dendritic cells maturation through toll-like receptor 4. International Immunopharmacology, 18(2), 236–243. Hu, J. L., Nie, S. P., Min, F. F., & Xie, M. Y. (2012). Polysaccharide from seeds of Plantago asiatica L. increases short-chain fatty acid production and fecal moisture along with
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Interactions between ascorbic acid and water soluble polysaccharide from the seeds of Plantago asiatica L.: Effects on polysaccharide physicochemical properties and stability
T
Ou-Ye Lia, Li Wanga, Xiao-Ying Liua, Jun-Yi Yina,b,∗∗, Shao-Ping Niea,∗ a State Key Laboratory of Food Science and Technology, China-Canada Joint Lab of Food Science and Technology (Nanchang), Nanchang University, Nanchang, Jiangxi Province, 330047, China b Department of Applied Biology and Chemical Technology, The HongKong Polytechnic University, Kowloon, Hong Kong, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Plantago asiatica L. Polysaccharide Ascorbic acid Apparent viscosity Molecular weight Hydroxyl radical
Viscous polysaccharide is one kind of main bioactive components in Plantago asiatica L. seeds. This study aims to investigate effects of various monosaccharides, disaccharides, especially ascorbic acid (VC), on apparent viscosity and physicochemical properties of crude water soluble polysaccharide from the seeds of P. asiatica L. (PLWEP-C), as well as its stability and degradation mechanism. Results showed that small molecules such as glucose, sucrose, maltose, and lactose had little effects on apparent viscosity of PLWEP-C, while VC could significantly reduce the viscosity. There was no obvious change in FT-IR spectra and monosaccharide composition of PLWEP-C after VC addition. Hydroxyl radicals were probably involved in the degradation of polysaccharide induced by VC, which was confirmed by glucose inhibition test. SEM observation showed that PLWEP-C was mainly in lamellar and filamentous appearance, and no great change was generated by VC. The solution stability of PLWEP-C can only be kept well, when VC was at low concentration and low temperature. In conclusion, VC could significantly reduce the viscosity of polysaccharide from the seeds of P. asiatica L., which was probably attributed to the reduction of molecular weight. Hydroxyl radicals participation were probably one of main reasons in the degradation process.
1. Introduction Polysaccharide is a class of biopolymer widely existed in natural resources, including terrestrial and marine plants, as well as some exogenous metabolites of bacteria. It is a very important material, widely applied in food, cosmetic, pharmaceutical, biomedicine and other industries. Structure of the polysaccharide is complicated, because it is linked by the attachment to different monosaccharides through diverse glycosidic bonds (Caffall & Mohnen, 2009; Yang & Zhang, 2009). Usually, polysaccharide solution exhibits viscous property and forms gel, which is influenced by polymer concentration, molecular weight, degree of branching, functional groups, and other non-polysaccharide chemicals (Wang & Cui, 2005). Meanwhile, the viscosity of polysaccharide has a great influence on its physicochemical properties and functions. In food industry, the texture and processing conditions of the product are closely related to the viscosity of the polymer.
Both macromolecules and small molecules exist in the food system. The interaction between them is an interesting topic (Lin et al., 2017; Takemasa & Nishinari, 2016). Polysaccharide can also interact with small molecular compounds, such as vitamins (Alyafeai & Böhm, 2018; Rodrigues & de Oliveira, 2012), polyphenols (Wang, Liu, Chen, & Chen, 2017) and other chemical compounds (Menchicchi et al., 2015; Watrelot, Schulz, & Kennedy, 2017), thereby affects the quality and function of polysaccharides. Vitamin C (ascorbic acid, VC) exists in many vegetables and fruits, and is widely used as an antioxidant. However, it can also act as a pro-oxidant at low concentration (Buettner & Jurkiewicz, 1996; Kivelä, Nyström, Salovaara, & Sontagstrohm, 2009; Yen, Duh, & Tsai, 2002). Hydroxyl radicals can be produced by the decomposition of hydrogen peroxide in Fenton reaction against the presence of VC (Guo, Yuan, Wu, Xie, & Yao, 2002), which is catalyzed by transition metals (Arts, Mombarg, Bekkum, & Sheldon, 1997; Duarte & Lunec, 2005; Haber & Weiss, 1934). Therefore, polysaccharide is prone to degradation when they coexist. Recent studies have shown
∗
Corresponding author. Corresponding author. State Key Laboratory of Food Science and Technology, China-Canada Joint Lab of Food Science and Technology (Nanchang), Nanchang University, Nanchang, Jiangxi Province, 330047, China. E-mail addresses: [email protected] (J.-Y. Yin), [email protected] (S.-P. Nie). ∗∗
https://doi.org/10.1016/j.foodhyd.2019.105351 Received 13 July 2018; Received in revised form 29 August 2019; Accepted 29 August 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Effects of fructose (a), glucose (b), lactose (c), sucrose (d) and maltose (e) on apparent viscosity of 0.5% PLWEP-C.
that the viscosity reduction of β-glucan solutions containing VC was due to degradation of β-glucan (Faure, Andersen, & Nyström, 2012; Mäkelä, Sontag-Strohm, & Maina, 2015; Reetta, Fred, & Tuula, 2009). Polysaccharide from the seeds of Plantago is usually composed of arabinose (Ara) and xylose (Xyl), whose backbone is linked by β-1,4Xylp residues (Fischer et al., 2004; Gong et al., 2015; Yin, Lin, et al., 2012). It is highly branched, where most of the Ara residues are distributed in its side chains. Health benefits of this polysaccharide are widely accepted, including relieving constipation (Ashraf, Pfeiffer, Park, Lof, & Quigley, 1997), reducing abdominal pain in children with irritable bowel syndrome (Shulman et al., 2016), lowering plasma lipids (Rodriguez-Moran, 1998; Solà et al., 2010), improving immunity (Huang, Nie, Jiang, & Xie, 2014) and promoting short chain fatty acid
production (Hu, Nie, Min, & Xie, 2012; Yadav, Sharma, Kapila, Malik, & Arora, 2016). The polysaccharide is a kind of hydrophilic colloid, which has strong water swelling ability leading to the formation of the viscous suspension or a gel (Qian, Cui, Wang, Goff, & Smith, 2009; Yin, Nie, et al., 2012; Yu et al., 2017). The pH value of its solution was another important condition (Farahnaky, Askari, Majzoobi, & Mesbahi, 2010). Kale et al. (Kale, Yadav, & Hanah, 2016) have reported that high molecular weight and low viscosity soluble polysaccharides, including maltodextrin, gum arabic, locust bean gum, and corn bio-fiber gum, could produce significant decreases in the viscosity of psyllium husk suspensions. However, investigation on the effect of non-polysaccharides on viscosity and physicochemical properties of psyllium polysaccharide is rare. According to our previous studies on the 2
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defatted residue (200 g) was extracted twice with boiling water (2 L) for 3 h. Combined aqueous extracts were concentrated to 1 L, and ethanol (95%, v/v) was added until the final concentration was 80% (v/v). It was kept at 4 °C overnight, and then centrifuged (4800 rpm, 15 min). The precipitate was collected and lyophilized to get crude polysaccharide. The crude polysaccharide was dissolved at a concentration of 5.0 mg/mL and centrifuged (4800 rpm, 15 min) again to remove the precipitate. Finally, the supernatant was lyophilized to get P. asiatica L. water extracted polysaccharide, named as PLWEP-C. PLWEP-C was determined to contain 55.1% neutral sugar, 20.3% uronic acid and 4.0% protein. 2.3. Apparent viscosity and gelling property of PLWEP-C Apparent viscosity of polysaccharide solutions were measured by an ARES G-2 Rheometer (TA Instruments, New Castle, DE, USA) using cone-and-plate (40 mm diameter with a gap of 0.046 mm). Temperature of 25.0 °C was controlled by a SR5 Peltier Circulator (TA Instruments, New Castle, DE, USA).
Fig. 2. Effect of VC on apparent viscosity of 0.5% PLWEP-C.
2.3.1. Apparent viscosity PLWEP-C or commercial arabinoxylan solution was dissolved in ultra-pure water at 60 °C. Then, VC was added into the polysaccharide solution at concentrations of 0.025, 0.05, 0.1 and 0.2% (w/v), successively. The final concentration of polysaccharide was kept at 0.5% (w/ v). At the same time, glucose was introduced to the study as a competitive hydroxyl radical scavenger (Buxton, Greenstock, Helman, & Ross, 1988), whose concentration was selected to be 1 M (appr. 20%, w/w). The effects of other small molecules including glucose, fructose, lactose, sucrose, and maltose on apparent viscosity of PLWEP-C were evaluated in the similar way. The concentrations of these small molecules were chosen as 0.025, 0.05, 1.0, 2.0 and 4.0%, successively. 2.3.2. Gelling property To determine the gelling properties of PLWEP-C before and after VC addition, PLWEP-C was completely dissolved in water. VC was added into the polysaccharide solution at final concentrations of 0.1, 0.2, 0.4 and 0.8% (w/v), successively. The final concentration of PLWEP-C was kept at 2.0% (w/v). The mixture was kept at 20 °C for more than 12 h. The range of frequency was 0.1-20 Hz at strain of 2% in the linear viscoelastic region.
Fig. 3. Effect of VC on apparent viscosity of 1% commercial arabinoxylan.
polysaccharide from seeds of P. asiatica L., we have found that some small molecular compounds have a unique influence on its viscosity. Therefore, this study aims to report different effects of monosaccharides, disaccharides, and ascorbic acid on physicochemical properties of water soluble polysaccharide from the seeds of P. asiatica L., which would guide the application.
2.4. Stability observation The effects of VC on stability of PLWEP-C at room temperature (23 °C) and low temperature (4 °C) were compared. The concentrations of VC were chosen as 0.025, 0.05, 0.1 and 0.2% (w/v), successively. Final concentration of PLWEP-C was kept at 0.5% (w/v), which contained 0.02% of NaN3 for bacterial inhibition. Photos of each sample were collected at different times during the storage period. Meanwhile, samples of PLWEP-C stored at 23 °C for 20 days was chosen to determine protein content (Bradford, 1976) and monosaccharide composition (Liu et al., 2017) of the precipitate, which was collected by centrifugation (4800 rpm, 15 min, twice).
2. Materials and methods 2.1. Materials and chemicals Seeds of P. asiatica L. were collected in November 2013 from Ji'an, Jiangxi Province, China. They were kept under dry conditions in dark until June 2015 for polysaccharide preparation. VC (≥99.0%), arabinoxylan (95%), and glucose (≥99.5%) were obtained from SigmaAldrich Shanghai Trading Co. Ltd (Shanghai, China). Other chemicals and reagents were of analytical grade.
2.5. Morphology observation by SEM The solid morphology of polysaccharide was analyzed by scanning electron microscopy (SEM). At first, polysaccharide solutions were prepared at the concentration of 0.5% which contained 0.025, 0.05, 0.1 and 0.2% of VC, successively. Then, each of them was diluted by 10 times and placed in liquid nitrogen for pre-freezing for 30 min. Finally, they were transferred to a freezer and lyophilized before test. The dried samples were placed on a sample stage, and coated with a thin layer of platinum. A JSM-6701F SEM (Jeol Company, Tokyo, Japan) was used
2.2. Polysaccharide preparation The preparation process of polysaccharide from the seeds of P. asiatica L. was conducted according to earlier report (Yin, Nie, Chao, Yin, & Xie, 2010) with some modifications. Primarily, the seeds of P. asiatica L. were defatted with ethanol (95%, v/v) and dried. Then, the 3
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Fig. 4. Inhibition effects of glucose addition on apparent viscosity of arabinoxylan added with different concentrations of VC.
Fig. 5. Effect of VC on storage modulus (G′) and loss modulus (G″) of PLWEP-C at 25.0 °C. Strain used in all the experiments was within the linear viscoelastic region where the gel structure was not damaged.
Fig. 7. Effect of VC on molecular weight of PLWEP-C determined by HPGPC. All the samples after the treatment with VC were dialyzed against distilled water, and freeze-dried. Polysaccharide concentration was chosen to be 0.5 mg/mL before membrane filtration for HPGPC analysis. Fig. 6. Effect of VC on FT-IR spectra of PLWEP-C. All the samples after the treatment with VC were dialyzed against distilled water, and freeze dried.
4
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chromatographic system. 2.6.3. Monosaccharide composition analysis Monosaccharide composition analysis was carried out according to the method reported by Liu et al. (Liu et al., 2017) with some modifications. Polysaccharide sample (5.0 mg) was hydrolyzed with 0.5 mL of 12 M H2SO4 in the iced water bath for 30 min, then diluted by water (2.5 mL) and hydrolyzed for another 2 h in a silicone oil bath (100 °C). The hydrolysate was diluted and analyzed by high performance anionexchange chromatography (Dionex ICS-5000, Thermo Fisher Scientific Massachusetts, USA) coupled with pulsed amperometric detection (PAD). A CarboPac™ PA20 guard column (4 mm × 50 mm) and a CarboPac™ PA20 analytical column (4 mm × 250 mm) were used. A Chromeleon 6.8 (Thermo Fisher Scientific, Massachusetts, USA) software was used for data collection and processing.
Fig. 8. SEM images of PLWEP-C (0.5%) added with 0.025, 0.05, 0.1 and 0.2% of VC. All the samples were completely mixed with VC at certain concentrations, freeze-dried and observed at magnification of 1000. The final concentration of polysaccharide in each group was kept at 0.05%.
3. Results and discussions 3.1. Effects of small molecules on apparent viscosity of PLWEP-C
for images collection at an accelerating voltage of 5 kV.
Fructose, glucose, lactose, sucrose and maltose were commonly used carbohydrates in food industry. Fig. 1 shows the effects of monosaccharides and disaccharides at concentrations from 0.025 to 4.0% (w/v) on apparent viscosity of PLWEP-C. PLWEP-C at the concentration of 0.5% displayed shear-thinning behavior (pseudoplastic) behavior. It can be found that fructose, glucose, lactose, sucrose and maltose had little effects on apparent viscosity of PLWEP-C at tested concentrations. VC is widely applied as antioxidant. Fig. 2 shows the effect of VC on apparent viscosity of PLWE-C. VC could reduce the apparent viscosity of PLWEP-C. When the concentration of added VC was 0.05%, PLWEP-C had the lowest viscosity. As the concentration of VC was over 0.05%, the viscosity of PLWEP-C increased with increasing of concentration of VC. When VC was removed from the polysaccharide after dialysis, the viscosity could not be recovered (data not shown). The viscosity of commercial arabinoxylan was much lower than PLWEP-C, while effect of VC on apparent viscosity of commercial arabinoxylan (Fig. 3) was similar to that of PLWEP-C. The greatest reductions in viscosity of polysaccharide (Figs. 2 and 3) were observed at the low concentrations of VC (0.025 and 0.05%), with progressively smaller reductions as the concentration was increased, until ultimately, reductions as the concentration of VC was increased to the highest value used (0.4%). VC has a multiplicity of antioxidant properties, but it can exert pro-oxidant effects at low concentration
2.6. Effect of VC on physicochemical properties of PLWEP-C after dialysis Polysaccharide solutions (0.5%, w/v) treated by VC at concentrations of 0.025, 0.05, 0.1 and 0.2% (w/v) were kept under stirring for 24 h, then dialyzed against ultra-pure water for three days to remove the excess VC in the mixture. After that, the samples were freeze-dried and applied for apparent viscosity, FT-IR spectrum, morphology, molecular weight, and monosaccharide composition analyses. 2.6.1. FT-IR observation FT-IR spectra of PLWEP-C and VC treated samples were recorded from 4000 cm−1 to 500 cm−1 by a Thermo Nicolet 5700 infrared spectrograph. Samples were dried prior to tableting with KBr powder. 2.6.2. Molecular weight determination by HPGPC Molecular weights of PLWEP-C and VC treated samples were detected by the high performance gel permeation chromatography (HPGPC) according to the previous reports (Feng, Yin, Nie, Wan, & Xie, 2016; Wang et al., 2017). Ultra-pure water containing 0.02% (w/v) NaN3 was used as mobile phase in the current study. Samples and dextran standards (1.0 mg/mL) dissolved in mobile phase were passed through a 0.22 μm membrane filter and then injected into the
Fig. 9. SEM images of PLWEP-C (0.5%) added with 0.025, 0.05, 0.1 and 0.2% of VC. All the samples after the treatment with VC were dialyzed against distilled water, freeze-dried and observed at magnification of 1000. The final concentration of polysaccharide in each group was kept at 0.05%. 5
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Fig. 10. Stability of 0.5% PLWEP-C added with different concentrations of VC under the temperature of 4 °C. All the solutions contain 0.02% NaN3 for bacterial inhibition. Number of 1, 2, 3, and 4 shown in the figure in black font represents the concentration of added VC was 0.025, 0.05, 0.1and 0.2%, successively.
Fig. 11. Stability of 0.5% PLWEP-C added with different concentrations of VC under the temperature of 23 °C. All the solutions contain 0.02% NaN3 for bacterial inhibition. Number of 1, 2, 3 and 4 shown in the figure in black font represents the concentration of added VC was 0.025, 0.05, 0.1 and 0.2%, successively. Table 1 Chemical component and monosaccharide composition of the precipitation from PLWEP-C and ascorbic acid treated samples (n = 3, mean ± SD). Precipitation from the solution
Protein (%,w/w)
Monosaccharide composition (%, w/w) Ara
PLWEP-C PLWEP-C+0.025%VC PLWEP-C+0.05%VC PLWEP-C+0.1%VC PLWEP-C+0.2%VC
n.d. n.d. n.d. n.d. n.d.
6.07 8.98 7.25 6.62 6.14
Gal ± ± ± ± ±
0.05 0.27 0.10 0.08 0.03
0.02 0.72 0.32 0.11 0.09
Xyl ± ± ± ± ±
0.01 0.01 0.02 0.02 0.01
28.01 32.05 28.67 27.08 28.96
Gala ± ± ± ± ±
1.35 1.01 1.26 0.40 0.19
3.76 4.17 4.65 3.75 3.73
Ara/Xyl ± ± ± ± ±
0.21 0.40 0.24 0.12 0.28
0.22 0.28 0.25 0.24 0.21
n.d., not determined.
Glucose was considered as a competitive hydroxyl radicals scavenger (Buxton et al., 1988; Reetta et al., 2009). Therefore, it was added into the solution combining with VC and arabinoxylan. Different concentrations of VC and glucose (1 M, 20 w/w %) were sequentially added to the arabinoxylan. The results (Fig. 4) showed that glucose could effectively inhibit the viscosity decrease caused by the addition of VC. It indicated that the hydroxyl radicals induced degradation of polysaccharide was inhibited by the addition of glucose. It was similar to viscosity change for PLWEP-C when glucose was added into the solution containing VC.
(Buettner & Jurkiewicz et al., 1996; Kivelä et al., 2009; Yen et al., 2002). It was probably because of the antioxidant property of VC at higher concentration, and pro-oxidant effect at low concentration. We have taken another test to reverse verify it. Hydroxyl radicals, one specific species of reactive oxygen species, have been established to scission polysaccharides (Fry, 1998). Fenton reaction, which is typically present in the aqueous phases of food, can produce aggressive hydroxyl radicals (Arts et al., 1997; Duarte & Lunec, 2005; Haber & Weiss, 1934; Slavin, Martini, Jacobs, & Marquart, 1999; Wardman & Candeias, 1996). In certain conditions, VC behaves as a pro-oxidant initiating the Fenton reaction due to the reducing capacity (Reactions 1 and 2), then produces aggressive hydroxyl radicals (Reaction 3). AH2 + 2Cu2+/Fe3+→ A + 2H+ + 2Cu+/Fe2+
(1)
AH2 + O2 → A + H2O2
(2)
Cu+/Fe2+ + H2O2 →˙OH + OH− + Cu2+/Fe3+
(3)
3.2. Effect of VC on viscoelastic properties of PLWEP-C Previous study (Hesarinejad et al., 2018; Yin, Nie, et al., 2012) showed that water extracted polysaccharide from the seeds of P. asiatica L. had weak-gel like structure at high concentration, as values of storage modulus (G′) were higher than those of loss modulus (G″) during 6
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observed. At the same time, PLWEP-C was firstly added with different concentrations of VC, then dialyzed against distilled water to remove those small molecules. Their morphological appearance was similar to each other. Much more flaky and curly aggregates appeared for the polysaccharide treated higher concentration of VC, but all samples contained linear morphology.
the whole experimental range. Effect of VC on viscoelastic properties of PLWEP-C was carried out at high polysaccharide concentration (2.0%), with the concentration ratios of VC to PLWEP-C fixed at 20, 10, 5 and 2.5, successively. Mechanical spectra of all samples (Fig. 5) showed that G′ was greater than G″ throughout the tested frequency range and they were both frequency dependent, suggesting the tested samples exhibited weak-gel structure. VC could reduce G′ values of polysaccharide, which was much lower than the original one in most cases except the additional concentration of VC reaching 0.8%. The gel strength was the lowest when VC concentration was 0.1%. With increasing concentration of VC, the gel strength increased.
3.6. Effect of VC on stability of PLWEP-C solution Stability of polysaccharide solution added with other compounds is an important issue, which affects its application. Therefore, effects of VC on stability of PLWEP-C solution at different storage conditions were investigated (Figs. 10 and 11). The original polysaccharide solution was in gray and a little yellow color. It was stable during the tested period both at 4 °C and 23 °C. The polysaccharide containing VC was much more stable at 4 °C. There was some precipitation observed when PLWEP-C containing VC was kept at 23 °C for 10 days, even when the concentration of VC was only 0.025%. We have determined the basic chemical composition of the precipitation from PLWEP-C and VC interaction, for 20 days at temperature of 23 °C. The results of the chemical component and monosaccharide composition are shown in Table 1. No protein was determined. We also determined the monosaccharide composition of precipitation for PLWEP-C and VC treated samples. The ratio of Ara/Xyl was similar to that of the untreated sample.
3.3. FT-IR observation Fig. 6 shows the effect of VC treatment on the structure of PLWEP-C by FT-IR. The strong and broad peak at around 3415 cm−1 can be assigned to stretching vibrations of hydroxyl groups. The weak peak at around 2926 cm−1 belonged to the stretching vibration of methyl group (Gong et al., 2015). Absorption band at 1730 cm−1 indicated the presence of uronic acid, while absorption at 1611 cm−1 and 1424 cm−1 arose from asymmetric and symmetric stretching vibrations of carboxylate were generated by carboxylic acid (Yin, Nie, Zhou, Wan, & Xie, 2010). A strong absorption at 1043 cm−1 was due to stretching vibration of the pyranose ring. An IR band at 899 cm−1 was the characteristic absorption of β-anomeric configuration (Yang & Zhang, 2009). It was obvious that all the above mentioned absorption peaks could be found in the polysaccharide before and after VC treatment, indicating the reservation of structural characteristics.
4. Conclusions Our study showed that the apparent viscosity of PLWEP-C could be changed by adding VC. When it was added at lower concentration, the apparent viscosity decreased significantly, then increased with the increase of VC concentration. Structural analysis by FT-IR and monosaccharide composition analysis indicated there was no significant difference in structural features between PLWEP-C and VC treated samples. SEM showed that the morphology also did not change a lot. However, great reduction was found in molecular weight of PLWEP-C after the treatment with VC. Change in viscosity of PLWEP-C might be attributed to the reduction of molecular weight. Stability of PLWEP-C solution decreased when VC was added. Macromolecules are susceptible to degradation induced by oxygen species, especially hydroxyl radicals, which belong to reactive oxygen species and can oxidize bio-macromolecules. In recent years, many researches on hydroxyl radicals degraded carbohydrates have been reported (Majzoobi, Radi, Farahnaky, & Tongdang, 2012; Wu et al., 2016). It was found that VC had degradation effects on wheat starch molecules especially after gelatinization (Majzoobi et al., 2012). Reetta et al. (Reetta et al., 2009) found that polysaccharides were sensitive to hydroxyl radicals induced depolymerization, and these radicals can be produced in cereal food systems. Reetta et al. (Reetta, Henniges, Sontag-Strohm, & Potthast, 2012) have further studied the β-glucan solution added with VC, which demonstrated that the β-glucan chain was oxidized during the addition of VC and posed a risk to the functionality of β-glucan in solution. Mäkelä et al. (Mäkelä et al., 2015) have proved that the oxidation of barley β-glucan resulted in significant degradation via glycosidic bond or cross-ring cleavage, which decreased both molecular weight and viscosity (Mäkelä et al., 2015). In this study, glucose was used as a competitive hydroxyl radical scavenger. The results showed that glucose could effectively inhibit the viscosity decrease of PLWEP-C caused by the addition of VC. It indicated that the hydroxyl radicals induced degradation of arabinoxylan by VC was inhibited by the addition of glucose. However, whether there are any changes of glycosidic bonds and which kinds of free radicals involved in the reaction should be further explored. In addition, functional properties of the polysaccharides before and after VC treatment should also be investigated. It would be helpful in
3.4. Molecular weight and monosaccharide composition Molecular weight distributions of PLWEP-C and VC treated samples are shown in Fig. 7. The weight-average molecular weight of PLWEP-C was 6.4 × 106, while those of PLWEP-C (0.5%) treated with 0.025, 0.05, 0.1 and 0.2% VC were 3.1 × 106, 3.2 × 106, 2.7 × 106, and 3.9 × 106, respectively. VC could reduce the weight-average molecular weight. This result was inconsistent with the change of apparent viscosity, as the molecular weight did not decrease with the decrease of VC concentration. Since HPGPC required membrane filtration before injection, we speculated that some samples were trapped during the filtration process, resulting in the loss of some polysaccharide fractions. Therefore, xylose was used as a standard to determine the polysaccharide content in the solution after filtration by colorimetric method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). Only 26.2% of polysaccharide in PLWEP-C was retained after filtration, which were 82.6%, 81.1%, 73.3%, and 63.1% for PLWEP-C treated with 0.025, 0.05, 0.1 and 0.2% VC, successively. It indicated that the lowest molecular weight of PLWEP-C treated with 0.1% VC was probably because of its lower mass retention rate than 0.025% and 0.05% VC treated samples. According to monosaccharide composition analysis results, PLWEPC was confirmed to contain Rha (0.66%), Ara (9.40%), Gal (1.96%), Glc (0.58%), Xyl (32.64%), GalA (2.07%) and GlcA (6.00%), which was similar to our previous reports (Liu et al., 2017). After VC treatment, there was no significant difference among the samples. 3.5. Morphology observation by SEM SEM images of PLWEP-C treated with VC before and after dialysis are shown in Figs. 8 and 9, respectively. The particles of PLWEP-C were irregular in shape. Some of them were in linear style, while others were flaky curly aggregates. When VC was added into PLWEP-C, the polysaccharide morphology changed a little, but not too much. A little more linear appearance was found in the polysaccharide when it was added with 0.025, 0.05 or 0.1% of VC. The appearance of PLWEP-C containing 0.2% VC was condensed, although some linear style could still be 7
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understanding the interactions between the polysaccharide from P. asiatica L. and VC, and promoting the development of polysaccharides in food and pharmaceutical industries.
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