Article pubs.acs.org/JAFC

Grafting of Gallic Acid onto Chitosan Enhances Antioxidant Activities and Alters Rheological Properties of the Copolymer Minhao Xie, Bing Hu, Yan Wang, and Xiaoxiong Zeng* College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China ABSTRACT: A new, simple, and effective method to graft gallic acid (GA) onto chitosan (CS) in aqueous solution in the presence of carbodiimide and hydroxybenzotriazole was developed. The grafting amount of GA reached as much as 209.9 mg/g of copolymer, which appears as the highest one among the reported literature, and the grafting degree of GA to CS was adjustable with modulation of the mass ratio of GA to CS. The covalent insertion of GA onto the polymeric backbones was confirmed by UV−vis and 1H NMR analyses. Grafting endowed the resulting copolymer GA-grafted-CS (GA-g-CS) with both the advantages of CS and GA. The antioxidant capacity of GA-g-CS was much higher than that of the plain CS examined by assays of DPPH, superoxide, and ABTS radicals scavenging activities, reducing power, chelating power, inhibition of lipid peroxidation, ferric reducing antioxidant potential, and β-carotene-linoleic acid assays. Particularly, GA-g-CS showed significantly higher antioxidant activity than GA in β-carotene-linoleic acid assay. Furthermore, the viscosity of GA-g-CS was significantly higher than that of CS. The present study developed a novel approach to synthesize GA-g-CS that could be a potential biomaterial in food industries. KEYWORDS: chitosan, gallic acid, grafting, antioxidant, rheological property

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biomedical, food, and chemical industries.22,23 In the chemical structure of CS, there are three types of reactive functional groups: amino groups and primary and secondary hydroxyl groups at C-2, C-3, and C-6 positions, respectively,24 which enable the grafting of a large variety of properly functionalized molecules to CS possible. 25 Therefore, numerous CS derivatives developed through grafting reactions, such as trimethylated, N-succinylated, thiolated, azidated, sugar-modified, cyclodextrin-linked, crown-ether-bound, and enzymatic modified CS, as well as their applications have been reported.26 As grafting phenolic compound onto polymer, it can be achieved through enzyme- or chemical reagent-mediated ways. The most widely used enzymes involved in such reactions are laccase,27 tyrosinase,28 and horseradish peroxidase.29 For the chemical reagents-mediated way, it mainly contains grafting procedures via free radicals or carbodiimides. Gallic acid (GA, 3,4,5-trihydroxy benzoic acid), an endogenous plant polyphenol, is found abundantly in tea, grapes, berries, and other fruits, as well as in wine. It exhibits a variety of biological activities such as antioxidant, antiinflammatory, and anticancer effects.30 These functions make it widely used in foods, drugs, and cosmetics. It is a useful approach to graft GA onto CS to reduce the intra- and intermolecular hydrogen bond networks and increase the antioxidant ability of CS.16 Although, the methods mentioned above are valid in grafting GA onto CS, they have certain deficiencies. Enzymatic grafting reactions depend on the oxidation of phenolic hydroxyl groups, which may damage the activities of the phenolic compounds. In accordance with

INTRODUCTION Grafting is an attractive approach to impart a variety of functional groups to a polymer, wherein monomers are covalently bonded onto the polymer chain.1 It allows for the development of new materials, combining the advantages of both grafted molecules and polymers and endowing them new characteristics for specific applications.2 Recently, synthesis of antioxidant-polymer conjugates by grafting of antioxidant molecules onto polymer chains has received much attention. It has been reported that the grafting of phenolic compounds can provide the polymers with new characteristics, such as enhanced mechanical and physiological properties,3 as well as the alternations in solubility, antioxidant, and emulsifying properties.4 Furthermore, the phenolic compounds grafted polymers exhibited amplified bioactivities compared to the corresponding free phenolic compounds. For example, the poly(ε-lysine)-catechin conjugate showed greatly improved inhibitory effects on the activities of disease-related enzymes than catechin monomers,5 and the gelatin-catechin conjugate exhibited an amplified inhibitory effect on the oxidation of low density lipoprotein.6 The novel antioxidant-biomacromolecule conjugates could be employed as new food additives, packing materials, and even functional foods. Phenolic compounds extracted from plants, including gallic acid, catechin, caffeic acid, tannic acid, ferulic acid, and quercetin have been grafted to biomacromolecules, such as chitosan (CS) and its derivatives, gelatin, alginate, and inulin.2,7−16 The synthesized antioxidant-polymer conjugates showed diverse bioactivities, including antioxidant,17 anticancer,18 and inhibitory effects on digestive enzymes,19 βsecretase,20 and food-borne pathogens.21 CS, a natural copolymer of β-(1 → 4)-linked D-glucosamine and N-acetylD-glucosamine produced by deacetylation of chitin, is currently receiving considerable attention for its potential applications in © 2014 American Chemical Society

Received: Revised: Accepted: Published: 9128

July 6, 2014 August 31, 2014 August 31, 2014 September 1, 2014 dx.doi.org/10.1021/jf503207s | J. Agric. Food Chem. 2014, 62, 9128−9136

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GA-g-CS I, II, and III, were prepared by changing the ratio of GA and EDC. Characterization of GA-g-CS. Structural characterization of GAg-CS was performed by UV−vis and NMR analysis. The UV−vis spectra were determined by a UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan) by scanning from 200 to 600 nm. 1H NMR spectra were recorded at 25 °C with samples dissolved in CD3COOD/D2O (v/v, 1%) by using AVANCE III 500 MHz NMR Spectrometer (Bruker, Switzerland). Determination of Phenolic Content. Phenolic content was determined according to the previous reported method.33 Briefly, 0.5 mL of GA-g-CS solution was mixed with 1.0 mL of Folin-Ciocalteau reagent for 5 min in the dark, followed by the addition of 2.0 mL 20% sodium carbonate (Na2CO3) solution. The mixture was shaken and kept at 30 °C for 1 h, absorbance (Abs) of which was measured at 747 nm by using a 722S Visible Spectrophotometer (Shnghai Jinghua Science & Technology Instruments Co., Ltd., China). GA was used as a standard. The grafting ratios of GA-g-CS were expressed as milligram GAE per gram of copolymer (mg GAE/g). Assay of Antioxidant Activity of GA-g-CS in Vitro. Assay of Scavenging Activity on DPPH Free Radicals. DPPH free radicals scavenging activity was determined according to the reported procedure34 with slight modifications. Briefly, GA, CS, and GA-g-CS (I, II, and III) were homogeneously dispersed in deionized water for preparation of a series of concentrations (0.125, 0.25, 0.5, 1.0, 1.5, and 2.0 mg/mL). The solutions were pipetted into a 96-well plate (50 μL/ well), and to each well, 200 μL of methanolic DPPH solution (0.4 mM) was added and mixed. After a 30 min reaction at room temperature in the dark, the Abs at 517 nm was measured by a microplate reader (BioTek Instruments Inc., Winooski, VT).

previous studies, the grafting substitution by chemical reagents is also limited. The reported maximum grafting ratio of GA onto CS was 128.3 mg GA equivalents (GAE)/g via free radicals generated by ascorbic acid and the hydrogen peroxidemediated method.19 For 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS)-mediated activated ester method, the maximum grafting ratio of GA onto CS was only about 65 mg GAE/g CS.31 Furthermore, the rheological potential of GA-grafted-CS (GA-g-CS) compared to free GA has seldom been studied. Therefore, the objectives of this work are to establish an efficient method to graft large amounts of GA onto CS and to study the influence of grafted GA on CS, especially the antioxidant activities and rheological properties. In the present study, a new synthetic method to implant GA to CS with significantly improved graft ratio was developed, in which CS was dissolved, assisted with hydroxybenzotriazole (HOBt), and the synthesized GA-g-CS was characterized by UV−vis spectroscopy and proton nuclear magnetic resonance (1H NMR) spectroscopy. Then, GA-g-CS with different degrees of substitution were prepared and investigated systematically with various in vitro antioxidant assays, including assays of scavenging activities on 2,2-diphenyl1-picrylhydrazyl (DPPH), superoxide, and 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, reducing power, chelating power, inhibition of lipid peroxidation, ferric reducing antioxidant potential, and β-carotenelinoleic acid assay. Finally, the viscosities of GA-g-CS with different grafting ratios were investigated.

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DPPH free radicals scavenging activity (%) ⎛ Abs1 − Abs2 ⎞ = ⎜1 − ⎟ × 100 Abs0 ⎝ ⎠

MATERIALS AND METHODS

Materials and Reagents. CS (viscosity-average molecular weight ∼1.5 × 105, degree of deacetylation ≥90.0%), EDC hydrochloride (EDC-HCl), Folin-Ciocalteau reagent were purchased from Kayon Biological Technology Co., Ltd. (Shanghai, China). GA, nitrotetrazolium blue (NBT) chloride, and HOBt were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). DPPH, βcarotene, linoleic acid, ABTS, phenazine methosulfate (PMS), 2,4,6tris(2-pyridyl)-s-triazine (TPTZ) were purchased from Sigma Chemical Co. (St. Louis, MO). Ferrozine (3-(2-pyridyl)-5,6diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt) was purchased from Aladdin Industrial Inc. (Shanghai, China). Reduced nicotinamide adenine dinucleotide disodium salt (NADH) was purchased from Roche Ltd. (Basel, Switerzaland). Deuterium oxide (D2O) and acetic acid-d4 (CD3COOD) for NMR were purchased from J&K Scientific Ltd. (Beijing, China). All of other reagents were of analytical grade. Synthesis of GA-g-CS. The synthesis of GA-g-CS was performed based on the reported one-pot method32 with some modifications. CS (0.303 g, 1.85 mmol) was stirred in deionized water (30.0 mL) with HOBt (0.282 g, 1.85 mmol) overnight until a clear solution was obtained. GA (0.311 g, 1.85 mmol) was introduced into the CS solution followed by the dropwise addition of an alcoholic solution of EDC-HCl (0.355 g, 1.85 mmol, 2.0 mL). The reaction was carried out for 24 h in ambient temperature and atmosphere. The resultant liquid was poured into dialysis tubes (MWCO 3500 Da) and dialyzed against deionized water for 48 h with eight changes of deionized water. The resulting solution was lyophilized to afford solid copolymer of GA-gCS. Thin layer chromatography (TLC) was performed to verify whether free GA was removed from the product of GA-g-CS. Briefly, silica plates with applied CS, GA, and GA-g-CS were developed with acetic acid−water−n-butanol (v/v/v, 1:4:5) as the mobile phase at laboratory temperature. After development and air-drying, the spots on the silica plate were detected by using iodine vapor. Blank CS, acting as a control, was prepared in the same conditions but in the absence of GA. GA-g-CS with different substitutions of GA, named as

where Abs0 is the Abs of the control (water instead of sample), Abs1 is the Abs of the sample, and Abs2 is the Abs of the sample under identical conditions as Abs1 with methanol instead of DPPH solution. Evaluation of Reducing Power. Reducing powers of the samples were determined by the previously described method35 with some modifications. The reactions were carried out in 96-well microplates, each well of which contained a mixture of 50 μL of sample solution, sodium phosphate buffer (PBS, 0.2 M, pH 6.6), and K3Fe(CN)6 solution (1%, w/v), and were incubated at 50 °C for 20 min. After the addition of 50 μL of trichloroacetic acid (10%, w/v) and 30 μL of FeCl3 (0.1%, w/v), the Abs was measured at 700 nm.

reducing power = Abs1 − Abs2 where Abs1 is the Abs of sample and Abs2 is the Abs of the sample under identical conditions as Abs1 with water instead of FeCl3 solution only. Assay of Scavenging Activity on Superoxide Radicals. Superoxide radicals scavenging activities of the samples were evaluated according to the literature35 with some modifications. NADH, NBT, and PMS were diluted in PBS (0.1 M, pH 7.4). The mixture of 50 μL of sample, NADH (468 mM), NBT (156 mM), and PMS (60 mM) was added in the well of the microplate and incubated at 25 °C for 5 min, and then the Abs was measured at 560 nm. superoxide radicals scavenging activity (%) ⎛ Abs1 − Abs2 ⎞ = ⎜1 − ⎟ × 100 Abs0 ⎝ ⎠ where Abs0 is the Abs of the control (water instead of sample solution), Abs1 is the Abs of the sample, and Abs2 is the Abs of the sample under identical conditions as Abs1 with 0.1 M phosphate buffer instead of NBT solution. Assay of Metal Chelating Ability. The metal chelating ability for Fe2+ was tested according to the reported method34 with some 9129

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Figure 1. Proposed mechanism for grafting gallic acid onto chitosan. modifications. Briefly, 50 μL of sample, 30 μL of FeCl2 (0.3 mM), and 120 μL of ferrozine (0.3 mM) solutions were mixed and incubated at 25 °C for 10 min, and then Abs at 562 nm was determined.

solution (20 mM) together. The mixture containing 40 μL of sample and 200 μL of FRAP solution was incubated at 25 °C for 10 min, and the Abs was detected at 593 nm. Assay of Scavenging Activity on ABTS Free Radicals. The preformed radicals of ABTS were generated by oxidation of ABTS (7.0 mM) with potassium persulfate (K2S2O8, 4.95 mM) for 12 h in the dark at room temperature.33 After generation, ABTS was diluted with PBS (0.2 M, pH 7.4) to an appropriate concentration, affording the working solution. The Abs of a mixture of a 20 μL sample and a 200 μL working solution was measured at 734 nm.

⎛ Abs1 − Abs2 ⎞ chelating activity (%) = ⎜1 − ⎟ × 100 Abs0 ⎝ ⎠ where Abs0 is the Abs of the control (water instead of sample), Abs1 is the Abs of the sample, and Abs2 is the Abs of the sample under identical conditions as Abs1 with deionized water instead of FeCl2 solution. Assay of Lipid Peroxidation Inhibition. The lipid peroxidation inhibitory effect was determined by thiobarbituric acid reactive substances (TBARS) assay.34 First, 1.0 g of mouse liver was homogenated in 100.0 mL purified water to afford lipid-rich media. Then, 0.2 mL of sample was mixed with 1.0 mL of liver homogenate, and 50 μL FeCl2 solution (0.1 mM) and H2O2 (0.1 mM) were added. After incubation at 37 °C for 1 h, the peroxidizing reaction was quenched by the addition of 0.3 mL of trichloroacetic acid (20%, w/v) and thiobarbituric acid (0.8%, w/v). The resulting mixture was incubated at 100 °C for 15 min and then centrifuged (4000 rpm, 10 min), and 200 μL of the supernatant was added to the well of the 96well microplate. Abs was measured at 532 nm.

ABTS free radicals scavenging activity (%) ⎛ Abs1 − Abs2 ⎞ = ⎜1 − ⎟ × 100 Abs0 ⎝ ⎠ where Abs0 is the Abs of the control (water instead of sample), Abs1 is the Abs of the sample, and Abs2 is the Abs of the sample only (PBS instead of ABTS). β-Carotene-Linoleic Acid Assay. GA-g-CS inhibiting peroxidation in a linoleic acid model was determined by a β-carotene bleaching test15 with some modifications. Briefly, 2.0 mL of β-carotene solution (1.0 mg/mL in chloroform) was added to 40 mg of linoleic acid and 400 mg of Tween 20. The mixture was evaporated by a rotary evaporator at 40 °C to remove chloroform. After that, the mixture was immediately diluted with 200.0 mL of distilled water and followed by ultrasonication to form an emulsion. The emulsion (1.25 mL) was transferred to the microcentrifuge tube containing 50 μL of sample. The tubes were then shaken gently and incubated at 45 °C for 1 h. The Abs of resulting liquid was measured at 470 nm by using the microplate reader.

lipid peroxidation inhibitory effect (%) ⎛ Abs1 − Abs2 ⎞ = ⎜1 − ⎟ × 100 Abs0 ⎝ ⎠ Where Abs0 is the Abs of the control (water instead of sample), Abs1 is the Abs of the sample, and Abs2 is the Abs of the sample only (water instead of liver homogenate). Assay of Total Antioxidant Capacity. The ferric reducing antioxidant potential (FRAP) was used to determine the total antioxidant capacity according to the previous procedure34 with some modifications. The FRAP working solution was obtained by mixing 100.0 mL of sodium acetic buffer (0.3 M, pH 3.6), 10.0 mL of TPTZ (10 mM, dissolved in 40 mM HCl), and 10.0 mL of FeCl3

⎛ Abs0 − Abs60 ⎞ ⎟ × 100 antioxidant activity (%) = ⎜1 − 0 Abs00 − Abs60 ⎝ ⎠ where Abs0 and Abs00 are the Abs measured at the initial incubation time for samples and control, respectively, whereas Abs60 and Abs060 are 9130

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the Abs of the samples and control, respectively, after incubation for 60 min. Analysis of Rheological Property. Rheological behavior of sample was measured on a MCR 301 Rheometer (Anton Paar GmbH, Austria) at a temperature of 20 °C. The samples, CS and GA-g-CS (I, II, and III), were dissolved in acetic acid solution (v/v, 1%) to a concentration of 1% (m/v). The viscosity was measured as a function of increasing shear rate, varying from 2 to 256 s−1. The viscosity at every point was recorded five times, and each sample was tested in triplicate. Statistical Analysis. Data were expressed as the mean ± standard deviation (SD) of triplicates. The least significant difference (LSD), Duncan’s multiple range test, and one-way analysis of variance (ANOVA) were used for multiple comparisons by SPSS 16.0. The difference was considered to be statistically significant if p < 0.05.

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RESULTS AND DISCUSSION Synthesis of GA-g-CS. CS was chosen as the polymeric backbone to synthesize the biomaterial containing the antioxidant groups of GA since it is natural, edible, and biocompatible and processes the amino groups of which can be grafted onto easily. For the grafting reaction, CS is generally dissolved in acetic acid solution. In the present study, however, CS was dissolved with the assistance of HOBt. As we know, HOBt is an organic compound used as a racemization suppressor and also for improving the yield in peptide synthesis. There are relatively intense hydrogen bonds or even ionic bonds between the amino groups of CS and the hydroxyl groups of HOBt.36 The transparence in the mixture of CS and HOBt means it is possible to modify CS in an aqueous system without the organic solvent, avoiding the side reactions resulting from the presence of acetic acid. As a result, CS was efficiently conjugated with GA in the present study via a watersoluble carbodiimide conjugating agent.37 The detailed reaction mechanism is depicted in Figure 1. EDC was protonated by HCl coexisting in the carbodiimide conjugating reagent, forming carbocation 1 that further reacted with the carboxyl group of GA to form O-acylisourea 2. In order to avoid the hydrolyzation of EDC to urea 4, EDC was dissolved in ethanol and added slowly into the solution of CS-HOBt and GA. When EDC was in excess, N-acylurea 3 could be by-produced. Herein, the presence of HOBt in combination with EDC is designed to reduce 3 and also to improve the reaction rate.32 The conjugation might occur at C-2 of CS to afford amide or at C-3 and C-6 of CS to afford ester linkages. Although the hydroxyl group is reactive, the reaction at C-3 can be omitted due to the steric hindrance.32 HOBt and byproduct urea 4 were removed from the resulting solution by dialysis against water. It has been reported that the binding constant (K) of HOBt with CS is 112.32 L/mol,38 which means HOBt may be present a little in the resulting product. The absence of HOBt in the target copolymer 5 was confirmed by 1H NMR. Characterization of GA-g-CS. The UV−vis spectra of GA, CS, and GA-g-CS (I, II and III) are displayed in Figure 2. During 240 to 500 nm, CS showed no absorption peak, and GA exhibited two characteristic absorption bands at 212 and 262 nm, respectively, assigned to the π-system of the benzene ring. In the spectra of GA-g-CS (I, II, and III), it was found that the UV−vis absorption peak at 262 nm shifted toward a longer wavelength (272 nm). It is consistent with the reported result for GA-g-carboxymethyl CS.14 The results indicated that GA was successfully grafted onto CS.2 The red shift might be attributed to the smaller amount of energy required for the

Figure 2. UV−vis spectra of chitosan (CS), gallic acid (GA), and GA grafted CS (GA-g-CS I, II, and III).

π−π* transition due to the longer conjugated system brought by the covalent linkage of GA with CS. Further characterization of GA-g-CS was performed by 1H NMR. As shown in Figure 3, GA showed only one peak at 7.1 ppm (H-a of benzene ring). CS exhibited a single peak at 3.1 ppm (H-2), multiple peaks at 3.6−3.9 ppm (H-3 to H-6), a small peak at 4.6 ppm (H-1), and a single peak at 2.0 ppm, representing the three protons of the N-acetyl group. The spectrum of GA-g-CS contained all the peaks of CS, and a new peak at around 7.1 ppm originated from GA, confirming again the successful grafting of GA onto CS. In addition, the peaks at 7.4−8.0 ppm, the signals of HOBt, did not appear in the spectrum, assuring that there was no HOBt remaining in the copolymer. The substitution of GA on CS could be evaluated by determining the phenolic groups of GA-g-CS. In the FolinCiocalteu reaction, phenolic compounds undergo a complex redox reaction with phosphotungstic and phosphomolybdic acids.39 The transfer of electrons at basic pH reduces the phosphomolybdic/phosphotungstic acid complexes to form chromogens, and then the color is developed. For GA-g-CS I, II, and III, these values were 209.9 ± 2.0, 117.8 ± 1.3, and 99.6 ± 0.7 mg GAE/g of dry copolymers, respectively. Interestingly, the 209.9 mg GAE/g copolymer is the highest substitution ratio of GA grafted onto CS among those that have been reported so far. As the reason, it might be due to the use of HOBt that resulted in more efficient grafting than traditional ways. In addition, it is meaningful to express the antioxidant potential in terms of phenolic content, since the activity of GA-g-CS conjugate is mainly originated from the phenolic groups in the polymeric backbone. Antioxidant Activity of GA-g-CS. CS has been exploited as a natural macromolecular antioxidant recently. However, three main obstacles including the poor solubility, chemical inertness based on the strong inter- and intramolecular hydrogen bonds network, and poor H atom-donating ability have to be overcome. Introduction of GA onto CS gives a solution to such problems, and GA-g-CS has been synthesized as a novel potential polysaccharide antioxidant. 40 The antioxidant activities of compounds are attributed to various mechanisms, such as breaking radical chain, prevention of introduction of initiating radicals, hindering metal-catalyzed initiation reactions, decomposition of lipid hydroperoxides, 9131

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Figure 3. 1H NMR spectra of (A) gallic acid, (B) chitosan, and (C) GA grafted CS (GA-g-CS).

abilities of all the three GA-g-CS were above 80%, and GA-g-CS I was equal to GA (p > 0.05) at a concentration of 2.0 mg/mL. The reducing power of a compound is a significant index of its potential antioxidant activity. As shown in Figure 4D, the reducing power of all samples increased with concentrations. The reducing power of GA-g-CS I was stronger than GA-g-CS II and GA-g-CS III (p < 0.05) and equal to GA at high concentrations (>1.0 mg/mL). Metal ion chelating activity plays a vital role in antioxidant mechanism since transition metals are known to catalyze the formation of ROS and aggravate oxidative stress.43 The Fe2+ chelating ability was determined by measuring the ironferrozine complex, and the results are summarized in Figure 4E. GA-g-CS showed much higher metal chelating activity than GA and CS. Especially, GA-g-CS I and GA-g-CS II showed stronger chelating capacity than GA-g-CS III (p < 0.05), except at a low concentration (0.125 mg/mL). Phenolics grafted onto CS lead to an exponential increase in iron-chelating ability and open a novel strategy for the development of a new generation of CS-based chelating therapeutic polymers for iron overload.44 Lipids, especially polyunsaturated fatty acids in the cell membrane, are vulnerable to both enzymes and nonenzymatic oxidants and are readily oxidized by free radicals, leading to membrane damage and eventually to various disorders and diseases.45 Antioxidant may directly react with initiator radicals or lipid peroxides involved in lipid peroxidation processes, and it may also inhibit the formation of active radicals. As shown in Figure 4F, the lipid peroxidation inhibition effect of GA-g-CS was about 50% at a high concentration, much higher than CS (p < 0.05) but lower than GA (p < 0.05). And the inhibition increased with the elevation of concentration. The β-Carotene−linoleic acid system is a rapid method for evaluation of antioxidants developed by Marco.46 In this method, the linoleic acid free radical formed attacks the highly unsaturated β-carotene molecules and rapidly bleached the orange color of β-carotene in the absence of an antioxidant.47 The lowest β-carotene discoloration rate exhibited the highest antioxidant activity. The inhibition effect of GA-g-CS was

singlet oxygen quenching, oxygen scavenging, and blocking the prooxidant effects by binding certain proteins containing catalytic metal sites.41 Therefore, various in vitro chemicalbased assays, such as assays of oxygen radical absorbance capacity (ORAC), scavenging activity on DPPH free radicals, and ferric reducing capacity, have been developed to determine antioxidant activity. Furthermore, a single in vitro chemical method is not enough to evaluate and compare their antioxidant properties by considering the complexity involved in their in vivo mechanisms of action. The results of antioxidant assays are displayed in Figure 4. The results showed that the antioxidant ability decreased in the order of GA-g-CS I, GA-g-CS II, GA-g-CS III, and CS. Obviously, GA-g-CS showed great antioxidant potential. In addition, the antioxidant capacities of all samples were wellcorrelated with the increase of concentrations and the amount of GA grafted onto the backbone. The DPPH and ABTS free radicals have been widely used for testing the preliminary radical scavenging capacity of plant extracts or antioxidant compounds. The DPPH and ABTS free radical scavenging activities of GA-g-CS are shown in Figure 4 (panels A and B), respectively. The results of the two assays were presented in a similar manner that the scavenging activities of samples decreased in order of GA, GA-g-CS I, GAg-CS II, GA-g-CS III, and CS at a low concentration corresponding to the quantities of GA contained, and there was no significant difference between the scavenging activities of GA-g-CS and GA at high concentrations. Superoxide radical, arising from the addition of one electron to dioxygen either through metabolic processes or following oxygen activation by physical irradiation, is considered as the primary reactive oxygen species (ROS). It can interact with other molecules to generate secondary ROS either directly or prevalently through enzyme- or metal-catalyzed processes, including hydroxyl radical, H2O2, and singlet oxygen.42 The superoxide radical scavenging activities of CS, GA-g-CS, and GA are shown in Figure 4C. All the samples exhibited scavenging activities on superoxide radicals. The scavenging 9132

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Figure 4. Scavenging activities on (A) 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, (B) 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical, and (C) superoxide radical. (D) reducing power, (E) chelating power, (F) lipid peroxidation, (G) linoleic acid autoxidation inhibition effect, and (H) total antioxidant capacity of gallic acid (GA, ■), chitosan (CS, red ●), GA-g-CS I (blue ▲), GA-g-CS II (pink ▼), GA-gCS III (green ◆), ethylene diamine tetraacetic acid (EDTA, blue ★). Data are presented as means ± SD of triplicates.

The assays used in the present study can roughly be classified into two types, depending upon the reactions involved: assays based on hydrogen atom transfer (HAT) reactions and the ones based on electron transfer (ET) reactions.48 The ETbased assays included the total phenol assay by Folin-Ciocalteu reagent, DPPH and ABTS radicals scavenging assays, reducing power, and total antioxidant potential assays with FRAP. The antioxidant activities measured by these assays are correlated with the reducing agent present in samples, the GA grafted

around 80% at 1.0 mg/mL (Figure 4G), higher than that of CS (p < 0.05) and notably even much better than GA (p < 0.05). The FRAP assay is based on the ability of antioxidant compounds to reduce complex (Fe (III)-TPTZ) to (Fe (II)TPTZ) that gives a blue color. The results of the FRAP assay are presented in Figure 4H. The activities increased in order of CS, GA-g-CS III, GA-g-CS II, GA-g-CS I, and GA (p < 0.05), related to the reductant (GA) content of the sample. 9133

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Figure 5. Proposed interaction between gallic acid−chitosan and free linoleic acid radicals.

Rheological Property of GA-g-CS. The flow curves of CS and GA-g-CS are shown in Figure 6. It was observed that GA-g-

onto CS in our case. It could easily be understood that the antioxidant potential obtained from these assays decreased in the order of GA, GA-g-CS I, GA-g-CS II, GA-g-CS III and CS, which was associated with concentration of sample. CS showed little antioxidant potential in our assays. However, previous studies reported that CS dissolved with acetic acid, HOBt, thiamine pyrophosphate (CS-TPP), and the ethylenediaminetetraacetic acid (CS-EDTA) solution possessed superoxide and hydroxyl radicals scavenging abilities.49 It was supposed that the antioxidant capacity of CS dissolved in pure water was suppressed due to its limited solubility. Notably, GA-g-CS showed stronger antioxidant activity than GA in the β-carotene-linoleic acid assay. β-Carotene-linoleic assay, different from DPPH assay, is a HAT-based antioxidant assay. It was observed that the extract of mushroom with great antioxidant potential in the DPPH assay did not perform well in β-carotene-linoleic acid assay, as variation of phenolic contents did not directly influence the antioxidant activity in the β-carotene-linoleic acid assay.50 During the oxidation in this model, an atom of hydrogen is abstracted from the active bisallylic methylene group of linoleic acid located at C-11 between two double bonds and then the formed pentadienyl free-radical attacks highly unsaturated β-carotene molecules in an effort to reacquire a hydrogen atom. As the β-carotene molecules lose their conjugation, the carotenoids lose their characteristic orange color in the absence of an antioxidant.51 It has been reported that CS could absorb fatty acids by electrostatic interaction, 52 and the antioxidant activity of phenolic compounds depends on various structural features such as the O−H bond dissociation energy, resonance delocalization of the phenoxyl radical, and the steric hindrance due to bulky groups substituting hydrogen in the aromatic ring.53 Considering that, we hypothesize that the interaction between GA-gCS and linoleic acid radicals could occur as illustrated in Figure 5, GA-g-CS attracting linoleic acid radicals by electrostatic affinity and quenching them with the GA groups rapidly. Furthermore, grafting GA onto CS developed amide linkages that enlarged the conjugated system of GA and contributed to an increase in electron donating groups, making GA-g-CS more stable than free GA. We suppose that GA-g-CS with swelling macromolecular structure could capture the linoleic acid free radicals, while small molecule GA could not. Linoleic acid radicals had larger size and had more potential for interacting with CS than superoxide radicals so that they were more easily captured. Those might be the reasons why GA-g-CS exhibited higher free radical scavenging ability than GA in the β-carotenelinoleic acid assay.

Figure 6. Viscosity of chitosan (CS) and gallic acid (GA) grafted CS (GA-g-CS I, II, and III).

CS (I, II, and III) was more viscous than plain CS significantly (p < 0.05). The viscosity of GA-g-CS decreased in order of GAg-CS III, GA-g-CS II, GA-g-CS I, and CS, and the viscosity of GA-g-CS III was 50 times higher than that of CS at 2 s−1 and 5 times higher at 256 s−1. Similar phenomenon had also been found in natural macromolecules. The polysaccharide-containing ferulic acid extracted from the seeds of Plantago asiatica L. exhibited much higher apparent viscosity compared to that of the polysaccharide depleted ferulic acid.54 Additionally, arabinoxylan samples with different ferulic acid contents had been employed to obtain gels by enzymatic covalent crosslinking of the phenolic acid.55 For GA-g-CS, the grafting of GA onto CS provides the possibility that links the different GA-gCS macromolecular chains by the formation of phenolic dimers or trimers and even the covalent junction between GA and amino group from glucosamine residue. The new bonds may lead to larger molecular weight, longer chain length, and a more viscous solution. Therefore, it was hypothesized that the high viscosity of GA-g-CS was attributed to the covalent crosslinking junctions produced by the oxidation of GA groups. For GA-g-CS (I, II and III), the viscosity of the sample decreased with the increase of the substituting degree. The GAg-CS III with a substituting degree of 99.6 mg GAE/g of dry 9134

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(4) Aewsiri, T.; Benjakul, S.; Visessanguan, W.; Eun, J. B.; Wierenga, P. A.; Gruppen, H. Antioxidative activity and emulsifying properties of cuttlefish skin gelatin modified by oxidised phenolic compounds. Food Chem. 2009, 117, 160−168. (5) Ihara, N.; Schmitz, S.; Kurisawa, M.; Chung, J. E.; Uyama, H.; Kobayashi, S. Amplification of inhibitory activity of catechin against disease-related enzymes by conjugation on poly(ε-lysine). Biomacromolecules 2004, 5, 1633−1636. (6) Chung, J. E.; Kurisawa, M.; Uyama, H.; Kobayashi, S. Enzymatic synthesis and antioxidant property of gelatin-catechin conjugates. Biotechnol. Lett. 2003, 25, 1993−1997. (7) Shiu, J. C.; Ho, M. H.; Yu, S. H.; Chao, A. C.; Su, Y. R.; Chen, W. J.; Chiang, Z. C.; Yang, W. P. Preparation and characterization of caffeic acid grafted chitosan/CPTMS hybrid scaffolds. Carbohydr. Polym. 2010, 79, 724−730. (8) Zhang, X. Q.; Do, M. D.; Casey, P.; Sulistio, A.; Qiao, G. G.; Lundin, L.; Lillford, P.; Kosaraju, S. Chemical cross-linking gelatin with natural phenolic compounds as studied by high-resolution NMR spectroscopy. Biomacromolecules 2010, 11, 1125−1132. (9) Spizzirri, U. G.; Parisi, O. I.; Iemma, F.; Cirillo, G.; Puoci, F.; Curcio, M.; Picci, N. Antioxidant-polysaccharide conjugates for food application by eco-friendly grafting procedure. Carbohydr. Polym. 2010, 79, 333−340. (10) Spizzirri, U. G.; Altimari, I.; Puoci, F.; Parisi, O. I.; Iemma, F.; Picci, N. Innovative antioxidant thermo-responsive hydrogels by radical grafting of catechin on inulin chain. Carbohydr. Polym. 2011, 84, 517−523. (11) Ren, J. M.; Li, Q.; Dong, F.; Feng, Y.; Guo, Z. Y. Phenolic antioxidants-functionalized quaternized chitosan: Synthesis and antioxidant properties. Int. J. Biol. Macromol. 2013, 53, 77−81. (12) Puoci, F.; Morelli, C.; Cirillo, G.; Curcio, M.; Parisi, O. I.; Maris, P.; Sisci, D.; Picci, N. Anticancer activity of a quercetin-based polymer towards HeLa cancer cells. Anticancer Res. 2012, 32, 2843−2847. (13) Pasanphan, W.; Chirachanchai, S. Conjugation of gallic acid onto chitosan: An approach for green and water-based antioxidant. Carbohydr. Polym. 2008, 72, 169−177. (14) Yu, S. H.; Mi, F. L.; Pang, J. C.; Jiang, S. C.; Kuo, T. H.; Wu, S. J.; Shyu, S. S. Preparation and characterization of radical and pHresponsive chitosan-gallic acid conjugate drug carriers. Carbohydr. Polym. 2011, 84, 794−802. (15) Curcio, M.; Puoci, F.; Iemma, F.; Parisi, O. I.; Cirillo, G.; Spizzirri, U. G.; Picci, N. Covalent insertion of antioxidant molecules on chitosan by a free radical grafting procedure. J. Agric. Food Chem. 2009, 57, 5933−5938. (16) Cho, Y. S.; Kim, S. K.; Ahn, C. B.; Je, J. Y. Preparation, characterization, and antioxidant properties of gallic acid-graftedchitosans. Carbohydr. Polym. 2011, 83, 1617−1622. (17) Casettari, L.; Gennari, L.; Angelino, D.; Ninfali, P.; Castagnino, E. ORAC of chitosan and its derivatives. Food Hydrocolloids 2012, 28, 243−247. (18) Vittorio, O.; Cirillo, G.; Iemma, F.; Di Turi, G.; Jacchetti, E.; Curcio, M.; Barbuti, S.; Funel, N.; Parisi, O. I.; Puoci, F.; Picci, N. Dextran-catechin conjugate: A potential treatment against the pancreatic ductal adenocarcinoma. Pharm. Res. 2012, 29, 2601−2614. (19) Liu, J.; Lu, J. F.; Kan, J.; Jin, C. H. Synthesis of chitosan-gallic acid conjugate: Structure characterization and in vitro anti-diabetic potential. Int. J. Biol. Macromol. 2013, 62, 321−329. (20) Eom, T. K.; Ryu, B.; Lee, J. K.; Byun, H. G.; Park, S. J.; Kim, S. K. β-Secretase inhibitory activity of phenolic acid conjugated chitooligosaccharides. J. Enzyme Inhib. Med. Chem. 2013, 28, 214−217. (21) Lee, D. S.; Je, J. Y. Gallic acid-grafted-chitosan inhibits foodborne pathogens by a membrane damage mechanism. J. Agric. Food Chem. 2013, 61, 6574−6579. (22) Rinaudo, M. Chitin and chitosan: Properties and applications. Prog. Polym. Sci. 2006, 31, 603−632. (23) No, H. K.; Meyers, S. P.; Prinyawiwatkul, W.; Xu, Z. Applications of chitosan for improvement of quality and shelf life of foods: A review. J. Food Sci. 2007, 72, R87−R100.

copolymers appeared more viscous than both the plain CS and copolymer with a higher degree of substitution (I or II). A similar tendency was also observed in GA-gelatin. The gel strength and modulus of viscosity (G″) increased with increasing GA concentration up to 20 mg/g dry gelatin and then decreased at further elevated GA concentrations.56 The results indicated that the viscosity of GA-g-CS was related to the grafting amount of GA. The introduction of GA onto CS takes many properties to GA-g-CS copolymer. On one hand, GA may reduce the intraand intermolecular hydrogen bond networks and eliminate crystal structure, but on the other, the grafting provides the sites to form covalent bonds and further affects the solubility and rheological property of CS. A balance may be achieved by regulating the grafted amount of GA. As the previous study suggested, GA-g-CS of a certain substitution was more soluble than plain CS, and higher substitution led to higher solubility in some range.13 In addition, reacting conditions, especially the oxygen content and pH, could be the main factors contributing to the oxidative polymerization of the copolymer. GA-g-CS with a different viscoelastic property and gelling capacity could be obtained by controlling oxidative conditions.55 The results suggest that GA-g-CS possesses the potential to be employed as thickener or gel-forming material in food industries. In conclusion, GA-g-CS with GA grafted up to 209.9 mg/g of the copolymer, strong antioxidant activity, and an adjustable rheological property was synthesized and characterized in the present study. The results suggested that high content of GA could be easily grafted onto CS by the developed one-pot method with EDC and HOBt. GA-g-CS, possessing the advantages of both GA and CS, showed greater antioxidant potential and stronger antioxidant power than GA in the βcarotene-linoleic acid assay. It was the first time to observe that polyphenol-polysaccharide conjugate exhibited stronger antioxidant power than its corresponding polyphenol. In addition, GA grafted onto CS altered the rheological property of the biopolymer, making GA-g-CS with a higher viscosity than plain CS, which could be adjustable through modifying the amount of grafted GA. Our present study developed a novel approach to synthesize GA-g-CS that could be explored as promising biomaterials in food industries.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-25-84396791. Funding

This work was supported by Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Bhattacharya, A.; Misra, B. N. Grafting: A versatile means to modify polymers - Techniques, factors and applications. Prog. Polym. Sci. 2004, 29, 767−814. (2) Spizzirri, U. G.; Iemma, F.; Puoci, F.; Cirillo, G.; Curcio, M.; Parisi, O. I.; Picci, N. Synthesis of antioxidant polymers by grafting of gallic acid and catechin on gelatin. Biomacromolecules 2009, 10, 1923− 1930. (3) Prodpran, T.; Benjakul, S.; Phatcharat, S. Effect of phenolic compounds on protein cross-linking and properties of film from fish myofibrillar protein. Int. J. Biol. Macromol. 2012, 51, 774−782. 9135

dx.doi.org/10.1021/jf503207s | J. Agric. Food Chem. 2014, 62, 9128−9136

Journal of Agricultural and Food Chemistry

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(24) Shi, C. M.; Zhu, Y.; Ran, X. Z.; Wang, M.; Su, Y. P.; Cheng, T. M. Therapeutic potential of chitosan and its derivatives in regenerative medicine. J. Surg. Res. 2006, 133, 185−192. (25) Prabaharan, M.; Mano, J. F. Chitosan derivatives bearing cyclodextrin cavities as novel adsorbent matrices. Carbohydr. Polym. 2006, 63, 153−166. (26) Kumar, M. N. V. R.; Muzzarelli, R. A. A.; Muzzarelli, C.; Sashiwa, H.; Domb, A. J. Chitosan chemistry and pharmaceutical perspectives. Chem. Rev. 2004, 104, 6017−6084. (27) Bozic, M.; Strancar, J.; Kokol, V. Laccase-initiated reaction between phenolic acids and chitosan. React. Funct. Polym. 2013, 73, 1377−1383. (28) Jus, S.; Stachel, I.; Fairhead, M.; Meyer, M.; Thony-Meyer, L.; Guebitz, G. M. Enzymatic cross-linking of gelatine with laccase and tyrosinase. Biocatal. Biotransform. 2012, 30, 86−95. (29) Liu, Y.; Sakai, S.; Taya, M. Impact of the composition of alginate and gelatin derivatives in bioconjugated hydrogels on the fabrication of cell sheets and spherical tissues with living cell sheaths. Acta Biomater. 2013, 9, 6616−6623. (30) Verma, S.; Singh, A.; Mishra, A. Gallic acid: Molecular rival of cancer. Environ. Toxicol. Pharmacol. 2013, 35, 473−485. (31) Schreiber, S. B.; Bozell, J. J.; Hayes, D. G.; Zivanovic, S. Introduction of primary antioxidant activity to chitosan for application as a multifunctional food packaging material. Food Hydrocolloids 2013, 33, 207−214. (32) Fangkangwanwong, J.; Akashi, M.; Kida, T.; Chirachanchai, S. One-pot synthesis in aqueous system for water-soluble chitosan-graftpoly(ethylene glycol) methyl ether. Biopolymers 2006, 82, 580−586. (33) Liu, L.; Sun, Y.; Laura, T.; Liang, X.; Ye, H.; Zeng, X. Determination of polyphenolic content and antioxidant activity of kudingcha made from Ilex kudingcha C.J. Tseng. Food Chem. 2009, 112, 35−41. (34) Liu, J.; Luo, J.; Ye, H.; Sun, Y.; Lu, Z.; Zeng, X. In vitro and in vivo antioxidant activity of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3. Carbohydr. Polym. 2010, 82, 1278−1283. (35) Liu, J.; Luo, J.; Ye, H.; Sun, Y.; Lu, Z.; Zeng, X. Production, characterization and antioxidant activities in vitro of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3. Carbohydr. Polym. 2009, 78, 275−281. (36) Opanasopit, P.; Techaarpornkul, S.; Rojanarata, T.; Ngawhirunpat, T.; Ruktanonchai, U. Nucleic acid delivery with chitosan hydroxybenzotriazole. Oligonucleotides 2010, 20, 127−136. (37) Fangkangwanwong, J.; Akashi, M.; Kida, T.; Chirachanchai, S. Chitosan-hydroxybenzotriazole aqueous solution: A novel water-based system for chitosan functionalization. Macromol. Rapid Commun. 2006, 27, 1039−1046. (38) Dai, B.; Peng, M.; Chen, Q. Interaction between chitosan and 1hydroxybenzotriazole studied by NMR spectroscopy. Chin. J. Magn. Reson. 2008, 4, 453−460. (39) Siripatrawan, U.; Harte, B. R. Physical properties and antioxidant activity of an active film from chitosan incorporated with green tea extract. Food Hydrocolloids 2010, 24, 770−775. (40) Pasanphan, W.; Buettner, G. R.; Chirachanchai, S. Chitosan gallate as a novel potential polysaccharide antioxidant: An EPR study. Carbohydr. Res. 2010, 345, 132−140. (41) Frankel, E. N.; Meyer, A. S. The problems of using onedimensional methods to evaluate multifunctional food and biological antioxidants. J. Sci. Food Agric. 2000, 80, 1925−1941. (42) Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T. D.; Mazur, M.; Telser, J. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 2007, 39, 44− 84. (43) Lu, J. M.; Lin, P. H.; Yao, Q. Z.; Chen, C. Y. Chemical and molecular mechanisms of antioxidants: Experimental approaches and model systems. J. Cell. Mol. Med. 2010, 14, 840−860. (44) Brzonova, I.; Steiner, W.; Zankel, A.; Nyanhongo, G. S.; Guebitz, G. M. Enzymatic synthesis of catechol and hydroxyl-carboxic

acid functionalized chitosan microspheres for iron overload therapy. Eur. J. Pharm. Biopharm. 2011, 79, 294−303. (45) Niki, E. Biomarkers of lipid peroxidation in clinical material. Biochim. Biophys. Acta, Gen. Subj. 2014, 1840, 809−817. (46) Marco, G. J. A rapid method for evaluation of antioxidants. J. Am. Oil Chem. Soc. 1968, 45, 594−598. (47) Abdille, M. H.; Singh, R. P.; Jayaprakasha, G. K.; Jena, B. S. Antioxidant activity of the extracts from Dillenia indica fruits. Food Chem. 2005, 90, 891−896. (48) Huang, D. J.; Ou, B. X.; Prior, R. L. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 2005, 53, 1841−1856. (49) Charernsriwilaiwat, N.; Opanasopit, P.; Rojanarata, T.; Ngawhirunpat, T. In vitro antioxidant activity of chitosan aqueous solution: Effect of salt form. Trop. J. Pharm. Res. 2012, 11, 235−242. (50) Vieira, P. A. F.; Gontijo, D. C.; Vieira, B. C.; Fontes, E. A. F.; de Assuncao, L. S.; Leite, J. P. V.; Oliveira, M. G. D.; Kasuya, M. C. M. Antioxidant activities, total phenolics and metal contents in Pleurotus ostreatus mushrooms enriched with iron, zinc or lithium. LWT−Food Sci. Technol. 2013, 54, 421−425. (51) Amarowicz, R.; Pegg, R. B.; Rahimi-Moghaddam, P.; Barl, B.; Weil, J. A. Free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Food Chem. 2004, 84, 551−562. (52) Xia, W.; Liu, P.; Zhang, J.; Chen, J. Biological activities of chitosan and chitooligosaccharides. Food Hydrocolloids 2011, 25, 170− 179. (53) Adelakun, O. E.; Kudanga, T.; Parker, A.; Green, I. R.; le RoesHill, M.; Burton, S. G. Laccase-catalyzed dimerization of ferulic acid amplifies antioxidant activity. J. Mol. Catal. B: Enzym. 2012, 74, 29−35. (54) Yin, J. Y.; Nie, S. P.; Li, J.; Li, C.; Cui, S. W.; Xie, M. Y. Mechanism of interactions between calcium and viscous polysaccharide from the seeds of Plantago asiatica L. J. Agric. Food Chem. 2012, 60, 7981−7987. (55) Carvajal-Millan, E.; Landillon, V.; Morel, M. H.; Rouau, X.; Doublier, J. L.; Micard, V. Arabinoxylan gels: Impact of the feruloylation degree on their structure and properties. Biomacromolecules 2005, 6, 309−317. (56) Yan, M. Y.; Li, B. F.; Zhao, X.; Yi, J. B. Physicochemical properties of gelatin gels from walleye pollock (Theragra chalcogramma) skin cross-linked by gallic acid and rutin. Food Hydrocolloids 2011, 25, 907−914.

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