lable at ScienceDirect

Food Hydrocolloids 39 (2014) 113e119

Contents lists avai

Food Hydrocolloids

journal homepage: www.elsevier .com/locate/ foodhyd

Rheological and antioxidant power studies of enzymatically graftedchitosan with a hydrophobic alkyl side chain

Leonor Zavaleta-Avejar a, Elsa Bosquez-Molina a,*, Miquel Gimeno b,Juan Pablo Pérez-Orozco c, Keiko Shirai a

aUniversidad Autónoma Metropolitana-Iztapalapa, Depto. de Biotecnología, San Rafael Atlixco 186, México D.F. C.P. 09340, MexicobDepto. Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México D.F., Mexicoc Instituto Tecnológico de Zacatepec, Depto. De Ingeniería Química y Bioquímica, Calzada Tecnológico 27, Zacatepec de Hidalgo, Morelos C.P. 62780, Mexico

a r t i c l e i n f o

Article history:Received 19 July 2013Accepted 21 December 2013

Keywords:Chitosan modificationEnzymatic graftingViscoelastic propertiesAntioxidant power

* Corresponding author. Tel.: þ52 5558044711; faxE-mail addresses: elbm@xanum.uam.mx (E. Bosqu

com (M. Gimeno).

0268-005X/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodhyd.2013.12.030

a b s t r a c t

The enzyme-mediated grafting of hydrophobic alkyl side chains onto Chitosan (Ch) has been successfullyachieved using octyl gallate and horseradish peroxidase. The properties of the resulting materials havebeen studied by rheology and electron paramagnetic resonance (EPR) spectroscopy in order to envisageits potential applications as bioactive food additive. The chemical structures of the octyl gallate-graftedCh were corroborated by ATR-FTIR, 1HRMN, viscosimetric molecular weight and z potential. The anti-oxidant capacity of materials determined by EPR show that functionalized Ch radical scavenging in-creases with grafting, which was related to the aromatic ring in the octyl gallate. The Ch with the highestgrafting exhibited an antioxidant capacity of 81%. Solutions of the materials in acetic acid and lactic acidexhibited a shear-thinning flow behavior which also increased with the grafting. The viscoelasticproperties of solutions were characterized by oscillatory shear measurement and the result showedfluid-like viscoelastic behavior. The elasticity of the solutions decreased with the plasticizer addition.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Currently, there is a growing interest in sustainable polymersparticularly in basic applications such as packaging, edible filmsand coatings which biodegrade under controlled conditions ofstorage in order to alleviate the growing global synthetic materialswaste problem. In this sense, the chitosan and its derivatives havedemonstrated great potential as biological packaging material dueto its inherent antimicrobial power, film-forming material as wellas their versatile physical and chemical properties as several re-searchers have highlighted in recent reviews (Dutta, Tripathi,Mehrotra, & Dutta, 2009; Vinsová & Vavríková, 2011).

As a result of the interest in this biomacromolecule, awide rangeof chitosan-based (Ch) materials have been proposed by means ofsuitable chemical or enzymatic modifications onto the Ch backboneto improve its solubility in water, antimicrobial properties, floccu-lant capacity, absorbency and adhesiveness, while remainingbiodegradable and biocompatible for potential applications inbiomedicine, food or water treatment. Chemical modifications

: þ52 5558044712.ez-Molina), mi_quel@yahoo.

All rights reserved.

mainly consisted in the insertion of small functional groups such asalkyl, carboxymethyl, saccharides or oligosacharides for increasingthe Ch solubility at neutral and alkaline pH without affecting itscationic character (Alves & Mano, 2008; Sashiwa & Aiba, 2004).Other compounds like cyclodextrins, and acids, such as p-amino-benzoic, lactobionic, sialic and polyacrylic have also been attachedto confer removal capacity of textile dyes, drug delivery, woundhealing, inhibition of microorganisms, antioxidant capacity andincreasing hydrophilicity (Calero, Muñoz, Ramírez, & Guerrero,2010; Kumar, Muzzarelli, Muzzarelli, Sashiwa, & Domb, 2004; Liu,Chen, & Pan, 2007; Siripatrawan & Harte, 2010; Xie, Xu, Wang, &Liu, 2002). Pasanphan, Buettner, and Chirachanchai (2010) graftedgallic acid onto Ch by a conjugated reaction with potential use asadditive in foodstuffs owing to improved solubility and antioxidantpower. Despite of the possibility to modify Ch by chemical means,enzymatic modifications offer alternative routes, especially inproducts to be applied in foodstuffs due to minimized hazardsassociated with toxic reagents, in addition to mild and environ-mentally friendly reaction conditions (Alves & Mano, 2008; Belalia,Grelier, Benaissa, & Coma, 2008; Chao, Shyu, Lin, & Mi, 2004; Curcioet al., 2009; Kumar et al., 2004; Sobahi, Abdelaal, & Makki, 2011;Xiao-Yi, Jian-Ping, Huai-Tian, Gang-Biao, & Ming-Hua, 2011).Research work on enzymatically modified Ch cover those reportedby Kumar, Smith, and Payne (1999) who used tyrosinase for

L. Zavaleta-Avejar et al. / Food Hydrocolloids 39 (2014) 113e119114

grafting a wide range of phenolic substrates onto Ch conferringsolubility in basic conditions and antioxidant capacity. Vachoud,Chen, Payne, and Vazquez-Duhalt (2001) demonstrated the abil-ity of horseradish peroxidase (HRP) to graft dodecyl gallate (DDG)onto Ch by generating radicals from the phenolic derivative to thereaction media to form stable linkages, mainly between Ch aminomoiety and aromatic carbons. Generally, peroxidases have severaladvantages over tyrosinase as broader substrate range and opti-mum pH for activity between 5 and 6, which is convenient in Chmodifications as it solubilizes at pH below 6.0.

Gallates are used as antioxidant additives in foods, cosmeticsand medicinal preparations as good H donors. Among them, theoctyl gallate (OG), unlike DDC and other synthetic antioxidants, hasnot been pointed as potential cancer precursor. The bulky structureof OG when grafted onto Ch might allow for the reducing of theintra- and intermolecular hydrogen bonding and the alkyl sidechain might provide hydrophobicity to Ch molecule associated tothickening properties in the bulk. The improved rheological char-acteristics might be related with the length of grafted side chains inaddition to pH, polymer concentration and external salt concen-tration (Desbrieres, 2004; Rinaudo, Auzely, Vallin, & Mullagaliev,2005). Therefore, the solution behavior of modified Ch is essentialto predict, design, and characterize in order to envisage its potentialof application. In particular, the linear viscoelastic properties ofaqueous solutions of Ch are closely related to their colloidalstructure while shear rate dependence of viscosity is associatedwith the structural modifications (Fujita & Kubo, 2002; Vartiainen,Rättö, Lantto, Nättinen, & Hurme, 2008).

However, despite of the aforementioned reports, there is noinformation regarding the applicability of Ch grafted with hydro-phobic alkyl side chains and in the present work, the aim was tostudy the rheological properties of the enzymatically produced Ch-g-OG samples as important parameter linked to the application ofthis materials in addition to chemical and antioxidantcharacterizations.

2. Materials and methods

2.1. Materials

Ch (Mv 486.33 kDa, 87.2% deacetylation), OG, horseradishperoxidase (HRP) type II, hydrogen peroxide (H2O2), glycerolanhydrous (Gly), lactic acid (L), deuterium oxide (D2O), methylalcohol, 1,1-diphenyl-2-picryl-hydrazyl (DPPH�), ninhydrin andninhydrin reagent were purchased from Sigma Aldrich (St. Louis,MO, USA). Acetone, sodium hydroxide (NaOH), hydrochloric acid(HCl), ethyl alcohol and glacial acetic acid (A), were purchased fromJ.T. Baker (Phillipsburg, NJ, USA). Deionized water (filtration systemMilli-Q) was used for the preparation of all solutions.

2.2. Enzymatic grafting

Ch-g-OG was performed following a modification of the methodreported by Vachoud et al. (2001). An HCl 2M (pHw2.5) solution ofnative Ch (1.6% wt/v) was stirred for 1 h. Then, the solution wasdilutedwith 433.3mL of phosphate buffer (0.1M, pH 5.5) to obtain afinal 0.3% wt/wt Ch concentration, Thereupon, the pH was raised to4.5 with 1 MNaOH.100 mL of the Ch solutionwas mixed separatelywith 25mL of twoOG solutions in acetone,10 and 20mM to obtain aratio of Ch:OG 80:20 (v/v) and 60:40 (v/v), respectively. HRP (1.4mgof enzyme/mLphosphate buffer pH5.5)was added, and the reactionwas initiated by adding 600 mL of H2O2 and it was sustainedwith sixsuccessive additions of 600 mL of H2O2 at 10 min intervals. After thelast addition of H2O2, the reaction mixture was stirred for 60 minfollowed by a centrifugation during 20 min at 7500 rpm and 20 �C.

The resulted material was kept at �80 �C in an ultra-freezer REVCO(USA) for 24 h, after that, it was lyophilized to obtain the corre-sponding two materials Ch-g-OG1 and Ch-g-OG2 as powders. Con-trol experiments were carried out without Ch addition. Productswere precipitated in 1/10 v/v of cold methanol as brown powders,filtered and dried in a vacuum oven prior to analyses.

2.3. Characterizations

2.3.1. 1HNMR analysisProton nuclear magnetic resonance (1HNMR) spectra were

recorded on a Bruker (AVANCE-III 500 (Germany)) instrument at200 MHz. Ch grafted samples were dissolved in D2O containing DCland 3-(trimethylsilyl)-propionic acid was used as internal referencestandard. Products from control experiments without Ch weredissolved in deuterated acetone and tetramethylsilane was used asinternal reference standard. Deacetylation degree (DD) of Ch wasdetermined according to Hirai, Odani, and Nakajima (1991) andthat for grafted samples following equation (1). OG incorporation asmolar% was determined according to equation (2).

DD% ¼

26641�

0BB@

1 =

3 HAc

1 =

6

ZH2�6 � 2�

�ZAlk=15

�1CCA3775� 100 (1)

molar% ¼

ZAlk=152

664Z

H2�6 � 2��Z

Alk=15

�

6

3775� 100 (2)

where !HAc is the residue of CH3, !H2e6 is the integration of themassive of Ch and !Alk is the integration of 6 H of the side alkylchain of OG.

2.3.2. ATR-FTIR analysisAttenuated total reflectance Fourier transform infrared (ATR-

FTIR) spectra were recorded in a Perkin Elmer ATR-FTIR 100 in-strument (Waltham, Massachusetts, USA) at a wavelength rangefrom 650 to 4000 cm�1 with 20 scans per sample.

2.3.3. z potentialz potential values were obtained from Ch-g-OG and Ch solutions

prepared with 1% w/v of the polymers into a 1% v/v acetic acid so-lution by stirring during 24 h at room temperature. Solutions werethendiluted to1:10mLwith 1% acetic acid solution and injected intothe chamber of a ZetaSizer ZEM5003 (Zetamaster, Malvern In-struments, UK). Smoluchowsky mathematical model was used toconvert the electrophoretic mobility measurements into z potentialvalues. Measurements were performed by triplicate.

2.3.4. Determination of amino group content by ninhydrin assayFree amine groups of functionalized Ch were determined ac-

cording to the method reported by Alonso et al. (2009). 0.1 mg/mLsolutions of Ch, Ch-g-OG1 and Ch-g-OG2 were dissolved in a so-lution of acetic acid 1% v/v with constant stirring for 24 h at roomtemperature. Then, 1 mL of ninhydrin reagent freshly prepared,were added to the sample solution. The mixtures were boiled inwater for 10 min and transferred to ice-water bath and paperfiltered through a Whatman No. 40. Then, the absorbance wasmeasured at 570 nm.

L. Zavaleta-Avejar et al. / Food Hydrocolloids 39 (2014) 113e119 115

2.3.5. Intrinsic viscosityIntrinsic viscosity was obtained in an Oswald viscometer at

25 �C. Stock solutions of Ch, Ch-g-OG1, and Ch-g-OG2 were pre-pared dissolving 0.1e0.3 g of each polymer in 25 mL of 1% v/vglacial acetic acid with the aid of stirring for 24 h (Bastos et al.,2010; Kasaai, 2007). Five dilutions of different concentration fromthe stock solution were made and the flow rates measured. Theintrinsic viscosity was calculated by the Huggins equation:

hsp

.C ¼ ½h� þ k1½h�2C (3)

where hsp is the specific viscosity, C is the solution concentration,[h] is the intrinsic viscosity and k1 is Huggins constant.

To know if oligomers are produced during control reaction(without Ch addition) the molecular weight was determined withthe Size exclusion chromatography (SEC) using chloroform (flow1 ml/min) in an HP 1050 chromatograph (HewlettePackard,Waldbronn, Germany); column PLGEL 10 mm MIXED-B LS. The UVdetection of OG was at 270 nm. The calibration curve was madewith polystyrene solutions.

2.4. Rheological measurements

Ch, Ch-g-OG1 and Ch-g-OG2 solutions (1% w/v) were preparedby dissolving 1 g of polymer in 100 mL acetic acid or lactic acid (1%v/v) under mechanical stirring during 24 h. These stocks solutionswere stored overnight at 5 �C, for further use. In order to analyzethe effect of plasticizer, 1% v/v of glycerol was added to 30 mL of thestocks solutions and mixed during 30 min under continuous stir-ring. Dynamic oscillatory and flow measurements were performedat 25 �C using a Paar-Physica USD 200 Rheometer (Anton Paar,Messtechnik, Stuttgart, Germany) fitted with a cone-plate geome-try with a gap of 0.05 mm. Storage modulus (G0) and the lossmodulus (G00) were recorded in the 0.01e10 Hz frequency range atstress amplitude of 0.2 Pa. Samples were individually loaded on themeasuring geometry and allowed to stand for 10 min prior to thetest. The apparent viscosity as a function of the shear rate wasdetermined in the range from 0.1 to 100 s�1.

Fig. 1. ATR-FTIR spectra of Ch, ChOG1, ChOG2.

2.5. Antioxidant power by EPR spectroscopy

The DPPH� is a relatively stable free radical which can be easilydetected by EPR; thus, this spectroscopy method becomes usefuland practical for the evaluation of antioxidant potential of ourmaterials. The antioxidant capacity was evaluated by plotting thefield against the intensity of free radical scavenging capacity.

EPR spectra were acquired in a Bruker model E-500 Elexsys EPRspectrometer set at field [G] 3436.15; 1024 pts; receiver gain, 60;microwave power, [w] 0.006395;modulation frequency,100,000 Hz;microwave frequency, [Hz] 9.778604 eþ09;modulation amplitude, [T]0.0001; time constant, [s] 0.01024; 4 scans. Ch-g-OG solutions (500,800, 1000, 1200 and 1500 g/kg DPPH) were reacted with 0.025 g/Lmethanolic DPPH� solution (2.9 g/ml). The mixture was stirred andallowed to react for 30 min at room temperature, and thereupontransferred to a quartz flat cell. The intensity of the remaining DPPH�

measured by EPR calculated using the following equation (4).

%RC ¼�h0 � hc=h0

�� 100 (4)

where %RC is the percentage of the radical scavenging capacity, hcand h0 are the peak heights belonging to the middle peak of theDPPH� spectrum with and without DPPH, respectively. Three rep-etitions were performed for each sample.

The %RC data were processed with the Probit analysis to obtainthe EC50 (NCSS software, 1997).

3. Results and discussion

3.1. ATR-FTIR, 1HNMR spectroscopies, and z potential

The grafting could be inferred by ATR-FTIR analyses where thenative Ch spectrum displays the characteristic absorption band at3354 and 3286 cm�1 assigned to eNH stretching (Fig. 1). In thesame manner for the other Ch groups i.e. the band at 2870.97 cm�1

owing to the stretching of CH3, as well as the bands at 1651 cm�1

and 1585 cm�1 of stretching and bending vibrations of the amide I(eNH2) in the acetylated units and CH3, respectively (Abugoch,Tapia, Villamán, Yazdani-Pedram, & Díaz-Dosque, 2011).Compared to Ch, the spectra of Ch-g-OG1 and Ch-g-OG2 exhibitedan increased absorption in the 2850e2930 cm�1 region, which isascribed to the symmetric and asymmetric stretching of methylenegroups. The presence of carbonyl signal was observed at 1716 cm�1

evidencing the grafting of OG. This signal is similar to thatdescribed by Vachoud et al. (2001) for the Ch-g-DDG and byPasanphan and Chirachanchai (2008) for the Ch-g-gallic acid.

As for the 1HNMR analyses, the spectra of Ch-g-OG1 and Ch-g-OG2 displayed signals at 2.0e2.1 ppm assigned to the aliphatichydrogens of alkyl side chain (Fig. 2). The DD% and OG incorpora-tion (molar%) from the integration of the characteristic signals on1HNMR spectra are shown in Table 1. The DD% of the Ch sampleused in this work (87.02%) is in the range (85e99%) reported forcommercial Chs and considered adequate for the insertion ofphenolic derivatives throughout the amino moiety (Bastos et al.,2010; Chen, Kumar, Harris, Smith, & Payne, 2000; Pang, Chen,Park, Cha, & Kennedy, 2007; Pasanphan et al., 2010). Regardingthe grafted samples, higher DD%was found in Ch-g-OG1 than Ch-g-OG2. This is in agreement with the aforementioned reports inwhich usually, the increase of phenolic initial concentration in-creases the insertion onto the Ch backbone. However, our z valuesindicated the opposite in agreement also with the free aminegroups determined by the ninhidrine (Table 1). Therefore, our re-sults might otherwise indicate the presence of side oligomeric OGchains from the Ch backbone or OG propagation non-bonded to thepolysaccharide in addition to the expected monomolecular OGgrafting, which is more evident at highest gallate concentration.Further evidence is found by the absence of signals downfield foraromatic hydrogens, which points out to aromatic carbon couplingamong OG units. Control experiments conducted without Chaddition partially support this assumption with maximum yield of23% of oligomers with molecular weights (Mn) always below 600 g/mol in all control experiments as inferred by SEC analyses. Chen

Fig. 2. 1HRMN spectra of (a) Ch, (b) ChOG1, (c) ChOG2.

L. Zavaleta-Avejar et al. / Food Hydrocolloids 39 (2014) 113e119116

Table 1Percentage of deacetylation, grafted degree, z potential, amine group content, intrinsic viscosity and Huggins constant of Ch, ChOG1 and ChOG2.

Sample % DD Molar% z potential (mV) Amino group content (Abs) [h] (dL/g) k1

Ch 87.02 � 0.53a e 80.75 � 2.19a 0.615 � 0.02a 16.914 � 0.04a 0.213 � 0.04a

ChOG1 83.62 � 0.46a 4.22 � 0.04a 67.70 � 0.70b 0.123 � 0.07b 6.903 � 0.03b 2.062 � 0.44bChOG2 80.90 � 0.98a 11.75 � 0.54a 73.55 � 0.21c 0.379 � 0.01c 5.271 � 0.06b 2.031 � 0.29b

Means with different letter in the same column are significantly different (p < 0.005).Ch ¼ Native chitosan, ChOG1 ¼ chitosan-g-octyl gallate, ChOG2 ¼ chitosan-g-octyl gallate.[h] ¼ intrinsic viscosity, k1 ¼ Huggins constant.

L. Zavaleta-Avejar et al. / Food Hydrocolloids 39 (2014) 113e119 117

et al., 2000 also gave evidences in their hexyloxyphenol graftingonto Ch studies for both oligomerization of the phenolic or theresult of species no longer having phenolic structures but quinonesattached to Ch. Considering formation of stable quinones from OGunlikely, the propagation of the phenolic derivatives up to someextent cannot be ruled out in this as well as other aforementionedstudies based on enzyme-mediated grafting of gallic acid or gallatesonto Ch.

In respect of the intrinsic viscosity, the values in Table 1demonstrate that the branched structure of functionalized Chhave a higher coil densities assuming compact conformations andthe Huggins constant values show an enhanced polymerepolymerinteraction indicating poorer solvent quality.

3.2. Rheological studies

Fig. 3 shows the apparent viscosity versus shear rate plot for theCh, Ch-g-OG1 and Ch-g-OG2 dispersed in acetic or lactic acid. Thenative Ch exhibited a Newtonian behavior which did not depend onthe type of acid solvent. The shear rate dependence of apparentviscosity for Ch solutions with different concentrations wasinvestigated by Payet, Ponton, Grossiord, and Agnely (2010). Theyreported a Newtonian plateau region at low shear rate and lowconcentration due to free movement of the polymeric chainsthrough the system unperturbed by the neighbors. FunctionalizedCh solutions showed a shear-thinning. The apparent viscosityvalues and shear-thinning character increased with the graftingdegree when using acetic acid as solvent. Shear-thinning behaviorof Ch-g-OG1 y Ch-g-OG2 solutions can be related in terms ofstructure and conformation of polymeric chains that form entan-glements in solutions. When the shear rate is increased, the rate ofdisentanglement exceeds the rate at new entanglements formationand this leads to a reduction in the cross-linking density and,

Fig. 3. Flow curves of chitosan solutions of different solvents. ChA (-); ChOG1A (C);ChOG2A (:); ChL (,); ChOG1L (B); ChOG2L (6). A, acetic acid; L, lactic acid.

consequently, the reduction in viscosity. When using lactic acid as asolvent, the apparent viscosity of Ch-g-OG1 and Ch-g-OG2 waslower than that of native Ch in all shear rate interval as conse-quence of the presence of the hydroxyl group into lactic acidstructure, which induces an electrolytic stability offering a lessresistance to flow due to interaction with the amino and hydroxylgroups. This trend is in agreement with that reported by severalauthors (El-Hefian, Elgannoudi, Mainal, & Yamaya, 2010; Hwang &Shin, 2000).

Themodel that best described the experimental data (R2> 0.98)of the flow properties of all chitosan solutions was the Oswalt-deWaele or power law equation:

h ¼ k _gn (5)

where h is the apparent viscosity, k is the consistency index, and n isthe flow behavior. The k value it is a measure of the materialstructure (i.e. material “body”) and n indicates the mechanicalstability. The Power law model’s parameters for the polymer so-lutions are reported in the Table 2. It is shown that the flowbehavior index (n) decreased with the grafting degree, reflectingthe shear-thinning nature of the solutions. When using acetic acidsolvent, the consistency index (k) increased according to thegrafting degree suggesting that intermolecular and/or intra-molecular links are intensified due to hydrophobic groups andstructural conformation of the polymer; whereas, when lactic acidis used, k in Ch-g-OG1 and Ch-g-OG2 was lower even than thatfound in native Ch, this fact can be attributed to the polyelectrolyteinstability which shows that these materials are more fluid in thissolvent.

Regardless of the type of solvent, the plasticizer increased theviscosity of native chitosan solutions exhibiting a non-Newtonianbehavior with a shearethinning character (Fig. 4). In the case offunctionalized Chs, the reduction in viscosity observed in the rangeof shear rate studied, could be explained by the interaction among

Table 2Consistency index (K) and flow behavior (n) of Ch, ChOG1 and ChOG2 solutions.

Solution n K R2

ChA 0.987 0.036 0.999ChOG1A 0.756 0.271 0.999ChOG2A 0.658 0.473 0.999ChAGly 0.875 0.348 0.999ChOG1AGly 0.887 0.053 0.999ChOG2AGly 0.797 0.132 0.999ChL 0.949 0.057 0.999ChOG1L 0.913 0.013 0.976ChOG2L 0.852 0.021 0.987ChLGly 0.861 0.423 0.999ChOG1LGly 0.889 0.061 0.999ChOG2LGly 0.789 0.147 0.999

Ch ¼ Native chitosan; ChOG1 ¼ chitosan-g-octyl gallate (4.22 molar %);ChOG2 ¼ chitosan-g-octyl gallate (11.75molar%); A, acetic acid; L, lactic acid, andGly, glycerol.

Fig. 4. Flow curves of chitosan solutions of different solvents with glycerol (Gly). ChAGly

(-); ChOG1AGly (C); ChOG2AGly (:); ChLGly (,); ChOG1LGly (B); ChOG2LGly (6). A,acetic acid; L, lactic acid.

L. Zavaleta-Avejar et al. / Food Hydrocolloids 39 (2014) 113e119118

glycerol hydroxyl groups with the amino and hydroxyl groupsdecreasing the molecular hydrodynamic volume.

The consistency index (k) values match with the results ob-tained in z potential, since the charge density of the polymermolecule (native Ch > Ch-g-OG2 > Ch-g-OG1) is associated to theavailability of the amino groups, which in turn interact with thehydroxyl groups of plasticizer, resulting in entanglements regard-less of the type of solvent. The higher charge density the higherentanglements and consequently, a higher k value.

The changes in G0 and G00 as a function of frequency of the nativeand functionalized Ch dispersed in acetic and lactic acid are shownin Fig. 5. At low (<0.10 Hz) frequencies it was observed a liquid-likebehavior, where the loss modulus (G00) is higher than the storagemodulus (G0). Above 3 Hz, it was observed a crossover of themoduliindicating a predominant elastic contribution. The high depen-dence of viscoelastic moduli with the frequency indicates a weakgel behavior. Regarding the Ch, Ch-g-OG1 and Ch-g-OG2 solutionsin lactic acid, it was observed a reduction in G0 at higher molar%.This can be explained as result of enhanced interactions betweenthe OG and hydroxyl groups of lactic acid, reducing the molecular

Fig. 5. Effect of acid type on: storage modulus (G0) and loss modulus (G00) as a functionof oscillatory frequency for chitosan and chitosan-g-octyl gallate solutions at 25 �C. Fullsymbol G0 and empty symbol G00 , ChA (-); ChOG1A (C); ChOG2A (:); ChL (A);ChOG1L (<); ChOG2L (*). A, acetic acid; L, lactic acid.

entanglement. When acetic acid was used, the opposite phenom-enon was observed, that is, the molecular entanglement increasedshowing a higher G0. This behavior is typical of polysaccharides ashas been reported by various authors (Bastos et al., 2010; El-Hefianet al., 2010; Hwang & Shin, 2000).

Fig. 6 shows the effect of the glycerol on the viscoelastic prop-erties. Since the chains mobility of the biopolymers increasedbecause of the plasticizer, the G0 value decreased whereas theviscous character increased (G00), avoiding the crossover of themoduli throughout the frequency range studied.

3.3. EPR spectroscopy analyses

Fig. 7 shows the comparative radical scavenging capacity ofsamples at 1500 g/kg DPPH�. The decrease of the signal intensity [G]indicates the ability of Ch-g-OG to scavenge DPPH� compared to thelow scavenging capacity of Ch (18.58%). Noteworthy, Pasanphanet al. (2010) reported that Ch has no antioxidant capacity, howev-er slight antioxidant capacity was found in this study, which mightbe due to the source, molecular weight and degree of deacetylationof the polysaccharide. The antioxidant capacity of the functional-ized samples against DPPH� increased with the molar% and con-centration assayed up to 81% when the concentration reached1500 g/kg DPPH�. The EC50 value (the antioxidant concentration toreduce the radical by 50%) is a good indicator to the antioxidantpower. The maximum EC50 for DPPH� scavenging was 964 g/kgDPPH� and 2180 g/kg DPPH� by Ch-g-OG2 and Ch-g-OG1,respectively.

Our results are comparable with those reported by Pasanphanand Chirachanchai (2008) who found a chitosan-g-gallic scav-enging capacity of 87% when used a maximum concentration of1200 mM. In a more recent study carried out with the same chitosanderivative, it was reported an 80% scavenging capacity using aconcentration of 17.6 mg/mL (Cho, Kim, Ahn, & Je, 2011). However,these results were determined using the spectrophotometric DPPH�

method and we found that EPR gives values which are about 15%higher. This is because EPR can specifically detect the free radicals;therefore, our results indicate that functionalized Ch-g-OG is a Chderivative with strong antioxidant power.

In summary, although both functionalized Chs showed goodfunctional properties, the Ch-g-OG2 have an antioxidant powercomparable to gallic acid, a significant antimicrobial potential due

Fig. 6. Effect of acid type and glycerol on: storage modulus (G0) and loss modulus (G00)as a function of oscillatory frequency for chitosan and chitosan-g-octyl gallate solu-tions at 25 �C. Full symbol G0 and empty symbol G00 , ChAGly (-); ChOG1AGly (C);ChOG2AGly (:); ChLGly (A); ChOG1LGly (<); ChOG2LGly (*).A, acetic acid; L, lactic acid;Gly, glycerol.

Fig. 7. EPR spectra methanolic solutions (1500 g/kg DPPH) Ch, ChOG1, and ChOG2.

L. Zavaleta-Avejar et al. / Food Hydrocolloids 39 (2014) 113e119 119

to the free amino groups determined as well as a good viscoelasticproperties and adhesiveness, which are important features forfuture studies as material with potential in the formulation foredible films or coatings and applications in food preservation.

4. Conclusions

In this work a functionalized Chwith an antioxidant-hydrophobicmolecule like the OG was enzymatically obtained with two percent-age of grafting (4.22 and 11.75%) as confirmed by 1HRMN and ATR-FTIR analyses. The functional and rheological properties are themost important features which must be characterized when a newmaterial is developed. Thus, in the present research the chitosan de-rivative with the best improved functional properties was obtainedwith the higher molar%, this material also showed the best antioxi-dant capacity with an EC50 of 964 g/kg of DPPH� as well as the highercontent of free amine groups (antimicrobial activity). On the otherhand, thismaterial in acetic acid solution owns viscoelastic propertieswhichprovide structural andmechanical stabilitywhich are desirableproperties for the manufacturing of biodegradable materials such asedible films or coatings, scaffolding cells, drug delivery material, andmoisturizer among others with potential application in food preser-vation, biomedicine or cosmetics.

The results of this work may also help to better understand theinfluence of the octyl gallate concentration, solvent, and the plas-ticizer for achieving desired or improved functional properties.

Acknowledgments

This research was partially carried out with the funding supportof UAM-Iztapalapa. The authors thank CONACyT for the scholarshipof Leonor Zavaleta Avejar during her Ph.D. studies. We also wouldlike to thank Dr. Alejandro Solano for EPR analyses at the Faculty ofChemistry of Universidad Nacional Autónoma de México. We wouldlike to thank SEP-Básica CONACyT Project 165757 for funding.

References

Abugoch, L. E., Tapia, C., Villamán, M. C., Yazdani-Pedram, M., & Díaz-Dosque, M.(2011). Characterization of quinoa protein-Ch blend edible films. Food Hydro-colloids, 25(5), 879e886.

Alonso, D., Gimeno, M., Olayo, R., Vázquez-Torres, H., Sépulveda-Sánchez, J. D., &Shirai, K. (2009). Cross-linking into UV-irradiated cellulose fibers for the prepa-ration of antimicrobial-finished textiles. Carbohydrate Polymers, 77, 536e543.

Alves, M. N., & Mano, F. J. (2008). Ch derivatives obtained by chemical modificationsfor biomedical and environmental applications. International Journal of Biolog-ical Macromolecules, 43, 401e414.

Bastos, S. D., Barreto, N. B., Souza, S. H., Bastos, M., Rocha-Leão, M. M. H.,Andrade, T. C., et al. (2010). Characterization of a Ch simple extracted fromBrazilian shrimps and its application to obtain insoluble complexes with acommercial whey protein isolate. Food Hydrocolloids, 24, 709e718.

Belalia, R., Grelier, S., Benaissa, M., & Coma, V. (2008). New biomaterials based onQuaternized Ch. Journal Agriculture Food Chemistry, 56, 1582e1588.

Calero, N., Muñoz, J., Ramírez, P., & Guerrero, A. (2010). Flow behaviour, linearviscoelasticity and surface properties of Ch aqueous solutions. Food Hydrocol-loids, 24, 659e666.

Chao, A. C., Shyu, S. S., Lin, Y. C., & Mi, F. L. (2004). Enzymatic grafting of carboxylgroups on to Cheto confer on Ch the property of a cationic dye adsorbent.Bioresource Technology, 91(2), 157e162.

Chen, T., Kumar, G., Harris, T. M., Smith, J. P., & Payne, F. G. (2000). Enzymaticgrafting of hexyloxyphenol onto Ch to alter surface and rheological properties.Biotechnolog and Bioengineering, 70(5), 564e573.

Cho, Y.-S., Kim, S.-K., Ahn, Ch-B., & Je, J.-Y. (2011). Preparation, characterization, andantioxidant properties of gallic acid-grafted-Chs. Carbohydrate Polymers, 83,1617e1622.

Curcio, M., Puoci, F., Iemma, F., Parisi, O., Cirillo, G., Spizzirri, U. G., et al. (2009).Covalent insertion of antioxidant molecules on Ch by a free radical graftingprocedure. Journal Agriculture Chemistry, 57, 5933e5938.

Desbrieres, J. (2004). Autoassociative natural derivatives: the alkylchitosan. Rheo-logical behavior and temperature stability. Polymer, 45, 3285e3295.

Dutta, P. K., Tripathi, S., Mehrotra, G. K., & Dutta, J. (2009). Perspective for chitosanbased antimicrobial films in food application. Review. Food Chemistry, 114,1173e1182.

El-Hefian, E. A., Elgannoudi, S. E., Mainal, A., & Yamaya, H. A. (2010). Characteriza-tion of chitosan in acetic acid: rheological and thermal studies. Turk JournalChemistry. http://dx.doi.org/10.3906/kim-0901-38.

Fujita, K., & Kubo, I. (2002). Antifungal activity of octyl gallate. International Journalof Food Microbiology, 79, 193e201.

Hirai, A., Odani, H., & Nakajima, A. (1991). Determination of degree of deacetylationof Ch by 1H NMR spectroscopy. Polymer Bulletin, 26, 87e94.

Hwang, J. K., & Shin, H. H. (2000). Rheological properties of Ch solutions. Korea-Australia Rheology Journal, 12(3/4), 175e179.

Kasaai, R. M. (2007). Calculation of MarkeHouwinkeSakurada (MHS) equationviscometric constants for Ch in any solventetemperature system using experi-mental reported viscometric constants data. Carbohydrate Polymers, 68, 477e488.

Kumar, M. N. V. R., Muzzarelli, R. A. A., Muzzarelli, C., Sashiwa, H., & Domb, A. J.(2004). Ch chemistry and pharmaceutical perspectives. Chemistry Review, 104,6017e6084.

Kumar, G., Smith, J. P., & Payne, F. G. (1999). Enzymatic grafting of a natural productonto Ch to confer water solubility under basic conditions. Biotechnology andBioengineering, 63, 154e165.

Liu, X., Chen, Q., & Pan, H. (2007). Rheological behavior of Ch derivative/cellulosepolyblends from N-methylmorpholine N-oxide/H2O solution. Journal of MaterialScience, 42(16), 6510e6514.

Pang, T. H., Chen, G. X., Park, J. H., Cha, S. D., & Kennedy, F. J. (2007). Preparation andrheological properties of deoxycholate-Ch and carboxymethyl-Ch in aqueoussystems. Carbohydrate Polymers, 69, 419e425.

Pasanphan, W., Buettner, G. R., & Chirachanchai, S. (2010). Ch gallate as potentialpolysaccharide antioxidant: an EPR study. Carbohydrate Research, 345, 132e140.

Pasanphan, W., & Chirachanchai, S. (2008). Conjugation of gallic acid onto Ch: anapproach for green and water-based antioxidant. Carbohydrate Polymers, 72,169e177.

Payet, L., Ponton, A., Grossiord, J.-L., & Agnely, F. (2010). Structural and rheologicalproperties of Ch semiinterpenetrated networks. The European Physical Journal E,32, 109e118. http://dx.doi.org/10.1140/epje/i2010-10602-7.

Rinaudo, M., Auzely, R., Vallin, C., & Mullagaliev. (2005). Specific interactions inmodified Ch system. Biomacromolecules, 6(5), 2396e2407.

Sashiwa, H., & Aiba, S. (2004). Chemically modified chitin and Ch as biomaterials.Progress in Polymer Science, 29, 887e908.

Siripatrawan, U., & Harte, B. R. (2010). Physical properties and antioxidant activity ofan active film from Ch incorporated with green tea extract. Food Hydrocolloids,24, 770e775.

Sobahi, T. R. A., Abdelaal, M. Y., & Makki, M. S. I. (2011). Chemical modification of Chfor metal ion removal. Arabian Journal of Chemistry. http://dx.doi.org/10.1016/j.arabjc.2010.12.011.

Vachoud, L., Chen, T., Payne, F. G., & Vazquez-Duhalt, R. (2001). Peroxidase catalyzedgrafting of gallate esters onto the polysaccharide Ch. Enzyme and MicrobialTechnology, 29, 380e385.

Vartiainen, J., Rättö, M., Lantto, R., Nättinen, K., & Hurme, E. (2008). Tyrosinase-catalysed grafting of food-grade gallates to Ch: surface properties of novelfunctional coatings. Packaging Technology and Science. http://dx.doi.org/10.1002/pts.813.

Vinsová, J., & Vavríková, E. (2011). Chitosan derivatives with antimicrobial, anti-tumour and antioxidant activities e a review. Current Pharmaceutical Design, 17,3596e3607.

Xiao-Yi, H., Jian-Ping, B., Huai-Tian, B., Gang-Biao, J., & Ming-Hua, Z. (2011). Removalof anionic dye eosin Y from aqueous solution using ethylenediamine modifiedCh. Carbohydrate Polymers, 84, 1350e1356.

Xie, W., Xu, P., Wang, W., & Liu, Q. (2002). Preparation and antibacterial activity of awater-soluble Ch derivative. Carbohydrate Polymers, 50, 35e40.