Research Article Physicochemical and Antioxidant …downloads.hindawi.com/journals/ijps/2015/974506.pdfResearch Article Physicochemical and Antioxidant Properties of Chitosan Films

Research ArticlePhysicochemical and Antioxidant Properties of Chitosan FilmsIncorporated with Cinnamon Oil

Marco A Loacutepez-Mata12 Saul Ruiz-Cruz1 Norma Patricia Silva-Beltraacuten12

Joseacute de Jesuacutes Ornelas-Paz3 Viacutector Manuel Ocantildeo-Higuera4 Francisco Rodriacuteguez-Feacutelix5

Luis A Cira-Chaacutevez1 C L Del-Toro-Saacutenchez6 and Keiko Shirai7

1Departamento de Biotecnologıa y Ciencias Alimentarias Instituto Tecnologico de Sonora 5 de Febrero 818 Sur85000 Ciudad Obregon SON Mexico2Departamento de Ciencias de la Salud Universidad de Sonora Campus Cajeme Boulevard Bordo NuevoEjido Providencia 85040 Cajeme SON Mexico3Centro de Investigacion en Alimentacion y Desarrollo AC Avenida Rıo Conchos sn Parque Industrial31570 Cuauhtemoc CHIH Mexico4Departamento de Ciencias Quımico Biologicas Universidad de Sonora Encinas y Rosales sn 83000 Hermosillo SON Mexico5Departamento de Investigacion y Posgrado en Alimentos Universidad de Sonora Boulevard Luis Encinas yRosales sn Colonia Centro 83000 Hermosillo SON Mexico6Departamento de Ciencias Medicas y de la Vida Centro Universitario de la Cienega Universidad de Guadalajara47810 Ocotlan JAL Mexico7Departamento de Biotecnologıa Laboratorio de Biopolimeros Universidad Autonoma MetropolitanaAvenida San Rafael Atlixco No 186 Colonia Vicentina 09340 Ciudad de Mexico DF Mexico

Correspondence should be addressed to Saul Ruiz-Cruz sruizitsonedumx

Received 18 February 2015 Revised 13 April 2015 Accepted 15 April 2015

Academic Editor Guiping Ma

Copyright copy 2015 Marco A Lopez-Mata et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Chitosan films (CF) with cinnamon bark oil (CO) incorporated at 0 (control) 025 05 and 10 vv were prepared by anemulsion method The films were characterized based on their physical properties (solubility water vapor permeability opticalproperty and microstructure) and antioxidant properties (DPPH ABTS and its protective effects on human erythrocytes) Theresults showed that the incorporation of 05 and 10 of CO into the CF significantly decreased its solubility to 22 of the control(119901 lt 005) The water vapor permeability of the CF-CO was significantly reduced to 40 with low concentrations of CO (025)incorporated into the CF In general the films presented a yellow coloration and an increase in transparency with the incorporationof CO into the CF It was also observed that the incorporation of CO increased the antioxidant activity between 60-fold and 145-fold compared to the control and the protective capacity against erythrocyte hemolysis increased by as much as 80

1 Introduction

Consumers now demand foods that contain low levels or arefree of synthetic chemical preservatives [1] Some alternativeshave aimed to use matrices based on proteins lipids andcarbohydrates that present film formation properties [2] Chi-tosan is a carbohydrate consisting of 120573-(1-4)-D-glucosamineand 120573-(1-4)-N-acetyl-D-glucosamine obtained from thethermo-alkaline deacetylation of chitin This biopolymer is

viscous biodegradable nontoxic antigenic antimicrobialand biocompatible [3] These characteristics make it suitablefor the development of diverse applications in medicineagriculture and the food industry [4] In the food industrychitosan has been used as a natural preservative due to itsfilm formation properties It has been shown that chitosanfilms can create a semipermeable barrier that can reducebreathing retardmicrobial growth and offer other protectivequalities in fruits and vegetables [5] In addition its use as

Hindawi Publishing CorporationInternational Journal of Polymer ScienceVolume 2015 Article ID 974506 8 pageshttpdxdoiorg1011552015974506

2 International Journal of Polymer Science

a preservative in food products has been considered safe[6] However due to its hydrophilic nature one of the maindisadvantages of the CF is its limited action as a barrieragainst humidity Some alternatives that have been used toimprove the properties of chitosan have been the incorpora-tion of diverse bioactive compounds such as vitamins lipidsminerals drugs proteins colorants and essential oils [7 8]

Cinnamon bark essential oil (CO) contains a great varietyof chemical compounds (mainly cinnamaldehyde cinnamicacid coumaric acid cinnamyl alcohol and eugenol) thatpresent antioxidant and antimicrobial activities in an individ-ual and collective manner [9] In the food industry CO hasbeen used as a flavoring and preservative [10 11] However itsdirect application to food as a preservative has been limitedas it can be lost during storage due to its high volatility andreactivity with diverse food components [12] The hydropho-bic nature of CO could be exploited to make CF a betterbarrier against humidity and in turn the chitosan matrixcould conserve the properties of CO which could improvethe physical and antioxidant characteristics of CF How-ever the incorporation of CO into CF could also gener-ate undesired changes within the CF Therefore the goalof this study was to characterize the physical (solubilitywater vapor solubility (WVP) and optical) antioxidant(DPPH (22-diphenyl-1-picrylhydrazyl) ABTS (221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and protec-tive effects on human erythrocytes) and microstructuralproperties of CF surfaces containing CO incorporated by anemulsion method

2 Materials and Methods

21 Chitosan Preparation Chitosan was obtained by thethermo-alkaline deacetylation of shrimp chitin accordingto the methodology proposed by Hongpattarakere andRiyaphan [13] For this purpose 1 g of chitin was homoge-nized with 15mL of NaOH (50wv) at 95∘C for 2 h Thedegree of chitosan acetylation obtained was 34 with anaverage molecular weight of 128 kDa The chitosan was char-acterized in the Polymers Laboratory of the BiotechnologyDepartment at the Metropolitan Autonomous UniversityMexico DF

22 FC Preparation and CO Incorporation A chitosan solu-tion was prepared by dissolving 04 g of chitosan in 20mLof acetic acid (1 vv) and homogenizing the solution for4min at 15500 rpm (Ika Ultra-turrax T 18 basic Germany)Subsequently glycerol (200120583L) was added as plasticizer andagitated under similar conditions Before the incorporationof the CO (cinnamon bark oil FCC Sigma-Aldrich) thechitosan solution was emulsified with tween 80 (1 vv) asa surfactant For this study 0 CO was used as the controland three levels of CO incorporation were tested 02505 and 10 (vv) CO in chitosan solution homogenized at15500 rpm for 3minThe resulting emulsion was centrifugedat 2000 g for 5min (Hermle Labortechnik Germany) toeliminate air bubbles The emulsion was then decanted intoglass Petri dishes and left to settle for 24 h at 37∘C until theformation of the films

23 Film Thickness The film thickness was measured witha manual micrometer (Mitutoyo Japan) with a precision of003mm Five measurements were randomly taken for eachfilmThemean thickness was calculated and used to estimatethe WVP and the transparency The thickness of the filmsranged from 0086 to 0127mm

24 Scanning Electron Microscopy (SEM) The surface mor-phology of the films was examined with a scanning electronmicroscope (JEOL JSM-5410LV) (Tokyo Japan) equippedwith an INCA system and an EDS (Energy Dispersive X-Ray) microanalysis detector (Oxford Instruments Bucking-hamshire UK) and operated at 20 kV For the SEM analysisthe specimens were cut to an adequate size and placed on acylindrical copper support (1 cm in diameter) The sampleswere coatedwith gold to facilitate conduction and prevent theaccumulation of charge under electron bombardment Theanalysis of the sample was performed under high vacuumconditions

25 Solubility The solubility determination was performedaccording to the procedure reported by Casariego et al [14]The films were cut into squares (20 times 20mm) and placedin a vacuum oven (35 kPa for 24 h) to determine the weightof the dry films Subsequently the films were immersed in10mL of tridistilled water in Petri dishes kept at 25∘C for 24 hwith occasional agitation After this time the filmswere driedagain in the vacuum oven until they reached constant weightto determine the weight of dry matter that was not dissolvedin the water The solubility was calculated based on

Solubility = (Mi minusMfMi) 100 (1)

where Mi represents the initial mass and Mf the final mass

26 Measurement of the Water Vapor Permeability (WVP)The WVP was determined gravimetrically based on theASTM E96-92 method [15] The films were sealed in theupper part of a 50mL glass cup which contained 10mL ofdistilled water (100RH 3168 kPa of WVP at 25∘C) Sub-sequently they were placed in a desiccator with anhydrouscalcium sulfate at 25∘C and 0RH (0 kPa ofWVP)The cupswere weighed at 3 h intervals for 12 hThe ideal state and uni-formity of theWVPconditionswere assumedwith a constantair circulation outside of the test cup For that purpose aminiature fan was used inside the desiccator The lost weightof the cup was plotted with respect to time to estimate theslope by a linear equation (the correlation coefficient was099) Once the slope was obtained the water vapor trans-mission rate (WVTR) was calculated by

WVTR = SlopeFilm area

(2)

The WVP of the films was calculated by multiplying theWVTR by the average thickness of the film (119866) and dividingby the difference in partial pressures inside (PA1) and outside(PA2) the cup as expressed in

WVP = WVTR(PA1 minus PA2)

119866 (3)

International Journal of Polymer Science 3

27 Optical Properties The color and transparency weredetermined with a UV-Vis spectrophotometer (Cintra 10EAustralia) For the color measurement three films of eachconcentration were chosen from which the average of fivesamples was taken The film color was expressed based onthe CIE model (119871lowast 119886lowast and 119887lowast) The transparency wasdetermined according to the method established by Hanand Floros [16] For that purpose the films were cut intorectangles (9 times 45mm) and placed inside the measuring cellof the spectrophotometer The transmittance was recorded at600 nm and the film transparency was calculated by

Transparency = log(119879600119866) (4)

where 119879600 is the transmittance at 600 nm and 119866 representsthe film thickness (mm)

The UV-Vis light (200ndash800 nm) transmission propertiesthrough the films were also determined

28 Antioxidant Activity The CF-CO (50mg) was homog-enized in 5mL of 80 vv methanol using a homogenizer(Ika Ultra-turrax T 18 basic Germany) at 5∘C for 5minat 11000 rpm Afterwards the sample was centrifuged at16000 g for 10min at 5∘C the supernatant was collected andfiltered through Whatman number 1 filter paper These filmextracts were used for the assays of DPPH ABTS and theprotective effect on human erythrocytes

The DPPH was determined according to the methoddescribed by S Moein and M R Moein [17] with certainmodifications A stock solution was prepared by mixing25mg of the DPPH radical in 100mL of pure methanol andwas adjusted to 07plusmn002 at 490 nm of absorbance Trolox (6-hydroxy-2578-tetramethylchroman-2-carboxylic acid) wasused as the standard and 80methanol as the blank reactantThe sample (5 120583L) was placed on a microplate and 245 120583L ofDPPH radical was added The mixture was kept in the darkfor 30min and the absorbance was then read in a microplatereader (iMark Bio-Rad Japan) at a wavelength of 490 nmThe inhibition percentage was calculated for each of the CF-CO films which indicates the capacity of the antioxidantto reduce the radical absorbance after the final incubationFinally the results were expressed in trolox equivalents pergram of film (mmol TEg)

The ABTS test was determined according to Re et al [18]The ABTS+ radical was generated by the interaction of 5mLof ABTS solution (7mM) and 88120583L of potassium persulfatesolution (0139mM) The test was performed by adding295 120583L of the ABTS+ solution and 5 120583L of the film extractOnce the components were mixed in the microplate theabsorbancewasmonitored at 734 nmThe inhibition percent-age was calculated and converted to trolox equivalents (TE)per gram of film (mmol TEg)

29 Evaluation of the Protective Effect on the Human Ery-throcyte The hemolysis induced by AAPH [221015840-azobis (2-amidinopropane hydrochloride)] was determined by themethod established by Lu et al [19] with certain modifica-tions First the erythrocytes were washed three times withphosphate-buffered saline (PBS pH = 74) Subsequently

a 5 erythrocyte suspension was prepared with PBS Forthe erythrocyte protection test 50120583L of erythrocytes at 5was placed with 50 120583L of film extract at 400mmol in a glasstube A control mixture was prepared in a similar manner butwithout the film extract (complete hemolysis) The sampleswere incubated at 37∘C with continuous agitation at 30 rpmin the dark for 3 h After the incubation the reaction mixturewas diluted with 1mL of PBS and centrifuged at 2000 gfor 5min The supernatant absorbance was read at 540 nmin a microplate reader (iMark Bio-Rad Japan) The resultswere expressed as the hemolysis inhibition percentage in thehuman erythrocytes which was calculated using

Hemolysis inhibition percentage

= (AAPH minusHM

AAPH)times 100

(5)

whereAAPH= absorbancewith complete hemolysis andHM= absorbance of the supernatant

210 Statistical Analysis Analysis of variance was applied tothe obtained data using CF with and without CO incorpo-ration as factors The transparency WVP solubility antiox-idant and antihemolytic activity were taken as responsevariables For the decision-making process theDuncan com-parison test was used (120572 = 005) All of the above analysis wasconducted using the NCSS statistical package 2000

3 Results and Discussion

31 Microstructure The surface appearance of the filmmicrostructure changed upon the addition of cinnamon oilFigure 1 shows SEM images of the CF alone and with incor-porated CO 10 where a surface without pores and withslight folds can be observed in the CF (control) Recently CFhas been reported to lack apparent pores [14] however otherauthors have documented the presence of pores uniformlydistributed on the film surface [20] It is known that in gen-eral this characteristic depends on the nature of the chitosan(acetylation degree and molecular weight) In contrast theCFs with CO presented a rough surface with pores of approx-imately 2-3 120583m in diameter The presence of these pores islikely due to the flocculation and coalescence of small dropsof emulsified cinnamon oil during the drying of the filmsimilar results have been reported by Peng and Li [21] andLopez-Mata et al [22] in films with carvacrol incorporated

32 Solubility The solubility is a very important propertyof the films that measures their resistance to water Figure 2shows the solubility percentages of the CFs with incorporatedCO where it can be observed that the incorporation ofCO into the CF significantly decreased (119901 lt 005) the CFsolubility This decrease was 15 for CF-CO 025 and 22for CF-CO 05 and 10 compared to the control It was alsoobserved that increasing the CO concentration from 05 to10 did not induce greater insolubility of the film The solu-bility percentage of the CF (41) is consistent with the rangespreviously published for CF (16 and 46) and reported byCasariego et al [14]When working with CF it is complicated


(a) (b)

(c)

Micropores

(d)

Figure 1 Scanning Electron Microscopy (SEM) pictures of chitosan film (a) and chitosan film with 10 incorporated cinnamon oil (b cand d) oriented on the dry side

CF CO-025 CO-050 CO-100

Solu

bilit

y (

)

0

10

20

30

40

50

a

bc c

Films

Figure 2 Solubility percentages values of CF and CF-CO films

to standardize a solubility value as the chitosan propertiescan vary depending on the source acetylation degree andmolecular weightTherefore the elevated solubility exhibitedby the control film could be influenced by the presence ofthe plasticizer used during the elaboration of the film and thechitosan functional groups [23] The solubility reduction ofthe films incorporated with CO could be due to a decreasein the hydrophobic nature influenced by the loss of its free

functional groups (amino and hydroxyl) The low affinitypresented by the films with incorporated CO is importantfor their application as coatings in food products with highmoisture contents

33 Water Vapor Permeability (WVP) The WVP is a prop-erty that indicates the rate at which water is lost througha film The WVP behavior of the studied films is shown inFigure 3 These results show that the incorporation of 025CO into CF gives a WVP of 053 plusmn 004 gmmkPa hm2 a40 decrease compared to the control (088 gmmkPa hm2)which is statistically significant The incorporation of 05and 10 of CO into CF did not show significant differ-ence compared with the control with values of 0816 and10178 gmmkPa hm2 respectively Sanchez-Gonzalez et al[1] established that increasing concentrations of essential oilsin food films do not necessarily translate to a linear reductionof theWVPThe reduction of theWVP at low concentrationsof incorporated CO could be due to the action of the diversecompounds that compose the CO which could as a wholeform a hydrophobic region in the film [24] However incre-mentally increasing the CO concentration can induce struc-tural changes in the film as can be observed in Figures 1(a)1(b) 1(c) and 1(d) where the control film is compared withthe CF-CO 10 Here a surface with a rougher appearanceand with the formation of pores is observed in the case of theCF-CO 10 It has been previously documented that the


CF CO-025 CO-050 CO-10000

02

04

06

08

10

12

a

b

a

a

Films

WV

P (g

mm

kPa

hm

2)

Figure 3 Water Vapor Permeability (WVP) of the CF and CF withthe incorporation of CO

introduction of cinnamaldehyde (the main component of theCO at asymp80) into the chitosan structure can react with theamino group of the C2 inducing the formation of a Schiffbase [25] This base can induce cross-linking of the chitosanpolymer and it has been observed that this cross-linkingcan induce the presence of pores in the films Also severalauthors have shown the properties of cinnamaldehyde as across-linking agent with chitosan [26 27] and these authorsreported that the introduction of aldehyde groups in chitosancan induce pore formation in the structure under controlledconditionsThe slight increase inWVP of the CFwith 10 ofCO could be due to the formation of pores which could onlyoccur in superficial parts of the film or in a partial mannerall pores do not necessarily form channels that connect bothsides of the film However it is also possible that thesepores arose from the instability of the emulsion drops whichescaped and left an apparent pore traceThese results are simi-lar to the report by Sun et al [28] who evaluated chitosan filmincorporated with various concentrations of complex of 120573-cyclodextrin and essential oils as eugenol cinnamaldehydeand carvacrol and Bonilla et al [29] who evaluated chitosanfilm with basil essential oil These authors showed that whenthe concentration of oils increased the WVP of the filmincreased and this increase was attributed to excessive oilsin the films which subsequently decreased the intermolec-ular forces between polymer chains that induce segmentalmotions and free space causing a more open matrix

34 Color Properties The measurement of the color param-eters is an important property in describing the appearanceof films Table 1 shows the effects of CO incorporationinto the CF where it can be observed that the CF withincorporated CO generally presented a yellow coloration(119887lowast) This coloration was significantly lower when CO wasincorporated at concentrations of 025 and 05 Howeverthe yellow coloration in the CF was intensified with a higherconcentration of incorporated CO (10) (119901 lt 005)Consequently this phenomenon could be considered normal

Table 1 Color parameters of CF and CF-CO films

Films 119871lowast

119886lowast

119887lowast

CF 6815 plusmn 073a minus012 plusmn 004a 625 plusmn 010a

CO-025 6789 plusmn 100a minus015 plusmn 002a 544 plusmn 009b

CO-050 6531 plusmn 099a 014 plusmn 001a 582 plusmn 008c

CO-100 5953 plusmn 042b 013 plusmn 004a 777 plusmn 005dlowastThe data represent the mean plusmn standard deviation of three determinationsand different letters in the same column indicate significant differences (119901 lt005)

in the control film as this coloration has been associatedwith the presence of repeated units of 120573-(1-4)-2-amino-2-deoxy-D-glucopyranose [20]The reduction of the colorationpresented in the films with 025 and 05 incorporated COcould be due to the competition of the diverse CO com-pounds when interacting with the functional groups of theCF however this effect could also be associatedwith the pres-ence of the surfactant (Tween 80) used during the elaborationof the film which can also form uniform structures with thechitosan [30]Therefore the increase in the yellow colorationof the CF with 10 CO could be due to the increasedcinnamaldehyde concentration and the possible formationof a Schiff base However the values of 119871lowast (luminosity andbrightness) significantly decreased in the CF with 10 COThis variation in the luminosity induced by the incorporationof CO could be due to the molecular alteration of chitosan

The transparency of the film is a desirable propertybecause the consumer needs to clearly see the productcovered by the film [31] The CFs with 05 and 10CO incorporated presented greater transparency than thecontrol (Table 2) which indicates that the incorporation ofCO can reduce the opacity of the CF However even withthis reduction the values indicate that the films are slightlyopaque Another evaluated parameter was the transmissionof light through the film where it could be observed that thefilmswith incorporatedCO and the control have the propertyof being able to block ultraviolet radiation in the region of280 nm in a transmission range of 015 to 002 How-ever the lowest transmission of the films was in the 200 nmregion (019ndash001)

35 Antioxidant Activity The antioxidant activity of thefilms was measured by two methods DPPH and ABTS Thecombination of two methods to measure the antioxidantactivity has been recommended for a better understandingof the extract antioxidant properties Figure 4 shows theantioxidant activity (DPPH) of the extracts of the studiedfilms A significant increase in the antioxidant capacity ofthe CF with CO incorporated at 05 (601 times) and 10(145 times) can be observed compared to the control Thisactivity can be attributed to the presence of the main COantioxidant which is eugenol however it could also be due tothe presence of cinnamaldehyde which has been reported tohave a lower activity than eugenol or to a synergetic effectamong chitosan eugenol and cinnamaldehyde [32] It hasbeen previously demonstrated that chitosan presents antiox-idant activity which has been attributed to the presence of


Table 2 Light transmittance () and transparency values of CF and CF-CO films

Films Wavelength (nm) Transparency valuelowast200 280 350 400 500 600 700 800

CF 019 015 2036 3046 3725 4020 4209 4347 266 plusmn 0012a

CO-025 008 017 1682 2851 3437 3664 3805 3907 269 plusmn 0008a

CO-050 001 002 1375 2719 3342 3601 3758 3871 256 plusmn 0010d

CO-100 000 002 525 1840 2556 2847 3017 3125 234 plusmn 0003clowastThe data represent the mean plusmn standard deviation of three determinations and different letters in the same column indicate significant differences (119901 lt 005)

CF CO-025 CO-050 CO-1000

2

4

6

8

10

12

14

DPP

H (m

mol

ET

g of

film

)

cc

b

a

Films

Figure 4 Antioxidant capacity of films extract determined byDPPHThe data are themean values of at least three determinationsThemean values represented by the bars for each type of films extractthat are indicated with a different letter are significantly different(119901 le 005)

nitrogen located at the C2 of the polymeric structure [33]The antioxidant activity tested by ABTS was similar to theresults of the DPPH testing method Therefore these resultsshow that the antioxidant capacity is increased between 61and 164 times for the CF with CO incorporated at 05 and10 respectively compared to the control (Figure 5)

36 Evaluation of the Protective Effect onHuman ErythrocytesFor this evaluation a radical initializer was used (AAPH)which generates peroxylalkoxy radicals in the presence ofoxygen at physiological temperature These secondary radi-cals are responsible for biological damage to cells Figure 6shows the protective effects of the CFs with incorporatedCO and the control extracts In general all films offered aprotective effect to the erythrocytes this effect providedmorethan 67 hemolysis inhibition The extract of the CFs with10 CO presented the greatest protective effect inhibitinghemolysis by up to 80 (119901 lt 005) It is important to highlightthat the control presented a higher capacity for hemolysisinhibition Previous studies have shown that chitosan with itspositively charged amino groups can interact with the nega-tive charges of the erythrocyte membrane and produce a pos-sible chitosan-erythrocyte aggregate that does not seriouslydamage the erythrocyte membrane [34] This aggregationmost likely forms a net that protect erythrocytes against the

CF CO-025 CO-050 CO-100

ABT

S (m

mol

ET

g of

film

)

0

2

4

6

8

10

12

14

16

18

20

cc

b

a

Films

Figure 5 Antioxidant capacity of films extract determined byABTSThe data are the mean values of at least three determinationsThemean values represented by the bars for each type of films extractthat are indicated with a different letter are significantly different(119901 le 005)

FilmsCF CO-025 CO-050 CO-100

Hem

olys

is in

hibi

tion

()

0

20

40

60

80

100

bb b a

Figure 6 Antioxidant capacity of films extract determined bypercentage of hemolysis inhibition The data are the mean values ofat least three determinations The mean values represented by thebars for each type of films extract that are indicated with a differentletter are significantly different (119901 le 005)

attacks of radicals generated by the AAPH In the case of theCF extract with the highest incorporated CO concentrationits greater hemolysis inhibition effect could be associated


with the probable presence of other biomolecules that mightinhibit the radicals in addition to the possible protective netformed by the chitosan-erythrocyte charges

4 Conclusions

In this study multiple concentrations of CO were incorpo-rated into CFs and their physical and antioxidant propertieswere evaluated The incorporation of CO was observed todecrease the CF solubility while WVP was only reducedat low incorporated CO concentrations (025) It was alsoobserved that all films presented a yellow coloration whichwas reduced at low incorporated CO concentrations butaccentuated at incorporated CO concentrations of 10Regarding the transparency it was observed that the incor-poration of CO can improve the transparency of the filmsand also act as a good blocker of ultraviolet radiation Theantioxidant activity of theCF incorporatedwithCO strength-ened the antioxidant activity of the films by 60 to 145 timescompared to the control and the filmswith 10CO incorpo-rated could protect erythrocytes against free radical attack byup to 80

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Financial support from PROMEP (ITSON-PTC-056 and1035122051) and ITSON (PROFAPI 0270 and 00401) isgratefully acknowledged

References

[1] L Sanchez-Gonzalez M Vargas C Gonzalez-Martınez AChiralt and M Chafer ldquoUse of essential oils in bioactive ediblecoatings a reviewrdquo Food Engineering Reviews vol 3 no 1 pp1ndash16 2011

[2] D Lin and Y Zhao ldquoInnovations in the development andapplication of edible coatings for fresh andminimally processedfruits and vegetablesrdquo Comprehensive Reviews in Food Scienceand Food Safety vol 6 no 3 pp 60ndash75 2007

[3] H K No S P Meyers W Prinyawiwatkul and Z Xu ldquoAppli-cations of chitosan for improvement of quality and shelf life offoods a reviewrdquo Journal of Food Science vol 72 no 5 pp R87ndashR100 2007

[4] G Cardenas P Anaya C von Plessing C Rojas and JSepulveda ldquoChitosan composite films Biomedical applica-tionsrdquo Journal of Materials Science Materials in Medicine vol19 no 6 pp 2397ndash2405 2008

[5] G A Gonzalez-Aguilar E Valenzuela-Soto J Lizardi-Mendozaet al ldquoEffect of chitosan coating in preventing deterioration andpreserving the quality of fresh-cut papaya lsquoMaradolrsquordquo Journal ofthe Science of Food andAgriculture vol 89 no 1 pp 15ndash23 2009

[6] M Friedman and V K Juneja ldquoReview of antimicrobial andantioxidative activities of chitosans in foodrdquo Journal of FoodProtection vol 73 no 9 pp 1737ndash1761 2010

[7] C A Campos L N Gerschenson and S K Flores ldquoDevelop-ment of edible films and coatings with antimicrobial activityrdquoFood and Bioprocess Technology vol 4 no 6 pp 849ndash875 2011

[8] V Giatrakou A Ntzimani and I N Savvaidis ldquoCombined chi-tosan-thyme treatments with modified atmosphere packagingon a ready-to-cook poultry productrdquo Journal of Food Protectionvol 73 no 4 pp 663ndash669 2010

[9] Y Ding E QWu C Liang et al ldquoDiscrimination of cinnamonbark and cinnamon twig samples sourced from various coun-tries using HPLC-based fingerprint analysisrdquo Food Chemistryvol 127 no 2 pp 755ndash760 2011

[10] L Shojun C Freitag and J J Morrell ldquoPreventing fungalattack of freshly sawn lumber using cinnamon extractsrdquo ForestProducts Journal vol 58 no 7-8 pp 77ndash81 2008

[11] B Shan Y-Z Cai J D Brooks and H Corke ldquoAntibacte-rial properties and major bioactive components of cinnamonstick (Cinnamomum burmannii) activity against foodbornepathogenic bacteriardquo Journal of Agricultural and Food Chem-istry vol 55 no 14 pp 5484ndash5490 2007

[12] C L del Toro-Sanchez J F Ayala-Zavala L Machi et alldquoControlled release of antifungal volatiles of thyme essential oilfrom 120573-cyclodextrin capsulesrdquo Journal of Inclusion Phenomenaand Macrocyclic Chemistry vol 67 no 3 pp 431ndash441 2010

[13] T Hongpattarakere and O Riyaphan ldquoEffect of deacetylationconditions on antimicrobial activity of chitosans prepared fromcarapace of black tiger shrimp (Penaeus monodon)rdquo Songk-lanakarin Journal of Science and Technology vol 30 no 1 pp1ndash9 2008

[14] A Casariego B W S Souza M A Cerqueira et al ldquoChitosanclay filmsrsquo properties as affected by biopolymer and clay micronanoparticlesrsquo concentrationsrdquo Food Hydrocolloids vol 23 no7 pp 1895ndash1902 2009

[15] V Guillard B Broyart C Bonazzi S Guilbert and N GontardldquoPreventing moisture transfer in a composite food using ediblefilms experimental and mathematical studyrdquo Journal of FoodScience vol 68 no 7 pp 2267ndash2277 2003

[16] J H Han and J D Floros ldquoCasting antimicrobial packagingfilms andmeasuring their physical properties and antimicrobialactivityrdquo Journal of Plastic Film and Sheeting vol 13 no 4 pp287ndash298 1997

[17] S Moein and M R Moein ldquoRelationship between antioxidantproperties and phenolics in Zhumeria majdaerdquo Journal ofMedicinal Plants Research vol 4 no 7 pp 517ndash521 2010

[18] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[19] J Lu Y Jin G Liu et al ldquoFlavonoids from the leaves of actinidiakolomiktardquo Chemistry of Natural Compounds vol 46 no 2 pp205ndash208 2010

[20] M Pereda A G Ponce N E Marcovich R A Ruseckaite andJ F Martucci ldquoChitosan-gelatin composites and bi-layer filmswith potential antimicrobial activityrdquo Food Hydrocolloids vol25 no 5 pp 1372ndash1381 2011

[21] Y Peng andY Li ldquoCombined effects of two kinds of essential oilson physical mechanical and structural properties of chitosanfilmsrdquo Food Hydrocolloids vol 36 pp 287ndash293 2014

[22] M A Lopez-Mata S Ruiz-Cruz N P Silva-Beltran J D JOrnelas-Paz P B Zamudio-Flores and S E Burruel-IbarraldquoPhysicochemical antimicrobial and antioxidant properties ofchitosan films incorporated with carvacrolrdquo Molecules vol 18no 11 pp 13735ndash13753 2013


[23] S Y Park K S Marsh and J W Rhim ldquoCharacteristics ofdifferent molecular weight chitosan films affected by the type oforganic solventsrdquo Journal of Food Science vol 67 no 1 pp 194ndash197 2002

[24] I Leceta P Guerrero and K de la Caba ldquoFunctional propertiesof chitosan-based filmsrdquo Carbohydrate Polymers vol 93 no 1pp 339ndash346 2013

[25] E P Azevedo and V Kumar ldquoRheological water uptake andcontrolled release properties of a novel self-gelling aldehydefunctionalized chitosanrdquo Carbohydrate Polymers vol 90 no 2pp 894ndash900 2012

[26] S M Ojagh M Rezaei S H Razavi and S M H HosseinildquoDevelopment and evaluation of a novel biodegradable filmmade from chitosan and cinnamon essential oil with low affinitytoward waterrdquo Food Chemistry vol 122 no 1 pp 161ndash166 2010

[27] L Higueras G Lopez-Carballo R Gavara and P Hernandez-Munoz ldquoReversible covalent immobilization of cinnamalde-hyde on chitosan films via schiff base formation and theirapplication in active food packagingrdquo Food and BioprocessTechnology vol 8 no 3 pp 526ndash538 2015

[28] X Sun S Sui C Ference et al ldquoAntimicrobial and mechanicalproperties of 120573-cyclodextrin inclusion with essential oils inchitosan filmsrdquo Journal of Agricultural and Food Chemistry vol62 no 35 pp 8914ndash8918 2014

[29] J Bonilla M Vargas L Atares and A Chiralt ldquoPhysicalproperties of chitosan-basil essential oil edible films as affectedby oil content and homogenization conditionsrdquo Procedia FoodScience vol 1 pp 50ndash56 2011

[30] Y Peng L Yin and Y Li ldquoCombined effects of lemon essentialoil and surfactants on physical and structural properties of chi-tosan filmsrdquo International Journal of Food Science amp Technologyvol 48 no 1 pp 44ndash50 2013

[31] C T Jutaporn C Suphitchaya andWThawien ldquoAntimicrobialactivity and characteristics of edible films incorporated withPhayom wood (Shorea tolura) extractrdquo International FoodResearch Journal vol 18 no 1 pp 39ndash54 2011

[32] S Mathew and T E Abraham ldquoStudies on the antioxidantactivities of cinnamon (Cinnamomum verum) bark extractsthrough various in vitro modelsrdquo Food Chemistry vol 94 no4 pp 520ndash528 2006

[33] P J Park J Y Je and S K Kim ldquoFree radical scavengingactivities of differently deacetylated chitosans using an ESRspectrometerrdquo Carbohydrate Polymers vol 55 no 1 pp 17ndash222004

[34] S Hirano M Zhang M Nakagawa and T Miyata ldquoWet spunchitosan-collagen fibers their chemical N-modifications andblood compatibilityrdquo Biomaterials vol 21 no 10 pp 997ndash10032000

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

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Polymer ScienceInternational Journal of



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CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


a preservative in food products has been considered safe[6] However due to its hydrophilic nature one of the maindisadvantages of the CF is its limited action as a barrieragainst humidity Some alternatives that have been used toimprove the properties of chitosan have been the incorpora-tion of diverse bioactive compounds such as vitamins lipidsminerals drugs proteins colorants and essential oils [7 8]

Cinnamon bark essential oil (CO) contains a great varietyof chemical compounds (mainly cinnamaldehyde cinnamicacid coumaric acid cinnamyl alcohol and eugenol) thatpresent antioxidant and antimicrobial activities in an individ-ual and collective manner [9] In the food industry CO hasbeen used as a flavoring and preservative [10 11] However itsdirect application to food as a preservative has been limitedas it can be lost during storage due to its high volatility andreactivity with diverse food components [12] The hydropho-bic nature of CO could be exploited to make CF a betterbarrier against humidity and in turn the chitosan matrixcould conserve the properties of CO which could improvethe physical and antioxidant characteristics of CF How-ever the incorporation of CO into CF could also gener-ate undesired changes within the CF Therefore the goalof this study was to characterize the physical (solubilitywater vapor solubility (WVP) and optical) antioxidant(DPPH (22-diphenyl-1-picrylhydrazyl) ABTS (221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and protec-tive effects on human erythrocytes) and microstructuralproperties of CF surfaces containing CO incorporated by anemulsion method

2 Materials and Methods

21 Chitosan Preparation Chitosan was obtained by thethermo-alkaline deacetylation of shrimp chitin accordingto the methodology proposed by Hongpattarakere andRiyaphan [13] For this purpose 1 g of chitin was homoge-nized with 15mL of NaOH (50wv) at 95∘C for 2 h Thedegree of chitosan acetylation obtained was 34 with anaverage molecular weight of 128 kDa The chitosan was char-acterized in the Polymers Laboratory of the BiotechnologyDepartment at the Metropolitan Autonomous UniversityMexico DF

22 FC Preparation and CO Incorporation A chitosan solu-tion was prepared by dissolving 04 g of chitosan in 20mLof acetic acid (1 vv) and homogenizing the solution for4min at 15500 rpm (Ika Ultra-turrax T 18 basic Germany)Subsequently glycerol (200120583L) was added as plasticizer andagitated under similar conditions Before the incorporationof the CO (cinnamon bark oil FCC Sigma-Aldrich) thechitosan solution was emulsified with tween 80 (1 vv) asa surfactant For this study 0 CO was used as the controland three levels of CO incorporation were tested 02505 and 10 (vv) CO in chitosan solution homogenized at15500 rpm for 3minThe resulting emulsion was centrifugedat 2000 g for 5min (Hermle Labortechnik Germany) toeliminate air bubbles The emulsion was then decanted intoglass Petri dishes and left to settle for 24 h at 37∘C until theformation of the films

23 Film Thickness The film thickness was measured witha manual micrometer (Mitutoyo Japan) with a precision of003mm Five measurements were randomly taken for eachfilmThemean thickness was calculated and used to estimatethe WVP and the transparency The thickness of the filmsranged from 0086 to 0127mm

24 Scanning Electron Microscopy (SEM) The surface mor-phology of the films was examined with a scanning electronmicroscope (JEOL JSM-5410LV) (Tokyo Japan) equippedwith an INCA system and an EDS (Energy Dispersive X-Ray) microanalysis detector (Oxford Instruments Bucking-hamshire UK) and operated at 20 kV For the SEM analysisthe specimens were cut to an adequate size and placed on acylindrical copper support (1 cm in diameter) The sampleswere coatedwith gold to facilitate conduction and prevent theaccumulation of charge under electron bombardment Theanalysis of the sample was performed under high vacuumconditions

25 Solubility The solubility determination was performedaccording to the procedure reported by Casariego et al [14]The films were cut into squares (20 times 20mm) and placedin a vacuum oven (35 kPa for 24 h) to determine the weightof the dry films Subsequently the films were immersed in10mL of tridistilled water in Petri dishes kept at 25∘C for 24 hwith occasional agitation After this time the filmswere driedagain in the vacuum oven until they reached constant weightto determine the weight of dry matter that was not dissolvedin the water The solubility was calculated based on

Solubility = (Mi minusMfMi) 100 (1)

where Mi represents the initial mass and Mf the final mass

26 Measurement of the Water Vapor Permeability (WVP)The WVP was determined gravimetrically based on theASTM E96-92 method [15] The films were sealed in theupper part of a 50mL glass cup which contained 10mL ofdistilled water (100RH 3168 kPa of WVP at 25∘C) Sub-sequently they were placed in a desiccator with anhydrouscalcium sulfate at 25∘C and 0RH (0 kPa ofWVP)The cupswere weighed at 3 h intervals for 12 hThe ideal state and uni-formity of theWVPconditionswere assumedwith a constantair circulation outside of the test cup For that purpose aminiature fan was used inside the desiccator The lost weightof the cup was plotted with respect to time to estimate theslope by a linear equation (the correlation coefficient was099) Once the slope was obtained the water vapor trans-mission rate (WVTR) was calculated by

WVTR = SlopeFilm area

(2)

The WVP of the films was calculated by multiplying theWVTR by the average thickness of the film (119866) and dividingby the difference in partial pressures inside (PA1) and outside(PA2) the cup as expressed in

WVP = WVTR(PA1 minus PA2)

119866 (3)












= (AAPH minusHM

AAPH)times 100

(5)







(a) (b)

(c)

Micropores

(d)


CF CO-025 CO-050 CO-100

Solu

bilit

y (

)

0

10

20

30

40

50

a

bc c

Films






CF CO-025 CO-050 CO-10000

02

04

06

08

10

12

a

b

a

a

Films

WV

P (g

mm

kPa

hm

2)





Films 119871lowast

119886lowast

119887lowast











CF 019 015 2036 3046 3725 4020 4209 4347 266 plusmn 0012a

CO-025 008 017 1682 2851 3437 3664 3805 3907 269 plusmn 0008a

CO-050 001 002 1375 2719 3342 3601 3758 3871 256 plusmn 0010d


CF CO-025 CO-050 CO-1000

2

4

6

8

10

12

14

DPP

H (m

mol

ET

g of

film

)

cc

b

a

Films




CF CO-025 CO-050 CO-100

ABT

S (m

mol

ET

g of

film

)

0

2

4

6

8

10

12

14

16

18

20

cc

b

a

Films



Hem

olys

is in

hibi

tion

()

0

20

40

60

80

100

bb b a





4 Conclusions




Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials














= (AAPH minusHM

AAPH)times 100

(5)







(a) (b)

(c)

Micropores

(d)


CF CO-025 CO-050 CO-100

Solu

bilit

y (

)

0

10

20

30

40

50

a

bc c

Films






CF CO-025 CO-050 CO-10000

02

04

06

08

10

12

a

b

a

a

Films

WV

P (g

mm

kPa

hm

2)





Films 119871lowast

119886lowast

119887lowast











CF 019 015 2036 3046 3725 4020 4209 4347 266 plusmn 0012a

CO-025 008 017 1682 2851 3437 3664 3805 3907 269 plusmn 0008a

CO-050 001 002 1375 2719 3342 3601 3758 3871 256 plusmn 0010d


CF CO-025 CO-050 CO-1000

2

4

6

8

10

12

14

DPP

H (m

mol

ET

g of

film

)

cc

b

a

Films




CF CO-025 CO-050 CO-100

ABT

S (m

mol

ET

g of

film

)

0

2

4

6

8

10

12

14

16

18

20

cc

b

a

Films



Hem

olys

is in

hibi

tion

()

0

20

40

60

80

100

bb b a





4 Conclusions




Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b)

(c)

Micropores

(d)


CF CO-025 CO-050 CO-100

Solu

bilit

y (

)

0

10

20

30

40

50

a

bc c

Films






CF CO-025 CO-050 CO-10000

02

04

06

08

10

12

a

b

a

a

Films

WV

P (g

mm

kPa

hm

2)





Films 119871lowast

119886lowast

119887lowast











CF 019 015 2036 3046 3725 4020 4209 4347 266 plusmn 0012a

CO-025 008 017 1682 2851 3437 3664 3805 3907 269 plusmn 0008a

CO-050 001 002 1375 2719 3342 3601 3758 3871 256 plusmn 0010d


CF CO-025 CO-050 CO-1000

2

4

6

8

10

12

14

DPP

H (m

mol

ET

g of

film

)

cc

b

a

Films




CF CO-025 CO-050 CO-100

ABT

S (m

mol

ET

g of

film

)

0

2

4

6

8

10

12

14

16

18

20

cc

b

a

Films



Hem

olys

is in

hibi

tion

()

0

20

40

60

80

100

bb b a





4 Conclusions




Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




CF CO-025 CO-050 CO-10000

02

04

06

08

10

12

a

b

a

a

Films

WV

P (g

mm

kPa

hm

2)





Films 119871lowast

119886lowast

119887lowast











CF 019 015 2036 3046 3725 4020 4209 4347 266 plusmn 0012a

CO-025 008 017 1682 2851 3437 3664 3805 3907 269 plusmn 0008a

CO-050 001 002 1375 2719 3342 3601 3758 3871 256 plusmn 0010d


CF CO-025 CO-050 CO-1000

2

4

6

8

10

12

14

DPP

H (m

mol

ET

g of

film

)

cc

b

a

Films




CF CO-025 CO-050 CO-100

ABT

S (m

mol

ET

g of

film

)

0

2

4

6

8

10

12

14

16

18

20

cc

b

a

Films



Hem

olys

is in

hibi

tion

()

0

20

40

60

80

100

bb b a





4 Conclusions




Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






CF 019 015 2036 3046 3725 4020 4209 4347 266 plusmn 0012a

CO-025 008 017 1682 2851 3437 3664 3805 3907 269 plusmn 0008a

CO-050 001 002 1375 2719 3342 3601 3758 3871 256 plusmn 0010d


CF CO-025 CO-050 CO-1000

2

4

6

8

10

12

14

DPP

H (m

mol

ET

g of

film

)

cc

b

a

Films




CF CO-025 CO-050 CO-100

ABT

S (m

mol

ET

g of

film

)

0

2

4

6

8

10

12

14

16

18

20

cc

b

a

Films



Hem

olys

is in

hibi

tion

()

0

20

40

60

80

100

bb b a





4 Conclusions




Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





4 Conclusions




Acknowledgments


References











































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Documents

Research Article Physicochemical and Antioxidant …downloads.hindawi.com/journals/ijps/2015/974506.pdfResearch Article Physicochemical and Antioxidant Properties of Chitosan Films