ORIGINAL PAPER

Nano Zinc Oxide-Loaded Calcium Alginate Filmswith Potential Antibacterial Properties

Sunil K. Bajpai & Navin Chand & Varsha Chaurasia

Received: 14 August 2010 /Accepted: 24 April 2011 /Published online: 11 May 2011# Springer Science+Business Media, LLC 2011

Abstract In this work, zinc oxide nanoparticles-loadedcalcium alginate films were investigated for their moistureuptake behavior at different temperatures. The equilibriumuptake data was interpreted quantitatively by GAB isothermmodels. The monolayer moisture contents were 0.301±0.003,0.0214±0.092, and 0.171±0.102 at 20, 30, and 37°C,respectively. The water vapor transmission rate was found tobe 0.816±0.143, 1.42±0.045, and 1.632±0.064 gs−1 m−2

respectively. For the moisture content range of 0.2 to 0.6,the net ΔH and ΔS values were found to be 22.73 to11.14 KJ/mol and 0.064 to 0.034 KJ/mol/K, respectively.The moisture uptake of films increased with water activitybut showed negative temperature dependence. The enthal-py of sorption (ΔH) and differential entropy (ΔS) weredetermined at different moisture content values, rangingfrom 0.2 to 0.6 g/g db. The two parameters showed ahigher degree of correlation. The equilibrium moisturecontent data was used to evaluate harmonic meantemperature Thm. Finally, the biocidal action of films wastested against model bacteria Escherichia coli.

Keywords Alginate . Zinc oxide nanoparticles .Watervapor permeability . GAB isotherm .E. coli

Introduction

In recent times, there has been a growing interest to developmaterials with film-forming capacity and also to loadantimicrobial agents helpful to maintain shelf life and improvefood safety. Antimicrobial packaging is one of the mostpromising active packaging systems, quite effective in killingor inhibiting spoilage and pathogenic micro-organismscausing contamination of food (Salleh et al. 2007). Antimi-crobial films are being used to control microbial growth infood ingredients. These films contain antimicrobial agents(Dutta et al. 2009) which belong to a wide range of organic/inorganic compounds (Eswaranandam et al. 2004), essentialoils (Chaibi et al. 1997), enzymes (Gucbilmez et al. 2007),fruit extracts (Conte et al. 2007), etc. These antibacterialagents possess great potential to inhibit microbial growth infood stuff. However, due to the development of new resistantstrains of bacteria to current antibiotics (Singh et al. 2008),these conventional antimicrobial agents have been losingtheir effectiveness. Thus, the current research is focused tosearch, new bactericides to reduce the harmful effects ofmicroorganism effectively. With the emergence of nanotech-nology, the search has now focused on the development ofnanostructure of coinage metals like silver, copper, zinc andgold as biocidal agents (Sondi and Salopek-Sondi 2004).The hiking rate of silver and gold metals has limited theiruse as antibacterial agents on industrial basis. As per severalrecent reports, ZnO nanoparticles have demonstrated fairantimicrobial activity (Yadav et al. 2006) and it has beenshown on the basis of preliminary growth analysis, that ZnOnanoparticles have higher antibacterial affects on microor-ganism like Staphylococcus aureus than any other metaloxide nanoparticles (Jones et al. 2007). Recently Tam et al.(2008) has studied, antibacterial activity of ZnO nanorodsprepared by hydrothermal method, and found that ZnO

S. K. Bajpai (*) :V. ChaurasiaPolymer Research Laboratory Department of Chemistry,Government Model Science College (Auton),Jabalpur, MP, Indiae-mail: sunil.mnlbpi@gmail.com

S. K. Bajpaie-mail: polymerlab@rediffmail.com

N. ChandAdvanced Materials and Processes Research Institute (AMPRI),formerly RRL Bhopal, CSIR,Bhopal, MP, India

Food Bioprocess Technol (2012) 5:1871–1881DOI 10.1007/s11947-011-0587-6

exhibited fair activity against Escherichia coli and Bacillusatrophaeus, though it was considerably more effective in thelater case (at 15 mM versus 5 mM concentration, respec-tively, showing zero viable cell count).

Antimicrobial films are usually made from proteins(Reinose et al. 2008), lipids, and polysaccharides whichare non-toxic, bio-compatible, and biodegradable. Recently,Bourtoom (2008) has reviewed the properties of ediblefilms that are produced using different types of naturalbiopolymers. Although a number of polysaccharides havebeen used for the preparation of edible films (Bruno et al.2008), unfortunately, this is not the case with calciumalginate which produces a good quality film when cross-linked with divalent metal ions like calcium (Olivas andBarbosa-Canovas 2008). Alginate is a structural polysac-charide, extracted from brown algae. On a molecular basis,it is composed of guluronic (G) and mannuronic (M) acidunits forming regions of M-blocks, G-blocks, and blocks ofalternating sequence (MG-blocks; Ertesvag and Valla1998). This is a significant biopolymer due to its importantfeatures such as biocompatibility, biodegradability, cost-effectiveness, and tendency to form gel via metal ionsinduced crosslinking at room temperature in an aqueousmedium. The ionotropic gelation results from specific andstrong interactions between Ca++ ions and carboxylategroups of guluronate blocks of alginate chains. This hasbeen well demonstrated by “egg-box” model (Braccine andPerez 2001). The present work describes a detailedinvestigation of moisture uptake behavior and water vaporpermeation properties of ZnO nanoparticles-loaded calciumalginate films. The films have also been investigated fortheir biocidal action against E. coli as model bacteria. Asper survey of literature, there are no reports on moistureuptake and antimicrobial behavior of zinc oxidenanoparticles-loaded calcium alginate films.

Materials and Methods

Materials

Sodium alginate (mol. mass 18,000 as determined visco-metrically (Bajpai and Tankhiwale 2007), G/M ratio −1.2 asper manufacturer specifications) was obtained from CentralDrug House, Mumbai, India. The crosslinker calciumchloride, nutrient agar, agar-agar type 1, nutrient broth,plasticizer glycerol, and other salts employed to preparesaturated salt solutions to create required relative humidities(RH), were purchased from E. Merck, Mumbai, India andwere analytical grade. Zinc chloride (analytical grade) andliquid ammonia were purchased from Hi Media Chemicals,Mumbai, India. The department of microbiology suppliedthe model bacteria E. coli whose source was contaminated

water and was pathogenic type. The double distilled waterwas used throughout the investigations.

Preparation of Zinc Oxide Nanoparticles

Zinc oxide nanoparticles were produced by hydrothermalapproach as described by Campo et al. (2009). In brief,0.2 M aqueous solution of sodium hydroxide was addeddrop-wise into 100 ml of 0.1 M zinc chloride solution underconstant stirring and the resulting solution was heated at60°C for 2 h. The reaction mixture was left standingovernight, filtered the next morning and precipitateobtained was kept in an electric oven at 60°C to ensurethe complete formation of zinc oxide nanoparticles.

Preparation of Zinc Oxide Nanoparticles-Loaded CalciumAlginate (ZNLCaA) Films

To 20 ml of 4% (w/v) solution of sodium alginate, definitequantity of zinc oxide nanoparticles was added and thesolution was mixed thoroughly under high stirring rate of160 rpm for 1 h (Remi equipments Pvt. Ltd, India). Now,1 ml of 5% calcium chloride solution (w/v) and 1 ml ofglycerol were added and the resulting mixture was furtherstirred for 30 min. The suspension was poured into Petridishes (9 cm) and kept in an air-circulated electric oven(Tempstar, India) at 50°C for 24 h. The resulting films,thus obtained, were placed in 5% calcium chloridesolution (w/v) for 1 min to further crosslink the films.Finally, the films were washed with distilled water andthen allowed to dry at 40°C till they obtained constantweight. It was observed that plain alginate films requirednearly 12 h for complete drying, whereas ZnO-loadedfilms took almost 11 to 12 h. The films were designated aszinc oxide nanoparticles-loaded calcium alginate(ZNLCaA) (25) where the number in parenthesis denotesthe amount of zinc oxide nanoparticles (in milligrams)present in 1 g of calcium alginate. The thickness of thefilms was measured using a micrometer and found to be(9.0±1.1)×10−5 m.

Characterization of Films

The morphological features of plain and zinc oxideloaded films were studied using a JOEL JSM840A(Japan) scanning electron microscope. DSC analysiswas performed with a Metter DSC-30 thermal analyzerwith PCaA and ZNLCaA (25). Film, of known weight(ca 2.4 mg), was taken in a sealed aluminum pan andheated from 40°C to 260°C at the heating rate of 10°Cper minute along with the constant flow of argon gas.The X-ray diffraction (XRD) pattern of nano zinc oxideloaded film was obtained with a PANalytical X’pert PRO

1872 Food Bioprocess Technol (2012) 5:1871–1881

MPDR X-ray diffractometer. The UV–Visible spectrumanalysis of the ZNLCaA (25) film was carried out bydissolving film in ethylenediamine tetra acetic acidsolution and recording spectrum in a UV–Visiblespectrophotometer (Shimadzu 6300) in the range of300–550 nm. The thermal history of the calcium alginatefilms, both plain nano zinc oxide loaded was evaluatedon a DuPont 2,100 TGA instrument with a heating rateof 10°C/min under 50 ml/min nitrogen flow. The sampleswere heated from room temperature to 1,000°C at a scanrate of 10°C/min.

Moisture Content Studies

The moisture sorption of films was determined gravimetri-cally at 20, 30, and 37°C (Alhamdan and Hassan 1999).Pre-weighed films were placed in Petri dishes inside glassdesiccators which contained different saturated salt solu-tions to provide a constant RH environment, ranging from3% to 98% (Oluwamukomi et al. 2008). The desiccatorswere placed inside a temperature-controlled incubator(Tempstar, India), set at a desired temperature. The sampleswere weighed at different time intervals using an electronicbalance (Denver, Germany) with an accuracy of 0.0001 g.Equilibrium was considered to have been obtained whenless than 1% weight change was found after two successivemeasurements. The dry mass of the film was determined at60°C in a vacuum oven using P2O5 as desiccant (Khalloufiet al. 2000). The three successive mass measurementswhich yielded constant weight during the drying processindicated the dry mass of the film. The equilibriummoisture contents were calculated on a dry basis using thefollowing expression.

EMC ¼ Hydrated mass� Dry mass

Dry massg=gfilm d:b: ð1Þ

The reported data of all the experiments carried out intriplicates, contains the average values.

Water Vapor Permeability Measurements

Water vapor transmission of films was measured at 20,30 and 37°C using the ASTM method (ASTM method E93–96 1993). The test cups were filled with 20 g of silicagel (desiccant) to produce a 0% RH below the film. Asample was placed in between the cups and the siliconcoated ring covered and held with four screws around thecircumference of the cups’. The air gap was at approxi-mately 1.5 cm in between the film surface and desiccant.The water vapor transmission rate (WVTR) of each filmwas measured at 100% RH. After taking initial weight ofthe test cup, it was placed in glass desiccators containing

distilled water to provide100% RH. The desiccators werekept in incubator (Tempstar, India) pre-maintained atrequired temperatures. The cups were weighed accuratelyat the regular time intervals of 30min. Three replicates of eachsample were measured.

The WVTR and other related parameters were calcu-lated by the following expressions (Bozdemir and Tutas2003):

WVTR ¼ $W

$t Ags�1m�2 ð2Þ

Permeance ¼ $W

$t A $Pgs�1m�2Pa�1 ð3Þ

Permeability ¼ $W #

$t A $Pgs�1m�1Pa�1 ð4Þ

where

ΔW/Δt Amount of water transmission per unit timeχ Film thicknessA Surface area of the film exposed to water vaporΔP Water vapor pressure difference across the film

Antibacterial Studies

The antibacterial efficacy of zinc oxide loaded calciumalginate film was studied both, quantitatively and qualita-tively, using the zone inhibition methods (Qin et al. 2006),with E. coli as the model bacteria.

In the “zone inhibition” method, 100 μl of the inoculasolution containing nearly 1.9×106 CFU per ml was addedto 5 ml of the appropriate soft agar, and sprayed onto hardagar plates. Circular pieces of plain and ZnO nanoparticles-loaded films, weighing 0.5826 and 0.6024 g, respectively,were cut from the test films (diameter=1.4 cm) and placedon the bacterial lawns. The plates were incubated for 24 hat 37°C in the appropriate aerobic incubation chamber andexamined visually for zones of inhibition around the filmdisks. The diameter of the zone was measured at two cross-sectional points and the average value was regarded as theinhibition zone.

Statistical Analysis

The GAB model was evaluated using non-linear regressionwhile the Henderson, Oswin, and Halsey models wereevaluated by linearized forms of the equations using SPSSfor windows version 6.0. The goodness-of-fit of the modelswere evaluated using the percent root mean square of error

Food Bioprocess Technol (2012) 5:1871–1881 1873

(% RMS) as prescribed by Wang and Brennan (1991)equation:

%RMS ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiX Mob�Mest

Mob

� �2

n

vuut� 100 ð5Þ

where

Mob Experimental valuesMest Predicted values andn No. of observations.

Results and Discussion

Formation of ZNLCaA (25) Films

In the present study, ZNLCaA (25) film was prepared bymixing the zinc oxide nanoparticles in aqueous solution ofsodium alginate, followed by Ca2+ ions-induced ionicgelation. These ions, due to their appropriate size, are saidto fit well within the “egg box” cavities of alginate chainsformed due to ionic-interaction between opposite charges(Braccine and Perez 2001). In a series of preliminaryexperiments, different volumes of 5% calcium chloridesolution (w/v) were added to 20.0 ml of alginate solution,under a constant stirring of 50 rpm. It was found thataddition of 1.0 ml of calcium chloride solution did not

result in formation of “lump” in alginate solution. TheseCa2+ ions crosslinked the alginate chains partially, thustransforming the solution into viscous gel. When this gelwas kept overnight, it was converted into a soft film. In thisfilm matrix, –OH and –COOH moiety of alginate mole-cules serve as templates for ZnO nanoparticles, thusensuring their almost homogeneous distribution. Finally,the film is again allowed to undergo crosslinking bydipping in 5% solution of calcium chloride (w/v) for aperiod of 60 s, to produce flexible film; although greatercrosslinking time resulted in a brittle film.

Characterization of Film

UV–Visible spectroscopy is also useful in characterizationof nanoparticles. In order to confirm the formation of ZnOnanoparticles, UV–Visible absorption spectrum wasrecorded as shown in Fig. 1 (inset). It can be seen that anabsorption peak at around 370 nm is noticed for the zincoxide nanoparticles due to the surface plasmon resonanceeffect which originate from the quantum size of zinc oxidenanoparticles. A well-defined surface plasmon resonance at380 nm has also been reported by Chindaduang et al.(2009).

Figure 1 shows X-ray diffraction pattern of plain andnano ZnO-loaded calcium alginate films. The values of 2θ,as displayed in Fig. 1b, are 31.6, 34.4, 36.0, 47.5, 56.6,63.0, and 69.2, respectively, which are almost identical with

Fig. 1 a Surface plasmon resonance spectrum for ZnO nanoparticles, b X-Ray diffraction pattern for ZNLCaA (25) film and c X-Ray diffractionpattern for PCaA film

1874 Food Bioprocess Technol (2012) 5:1871–1881

the reported values (JCPDS76–0704). These values alsoresembled those obtained by Li et al. (Li et al. 2008) whoreported 2θ values of 31.7, 34.4, 36.2, 47.5, 56.6, 62.8,67.9, 69.1, and 89.6° for reflections at (100), (002), (101),(102), (110), (103), (200), (112), and (201) planes. It is alsoobserved that plain film does not contain sharp peaks andindicates amorphous nature which suggests that presence ofZnO nanoparticles effect the crystallinity of the film. InFig. 1c, there is a broad diffused peak at 2θ value of 39.2,which is characteristic peak of calcium alginate. Similarpeak is also present in diffractogram of nano ZnO-loadedfilm. Due to the presence of ZnO nanoparticles the abovepeak is narrow and sharp owing to the enhanced crystal-linity of the film. There is also a possibility of linkagebetween hydrogen of –OH groups of alginate and electronrich oxygen of ZnO. However, some additional peaks arestill unidentified in the XRD spectrum. These peaks may bedue to crystallinity produced from strong electrostaticinteraction among ZnO nanoparticles and hydroxyls ofalginate chains.

The size of the nanoparticles was determined byrecording the TEM image of the film, as depicted inFig. 2. It is seen that particles are almost homogeneouslydispersed within the film matrix, with nearly 60% particlesexhibiting an average diameter of 54 nm.

Thermogravimetric Analysis

The presence of nano zinc oxide in the film was furtherconfirmed by thermogravimetric analysis. The resultsdepicted in Fig. 3a reveal that PCaA film exhibits greaterweight loss at the rate of 7.992%/min at 216.43°C which isin fair agreement with the observations reported by others

(Caykara et al. 2005) that sodium alginate film undergoesthermal decomposition in the range of 220 to 293°C. It isalso noticed that combustion of residues takes place beyond700°C. The thermogram of nano ZnO-loaded film is alsodisplayed in Fig. 3b. The film exhibits a weight loss at210.14°C and 233.66°C at the rates of 6.883%/min and7.245%/min respectively. The film also exhibits maximumweight loss in the temperature range of 180 to 300°C. Theobserved greater weight loss at 210°C could also beattributed to decomposition at alginate chains whereasfurther weight loss at 233°C could be due to reduction ofZnO by carbon.

DSC Analysis

The effect of presence of zinc oxide nanoparticles on thecrystallinity of the resulting calcium alginate film wasinvestigated by DSC measurements. The results of DSCanalysis are depicted in Fig. 4. It is seen that the glasstransition temperature Tg for PCaA and ZNLCaA (25) filmsare nearly 117.2 and 111.8°C, respectively. The lower valueof Tg may be attributed to the fact that zinc oxidenanoparticles act as plasticizer, increase the movement ofmacromolecular alginate chains. Moreover, their presencealso reduces the electrostatic attraction among the alginate

Fig. 3 Differential thermograms for a PCaA and b ZNLCaA (25) film

Fig. 2 TEM image of ZNLCaA (25) film

Food Bioprocess Technol (2012) 5:1871–1881 1875

chains, thus lowering the amount of thermal energyrequired for transformation of film from glassy to rubberystate. In addition, water molecules, present in the film, mayalso act as plasticizer (Bourtoom and Chinnan 2008). Themelting temperatures of plain and zinc oxide loaded filmwere found to be 138.0 and 153.2°C, respectively. Theobserved increase is attributable to the increase in degree ofcrystallinity owing to increased crosslinking by Zn2+ ionspresent in the poly-L-guluronic acid (poly G) blocks ofalginate chains. There have been several reports in whichalginate has been crosslinked with Zn(II) ions (Mutasem etal. 2008). The sharp decrease in width of the endothermicpeak is also an indication of increased degree of crystallinityof the nano ZnO-loaded films. The enthalpy of fusion, ΔHm

for the above samples was 565 and 132.98 Jg−1, respectively.Relatively lower values of ΔHm for zinc oxide nanoparticles-loaded calcium alginate film may be due to fact that the retrogradation of alginate molecules during their storage period issuppressed, thus lowering the ΔHm for these films.

Equilibrium Moisture Sorption Isotherms

GAB Isotherm

The GAB model (Shankar et al. 2010) is used frequently todescribe moisture uptake behavior of food products. It has atheoretical background and is applicable over almost entirewater activity range (Yao Clement and Tano 2008). TheGAB equation is normally given as

M ¼ MoCKaw1� Kawð Þ 1� Kaw þ CKawð Þ½ � ð6Þ

where M is the moisture content of material on a dry basis(in kilograms water per kilograms dry solid), aw is wateractivity, C is the Guggenheim constant related to heat ofsorption, K is the kinetic constant related to multilayer

sorption and Mo is the monolayer sorption capacity. Aftertransformation, the GAB equation has an equivalent formto the Hailwood and Horrobin (1946) equation:

awMo

¼ aa2w þ baw þ l ð7Þ

The following relations occur between the parameters ofthe above equation and those of the GAB model:

Mo ¼ 1ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffib2 � 4al

p ð8Þ

C ¼2ðþ

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffib2 � 4alÞ

q

�b þffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffib2 � 4al

p ð9Þ

K ¼ �bþffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffib2 � 4al

p

2lð10Þ

In order to evaluate parameters α, β, and 1, aw/w values areplotted against aw to yield a quadratic curve of polynomialfunction. The values of α, β, and 1, so obtained, are used tocalculate GAB parameters Mo, K, and C.

The equilibrium moisture uptake of ZNLCaA (25) filmwas investigated as a function of water activity at 20, 30, and37°C (data not displayed). The curves obtained were typicaltype II sigmoidal and equilibrium moisture content (EMC)dependence. For the low and moderate water activities, theEMC increased linearly while it increased sharply in region ofhigher water activity. The observed negative temperatureeffect was attributable to enhanced kinetic energies of watervapor molecules (Oluwamukomi 2009). The parameters forGAB model are given in Table 1

A close look at the data reveals that monolayer sorptioncapacities Mo are 0.301±0.003, 0.214±0.092, and 0.171±

Fig. 4 Thermograms obtainedfor DSC analysis of aZNLCaA (25) and b PCaAfilm

1876 Food Bioprocess Technol (2012) 5:1871–1881

0.102 kg kg−1 dry solid at 20, 30, and 37°C, respectively.With the increase in temperature, the enhanced kineticenergy of sorbed water molecule with temperature, thusallowing departure of vapor from the sorption sites. In orderto know the effect of presence of ZnO nanoparticles on themoisture uptake behavior of film, isotherms were alsoobtained for PCaA film at the same temperatures. Thevalues of Mo, monolayer sorption capacity, at 20, 30, and37°C were found to be 0.292±0.020, 0.276±0.102, and0.092±0.052 kg kg−1 dry solid, respectively, which arelower than those obtained with zinc oxide nanoparticles-loaded films. The relatively higher values of Mo for zincoxide nanoparticles-loaded film may be due to fact thathighly electronegative oxygen of zinc oxide nanoparticlesalso acts as binding site for sorption of water vapormolecules.

The value of GAB constant K provides a measure of theinteractions between the molecules in multi layers with theadsorbents and tends to fall between the energy value of themolecules in the monolayer and that of bulk liquid. When Kis unity, the multi layers have properties of bulk water(Perez-Alonso et al. 2006), and the sorption behavior couldbe modeled by the BET. It is seen from Table 1 that at 30and 37°C the GAB constant K is unity. This indicates thatmultilayer has bulk liquid properties. Similar results arealso reported elsewhere (Yao Clement and Tano 2008).

It is assumed that adsorbate–adsorbent interactions,being exothermic, are favored at lower temperature, thuscausing an increase in parameter C with temperature (Gabaset al. 2007). However, an opposite trend was observed inthis study. From analysis of the data from Table 1 resultsthat C increases with temperature increase. As the valuesobtained were greater than 2, this supported the observationthat sorption isotherms were sigmoidal type II (Farahnakyet al. 2009).

Effect of Temperature on Water Vapor Permeability

The main function of food packaging film is minimizing themoisture transfer between the food and the surroundingatmosphere, and therefore water vapor permeability shouldbe as low as possible (Kim et al. 2003). Studies, focusingon the water barrier properties of films, have also beenreported in recent past (Hong and Krochta 2004).

In the present study, dynamic uptake of moisture byZNLCaA film was investigated at 20, 30, and 37°C underthe relative humidity of 100% (i.e., aw=1). The results, asdepicted in Fig. 5, clearly indicate that moisture content,permeated through films, increases with temperature. Thisis due to the basis of the fact that with the increase intemperature the kinetic energy of permeating molecules andmovement of polymeric segments increase, thus resulting inenhanced diffusivity of water vapor molecules. Moreover,the increased temperature also favors desorption of watervapor molecules that have traveled along the width of thefilm and reached other end. The various transmissionparameters, as calculated using Eqs. 2, 3, and 4 have beengiven in Table 2. The values displayed also favor ourarguments.

To check if presence of zinc oxide nanoparticles alsoaffects the water vapor permeability, we also determined thesame for plains films at 20, 30, and 37°C. The water vaportransmission rates for plains films were found to be 0.61±0.02, 1.22±0.12, and 1.52±0.24 gs−1 m−2, respectively,which were slightly lower than those obtained for ZnOnanoparticles-loaded films. This could possibly be due tothe fact that plain calcium alginate films have greater

Temp (°C) GAB parameters

Mo C K % RMS

20 0.301±0.003 6.66±0.073 0.84±0.063 5.58

30 0.214±0.092 9.09±0.092 0.88±0.084 6.06

37 0.171±0.102 12.50±0.102 0.90±0.059 7.21

Table 1 Various isothermsparameters obtained for mois-ture sorption by ZNLCaA (20)films at different temperatures

Fig. 5 Kinetics of water vapor permeation through ZNLCaA (25)film at different temperatures (inset—plot between ln aw and 1/T forevaluation of ΔH and ΔS)

Food Bioprocess Technol (2012) 5:1871–1881 1877

degree of crosslinking of alginate chains by Ca2+ ions, thusresulting in slower transmission of water vapor moleculesthrough the films, while as nano ZnO-loaded films exhibitlower degree of crosslinking due to presence of ZnOnanoparticles which shield the electrostatic attractionbetween Ca2+ ions and the carboxylate groups of alginatechains. This results in relatively faster permeation of watervapor through the films.

Enthalpy of Sorption

The net enthalpy of sorption, enthalpy of sorption (ΔH) is agood measure of the interaction of water vapor with thesolid substrate. It is also known as binding energy ofsorption and defined as the difference ΔQ−ΔHvap whereΔQ is the total heat of sorption and ΔHvap is heat ofvaporization of water at given temperature. It can beconsidered as a measure of intermolecular attractive forcesbetween the sorption sites and water vapors, and can bedetermined using Clausius–Clapeyron equation (Bellaghaet al. 2008) given below:

ln aw ¼ � $H

RTþ $S

Rð19Þ

where ΔH is the net enthalpy of sorption (kJ/mol), R is gasconstant (R=8.314 J/mol/K) and ΔS is the differentialentropy (kJ/mol/K). Water activity was plotted againstreciprocal of absolute temperatures at various moisturecontents as shown in Fig. 5 (inset). The sorption isosteresshowed smooth straight lines thus confirming that Eq. 19fitted the experimental data. For the moisture content rangeof 0.2 to 0.6, the net ΔH values were obtained in the rangeof 22.73 to 11.14 kJ/mol (Fig. 6). The positive values ofΔH suggest an easy physical sorption of water moleculesforming a mono molecular layer. Finally, the ΔH values, asa function of moisture content are seen in Fig. 6. It is clearthat ΔH values decrease with increase in moisture content .The reason is that initially, sorption occurs on the mostactive available sites, giving rise to high interaction energy.As soon as these sites become occupied, sorption occurs onthe less active site and gives rise to lower heat of sorption.At low moisture content, the higher values of ΔH were dueto strong interactions between water molecules and hydro-philic group of alginate. Using the intercept of linear plots,shown in Fig. 5 (inset), differential entropy of sorption, ΔS,

were also evaluated. For the moisture content range givenabove, the ΔS values were obtained in the range of 0.064 to0.034 kJ/mol/K. The values of ΔH and ΔS were relatedaccording to the following equation:

$H ¼ TB$S þ $GB ð20Þ

where TB is the isokinetic temperature and ΔGB is the freeenergy at TB.

Isokinetic temperature represents the temperature atwhich all reaction rates proceed at the same rate. Figure 6(inset) shows the linear plot obtained between ΔH and ΔSwith a fair regression of 0.998. Using the slope of linearplot, TB was found to be 376.9 K. Now, since there ishigher degree of correlation between ΔH and ΔS, thecompensation theory may be assumed to be valid forsorption (Sharma et al. 2009). In order to confirm theexistence of compensation, a test has been proposed (Kruget al. 1976) to compare the isokinetic temperature with theharmonic mean temperature, Thm, defined as,

Thm ¼ nPnið1=TÞ

ð21Þ

Parameters Temperatures (°C)

20 30 37

WVTR (g s−1 m−2) 0.816±0.143 1.428±0.045 1.632±0.064

Permeance (g s−1 m−2Pa−1) (13.0±0.414) 10−5 (22.76±0.203)10−5 (26.0±0.26) 10−5

Permeability (g s−1 m−1Pa−1) (11.71±0.16) 10−9 (20.49±0.18) 10−9 (23.4±0.19) 10−9

Table 2 Various permeationparameters obtained for trans-port of water vapors throughZNLCaA (20) films at differenttemperatures

Fig. 6 Plot between moisture content and enthalpy of sorption. Inset—correlation between enthalpy and entropy of sorption

1878 Food Bioprocess Technol (2012) 5:1871–1881

where n is the total number of isotherms used. Theharmonic mean temperature was calculated to be 302.0 K,which was significantly different from the value of TB (i.e.376. 9 K), thus confirming the suitability of the isokinetictheory of water sorption of ZnO-loaded calcium alginatefilms. According to Leffler (1955), if TB>Thm the sorptionprocess is enthalpy controlled and if TB<Thm then sorptionprocess would be enthalpy driven. In this study, the moistureuptake mechanism seems to be enthalpy (i.e., isosteric heatof sorption) controlled because TB (376. 9 K)>Thm(302.0 K). The mechanism also suggests that film micro-structure shall remain stable throughout the temperaturerange studied.

Antibacterial Action of ZNLCaA Films

The antimicrobial efficacy of ZNLCaA film was investi-gated using zone of inhibition method. Petri plates weresupplemented with circular pieces (1.4 cm) of PCaA andZNLCaA (25) films and culture were grown. The results, asshown in Fig. 7, reveal that the Petri dish, containing plainfilm, demonstrates dense colony of bacterial cells, whereasthere is a clear zone of inhibition around the circular filmsloaded with zinc oxide nanoparticles. The average diameterof the zones, measured longitudinally and transversely, wasaround 1.7 cm. Time-dependent growth of bacterialcolonies in nutrient broth supplemented with pieces ofplain as well as nano zinc oxide loaded films were alsocompared. However, the differences in the microbialcontents were minimal and could not be considered asrelevant. This could possibly be due to the fact that timespan of killing kinetics was nearly 3 to 4 h and it might benot sufficient for ZnO nanoparticles to show inhibitoryaction appreciably.

As per available literature, the mechanism of actionof zinc oxide nanoparticles is different from that ofsilver nanoparticles, which involves release of silver assilver ions on coming into contact with moisture. The

mechanism of antibacterial action of ZnO nanoparticleshas already been reported in the literature. Whencoming in contact with bacterial cells in the presenceof moisture, ZnO nanoparticles produce reactive oxygenspecies such as hydroxyl radicals, super oxides, andH2O2 (Padmawathy and Vijayaraghavan 2008). Since, thehydroxyl radicals and super oxides are negativelycharged particles; they do not penetrate into the cellmembrane but remain in direct contact with the outersurface of bacteria and cause severe damage to proteins,lipids, and DNA (Kohen and Nyska 2002). However,H2O2 penetrates the cell membrane and kills the bacteria(Fang et al. 2006).

Conclusion

This study concludes that the moisture uptake behaviorof zinc oxide nanoparticles-loaded alginate film is bestinterpreted by the Oswin isotherm model. The moistureuptake data, obtained at different temperatures enables tocalculate isosteric heat and differential entropy ofabsorption. The presence of zinc oxide nanoparticleswithin the films causes a decrease in glass transitiontemperature of the film. However, there is no noticeabledifference in thermal stabilities of the two films. SinceZnO has been used in food industries, its incorporationas nanoparticles into alginate films can be advantages inso many ways. As nanoparticles provide much greatersurface area, they can perform their antibacterial actionin much more pronounced way. These films are not onlyantibacterial, but they can also be exploited as a sourceto release ZnO as zinc supplements (Shi et al. 2008).However, a detailed study is needed to investigate otherphysico-chemical properties of these films such asmechanical strength, aroma-barrier efficiency, color fast-ness, etc. before their industrial use in food packagingindustries.

Fig. 7 Zone of inhibition inPetri plates supplemented witha PCaA films and b ZNLCaA(25) films

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Acknowledgment We thank Dr. O. P. Sharma, Prof. and Head of theDepartment of the Chemistry, at Govt. Model Science College, Jabalpur,India, to provide experimental facilities. We are also thankful to Dr.Rajshree Kapoor, Head of the Department of English, for proof readingand making appropriate corrections throughout the manuscript.

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