Journal of Food Engineering 91 (2009) 468–473

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Journal of Food Engineering

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Effect of nano-clay type on the physical and antimicrobial properties of wheyprotein isolate/clay composite films

Rungsinee Sothornvit a, Jong-Whan Rhim b, Seok-In Hong c,*

a Department of Food Engineering, Faculty of Engineering at Kamphaengsaen, Kasetsart University, Kamphaengsaen Campus, Nakhonpathom 73140, Thailandb Department of Food Engineering, Mokpo National University, 560 Muanro, Chungkyemyon, Muangun 534-729, Jeonnam, Republic of Koreac Korea Food Research Institute, 516 Baekhyun, Bundang, Seongnam 463-746, Kyonggi, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 15 June 2008Received in revised form 23 September2008Accepted 25 September 2008Available online 7 October 2008

Keywords:Whey protein isolateComposite filmsMontmorilloniteOrganoclaysPhysical propertiesAntimicrobial activity

0260-8774/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.jfoodeng.2008.09.026

* Corresponding author. Tel.: +82 31 780 9053; faxE-mail addresses: sihong@kfri.re.kr, hsikfri@chollia

Whey protein isolate (WPI)-based composite films with three different types of nano-clays, Cloisite Na+,Cloisite 20A, and Cloisite 30B, were prepared using a solution casting method, and their physical and anti-microbial properties were determined in order to better understand the effect of nano-clay type on filmproperties. The resulting films exhibited an opaque appearance and haze, and the degree of this effectdepended on type of nano-clays added. However, these films displayed a similar gloss and slightly lowertransparency relative to transparent neat WPI film. The type of nano-clay used significantly influencedthe tensile and the water vapor barrier properties of the composite films with the exception of Cloisite30B, which had no negative effect. In addition, the WPI/Cloisite 30B composite films showed a benefi-cially bacteriostatic effect against Gram-positive bacteria, Listeria monocytogenes.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

The functional properties of biopolymer-based edible films orcoatings have been shown to act as barrier to solute and gas andenhance food quality and shelf life (Krochta et al., 1994; Genna-dios, 2002). However, these films do not display good mechanicaland water vapor barrier properties due to their hydrophilic charac-teristics. To overcome these issues, a new approach has beendeveloped, which use hybrid materials consisting of polymersand layered silicates (Giannelis, 1996). Layered silicates, such asmontmorillonite (MMT) clay mineral, result from the stackedarrangement of negatively charged silicate layers and contain aplatelet thickness of about 1 nm with a high aspect ratio (ratio oflength to thickness) (Sorrentino et al., 2007). The layered silicatefilled polymer composites exhibit extraordinary enhancement ofmechanical, thermal and physicochemical properties at a low levelof filler concentration in comparison to pure polymer and conven-tional microcomposites (Uyama et al., 2003). In particular, thesenanocomposites have excellent barrier properties because thepresence of clay layers delays the diffusing molecule pathwaydue to tortuosity (Bharadwaj, 2001; Sorrentino et al., 2006).

However, most work done on polymer/clay nanocomposites hasfocused mainly on synthetic polymers (Alexandre and Dubois,

ll rights reserved.

: +82 31 709 9876.n.net (S.-I. Hong).

2000; Ray and Okamoto, 2003). Biopolymer-based nanocompos-ites, in contrast, have been examined in only a few studies (Pandeyet al., 2005; Rhim and Ng, 2007). Some of the works done with bio-polymer-based nanocomposites were based on starch or polysac-charides, such as wheat and maize starch (McGlashan and Halley,2003), thermoplastic starch (Park et al., 2003), and chitosan (Linet al., 2005; Xu et al., 2006; Rhim et al., 2006; Gunister et al.,2007). A few studies on protein-based nanocomposites have beenpublished, including soy protein (Dean and Yu, 2005; Rhim et al.,2005), whey protein (Hedenqvist et al., 2006), and wheat gluten(Olabarrieta et al., 2006). Most of the biopolymer-based nanocom-posites have shown appreciable improvements in mechanical andbarrier properties compared to the counterpart biopolymer films.

Whey protein has received much attention for its potential useas an edible film and coating because it has been shown to maketransparent films and coatings that can act as excellent oxygenbarriers and provide certain mechanical properties (Sothornvitand Krochta, 2000, 2005). Unlike chitosan film, whey protein filmshave not shown any antimicrobial activity; therefore, incorpora-tion of antimicrobial agents, such as sorbic acid, p-aminobenzoicacid (Cagri et al., 2001), and lysozyme (Min et al., 2008), is neededto impart this property. Recently, Rhim et al. (2006) found thatchitosan-based nanocomposite films blended with some organi-cally modified MMT, such as Cloisite 30B, exhibited antimicrobialactivity against Gram-positive bacteria. They postulated that theantimicrobial action came from the quaternary ammonium salt

R. Sothornvit et al. / Journal of Food Engineering 91 (2009) 468–473 469

of the organically modified nano-clay. Therefore, it is of interest tofurther investigate the effect of other types of nano-clays on theperformance of nanocomposites prepared with readily availablebiopolymers like WPI.

The main objective of this study was to determine the effects ofdifferent types of nano-clays on film properties, such as optical,tensile, and water vapor barrier properties, as well as antimicrobialactivity against the selected food-borne pathogenic bacteria ofWPI-based composite films.

2. Materials and methods

2.1. Materials

BiPro WPI (97.7% protein) was supplied by Davisco Foods Inter-national (Le Sueur, MN) and three types of montmorillonite (MMT)nano-clays including a unmodified MMT (Cloisite Na+) and twoorganically modified MMTs (Cloisite 20A and Cloisite 30B) wereobtained from Southern Clay Co. (Gonzales, TX). The organicmodifiers of the Cloisite 20A and 30B are reportedly dimethyldehydrogenated tallow quaternary ammonium and methyl tallowbis-2-hydroxyethyl quaternary ammonium, respectively. The gen-eral characteristics of these MMT products are listed in Table 1.Glycerol, a plasticizer, was purchased from Aldrich, Milwaukee,WI.

2.2. Film preparation

WPI and WPI/clay composite films were prepared using a solu-tion or a solution/intercalation method (Rhim and Ng, 2007). WPIfilm solutions were prepared by dissolving 10 g of WPI in 100 mldistilled water with 5 g of glycerol. For the preparation of WPI/claycomposite film mixtures, 5% (w/w, relative to WPI) of nano-clays,at which level clay content is commonly used for preparation ofnanocomposites with biopolymers (Rhim et al., 2006), were used.At first a precisely weighed nano-clay (0.5 g) was dispersed withdistilled water (100 ml) and stirred using a magnetic stirrer over-night to reach complete hydration/swelling; however, Cloisite20A (hydrophobic nano-clay) required additional dispersion usinga probe sonicator (VCX-500, Sonics & Materials, Inc., Newtown, CT)for 2 h before stirring overnight. Then 10 g of WPI was added to ob-tain an aqueous solution, followed by adding 5 g of glycerol. All thefilm-forming mixtures were heated to 90 �C for 30 min in a waterbath, cooled to room temperature, and degassed using a bath-type

Table 1Characteristics of different commercial montmorillonite-based nano-clays.

MMT type Characteristics

Cloisite Na+ C

Organic modifier None Dq

Structural formula Na0.33(Al1.67Mg0.33)Si4O10(OH)2

Modifier concentration – 9Moisture content 4–9% <Particle size (90% <) 13 lm 1Color Off-white ODensity 2.86 g/ml 1Relative hydrophobicity Hydrophilic S

Data from the manufacturer (Southern Clay Co., Gonzales, TX, USA).T: beef tallow (�65% C18, �30% C16, �5% C14); HT: hydrogenated tallow; M: CH3.

ultrasound sonicator. The film-forming mixtures were then castedonto leveled Teflon-coated glass plates (24 � 30 cm) framed at foursides. The cast plates were dried at ambient temperature (22 ± 2 �C,50 ± 5% RH) for 2 days and then the films were peeled off from theglass plates.

2.3. Film thickness and conditioning

Film thickness was measured using a hand-held micrometer(No. 7326, Mitutoyo Manufacturing Co., Ltd., Tokyo, Japan) to thenearest 0.00254 mm (0.0001 in). Five thickness measurementswere taken on each testing specimen and the average value wasused in tensile strength (TS) and water vapor permeability (WVP)calculations as well as determining transparency property. All filmsamples were preconditioned for at least 48 h in a constant-tem-perature humidity chamber at 25 �C and 50% relative humidity(RH) before testing. Three replications were used to determineeach film property.

2.4. Optical properties (color, gloss, haze, and transparency)

Color values of WPI-based composite films were measuredusing a colorimeter (Minolta, CR-200, Tokyo, Japan). A white stan-dard color plate (L* = 97.75, a* = �0.49 and b* = 1.96) was used asthe background for color measurements. CIE system (L*, a* andb*) values were averaged from three readings for each sample.The total color difference (DE) was calculated as follows:

DE ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðDL�Þ2 þ ðDa�Þ2 þ ðDb�Þ2

q

The results were also expressed as DE values with the control WPIfilms as reference.

The gloss of WPI-based composite films was determined at inci-dence angles of 20o (G20) and 60o (G60) using a reflectance meter(BYK Gardner, micro-TRI-Gloss, Silver Spring, MD) in accordancewith ASTM D523, and reported as a gloss unit (GU; % of standard),based on three readings for each sample. A highly polished planesurface of black glass with a refractive index of 1.567 served asthe primary gloss standard and was assigned to an arbitrary glossvalue of 100, which differed depending on the angle used. The sam-ple was placed on the matte surface of black acrylic plates (TAPPlastics, Sacramento, CA), which have 0.2 and 3.0 GU for the 20o

and 60o angles, respectively, at room temperature (22 ± 2 �C,50 ± 5% RH).

loisite 20A Cloisite 30B

imethyl dihydrogenated tallow,uaternary ammonium (2M2HT)

Methyl tallow, bis-2-hydroxyethyl,quaternary ammonium (MT2EtOH)

5 meq/100 g clay 90 meq/100 g clay2% <2%3 lm 13 lmff-white Off-white.77 g/ml 1.98 g/mltrongly hydrophobic Less hydrophobic

470 R. Sothornvit et al. / Journal of Food Engineering 91 (2009) 468–473

The haze index (H) was defined as the difference in the glossvalues measured at an angle of 60o and 20o (H = G60�G20).

The transparency of WPI-based composite films was deter-mined by measuring the percent transmittance (%T) at 600 nmusing a spectrophotometer (Jasco, V-550 Series, Tokyo, Japan)according to ASTM D1746. The transparency (T600) was calculatedfrom the following equation:

T600 ¼ðlog %TÞ

b

where b is the film thickness (mm).

2.5. Tensile properties

For the tensile tests, film specimens were cut into rectangularshapes that were 2.54 cm wide and 15 cm long using a precisiondouble-blade cutter (model LB02/A, Metrotec, S.A., San Sebastian,Spain). The tensile strength (TS), elastic modulus (EM) and elonga-tion at break (E) of the films were determined using an InstronUniversal Testing Machine (Model 5565, Instron EngineeringCorporation, Canton, MA) with 50 N load cell according to theASTM-D882 standard method. The initial gauge separation andthe crosshead speed were set to 50 mm and 50 mm/min,respectively.

2.6. Water vapor permeability

The water vapor permeability (WVP) of WPI-based compositefilms was determined using a gravimetric cup method accordingto the ASTM E96-95. The WVP of the films was calculated usingthe following equation:

WVP ¼WVTR � thicknessDP

where WVTR is the steady state water vapor transmission rate,thickness is the film thickness and DP is the water vapor partialpressure difference across the two sides of the film. The actual DPvalue between both sides of the film was calculated using the meth-od of Gennadios et al. (1994).

2.7. Antimicrobial activity

Antimicrobial activity of the WPI films was assessed using dy-namic shake flask tests (Paik et al., 1998; Appendini and Hotchkiss,2002) against two types of pathogenic bacteria, Listeria monocytog-enes ATCC-19111 and Escherichia coli O157:H7 ATCC-11775. Filmswere cut into square pieces (10 � 10 cm) and placed in individualsterile flasks. The Gram-positive bacteria, L. monocytogenes and theGram-negative bacteria, E. coli O157:H7 stock cultures were grownin tryptic soy (TS) broth (Difco Lab, BD) and incubated aerobicallyfor 16 h at 37 �C and 30 �C, respectively. Each 30 ml tube of bacte-rial cell culture was centrifuged for 3 min at 4 �C and 7000g. Theresulting centrifuged samples were then decanted, washed with0.1 M KH2PO4–NaOH buffer (pH 7.0), centrifuged for 3 min, anddecanted a second time. The cell pellet was placed into 100 ml ofbrain heart infusion (BHI) broth (Difco Lab) for L. monocytogenesor TS broth for E. coli O157:H7 and diluted to 1/10 of the originalbroth concentration with 900 ml of sterile distilled water to obtainan inoculum of �(1.0�2.5) � 107 colony-forming units (CFU)/ml.The 100 ml of the inoculum was aseptically added to each of theflasks containing the sample films. The flasks were placed in anorbital shaker maintained at 30 �C and rotated at 50 rpm. Aliquotsof 1 ml of the cell suspension were periodically taken from theflasks, diluted serially with 0.1 g/100 ml peptone solution, and pla-ted in duplicate on BHI agar for L. monocytogenes cells or on TS agarfor E. coli O157:H7 cells. The plates were incubated in an aerobic

chamber for 2 days at 37 �C for BHI agar and at 30 �C for TS agar.The number of colonies on each plate was counted and reportedas CFU/ml. Two replications were used to determine the antimicro-bial activity.

2.8. Statistical analysis

A completely randomized experimental design was used to testthe type of MMTs. Two or three replications were used to deter-mine each property. SPSS 11.0 for Windows (SPSS Inc., Chicago,IL) was used to test analysis of variance (ANOVA) and a Duncan’smultiple range test was used to determine the significant differ-ence between treatments at a 95% confidence level.

3. Results and discussion

3.1. Film formation

The control WPI films were found to be flexible and transpar-ent; however, the transparency of WPI/clay composite films wasaffected by blending with nano-clays. In preparation of film-form-ing mixture, type of nano-clays greatly influences the degree ofdispersion in the mixture. Among the clays tested, the unmodifiedMMT (Cloisite Na+) and the less hydrophobic organoclay (Cloisite30B) were well dispersed in the film-forming mixture by simplemixing with a magnetic stirrer. However, the hydrophobic organo-clay (Cloiste 20A) was hardly dispersed without further treatmentwith ultrasonication. The transparent WPI film solution became amilky suspension when nano-clay particles were added. This wasespecially true for Cloisite 30B and Cloisite 20A. The WPI/clay com-posite films were also flexible and had a slight yellowish milkyappearance. The surface of the WPI/Cloisite 30B composite filmshad a shiny and opaque appearance, which affected the gloss val-ues of films, while the surface of the WPI/Cloisite 20A films wastransparent. The small amount of the hydrophobic organoclay thatwas hardly dispersed in the film did not seem to affect the trans-parency of WPI films, since there is probably low compatibility be-tween the clay and the WPI matrix. However, Cloisite Na+ was welldispersed in the WPI solution and the films surface was transpar-ent. The average thicknesses of WPI and WPI/clay composite filmswere 0.17 ± 0.02 mm.

3.2. Optical properties (color, gloss, haze, and transparency)

The optical properties of the WPI and WPI/clay composite films,determined by CIE color values, specular gloss, haziness, and trans-parency, are shown in Table 2. In general, the optical properties ofthe WPI films were significantly (p < 0.05) influenced by blendingwith the nano-clays. Type of nano-clays incorporated into theWPI films also affected color and DE significantly (p 6 0.05). Cloi-site Na+ and 30B incorporated WPI films had similar color values,while blending with Cloisite 20A showed the lowest L* and a* val-ues but the highest b* and DE values. This may be attributed to thehigh surface hydrophobicity of Cloisite 20A (Park et al., 2002),which could hinder complete dispersion into a hydrophilic WPIfilm solution. Such incomplete dispersion of Cloisite 20A in aWPI film matrix appears to make off-white color of the clay itselfprominent, probably resulting in the higher b* and DE values ofthe Cloisite 20A incorporated film. Generally, intercalation be-tween biopolymers and nano-clays is known to depend on thecompatibility and the surface polarities of both components (Parket al., 2002).

Gloss is a measure of the capability for a film surface to reflectlight. A film surface reflects some light at an angle equal to the an-gle of incidence (specularly reflected), and some light in other

Table 3Tensile properties of WPI and WPI/clay composite films.a

Film type TS (MPa) EM (MPa) E (%)

WPI 3.40 ± 0.58a 171.8 ± 14.3a 50.9 ± 12.5aWPI/Cloisite Na+ 2.98 ± 0.29a 109.3 ± 18.0b 42.4 ± 7.6abWPI/Cloisite 30B 3.29 ± 0.10a 162.6 ± 37.9a 51.7 ± 4.8aWPI/Cloisite 20A 1.55 ± 0.32b 115.5 ± 13.5b 29.1 ± 9.0b

a Means of three replicates with standard deviations. Any two means in the samecolumn followed by the same letter are not significantly (p > 0.05) differentaccording to Duncan’s multiple range tests.

Table 2Optical properties of WPI and WPI/clay composite films.a

Film type L* a* b* DE Gloss (GU, @60�) H (G60�G20) T600 (log(%T)/mm)

WPI 97.09 ± 0.47a �0.42 ± 0.18a 3.54 ± 0.78c – 66.20 ± 5.84b 57.02 ± 5.54b 14.38 ± 4.88aWPI/Cloisite Na+ 95.62 ± 0.79b �0.61 ± 0.10b 4.81 ± 0.29b 1.98 ± 0.77b 74.11 ± 4.03a 63.41 ± 2.53a 10.00 ± 0.18bWPI/Cloisite 30B 95.96 ± 0.39b �0.64 ± 0.11b 4.54 ± 0.46b 1.53 ± 0.60b 58.04 ± 6.08c 48.52 ± 4.17c 6.30 ± 0.49cWPI/Cloisite 20A 94.30 ± 0.76c �0.81 ± 0.13c 5.32 ± 0.36a 3.33 ± 0.83a 63.48 ± 3.14b 54.47 ± 3.06b 10.19 ± 1.22b

a Means of three replicates with standard deviations. Any two means in the same column followed by the same letter are not significantly (p > 0.05) different according toDuncan’s multiple range tests.

Table 4Water vapor permeability of WPI and WPI/clay composite films.a

Film type WVP (�109 g m/m2 s Pa) RHI (%)

WPI 66.0 ± 3.6a 68.8 ± 1.3bWPI/Cloisite Na+ 47.1 ± 3.3b 72.2 ± 0.9aWPI/Cloisite 30B 55.6 ± 7.5ab 70.1 ± 0.8bWPI/Cloisite 20A 64.8 ± 4.2a 71.1 ± 1.4ab

a Means of three replicates with standard deviations. Any two means in the samecolumn followed by the same letter are not significantly (p > 0.05) differentaccording to Duncan’s multiple range tests.

R. Sothornvit et al. / Journal of Food Engineering 91 (2009) 468–473 471

directions (diffusely reflected) (Kigle-Boeckler, 1996). Unlike com-monly used synthetic plastic films, most biopolymer films havesemi-gloss surface (35–70 GU at 60o) that not only reflect lightthrough specular reflection, but also in other directions throughdiffuse reflection, which results in the appearance of a less glossysurface (Trezza and Krochta, 2001). Except WPI/Cloisite Na+ com-posite films, which was the glossiest among the films tested, WPIand the other WPI/clay composite films were classified as semi-gloss films. Interestingly, blending with Cloisite Na+ improvedthe gloss value of the WPI film. However, the gloss value afterblending with Cloisite 20A remained the same, while the gloss va-lue of WPI/Cloisite 30B composite films decreased significantly(p < 0.05) compared to the control WPI films. The decrease in glossmay be attributed to chemical heterogeneity and surface rough-ness (Trezza and Krochta, 2001) as well as the hydrophilic/hydro-phobic nature of the nano-clays. The order in the degree ofhydrophobicity of the nano-clays tested in this study is CloisiteNa+ < 30B < 20A. This implies that good dispersion and high com-patibility between the polymer and clay particles can enhancethe gloss value. In this study, Cloisite Na+ was more compatibleand better dispersed in WPI than Cloisite 30B. On the other hand,Cloisite 30B which is less hydrophobic produced less glossy surfaceof the composite film, presumably due to its chemical heterogene-ity, than Cloisite 20A.

The haze of packaging films is caused by irregularities in thereflecting surface, which is influenced by specific parameters dur-ing film formation, such as the degree of dispersion, additive types,compatibility of components, etc. Thus, it is considered as a mea-sure of the irregularity and heterogeneity of the surface of films.Interestingly, incorporation of Cloisite 30B lowered the haze indexindicating that this nano-clay reduces the irregularity of the WPIfilms. Among the nano-clays tested, Cloisite 30B decreased thehaze index of WPI-based films more than any other type of MMTs.Such improvement in optical properties by blending with nano-scale MMTs was also observed in PVC/clay nanocomposites (Wanet al., 2003). Cloisite Na+, in contrast, did not reduce the haze indexof WPI-based composite films. It may be attributed to a much high-er gloss value at 60� of WPI/Cloisite Na+ composite films than theothers, considering that the composite films had similar gloss val-ues of 9.0–10.7 GU at 20� regardless of nano-clay types.

The transparency of WPI-based films decreased significantly(p < 0.05) depending on type of MMTs used. The opaque appear-ance of WPI-based composite films obstructs the transmission oflight through the films, which results in the observed decrease infilm transparency. WPI/clay composite films containing CloisiteNa+ or Cloisite 20A were more transparent than those with Cloisite30B. This may be due to the interactions between sulfated ash(1.54% as manufacturer’s report) contained in WPI and the twoethoxy groups of Cloisite 30B. Nonetheless, transparency valuesshowed a similar trend as gloss values.

3.3. Tensile properties

The tensile properties of the WPI films decreased after blendingwith nano-clays depending on the clay types used (Table 3). The

tensile properties of the WPI/Cloisite Na+ or 30B composite filmsdid not significantly decrease (p < 0.05), except the EM of theWPI/Cloisite Na+ composite films, which decreased remarkablycompared to the control WPI films. The decreased EM of the WPIfilms after blending with Cloisite Na+ may be associated with theincreased film thickness (data not shown). On the other hand, allthe tensile properties of the WPI/Cloisite 20A composite films de-creased significantly (p < 0.05). This may also be due to the incom-plete dispersion of the nano-clay (Cloisite 20A) into the polymermatrix, which is caused by the incompatibility of hydrophobicnano-clay with hydrophilic biopolymer. In contrast, the TS or EMof biopolymer-based nanocomposite films such as corn starch/Cloisite Na+ (Pandey and Singh, 2005), thermoplastic starch(TPS)/Cloisite 30B (Park et al., 2002, 2003), wheat or maizestarch/MMT nanocomposites (McGlashan and Halley, 2003), andchitosan-based nanocomposite films (Rhim et al., 2006) were re-ported to be improved by the formation of the nanocomposite.The increase in the TS and EM of such bio-nanocomposite filmscan be attributed to the high rigidity and aspect ratio of thenano-clay as well as the high affinity between biopolymer andnano-clay (Rhim et al., 2006). For the nano-clays tested in the pres-ent study, the tensile properties of WPI/clay composite films de-creased compared to the control WPI films, indirectly indicatingthat a nanocomposite was not formed between the clay and thepolymer matrix, but rather the nano-clay worked as a filler forthe composite films (Rhim et al., 2008).

3.4. Water vapor permeability

The water vapor barrier properties of WPI-based films were sig-nificantly improved by blending small amounts of nano-clays (5%)

Time (h)0 2 4 6 8 10

Via

ble

cell

coun

t (C

FU/m

l)

107

108

109

1010

WPIWPI/Cloisite Na+

WPI/Cloisite 20A WPI/Cloisite 30B

Time (h)0 2 4 6 8 10

Via

ble

cell

coun

t (C

FU/m

l)

107

108

109

1010

WPI WPI/Cloisite Na+

WPI/Cloisite 20A WPI/Cloisite 30B

A B

Fig. 1. Antimicrobial activity of WPI and WPI/clay composite films against (A) E. coli O157:H7 and (B) L. monocytogenes as determined by a viable cell count method. Errorbars shown represent standard deviations.

472 R. Sothornvit et al. / Journal of Food Engineering 91 (2009) 468–473

as shown in Table 4. The WVP and actual RH underneath the film(RHI) of WPI/clay composite films changed with type of nano-claysused. The WVP of WPI/Cloisite Na+ composite films decreased themost followed by WPI/Cloisite 30B and WPI/Cloisite 20A. In con-trast, the RHI of WPI/clay composite films increased the sameway as the WVP of the composite films. Interestingly, the orderof improvement in water vapor barrier properties reflected thehydrophilicity/hydrophobicity of the nano-clays. The unmodifiedMMT (Cloisite Na+), the most hydrophilic in nature, was greatlycompatible with the hydrophilic WPI film solution and very welldispersed in the polymer matrix, resulting in the best water vaporbarrier properties of WPI-based films among the MMTs tested.However, the organically modified MMTs (hydrophobic Cloisite20A and less hydrophobic Cloisite 30B) did not significantly im-prove the water vapor barrier properties of WPI-based films, whichcould be attributed to their low compatibility and dispersion in theWPI films. Park et al. (2002) also reported that the water vapor bar-rier property of TPS/Cloisite Na+ nanocomposite films was betterthan that of TPS/Cloisite 30B nanocomposite films. It has been wellestablished that the layered structure of nano-clays obstructs thetransmission of water vapor through the film matrix or delaysthe diffusing water vapor pathway due to tortuosity (Bharadwaj,2001; Park et al., 2003; Sorrentino et al., 2006). A decrease inWVP of clay/nanocomposite films has been frequently observedwith various biopolymer films such as TPS/Cloisite Na+ (Parket al., 2002), cellulose acetate/Cloisite 30B (Park et al., 2004), chito-san/Cloisite Na+ nanocomposite films (Rhim et al., 2006), and soyprotein isolate (SPI)/clays (Rhim et al., 2005).

3.5. Antimicrobial activity

The antimicrobial activity of WPI/nano-clay composite films de-pended on type of MMTs and microorganisms tested (Fig. 1). As ex-pected, the control WPI films showed no antimicrobial activityagainst both Gram-negative (E. coli O157:H7) and positive bacteria(L. monocytogenes). Incorporation of Cloisite 30B in WPI filmsshowed a distinctive bacteriostatic effect against Gram-positivebacteria, however, it did not show any antimicrobial effect againstGram-negative bacteria. On the contrary, WPI/Cloisite Na+ andWPI/Cloisite 20A films did not show any antimicrobial activity.Rhim et al. (2006) also found that a chitosan/Cloisite 30B nano-composite films exhibited bactericidal effects against the Gram-positive bacteria Staphylococcus aureus and L. monocytogenes, and

bacteriostatic effects against the Gram-negative bacteria Salmo-nella typhimurium and E. coli. They explained the antimicrobialactivity of the composite films might be attributed to the quater-nary ammonium group in the silicate layer of Cloisite 30B, whichdisrupts bacterial cell membranes and causes cell lysis (Rhimet al., 2006). Hong and Rhim (2008) recently tested the antimicro-bial activity of nano-clays alone, including Cloisite Na+, Cloisite20A, and Cloisite 30B. They found that Cloisite 30B has a strongantimicrobial activity against both Gram-positive and negativebacteria, however, Cloisite 20A exhibit bacteriostatic activityagainst only Gram-positive bacteria, and Cloisite Na+ did not showany antimicrobial activity. Rhim et al. (2008) also demonstratedthat Cloisite 30B incorporated poly(lactide) (PLA) films displayedbacteriostatic activity against the Gram-positive bacteria L. mono-cytogenes. In the present study, similar phenomena of bacterio-static action of Cloisite 30B incorporated WPI films wereobserved against L. monocytogenes.

These results imply that nano-clays may be incorporated intopolymer matrix not only as a filler to improve film properties butalso as an antimicrobial agent to provide functional properties.This is especially true for WPI/Cloisite 30B composite films, whichhas the potential for antimicrobial packaging to control growth ofsome pathogenic bacteria. This property will benefit applicationsfor a variety of foods such as meat, fish, poultry, cereals, cheese,fruits and vegetables (Han, 2005; Labuza and Breene, 1988; Chaand Chinnan, 2004; Cagri et al., 2004).

4. Conclusions

Incorporation of small amount of nano-clays into WPI films wasshown to change the film properties. The optical properties of theWPI composite films changed more or less depending on nano-claytype used, when compared to the control WPI film. However, theWPI/nano-clay composite films decreased water vapor permeationto an extent, indicating that some nano-clays effectively increasethe water vapor barrier properties of the film. Although no signif-icant improvement in mechanical properties was observed for theWPI composite films, incorporation of Cloisite 30B into WPI filmsexhibited a remarkably significant bacteriostatic effect againstGram-positive bacteria, L. monocytogenes. The WPI/nano-clay com-posite films have a great potential for application in food packagingfor extending the shelf life, improving quality, and enhancingsafety of food packaged with them.

R. Sothornvit et al. / Journal of Food Engineering 91 (2009) 468–473 473

Acknowledgements

This work was supported by the Postdoctoral Fellowship Pro-gram of the Korea Science and Engineering Foundation and by agrant from the Food Nanotechnology Development Project of theKorea Food Research Institute.

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