Radiation Physics and Chemistry 63 (2002) 821–825

Use of g-irradiation cross-linking to improve the water vaporpermeability and the chemical stability of milk protein films

B. Ouattaraa,b, L.T. Canha,b, C. Vachona,b, M.A. Mateescuc, M. Lacroixa,b,*aCanadian Irradiation Center (CIC), 535 Cartier blvd West, Laval, Quebec, Canada H7V 3S8

b INRS-Institut Armand-Frappier, Research Center in Microbiology and Biotechnology, 531des Prairies blvd,

Laval, Qu!ebec, Canada H7V 1B7cDepartment of Chemistry and Biochemistry, Universit!e du Qu!ebec "a Montr!eal, Case Postale 8888, Succursale Centre ville,

Montr!eal, Qu!ebec, Canada H3C 3P8

Abstract

g-irradiation was used to produce free-standing cross-linked milk proteins. Film forming solutions were prepared

according to a method previously developed in our laboratory using calcium caseinate (cas) with various proportions of

whey protein isolate (wpi) or whey protein concentrate (wpc). The following caseinate–whey protein (cas:wp) ratio were

prepared: 100:0, 75:25, 50:50, 25:75, and 0:100. The WVP of the films was determined gravimetrically at 231C using a

modified ASTM procedure. Molecular properties characterization was performed by size exclusion chromatography

(SEC). Results showed significant (pp0:05) reduction of the WVP of protein films for the following formulations:

cas:wpi or cas:wpc (100:0); cas:wpi (25:75); cas:wpc (25:75); and cas:wpc (0:100). Mixture of cas and wpi produced a

synergistic effect. The strongest combined effect was obtained for cas:wpi (25:75) formulation with permeability values

of 2.07 and 1.38 gmm/m2 dmm Hg for unirradiated and irradiated samples, respectively. g-irradiation also induced a

substantial increase of high molecular weight protein components in film forming solutions. The predominant fraction

was X10� 106Da for irradiated film forming solutions, compared to less than 0.2� 106Da for native unirradiated

solutions. r 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Irradiation; Cross-linking; Protein films

1. Introduction

During the last 10 years, biopolymers from renewable

sources (proteins, carbohydrates, and lipids) have gained

considerable research interests. Such films can be used

as food coating or stand-alone film wrap to retard

unwanted mass transfert in food products (Debeaufort

et al., 1998; Lim et al., 1999; Kester and Fennema,

1986). In general, protein films are excellent oxygen and

carbon dioxide barriers (Lim et al., 1999; McHugh and

Krochta, 1994). However, protein films are highly

hydrophilic and tend to absorb large quantities of water

under elevated relative humidity conditions. As a

consequence, their mechanical properties are weakened

and their water vapor permeabilities are increased.

Krochta and De Muller-Johnson (1997) reported WVP

values of 10–100 gmm/m2 kPa for casein and whey

protein films compared to 0.1–10 gmm/m2 kPa for

methylcellulose, hydroxypropyl methyl cellulose, and

cellulose acetate films.

Current approaches to extend functional and mechan-

ical properties of these films, include (i) incorporation of

hydrophobic compounds such as lipids in the film

forming solutions has improved the WVP of resulting

whey protein films (McHugh and Krochta, 1994;

*Corresponding author. Canadian Irradiation Center (CIC),

INRS-Institut Armand-Frappier, 531 Boulevard des Prairies,

Building 22, Laval, Qu!ebec, Canada H7V 1B7. Tel.: +1-450-

687-5010x4489; fax: +1-450-687-5792.

E-mail address: monique.lacroix@inrs-iaf.uquebec.ca

(M. Lacroix).

0969-806X/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.

PII: S 0 9 6 9 - 8 0 6 X ( 0 1 ) 0 0 5 7 3 - 4

Shellhammer and Krochta, 1997); (ii) optimization of

the interactions between polymers (protein–protein

interactions, charge–charge electrostatic complexes be-

tween proteins and polysaccharides) and (iii) formation

of cross-links through physical, chemical, or enzymatic

treatments (Miller et al., 1997; Ghorpade et al., 1995; Li

and Chen, 1999). As reported by Urbain (1986),

g-irradiation can also affect proteins by causing con-

formational changes, oxidation of amino acids, forma-

tion of protein free radicals, and recombination and

polymerization reactions. Based on that report, attempts

have been made to establish the suitability of irradiation

for the development of cross-linked protein solutions for

edible/biodegradable packaging applications. The ob-

jective was to evaluate the effect of g-irradiation on the

water vapor permeability and the chemical character-

istics of protein-based films (modification of protein

molecular weight, resistance to microbial and enzymatic

biodegradation).

2. Materials and methods

2.1. Film preparation

Calcium caseinate (cas) (New Zealand Milk Product

Inc., Santa Rosa, CA, USA) and whey protein

concentrate (wpc) (Saputo Cheese Ltd., Montreal,

Quebec, Canada) or whey protein isolate (wpi) (Food

Research and Development Centre, St-Hyacinthe, Qu-!ebec, Canada) were mixed to obtain various cas:wp

ratios (100:0, 75:25, 50:50, 25:75, and 0:100). Unirra-

diated films were obtained by casting the film forming

solutions onto a smooth petri plate (8.5 cm, ID) and

dried overnight at 201C711C in a climatic chamber

with a relative humidity of 45–50%. Irradiated

films were obtained after irradiation of the film

forming solution at a total dose of 32 kGy in a 60Co

underwater calibrator unit (UC-15b) (MSD Nordion,

Laval, Qu!ebec, Canada) with a mean dose rate of

17.33 kGy/h.

2.2. Film thickness

The film thickness was determined using a digimatic

indicator micrometer (Mitutoyo, Tokyo, Japan). Mea-

surements were taken at five locations and the mean

values were used for permeability calculations. The

thickness of the films were ranged from 66 to 95mmdepending on the formulation.

2.3. Permeability measurement

WVP of the films was determined gravimetrically at

two relative humidity (100% and 56%) and one

incubation temperature (231C) using a modified ASTM

(1983) procedure. The test films were sealed to glass cups

containing dehydrated phosphorus pentoxide crystals

(Sigma Chemicals, St-Louis, MO, USA) with exposed

film area of 13.40 cm2. The cups were placed in

desiccators and stored at 231C under 100% relative

humidity or 56% relative humidity. The water vapor

transferred through the film and absorbed by the

desiccant was determined by the weight gain of the

phosphorus pentoxide. The permeability values were

calculated as described by Gontard et al. (1992) using

the following equation:

WVP ¼ ðWX Þ=ATðP12P2Þ;

where W is the weight gain of the cups (g), T is the time

(d), X is the film thickness (mm), A is the exposed area

of the film (m2) and P22P1 is the water vapor pressure

differential across the film (32.23mm Hg for 100%

relative humidity and 9.82mm Hg for 56% relative

humidity).

2.4. Size-exclusion chromatography (SEC)

A Varian model Vista 5500 High Performance

Liquid Chromatograph equipped with a Varian Auto-

sampler model 9090 were used for the SE-HPLC study

on the soluble protein fractions. Two progel columns

(model TSK PWH and GMPW, Supelco, Bellefonte,

PA, USA) followed by two hydrogel columns (model

2000 and 500, Waters, Mississauga, ON, Canada) were

used for the molecular weight determination of cross-

linked and noncross-linked proteins. The total molecu-

lar weight exclusion limit was 25� 106Da based on

linear polyethylene glycol. The molecular weight cali-

bration curve was established using a set of protein

molecular weight markers MW-GF-1000 (Sigma Che-

micals, St-Louis, MO, USA) ranging from 2� 106 to

29 000Da.

2.5. Biodegradation tests

Enzymatic biodegradation tests were performed by

immersing film samples in a pH 7.5 potassium phos-

phate buffer containing 1% pancreatin (Fisher Scientific

Company, New Jersey, USA). Film samples were

removed every 15min during storage and dried at

1001C for 3 h in a model 019 vacuum oven (Precision

Scientific, Chicago, IL, USA) to determine the yield of

recovery. Microbiological biodegradation was deter-

mined in the same manner, but the films samples were

immersing in sterile saline (NaCl, 8.5 g/l) in presence of

Streptococcus thermophilus, and incubated at 371C.

Sample of solution were taken periodically and analyzed

for soluble nitrogen (N) using a LecoFP-428 combustion

oven apparatus (Leco Corporation, St-Joseph, MI,

USA).

B. Ouattara et al. / Radiation Physics and Chemistry 63 (2002) 821–825822

2.6. Statistical analysis

All the statistical calculations were performed using

the software SPSS (SPSS Inc. Chicago, IL). A reduced

two-level factorial design was used to evaluate main

effects of irradiation and cas:wp ratios as well as

interaction effects on the WVP of films. The least

significant difference test was used to determine sig-

nificant differences between casein/whey protein ratios.

Differences between irradiated and unirradiated samples

were determined using the Student t-test. Differences

between means were considered significant when

(pp0:05).

3. Results

3.1. Water vapor permeability

The effect of g-irradiation on the WVP of cas:wpi and

cas:wpc films are presented in Fig. 1. At 100% RH,

g-irradiation produced signficant reduction of WVP in

the following formulations: cas:wpc (100:0)\~; cas:wpc

(0:100)\~; cas:wpi (25:75)\~; and cas:wpi (100:0). The

strongest effect was obtained for the cas:wpi (25:75)

formulation with permeability values of 2.07 and

1.38 gmm/m2 dmm Hg for unirradiated and irriadiated

samples, respectively. At 56% RH, similar reduction of

WVP was observed in irradiated films, but significant

effects were observed only for the cas:wpi or cas:wpc

(100:0).

3.2. Size-exclusion high performance liquid

chromatography

For better understanding of the effect of irradiation

and mixing cas with whey protein, the soluble fractions

of the film forming solutions were compared. The SEC

patterns of cas:wpi (25:75) are presented in Fig. 2. In

both cases g-irradiation of proteins increased the

*

*

*

*

*

Cas:wp ratios

100:0 25:75 50:50 25:75 0:100

100:0 25:75 50:50 25:75 0:100

A

g.m

m/m

².d.m

m.H

gg.

mm

/m².d

.mm

.Hg

0

2

4

6

0

2

4

6

UnirradiatedIrradiated

UnirradiatedIrradiated

WPC

WPI

Irradiated

WPI

WPC UnirradiatedIrradiated

WPC

0

2

4

6

0

2

4

6

*

100:0 25:75 50:50 25:75 0:100

Cas:wp ratios

UnirradiatedIrradiated

WPI

g.m

m/m

².d.m

m.H

g

UnirradiatedIrradiated

WPC

100:0 25:75 50:50 25:75 0:100

g.m

m/m

².d.m

m.H

g

B

Fig. 1. Effet of g-irradiation on the WVP of cas:wpi and cas:wpc films at 100% (A) or 56% (B) RH (*) significant differences (pp0:05)between irradiated and unirradiated films.

Fig. 2. SEC curves of protein molecular weight. (A) cas:wpi

(100:0); (B) cas:wpi (25:75).

B. Ouattara et al. / Radiation Physics and Chemistry 63 (2002) 821–825 823

molecular weight 10–20 fold Based on the protein

calibration curve, the predominant of unirradiated

solutions were approximately 200 kDa for cas:wpi

(100:0) formulation and 500 kDa for cas:wpi (25:75)

formulation. With g-irradiation, molecular weight of

the soluble protein fraction increased to 2000 kDa

for cas:wpi (100:0) and to 10 000 kDa for cas:wpi

(25:75).

3.3. Biodegradation

In enzymatic biodegradation, 40% of the film cross-

linked by g-irradiation were recovered after 100 h while

control films and heat cross-linked films were completely

destroyed after 80 h (Fig. 3). In microbiological biode-

gradation evaluation, the percentage of soluble N

increased for control films to reach 0.30% at day 70.

In the fims cross-linked by g-rradiation, the percentage

of soluble N was stable at 0.10% during all the

experimental period (Fig. 4).

4. Discussion

The functional and barriers properties of protein films

reflect their molecular structure and the extent of

interactions occurring between the components of the

film forming solution. In particular, cohesion between

protein peptides or between proteins and other biopo-

lymers such as polysaccharides and lipids is one of the

most important factors which affect the barrier proper-

ties of edible films (Brault et al., 1997; Kester and

Fennema, 1986; Gennadios et al., 1993). The present

study showed that g-irradiation reduced significantly

(pp0:05) the WVP of protein films made from mixtures

of cas and wpc or wpi. These results are consistent with

several previous reports and can be attributed to the

hydrophilic nature of protein films. Leman and Kinsella

(1989) indicated that caseins have a high proline content

uniformly distributed though the polypeptide chain that

limits a-helix and b-sheet formation and results in

individual casein having relative open flexible structure.

As a result, interactions with water molecules may be

considerably increased in moist environments. Casein

also contains amino acids with polar side chains such as

tyrosine and cysteine which are able to establish

hydrogen bonds with water molecules (Cheftel et al.,

1985). The relationship between the molecular structure

of casein and the WVP of casein-based edible films has

also been established by Tomasula et al. (1998).

The improvement of barrier properties and resistance

to microbial and enzymatic biodegradation by g-irradiation is indicative of more cohesion between

polypeptide chains. As previously reported by Brault

et al. (1997), irradiation of aqueous protein solutions

generates hydroxyl radicals which react with aromatic

residues to form covalent bonds. For example, tyrosine

can react with hydroxyl groups and lead to the

formation of bityrosine between protein chains. In a

previous study, Ressouany et al. (1998) irradiated

calcium caeinate film forming solutions and obtained

significant increase of the concentration of bityrosine

and better mechanical properties of resulting films. The

results of SEC reported here confirmed the relationship

between g-irradiation of protein solutions and reduction

of the WVP of resulting films. According to several

previous studies (Brault et al., 1997; Davies and

Delsignore, 1987; Mezgheni et al., 1998; Ressouany

et al., 1998), amino acids present in protein solutions can

absorb radiations and recombine to form convalent

cross-links. Therefore, it can be assumed that the

formation of high molecular weight proteins in films

forming solutions may be responsible for the reduction

of the WVP, mainly by reducing the absorption of water

molecules into the polymeric matrix and the diffusion

through the film. Our results, however, differed from

those of Gennadios et al. (1998) who increased tensile

strength of soy protein films through ultraviolet

1

2

3

1000

20

40

60

80

100

0

Yie

ld o

f re

cove

ry (

%)

Time (mn)

20 40 60 80

Fig. 3. Biodegradation of protein-based films in presence of

pancreatin. (1) Control; (2) heat cross-linking; (3) g-irradiationcross-linking.

0 80

UnirradiatedIrradiated

0

0.10

0.20

0.30

Sol

uble

N (

%)

Time (d)20 40 60

Fig. 4. Biodegradation of protein-based films in presence of S.

thermophilus.

B. Ouattara et al. / Radiation Physics and Chemistry 63 (2002) 821–825824

radiation but failed to reduce the WVP. Apparently,

higher irradiation doses and extensive cross-linking is

necessary to affect the WVP of hydrophilic protein films.

In our study the films were irradiated at a total dose of

32 kGy. Ressouany et al. (1998) found that a dose of

16 kGy was not high enough to produce significant

cross-linking.

5. Conclusion

g-irradiation significantly (pp0:05) reduced the WVP

and increased the resistance to microbial and enzymatic

biodegradation. Results showed significant (pp0:05)reduction of the WVP of protein films for the following

formulations: cas:wpi or cas:wpc (100:0), cas:wpi

(25:75), cas:wpc (25:75), and cas:wpc (0:100). Mixture

of cas and wpi produced a synergistic effect. The

strongest combined effect was obtained for cas:wpi

(25:75) formulation with permeability values of 2.07 and

1.38 gmm/m2 dmm Hg for unirradiated and irradiated

samples, respectively. An increase of the concentration

of high molecular weight proteins in the film forming

solution was also observed. Two hypotheses may

explain the effect on g-irradiation: (i) a participation of

more molecular residues in intermolecular interactions

when protein with different physicochemical properties

and (ii) the formation of inter- and/or intra-molecular

convalent cross-links in the film forming solutions.

Acknowledgements

This work was funded by the FCAR-CQVB-Novalait

program, Department of Agriculture, Fisheries and

Food of the province of Quebec (CORPAQ program)

and by the Institut Armand-Frappier for granting a

postdoctoral fellowship to BO. Authors are grateful to

MDS Nordion Inc. for irradiation operations.

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