Use of g-irradiation cross-linkingto improve the water vapor permeability and the chemical stability of milk protein films

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1 Radiation Physics and Chemistry 3 () Use of g-irradiation cross-linkingto improve the water vapor permeability and the chemical stability of milk protein films B. Ouattara a,b, L.T. Canh a,b, C. Vachon a,b, M.A. Mateescu c, M. Lacroix a,b, a Canadian 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 1B7 c Department of Chemistry and Biochemistry, Universit!e duqu!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-standingcross-linked milk proteins. Film formingsolutions were prepared accordingto a method previously developed in our laboratory usingcalcium caseinate (cas) with various proportions of whey protein isolate (wpi) or whey protein concentrate (wpc). The followingcaseinate whey protein (cas:wp) ratio were prepared: 1:, 75:5, 5:5, 5:75, and :1. The WVP of the films was determined gravimetrically at 31C usinga modified ASTM procedure. Molecular properties characterization was performed by size exclusion chromatography (SEC). Results showed significant (pp:5) reduction of the WVP of protein films for the followingformulations: cas:wpi or cas:wpc (1:); cas:wpi (5:75); cas:wpc (5:75); and cas:wpc (:1). Mixture of cas and wpi produced a synergistic effect. The strongest combined effect was obtained for cas:wpi (5:75) formulation with permeability values of.7 and 1.38 gmm/m d mm Hgfor 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 X1 1 Da for irradiated film formingsolutions, compared to less than. 1 Da for native unirradiated solutions. r Elsevier Science Ltd. All rights reserved. Keywords: Irradiation; Cross-linking; Protein films 1. Introduction Duringthe last 1 years, biopolymers from renewable sources (proteins, carbohydrates, and lipids) have gained considerable research interests. Such films can be used as food coatingor stand-alone film wrap to retard unwanted mass transfert in food products (Debeaufort et al., 1998; Lim et al., 1999; Kester and Fennema, 198). In general, protein films are excellent oxygen and Correspondingauthor. Canadian Irradiation Center (CIC), INRS-Institut Armand-Frappier, 531 Boulevard des Prairies, Building, Laval, Qu!ebec, Canada H7V 1B7. Tel.: x89; fax: address: monique.lacroix@inrs-iaf.uquebec.ca (M. Lacroix). carbon dioxide barriers (Lim et al., 1999; McHugh and Krochta, 199). 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 1 1 gmm/m kpa for casein and whey protein films compared to.1 1 gmm/m kpa for methylcellulose, hydroxypropyl methyl cellulose, and cellulose acetate films. Current approaches to extend functional and mechanical properties of these films, include (i) incorporation of hydrophobic compounds such as lipids in the film formingsolutions has improved the WVP of resulting whey protein films (McHugh and Krochta, 199; 99-8X//$ - see front matter r Elsevier Science Ltd. All rights reserved. PII: S 99-8X(1)573-

2 8 B. Ouattara et al. / Radiation Physics and Chemistry 3 () Shellhammer and Krochta, 1997); (ii) optimization of the interactions between polymers (protein protein interactions, charge charge electrostatic complexes between 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 (198), g-irradiation can also affect proteins by causingconformational changes, oxidation of amino acids, formation 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 objective was to evaluate the effect of g-irradiation on the water vapor permeability and the chemical characteristics of protein-based films (modification of protein molecular weight, resistance to microbial and enzymatic biodegradation).. Materials and methods.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 (1:, 75:5, 5:5, 5:75, and :1). films were obtained by castingthe film forming solutions onto a smooth petri plate (8.5 cm, ID) and dried overnight at 1C711C in a climatic chamber with a relative humidity of 5 5%. films were obtained after irradiation of the film formingsolution at a total dose of 3 kgy in a Co underwater calibrator unit (UC-15b) (MSD Nordion, Laval, Qu!ebec, Canada) with a mean dose rate of kgy/h... Film thickness The film thickness was determined usinga digimatic indicator micrometer (Mitutoyo, Tokyo, Japan). Measurements were taken at five locations and the mean values were used for permeability calculations. The thickness of the films were ranged from to 95 mm dependingon the formulation..3. Permeability measurement WVP of the films was determined gravimetrically at two relative humidity (1% and 5%) and one incubation temperature (31C) usinga modified ASTM (1983) procedure. The test films were sealed to glass cups containingdehydrated phosphorus pentoxide crystals (Sigma Chemicals, St-Louis, MO, USA) with exposed film area of 13. cm. The cups were placed in desiccators and stored at 31C under 1% relative humidity or 5% 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. (199) using the followingequation: WVP ¼ðWX Þ=ATðP1PÞ; 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 (m ) and PP1 is the water vapor pressure differential across the film (3.3 mm Hgfor 1% relative humidity and 9.8 mm Hgfor 5% relative humidity)... Size-exclusion chromatography (SEC) A Varian model Vista 55 High Performance Liquid Chromatograph equipped with a Varian Autosampler model 99 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 and 5, Waters, Mississauga, ON, Canada) were used for the molecular weight determination of crosslinked and noncross-linked proteins. The total molecular weight exclusion limit was 5 1 Da based on linear polyethylene glycol. The molecular weight calibration curve was established usinga set of protein molecular weight markers MW-GF-1 (Sigma Chemicals, St-Louis, MO, USA) ranging from 1 to 9 Da..5. Biodegradation tests Enzymatic biodegradation tests were performed by immersingfilm samples in a ph 7.5 potassium phosphate buffer containing1% pancreatin (Fisher Scientific Company, New Jersey, USA). Film samples were removed every 15 min duringstorage and dried at 11C for 3 h in a model 19 vacuum oven (Precision Scientific, Chicago, IL, USA) to determine the yield of recovery. Microbiological biodegradation was determined in the same manner, but the films samples were immersingin 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-8 combustion oven apparatus (Leco Corporation, St-Joseph, MI, USA).

3 B. Ouattara et al. / Radiation Physics and Chemistry 3 () 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 significant differences between casein/whey protein ratios. Differences between irradiated and unirradiated samples were determined usingthe Student t-test. Differences between means were considered significant when (pp:5). 3.. Size-exclusion high performance liquid chromatography For better understandingof the effect of irradiation and mixingcas with whey protein, the soluble fractions of the film formingsolutions were compared. The SEC patterns of cas:wpi (5:75) are presented in Fig.. In both cases g-irradiation of proteins increased the 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 1% RH, g-irradiation produced signficant reduction of WVP in the followingformulations: cas:wpc (1:)\~; cas:wpc (:1)\~; cas:wpi (5:75)\~; and cas:wpi (1:). The strongest effect was obtained for the cas:wpi (5:75) formulation with permeability values of.7 and 1.38 gmm/m d mm Hgfor unirradiated and irriadiated samples, respectively. At 5% RH, similar reduction of WVP was observed in irradiated films, but significant effects were observed only for the cas:wpi or cas:wpc (1:). Fig.. SEC curves of protein molecular weight. (A) cas:wpi (1:); (B) cas:wpi (5:75). g.mm/m².d.mm.hg g.mm/m².d.mm.hg 1: 5:75 5:5 5:75 :1 WPI WPC 1: 5:75 5:5 5:75 :1 Cas:wp ratios A g.mm/m².d.mm.hg g.mm/m².d.mm.hg WPI WPC 1: 5:75 5:5 5:75 :1 1: 5:75 5:5 5:75 :1 Cas:wp ratios Fig. 1. Effet of g-irradiation on the WVP of cas:wpi and cas:wpc films at 1% (A) or 5% (B) RH () significant differences (pp:5) between irradiated and unirradiated films. B

4 8 B. Ouattara et al. / Radiation Physics and Chemistry 3 () molecular weight 1 fold Based on the protein calibration curve, the predominant of unirradiated solutions were approximately kda for cas:wpi (1:) formulation and 5 kda for cas:wpi (5:75) formulation. With g-irradiation, molecular weight of the soluble protein fraction increased to kda for cas:wpi (1:) and to 1 kda for cas:wpi (5:75) Biodegradation In enzymatic biodegradation, % of the film crosslinked by g-irradiation were recovered after 1 h while control films and heat cross-linked films were completely destroyed after 8 h (Fig. 3). In microbiological biodegradation evaluation, the percentage of soluble N increased for control films to reach.3% at day 7. In the fims cross-linked by g-rradiation, the percentage of soluble N was stable at.1% duringall the experimental period (Fig. ). Yield of recovery (%) Time (mn) 3 1 Fig. 3. Biodegradation of protein-based films in presence of pancreatin. (1) Control; () heat cross-linking; (3) g-irradiation cross-linking. Soluble N (%) Time (d) Fig.. Biodegradation of protein-based films in presence of S. thermophilus.. Discussion The functional and barriers properties of protein films reflect their molecular structure and the extent of interactions occurringbetween the components of the film formingsolution. In particular, cohesion between protein peptides or between proteins and other biopolymers such as polysaccharides and lipids is one of the most important factors which affect the barrier properties of edible films (Brault et al., 1997; Kester and Fennema, 198; Gennadios et al., 1993). The present study showed that g-irradiation reduced significantly (pp:5) 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 havingrelative 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 formingsolutions and obtained significant increase of the concentration of bityrosine and better mechanical properties of resultingfilms. The results of SEC reported here confirmed the relationship between g-irradiation of protein solutions and reduction of the WVP of resultingfilms. Accordingto 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 formingsolutions may be responsible for the reduction of the WVP, mainly by reducingthe 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

5 B. Ouattara et al. / Radiation Physics and Chemistry 3 () 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 3 kgy. Ressouany et al. (1998) found that a dose of 1 kgy was not high enough to produce significant cross-linking. 5. Conclusion g-irradiation significantly (pp:5) reduced the WVP and increased the resistance to microbial and enzymatic biodegradation. Results showed significant (pp:5) reduction of the WVP of protein films for the following formulations: cas:wpi or cas:wpc (1:), cas:wpi (5:75), cas:wpc (5:75), and cas:wpc (:1). Mixture of cas and wpi produced a synergistic effect. The strongest combined effect was obtained for cas:wpi (5:75) formulation with permeability values of.7 and 1.38 gmm/m d mm Hgfor 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 formingsolutions. 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. References ASTM, Standard test method for water vapor transmission of materials, method 15.9:E9. American Society of Testingand Material. Philadelphia, PA. Brault, D., D Aprano, G., Lacroix, M., Formation of free-standingsterilized films from irradiated caseinates. J. Agric. Food Chem. 5, Cheftel, J.C., Cuq, J.-L., Lorient, D., Amino acids, peptides, and proteins. In: Fennema, O.R. (Ed.), Food Chemistry. Marcel Dekker, Inc., New York, pp Davies, J.A., Delsignore, M.E., Protein damage and degradation by oxygen radicals. III. Modification of secondary and tertiary structure. J. Biol. Chem., Debeaufort, F., Quezada-Gallo, J.-A., Voilley, A., Edible films and coatings: tomorrow s packaging: a review. Crit. Rev. Food Sci. 38, Gennadios, A., Weller, C.L., Testin, R.F., Property modification of edible wheat, gluten-based films. Trans. ASAE 3, 5 7. Gennadios, A., Rhim, J.W., Handa, A., Weller, C.L., Hanna, M.A., Ultraviolet radiation affects physical and molecular properties of soy protein films. J. Food Sci. 3, 5 8. Ghorpade, V.M., Li, H., Gennadios, A., Hanna, M.A., Chemically modified soy protein films. Trans. ASAE 38, Gontard, N., Guilbert, S., Cuq, J.-L., 199. Edible gluten films: influence of the main process variables on film properties usingresponse surface methodology. J. Food Sci. 57, , 199. Kester, J.J., Fennema, O.R., 198. Edible films and coatings: a review. Food Technol., Krochta, J.M., De Muller-Johnson, C., Edible biodegradable polymer films: challenge and opportunities. Food Technol. 51, 7. Leman, J., Kinsella, J.E., Surface activity, film formation, and emulsifyingproperties of milk proteins. Crit. Rev. Food Sci. Nutr. 8, Li, J., Chen, H., Transglutaminase catalyzed crosslinking on functional properties of whey protein-based edible films. Book of Abstracts, IFT Annual MeetingJuly 8, Chicago, IL, USA. Lim, L.-T., Mine, Y., Tung, M.A., Barrier and tensile properties of transglutaminase cross-linked gelatin films as affected by relative humidity, temperature, and glycerol content. J. Food Sci., 1. McHugh, T.H., Krochta, J.M., 199. Water vapor permeability properties of edible whey protein lipid emulsion films. J. Am. Oil Chem. Soc. 71, Mezgheni, E., D Aprano, G., Lacroix, M., Formation of sterilized edible films based on caseinates: effects of calcium and plasticizers. J. Agric. Food Chem., Miller, K.S., Chiang, M.T., Krochta, J.M., Heat curing of whey protein films. J. Food Sci., Ressouany, M., Vachon, C., Lacroix, M., Irradiation dose and calcium effect on the mechanical properties of cross-linked caseinate films. J. Agric. Food Chem., Shellhammer, T.H., Krochta, J.M., Whey protein emulsion films performance as affected by lipid type and amount. J. Food Sci., Tomasula, P.M., Parris, N., Yee, W., Coffin, D., Properties of films made from CO -precipitated casein. J. Agric. Food Chem., 7 7. Urbain, W.M., 198. Biological effects of ionizing radiation. In: Schweigert, B.S. (Ed.), Food Irradiation. Academic Press Inc., Orlando, FL, pp