Effect of Cross-Linking on Microstructure and Physical Performance of

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Effect of Cross-Linking on Microstructure and Physical Performance of Casein Protein Arun Ghosh,*,† M. Azam Ali,*,† and George J. Dias‡ AgResearch Limited, Lincoln Research Centre, Springs Road and Gerald Street, Lincoln, Canterbury 8140, New Zealand, and Department of Anatomy and Structural Biology, University of Otago, 270 Great King Street, Dunedin 9016, New Zealand Received November 21, 2008; Revised Manuscript Received April 12, 2009

The development of advanced materials from biorenewable protein biopolymers requires the generation of more exogenous bonds to maintain the microstructure and durability in the final products. Casein is the main protein of milk, representing about 80% of the total protein. In the present investigation the casein protein was solubilized and/or emulsified in aqueous alkaline solutions, and 2D films and 3D matrices were produced. The effects of silane (3-aminopropyl triethoxy silane), DL-glyceraldehyde and glutaraldehyde on tensile properties and water swelling/absorption of 2D casein films and also the microstructure of the freeze-dried 3D matrices were analyzed. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that there were no significant changes in the molecular weight (19-23.9 kDa) of the casein proteins on exposure to alkaline solutions of sodium hydroxide and silane. The casein films produced without glycerol plasticizer and with heat treatment (130 °C for 18 h) were fragile. However, the fragile films were transformed into ductile and tough materials on exposure to moisture (i.e., conditioned for one week at 50 ( 2% relative humidity and 22 ( 2 °C) and showed a maximum average tensile strength of 49-52 MPa and modulus of 1107-1391 MPa. The chemical crosslinkers (i.e., DL-glyceraldehyde and glutaraldehyde) improved the microstructure of glycerol plasticized casein protein, when analyzed under scanning electron microscope (SEM). Furthermore, these chemical cross-linking agents enhanced the mechanical properties and water resistant properties of casein films.

Introduction Cross-linking is an important step for protein biopolymers, which influences the microstructure and physical performances of the biopolymers used for food, medical, and industrial applications. Chemical cross-linking methods typically use bifunctional chemicals (e.g., glutaraldehyde, formaldehyde, carbodiimides, polyepoxy compounds, acyl azide, etc.) that interact with the functional groups of proteins, such as the ε-amino function in lysine and hydroxylysine or the carboxyl group in aspartic and glutamic acids.1 Physical cross-linking methods such as heating, drying, and irradiation are also commonly applied to biopolymers. Growing environmental concerns have led to increased emphasis on research and development of biodegradable materials based on renewable biological resources for biomedical and industrial applications. The naturally occurring biopolymers such as collagen or gelatin, chitin or chitosan, zein, soy protein, wheat gluten, starch, cellulose, and so on are used in the production of composite films and coatings.2,3 Excellent bioplastics may be developed from biologically derived polymers by proper blending and processing techniques. Their functionalities can also be tailored by plasticizers, cross-linkers, and other additives. Casein is the main protein of milk, accounting for approximately 80% of the total protein content. It is a phosphoprotein, which can be separated into various electrophoretic fractions such as Rs-casein, κ-casein, β-casein, and γ-casein. These protein fractions differ in primary, secondary, and tertiary structure and molecular weight.4,5 * To whom correspondence should be addressed. Phone: +64 3 3218751. Fax: +64 3 3218811. E-mail: [email protected] (A.G.); [email protected] (A.A.). † AgResearch Limited. ‡ University of Otago.

There are numbers of reports in the literature on casein based edible films.6-11 Caseins have also been blended and grafted with other polymers and cross-linkers or monomers for improving functional properties such as mechanical, water resistant, thermal stability, and barrier properties.12-16 Presently, there are numerous applications of casein as coating, adhesive, and packaging materials in the paper, leather, textile, and food industries.4 Gawryla et al.17 have produced clay reinforced casein-based lightweight aerogel composites by freeze-drying technique and found the mechanical properties similar to other synthetic polymer/clay aerogel composites. The analyses of microstructural morphology and physical performances of casein protein cross-linked by heat treatment and chemical cross-linkers have not been reported earlier in the literature. This article presents the effects of chemical agents (i.e., glutaraldehyde, DL-glyceraldehyde, and 3-aminopropyl triethoxy silane) and heat treatment on cross-linking characteristics, and consequently, the changes of microstructure, mechanical and water swelling, or absorption properties of casein protein. In the present study, casein protein solutions were prepared separately in aqueous solutions of sodium hydroxide and 3-aminopropyl triethoxy silane. At the first stage 2D films were made from the protein solutions by plasticizing with different wt% of glycerol, and the effect of severe heat treatment (130 °C/18 h) on physical properties was analyzed. The objective at this stage was to select an appropriate composition of plasticized casein protein that may be useful for developing technically valuable products such as ductile or flexible films, scaffolds, and so on. In the next stage, different chemical cross-linkers (e.g., glutaraldehyde and DL-glyceraldehyde) were applied to improve the physical performance of plasticized casein protein.

10.1021/bm801341x CCC: $40.75  2009 American Chemical Society Published on Web 05/12/2009

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Table 1. Preparation of Chemically Cross-Linked Casein Filmsa sample code

A-00

A-10

A-20

A-30

casein protein, g (casein in NaOH/H2O solution) casein protein, g (casein in silane/H2O solution) glycerol, g glyceraldehyde, g glutaraldehyde, g

10

9

8

7

0

1

2

3

B-00

B-10

B-20

B-30

10 0

9 1

8 2

7 3

A-30-GC

A-30-GT

7

7

3 0.2

3 0.2

a

In text “A-xx-C, B-xx-C”, “A-xx-H, B-xx-H”, and “A-xx-HM, B-xx-HM” represent the control, heat-treated, and heat followed by moisture treated casein films, respectively.xx indicates two digit numerical numbers such as 00, 10, 20, and 30, which correspond to glycerol content.

Experimental Section Materials. Bovine milk derived casein (technical grade) was supplied by Sigma-Aldrich New Zealand Ltd. The molecular weights of casein subunits are in the range of 19-25 kDa.18 DL-Glyceraldehyde, glutaraldehyde solution (50 wt % in H2O), and 3-aminopropyl triethoxy silane were also supplied by Sigma-Aldrich New Zealand Ltd. Trishydrochloric acid (1 M), glycerol, dithiothreitol, and sodium hydroxide were supplied by BDH Chemicals, U.K. Sodium dodecyl sulfate (SDS) buffer, phosphoric acid, and ammonium sulfate (analytical grade) were purchased from Merck, Germany. Bromophenol blue (2%) was supplied by M&B Chemicals, U.K. Criterion Precast gel, Precision Plus Protein Standard (10-250 kDa), and Coomassie Brilliant Blue G-250 were purchased from Bio-Rad, U.S.A. Casein Solution and Film Preparation. Casein was solubilized and/ or emulsified separately by adding into an aqueous solution of 0.2 wt % (0.05 N) sodium hydroxide or 1 wt % of 3-aminopropyl triethoxy silane at 80 °C for about 3 h. The total protein content in each solution was kept constant at 10 wt %. The protein solution was then mixed with glycerol plasticizer for 10 min at 80 °C according to the formulation shown in Table 1. Each sample solution was casted on polystyrene Petri-dish and dried under airflow at room temperature (22 ( 2 °C). The average thickness of the films was in the range of 0.15 to 0.25 mm. For analyzing the effect of heat treatment on physical properties, each film was kept at 130 °C for 18 h in an oven. The heat-treated films were then exposed to 50 ( 2% relative humidity at 22 ( 2 °C for examination of the effect of moisture on mechanical properties of these films. Preparation of Chemically Cross-Linked Casein Films. The effect of aldehyde cross-linkers (i.e., glutaraldehyde and DL-glyceraldehyde) was studied on 30 wt % glycerol plasticized casein protein sample. First, 10 wt % casein solution was prepared using 0.2 wt % (0.05 N) NaOH as described above. The casein solution was then mixed with glycerol plasticizer at a ratio of 70:30 (dry weight basis) for 10 min at 80 °C and cooled down to room temperature. The chemical cross-linking agents (i.e., DL-glyceraldehyde and glutaraldehyde) were then added to the plasticized casein solution at the ratio of 1:50 (dry weight basis) and mixed for 10 min. The solution was then casted on a Petri-dish and dried under airflow at 22 ( 2 °C. A portion of each dried sample was post cured by heat treatment at 100 °C for 2 h. SDS-PAGE Analysis. SDS-PAGE was performed to analyze the effect of alkaline solutions on the molecular structure of casein proteins. A total of 10 µL of each protein solution (10 wt % protein content) was mixed with 100 µL of SDS buffer and then briefly heated to near boiling, which reduces disulfide linkages and breaks the quaternary protein structure. Similarly, 1 mg control casein protein was dissolved in 200 µL SDS buffer solution. Each protein solution was then injected at the loadings of 5, 10, and 15 µL in different lanes (from left to right) at one end of a layer of polyacrylamide gel (Bio-Rad’s Criterion Precast Gel). The Precision Plus Protein standard (10-250 kDa) as a marker was also injected at the loading of 10 µL in the outmost left and right lanes of the gel and the electrophoresis was carried out at 200 V for about 60 min. Then the gel was treated with 100 mL of aqueous solution of 10% acetic acid and 40% ethanol, and the protein was stained by adding 200 mL of blue silVer dye19 and digitally imaged. Measurement of Mechanical Properties. The mechanical properties such as tensile strength, modulus, and elongation at break of the casein films were measured using an INSTRON universal testing machine (model 4202) having a 100 N load cell with extension rate of 20 mm/min and

Figure 1. SDS-PAGE molecular weight band patterns of bovine casein proteins (lanes 1, 2, and 3 represent the injection of 5, 10, and 15 µL protein solution, respectively).

gauge length of 20 mm. Prior to analysis of the tensile properties, the casein films (formulations shown in Table 1) were first conditioned under standard atmospheric conditions (i.e., 50 ( 2% relative humidity and 22 ( 2 °C) for 24 h, and the heat-treated films were exposed to the above standard atmospheric conditions for 6 h and 1 week. Measurement of Water Absorption Properties. The water absorption properties of the casein films were expressed as percentage increase of weight after immersion in deionized water for a predetermined time. The dry films were weighed and then immersed in deionized water at room temperature (22 ( 2 °C). The swollen films were then removed from water at predetermined times, blotted dry, and weighed. The percentage increase of weight was calculated using the following equation.

∆W )

Wt - W0 × 100(%) W0

(1)

W0 and Wt indicate the weight of the film before and after immersion in water at a predetermined time. Analysis of Microstructure of Casein Protein. The selected glycerol plasticized casein protein solutions (i.e., A-30, A-30-GC, A-30GT, and B-30 formulations shown in Table 1) were frozen at -86 °C for about a week and then freeze-dried at about -80 °C for another week under vacuum using a Dura-Dry. The freeze-dried samples were gold coated and viewed at ×350 magnification with a Field Emission Scanning Electron Microscope (JEOL JSM-7000F).

Results and Discussion SDS-PAGE Analysis of Casein Protein. Casein derived from bovine milk contains four major protein subunits such as Rs1-casein (38%), Rs2-casein (10%), β-casein (36%), and κ-casein (13%) and a minor constituent, γ-casein (3%). Each subunit varies in amino acid composition, molecular weight (19-23.9 kDa), isoelectric point, and hydrophilicity.4,5 The SDS-PAGE image (Figure 1) exhibits similar band patterns for the control casein and silane-treated casein protein, indicating that silane did not have any significant effect on the physico-

8(2 17 ( 1 26 ( 4 28 ( 7 6(2 15 ( 7 18 ( 11 31 ( 8 ND ND 11 ( 4 17 ( 1 ND ND 4(2 20 ( 4

control (air dry)

ND 693 ( 38 319 ( 11 38 ( 4 ND 1004 ( 40 341 ( 8 35 ( 17 52 ( 0.2 21 ( 0.3 18 ( 0.3 17 ( 0.6 49 ( 3 33 ( 2 18 ( 0.7 10 ( 0.5 ND* 36 ( 0.4 13 ( 2 3.6 ( 0.2 ND 48 ( 2 16 ( 0.4 3 ( 0.7 A-00 A-10 A-20 A-30 B-00 B-10 B-20 B-30

ND ND 31 ( 2 20 ( 2 ND ND 34 ( 2 18 ( 1.5

control (air dry) samples

* ND ) not determined.

control (air dry)

ND 25 ( 9 51 ( 15 134 ( 9 ND 8(3 64 ( 5 94 ( 3 1107 ( 11 497 ( 40 392 ( 54 386 ( 68 1391 ( 48 765 ( 109 380 ( 28 197 ( 22

moisture (50 ( 2% RH, 22 ( 2 °C, 1 week) moisture (50 ( 2% RH, 22 ( 2 °C, 6 h) moisture (50 ( 2% RH, 22 ( 2 °C, 6 h)

moisture (50 ( 2% RH, 22 ( 2 °C, 1 week)

ND ND 674 ( 25 473 ( 15 ND ND 916 ( 82 408 ( 57

moisture (50 ( 2% RH, 22 ( 2 °C, 1 week) moisture (50 ( 2% RH, 22 ( 2 °C, 6 h)

heat treatment (130 °C/18 h)

elongation at break (%) tensile modulus (MPa)

heat treatment (130 °C/18 h) heat treatment (130 °C/18 h)

tensile strength (MPa)

chemical properties of casein solution. Both control and silane treated casein solutions show highly dense bands around 20-24 kDa along with some weak bands below 20 kDa and around 50-70 kDa or higher. The weak bands above molecular weight of 50 kDa are presumably due to formation of a complex aggregation of casein proteins through calcium phosphate bridges, corresponding to the so-called “native casein micelles”.20 The properties such as particle diameter and zeta potential of the casein micelles are not affected significantly in a weak alkaline environment such as silane solution, however, in a stronger alkaline (e.g., NaOH) solution the leakage of calcium phosphate bridges can happen and consequently partial destructuration and restructuration of casein micelles are observed,20 resulting in a more transparent solution of casein protein with partial weakening of the molecular weight bands around 50 kDa or higher. Casein used in the present study was of a technical grade, and therefore, contained relatively high protein impurities and fatty acids.18 The milk proteins such as bovine serum albumin, immunoglobulins and R-lactalbumin have molecular weights of 66.4 kDa, 50-70 kDa, and 14.2 kDa, respectively.21 Therefore, it is assumed that the broad and weak band patterns around 50-70 kDa or higher may also be partially due to the presence of bovine serum albumin and/or immunoglobulins as impurities while the bands around 10-20 kDa may be due to the presence of low molecular weight proteins (e.g., R-lactalbumin) as impurities in casein. Mechanical Properties of Casein Films. The mechanical properties such as tensile strength, modulus and elongation at break (EAB) of different casein films treated under different conditions are summarized in Table 2. The films obtained after room temperature (22 ( 2 °C) air drying are considered as controls. The casein films produced without plasticizers (control A-00 and B-00) were so brittle that it was not possible to determine the tensile properties of these films. Therefore, the effect of glycerol as a plasticizer on the properties of casein films was studied. The role of a plasticizer is to reduce polymer-polymer chain secondary bonding and increase the mobility of the macromolecules due to the augmentation of the free volume and, consequently, lower the glass-to-rubber transition temperature of the polymers.22,23 A plasticizer is expected to reduce the modulus, tensile strength, hardness, brittleness, and so on, of a material, while it increases its flexibility, extensibility, toughness, and numerous other characteristics. In general, the plasticizer is incorporated into the amorphous parts of polymers, while the structure and size of any crystalline part remains unaffected.22 Siew et al.24 have studied the molecular conformations of protein chain structure in solution and film phases of plasticized sodium caseinate using Infrared spectroscopy. The casein proteins are generally classified as unordered proteins that contain few R-helical and β-structures, and random and turn conformation (unordered structure) as a dominating feature of protein chain structure in aqueous solution. However, the protein chains become more ordered in the condensed phase, resulting in increased molecular packing. The addition of plasticizer alters the distribution of various protein chain conformations. Specifically, the random coil structures of casein protein are transformed into helical structures, resulting in a more open molecular network.24 Polyols are considered as efficient plasticizers for protein based materials because of their ability to reduce the intermolecular hydrogen bonding by increasing intermolecular spacing between protein chains. The sodium hydroxide solution derived control film with 10 wt % glycerol plasticizer (sample A-10C) showed tensile strength of 36 ( 0.4 MPa, modulus of 693 ( 38 MPa and EAB of 25 ( 9% (Table 2). Further addition of glycerol plasticizer (20% and 30 wt %) decreased the tensile


Table 2. Heat and Moisture Treatment Effect on Tensile Properties of Casein Films with Different Glycerol Content

Cross-Linking of Casein Protein

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Figure 2. Simplified probable cross-linking mechanisms of casein protein with (a) silane treatment, (b) heat treatment, and (c) dicarbonyl compounds (gray circle is a symbol of protein).

strength and modulus and increased the elongation at break, resulting in films that were increasingly flexible. The film generated with 10 wt % glycerol using silane showed higher tensile properties (strength of 48 ( 2 MPa, modulus of 1004 ( 40 MPa, and EAB of 8 ( 3%) than that prepared in NaOH. It is reported that the adhesion or cross-linking by functional silane is promoted by interdiffusion of the polymerized silane chain with the main polymer chain, resulting in an interpenetrating polymer network (IPN).25,26 Furthermore, there is a possibility of hydrogen bonding interactions of the silanol with several protein functionalities including the carboxylic acid of aspartic or glutamic acids (Figure 2a). However, at higher glycerol content (20 and 30 wt %), both NaOH- and silane-treated casein protein films exhibited similar tensile properties (Table 2), indicating that the reactivity of silane as a coupling or cross-linking agent is reduced with increasing glycerol concentration. It is assumed that there is migration of the cross-linking agent (i.e., silane) from the protein phase to the glycerol plasticizer, which results in dilution of silane concentration in the solution and thereby the reactivity of silane as a cross-linker is reduced. A similar depletion of cross-linking reactivity through migration of a curing agent (dicumyl peroxide and sulfur) from a virgin rubber matrix to a filler of ground rubber vulcanizate has been discussed elsewhere.27 The heat treatment (130 °C for 18 h) significantly increased the tensile strength and modulus, and decreased the EAB of the casein films (Table 2). The increases of tensile strength and modulus were about 2-3-fold for films with 20 wt % glycerol and about 5-10fold for films with 30 wt % glycerol, and the EABs were also decreased significantly (these samples were conditioned for 6 h at 50 ( 2% relative humidity and 22 ( 2 °C after heat treatment). This is because the heat treatment can produce isopeptide crosslinks between the protein chains, which are formed by the condensation reaction of the ε-amino group of a lysine residue with

the carboxylic and amide groups of aspartic and glutamic acid residues.28,29 The possible cross-linking mechanism by heat treatment is shown in Figure 2b. In addition, under severe heat treatment (e.g., 130 °C/18 h), there is a possibility of formation of additional denatured thermal cross-linked networks through the chemical interaction of carboxylic acid groups of aspartic or glutamic acid residues of casein protein with the hydroxyl groups of glycerol. It is reported elsewhere30,31 that under heating conditions glycerol participates in thermal cross-linking reactions with proteins that stabilize the whole protein systems and improve their mechanical strength. The unreacted glycerol and absorbed moisture molecules after heat treatment may act as plasticizing agents in casein protein. In the case of films prepared using silane, further cross-linking occurs under heating. Indeed, the hydrolyzed form of 3-aminopropyl triethoxy silane acts as a reactive bifunctional cross-linker under high temperature conditions. It is assumed that under severe heating condition the amine and free hydroxyl group of the polymerized silanol interact with some protein functional groups such as aspartic and glutamic acid residues, and form covalent cross-linking structures with casein protein as depicted in Figure 2a.28,29,32-34 The films without and with 10 wt % glycerol were brittle and fractured during preparation of tensile specimens. The moisture had considerable effect on improving the flexibility and ductility of the heat treated films. The heat treated films, which were brittle (A-00H, A-10H, B-00H, and B-10H), became more ductile on being exposed to 50 ( 2% relative humidity in about a week. Exposure to moisture significantly decreased tensile strength and modulus; however, these properties were still significantly higher compared to the respective control films. More interestingly, the films with 30 wt % plasticizer exhibited dramatic improvement of tensile properties and ductility on heat treatment followed by moisture absorption. For example, only air-dried casein films with 30 wt % glycerol plasticizer showed



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Figure 3. Effect of heat and moisture treatment on stress-strain behavior of casein film plasticized with 30 wt % glycerol. Figure 4. Effect of chemical cross-linking on stress-strain behavior of casein film plasticized with 30 wt % glycerol.

average tensile strength of 3-4 MPa, modulus of 35-38 MPa and EAB of 94-134%, and after heat treatment followed by moisture absorption for one week the same casein films (with 30% glycerol) exhibited average tensile strength of 10-17 MPa, modulus of 197-386 MPa and EAB of 28-31%, depending on the alkaline solution applied (Table 2). As discussed above, the unreacted glycerol and absorbed moisture molecules may act as plasticizing agents in heat treated casein films when exposed to atmospheric conditions of 50 ( 2% relative humidity. The stress-strain plots of the casein films with 30 wt % glycerol plasticizer are represented in Figure 3. Interestingly, the casein film derived from silane solution (B-30HM film) was more ductile compared to film derived from sodium hydroxide solution (i.e., A-30HM film), presumably due to increased moisture absorption by silane. For analyzing the effect of chemical cross-linking agents (i.e., DL-glyceraldehyde and glutaraldehyde) on mechanical properties of casein protein, films were produced using 0.05 N NaOH solution of casein and plasticized with 30 wt % glycerol. These chemical cross-linkers were not added to casein protein solubilized in silane solution, because silane itself acts as a crosslinking agent between protein chains. DL-Glyceraldehyde significantly (ca. 100%) improved the tensile strength and EAB without affecting tensile modulus of plasticized casein protein. In contrast, glutaraldehyde significantly improved the tensile modulus (ca. 100%), moderately increased the tensile strength (ca. 44%) and marginally affected the EAB values of plasticized casein protein (Table 3). There were significant enhancement of tensile modulus and a moderate increase of tensile strength and moderate to significant drop of EAB of casein films due to postcuring at 100 °C for 2 h. The stress-strain patterns indicate that 30 wt % glycerol plasticized casein protein acts as a flexible plastic without and with chemical cross-linking (Figure 4). However, DL-glyceraldehyde cross-linked plasticized casein

behaves more like a rubber-toughen flexible plastic. Glutaraldehyde is a dicarbonyl compound with two reactive moieties and it can readily react with a large number of available amino groups present in a protein molecule, resulting in a more tightly cross-linked network. On the other hand, glyceraldehyde or formaldehyde, with one carbonyl group per molecule, reacts less effectively at ambient temperature.1,35,36 The high reactivity of glutaraldehyde results in higher cross-linking density (i.e., higher tensile modulus or stiffness) in casein protein compared to the light cross-linking (i.e., lower modulus or stiffness) achieved with DL-glyceraldehyde at ambient condition. There is no consensus regarding the exact cross-linking mechanism between protein chains. It is generally believed that the reactive groups (i.e., aldehyde, isocyanate, azide, etc.) of cross-linkers mostly interact with the ε-amino function of lysine and hydroxylysine or the carboxyl group of aspartic and glutamic acids of the protein.1 Casein contains average 7-8 wt % lysine, 6-7 wt % aspartic acid, and 18-20 wt % glutamic acid moieties in the protein structure.37-39 In the present study, DL-glyceraldehyde and glutaraldehyde were added at the concentration of 2 g per 70 g casein (Table 1). The molar mass of lysine, DLglyceraldehyde and glutaraldehyde are 146.19, 90.08, and 100.12 g mol-1, respectively. Thus, in a 70 g casein sample (considering average lysine content of 7.5 wt %) the calculated molar ratios of lysine to DL-glyceraldehyde and lysine to glutaraldehyde are 1.6 and 1.8, respectively; these are equivalent to number ratios of lysine -amino to aldehyde functionalities of 1.6 and 0.9 for DL-glyceraldehyde (monoaldehyde) and glutaraldehyde (dialdehyde) cross-linked casein films, respectively. Therefore, considering a cross-linking efficiency of 100% in the systems, it is theoretically predicted that the DL-glyceraldehyde treated casein film has lower cross-linking density compared to glutaraldehyde treated casein film, and this is manifested in the

Table 3. Tensile Properties of Chemically Cross-Linked Casein Films with Glycerol Content of 30 wt % tensile strength (MPa) samples

control (air dry)


A-30 (no cross-linker) A-30-GC (2 wt % glyceraldehyde) A-30-GT (2 wt % glutaraldehyde)

3.6 ( 0.4 7.2 ( 1.2 5.2 ( 0.2

4.6 ( 0.3 7.7 ( 1.0 6.7 ( 0.5

tensile modulus (MPa)

elongation at break (%)

control (air dry)


control (air dry)


38 ( 5 38 ( 6 76 ( 6

93 ( 6 129 ( 19 131 ( 27

134 ( 9 235 ( 66 119 ( 23

83 ( 18 129 ( 20 94 ( 25

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Figure 5. Water absorption characteristics of casein films without glycerol plasticizer.

Figure 6. Water absorption characteristics of chemically cross-linked casein films plasticized with 30 wt % glycerol.

present studies of physical and microstructural properties of casein protein. However, in practice these cross-linkers do not exhibit 100% cross-linking efficiency, because they contain significant low percentages of free carbonyl compound in the aqueous solution. At room temperature, a minor fraction of DLglyceraldehyde exists in the aldehyde form and the remaining major portion is found in the cyclic dimeric hemiacetal form. Infrared spectroscopy along with 13C and 1H NMR have revealed the presence of 1% or less free carbonyl band in DL-glyceraldehyde as determined from the intensity in the solid state.40 However, with time or through heating, carbonyl bands appeared and increased in intensity, indicating the dissociation of the dimeric form in the glyceraldehyde solution.41 Possibly it forms malondialdehyde as an active dicarbonyl compound for protein cross-linking,35 and, therefore, DL-glyceraldehyde cross-linked casein showed increased tensile modulus on heating as observed in the present study. The aqueous solution of glutaraldehyde, on the other hand, consists of a mixture of free aldehyde, mono and dehydrated monomeric glutaraldehyde, monomeric, and polymeric cyclic hemiacetals and various R,β-unsaturated polymers; studies have revealed that the aqueous glutaraldehyde can polymerize easily at alkaline pH to yield an unsaturated product.36 The dicarbonyl compounds undergo simple condensation reaction with ε-amino group of lysine residue of protein chain and finally it leads to the Maillard reaction.28,29 The probable simplified mechanism of Maillard reaction is depicted in Figure 2c. Gerrard et al.35 have demonstrated the cross-linking reactivity of various carbonyl compounds with a model protein, ribonuclease (RNase), and observed that glutaraldehyde crosslinks protein almost instantaneously, accompanied by a dramatic loss in the availability of free lysine residues in the protein, whereas glyceraldehyde reacts with protein less readily and the extent of lysine loss is considerably less. Water Absorption Properties of Casein Films. The water absorption, or swelling characteristic, of a film is strongly influenced by the cross-linked microstructure of the polymers and the presence of hydrophilic groups. The percent weight changes of casein films (without plasticizer) after immersion in deionized water at predetermined times (0-320 min) are represented in Figure 5. The casein film made from aqueous NaOH solution (film A-00C) swelled to about 70 wt % on immersion in water and then collapsed and dissolved rapidly.

The heat treatment (130 °C for 18 h) significantly improved the stability of the film (A-00H) in water and increased the percent of water absorption (ca. 280% weight increase after 200 min of immersion in water) by the film. After a certain time, the film slowly collapsed and lost its structural integrity. In contrast, silane-treated casein films were more stable and swelled less in water. The control film derived from silane solution (film B-00C) slowly absorbed water compared to the films derived from NaOH solutions (films A-00C and A-00H). The heat treatment on silane solution derived film (B-00H) significantly improved its water-resistant properties, and the film did not show any swelling in water. The improved water-resistant properties of silane-treated films may be attributed to the strong hydrogen bonding and covalent interactions of casein proteins with silane (Figure 2a), resulting in cross-linked protein polymer networks that help retain the structural integrity of the films during the swelling process. Rouilly et al.42 have studied the water swelling properties of thermally extruded films of plasticized sun flower protein isolate and found that the films remained insoluble in water for 24 h with average swelling of 186 wt %. Lu et al.43 have reported the water swelling characteristics of thermoplastic sheets of soy protein isolate (SPI) reinforced by chitin whiskers; the SPI sheets absorbed about 40 wt % water, which decreased with increasing chitin whisker in the composite sheets. The effects of aldehyde based cross-linkers (DL-glyceraldehyde and glutaraldehyde) on the swelling properties of 30 wt % glycerol plasticized casein films in water are shown in Figure 6. Before heat treatment (at 100 °C for 2 h), the DLglyceraldehyde cross-linked casein film (A-30-GC Control) exhibited higher swelling (ca. 500 wt %) compared to glutaraldehyde cross-linked casein film (A-30-GT Control, ca. 400 wt %), which may be due to higher cross-linking density in the glutaraldehyde cross-linked sample as discussed above. There is a significant drop of water swelling of both films due to heat treatment or postcuring at 100 °C for 2 h, and they showed maximum swelling in the range of 140-170 wt %. The swollen forms of the chemically cross-linked casein protein films retained their structural integrity in water and their weight continued to remain constant over 6 h. Pierro et al.44 have reported the swelling characteristics of the transglutaminase cross-linked films of chitosan/whey protein blends and observed that transglutaminase significantly decreases the equilibrium



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Figure 7. SEM photomicrographs of 3D matrices of casein protein plasticized with 30 wt % glycerol: (a) 0.2 wt % NaOH, (b) 0.2 wt % NaOH and 2 wt % glyceraldehyde, (c) 0.2 wt % NaOH and 2 wt % glutaraldehyde, and (d) 1 wt % silane.

swelling of the films and this decrease of swelling is higher with increasing protein concentration. Fernańdez et al.45 have found that the degree of swelling in the equilibrium of agarose hydrogel increases with increasing the concentration of polyacrylamide as a cross-linker; a large swelling of hydrogel eventually causes a destruction of the agarose gels into microgels, which remained trapped within the polyacrylamide network. However, the present swelling experiment reveals that casein protein requires more efficient cross-linking techniques to improve its resistance to water swelling and retain its structural integrity and mechanical strength. Microstructural Morphology of Casein Protein. There is a considerable number of studies reporting SEM microstructure analysis of scaffolds or 3D matrices produced by freeze-drying technique for tissue engineering applications.46-50 In this investigation the SEM photomicrographs representing the microstructures of the freeze-dried 3D matrices of casein are shown in Figure 7; the effects of chemical cross-linking on the matrix microstructure of glycerol plasticized casein protein are distinctly visible. The 3D matrix of casein protein, generated from 0.05 N NaOH solution without cross-linking additive, exhibits nearly parallel and separated sheets, indicating that the 3D scaffold or matrix without cross-linker is fragile (Figure 7a). The addition of DL-glyceraldehyde as a cross-linker induces a microstructure where the parallel plates are interconnected and some porosity is generated (Figure 7b). This interconnection between the sheets is denser when glutaraldehyde is used as a cross-linking additive (Figure 7c). These microstructural features of the 3D matrices may be explained by the fact that the addition of chemical crosslinking agents increases the casein solution viscosity and restrains the aggregation of protein molecules, thereby the dicarbonyl cross-linked casein 3D matrix exhibits a porous and interconnected sheet-like structure. These structural morphologies clearly demonstrate that glutaraldehyde is a more efficient

cross-linker compared to DL-glyceraldehyde, and this is strongly reflected on the mechanical and water swelling characteristics of the respective casein films as discussed above. A similar microstructural morphology of the scaffolds made with fibroin and collagen proteins by freeze-drying method has been reported elsewhere.46 Interestingly, the 3D matrix of casein protein, produced from 1 wt % silane (3-aminopropyl triethoxy silane) solution, exhibits a different morphology (similar to a cactus tree), where several needle-like structures are attached to major parallel plates (Figure 7d). This structural morphology is presumably due to more chemical reactivity of polymerized form of the hydrolyzed 3-aminopropyl triethoxy silane with protein molecule, compared to dicarbonyl cross-linkers at room temperature conditions.

Conclusions The SDS-PAGE analysis revealed that the average molecular weight (19-23.9 kDa) of casein proteins remain unaffected by the treatment with aqueous solutions of silane and sodium hydroxide. The study of the mechanical properties of the casein films showed that casein protein without or with low loading (0-10 wt %) of glycerol plasticizer is brittle or fragile, which is an inherent characteristic of most natural biopolymers. The tensile properties of the 30 wt % glycerol plasticized casein films indicated the potential for developing technologically advanced materials including 2D films and 3D scaffolds. The heat treatment (130 °C for 18 h) dramatically improved tensile strength and modulus, changing the casein films into fragile plastic materials depending on glycerol content. Moisture absorption by the heat treated films resulted in tough and ductile films, which showed significantly higher tensile strength and modulus than the corresponding control films (before heat treatment). The chemical additives, such as DL-glyceraldehyde,

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glutaraldehyde, and silane, significantly improved the mechanical and water-resistant properties of the glycerol plasticized casein protein films. The stress-strain patterns showed that DLglyceraldehyde generates more rubber-toughen flexible casein based plastic material compared to glutaraldehyde at a similar loading of 2 g/100 g plasticized casein. These cross-linking characteristics of casein protein were distinctly observed in the scanning electron photomicrographs of the freeze-dried 3D matrices, where the parallel sheets were interconnected and formed a porous structure when glutaraldehyde or DL-glyceraldehyde was used as a cross-linker. Finally, this investigation reveals that the casein protein based advanced materials may be developed by tailoring their crosslinking chemistry depending on applications. These materials may have several potential applications including the fabrication of bioscaffolds, foams, contact lenses, drug delivery capsules, and numerous other technological applications. Acknowledgment. This research was funded by Foundation for Research, Science and Technology (FRST, New Zealand) and AgResearch-University of Otago Collaborative Research Fund.

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