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Effect of Nutraceuticals on Physico-Chemical Properties of Sodium Caseinate Films Plasticized with Glycerol
Elizabeth Lima-Lima, Susana Altamirano-Romo, Roco Rivas-Araiza, Gabriel Luna-Barcenas, and Cristina Perez-Perez

CONTENTS Introduction ........................................................................................................ 445 Materials and Methods ..................................................................................... 447 Materials ...................................................................................................... 447 Preparation of Film-Forming Solutions.................................................. 447 Film Formation ........................................................................................... 447 Measurement of Film Thickness and Physical Properties .................. 448 Water Vapor Permeability......................................................................... 448 Mechanical Properties ............................................................................... 448 Differential Scanning Calorimetry (DSC)............................................... 448 Statistical Analysis ..................................................................................... 449 Results and Discussion ..................................................................................... 449 Water Vapor Permeability......................................................................... 449 Mechanical Properties ............................................................................... 450 Differential Scanning Calorimetry (DSC)............................................... 450 Conclusions......................................................................................................... 452 References ........................................................................................................... 453

Introduction
There has been a resurgence of interest in recent years in the development of edible lms for food (Debeaufort et al., 1998). Several studies have indicated the potential of milk proteins for use in edible lms. For instance, caseinates easily form lms from aqueous solutions because of their random-coil

445

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nature and ability to form extensive intermolecular hydrogen, electrostatic, and hydrophobic bonds, resulting in an increase of the interchain cohesion (McHugh and Krochta, 1994). Moreover, edible lms based on milk proteins were reported to be avorless, tasteless, and exible, and depending on the formulation, they varied from transparent to translucent (Chen, 1995). Hydrophilic polymers, such as proteins that contain polar groups that provide hydrogen bonding, will absorb water from the surrounding air or from the food product they contain. Glycerol, a polyol, is well known for its plasticizing effects and its use in food technology. Glycerol was mixed at different concentrations to sodium caseinate solutions, generating lms with good properties. Among the lms investigated, edible lms based on proteins showed the best mechanical properties. However, their barrier properties are variable (Kester and Fennema, 1986). The increase of cohesion between protein polypeptide chains was thought to be effective toward the improvement of the barrier properties of the lms. For instance, the presence of calcium was reported to decrease the water permeability of caseinate-based lm (Avena-Bustillos and Krochta, 1993). The milk proteins as edible and/or biodegradable lms could be interesting because of their favorable functional properties as well as their food nutritional value. Sodium caseinate (SC) has the capability to carry nutraceuticals (Gontard et al., 1992; McHugh et al., 1993). The concepts of incorporating nutraceuticals into edible lms to enhance the nutritional value of foods have been discussed, but few studies have been reported. Nutraceuticals are chemicals found as natural components offoods or other ingestible forms that have been determined to be benecial to the human body in preventing or treating one or more diseases or improving physiological performance (Alvdrez-Morales et al., 2002). Calcium and vitamin C are important nutraceuticals as they play signicant roles in the human body to prevent certain diseases (Elliot, 1998; Pszezola, 1998). The lm properties depend strongly on lm composition, its formation, and the methods of its application to the products (Arvanitoyannis and Biliaderis, 1998; Debeaufort et al., 1998). When calcium is added to protein-based lms, the calcium ions may induce static cross-linking and the level of ionic strength may affect protein microstructure and interactions, which would in turn impact the mechanical and barrier properties (Kester and Fennema, 1986; Avena-Bustillos and Krochta, 1993; Mezgheni et al., 1998). Calcium has been one of the most difcult minerals to add to foods because of its high DRI value, its low solubility at neutral pH, and the bitter taste of some calcium salts. Mei and Zhao (2002) identied that Gluconal Cal (GC; Glucona America Inc., Janesville, WI, USA), a mixture of calcium lactate and calcium gluconate, has high nutritional value, good bioavailability and water solubility, and neutral taste and successfully incorporated it into edible coatings. However, few studies (Mei and Zhao, 2002; Mei and Zhao, 2003; Park and Zhao, 2004)

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have been reported. The performance of vitamin C in edible lms has not been evaluated. Our objectives in this study were to formulate SC lm solutions that can carry high concentrations of calcium and vitamin C, and to investigate the impacts of calcium and vitamin C on the physico-chemical properties of the lms.

Materials and Methods

Materials The materials used for lm formation include SC (Sigma C-8654, St. Louis, MO, USA) containing approx. 96% protein, and 4% ashes (dry basis); glycerol plasticizer (G: Sigma G-6279, St. Louis, MO, USA), serving as plasticizer in all lm-forming solutions, GC (Glucona America Inc.), a mixture of calcium lactate and calcium gluconate with water solubility up to 40 g/100 ml and neutral taste, and ascorbic acid (VC; Fermont. Productos Quimicos Monterrey, Mty, N.L., Mexico). All chemicals used were of reagent grade. Preparation of Film-Forming Solutions Aqueous solutions of 10% (w/w) SC were made with addition of 0 to 30% (w/w SC) G in the mixture. The addition nutraceuticals were 5 and 10% (w/w SC) GC, and 0.01% (w/w SC) VC in the mixture. SC and glycerol were mixed in distilled water. Solutions were heated to 60 ^ 28C approximately for 1 h while being stirred continuously. The samples containing nutraceuticals required the most time to obtain complete dissolution of the substances. The solutions were placed in a shaking water bath with a magnetic stirrer. Finally the solutions were dried over PVC plates at room temperature. Film Formation Different sizes of lms were formed. The samples were cut to measure lm properties and to establish a lm thickness control equation. The smalldiameter lms were used for the water vapor permeability (WVP) and puncture strength (PS); large ones for tensile strength and elongation at break (%) measurement. The volume of solution was added over PVC plates at volume constant using a thin layer chromatography spreader. The plates were placed on a leveled surface to achieve uniform lms and dried at room condition (25 ^ 28C and 58 ^ 3% HR) for 24 to 48 h. Films used for each property measurement were manufactured and stored under the same temperature but at different relative humidity conditions.

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Measurement of Film Thickness and Physical Properties The thickness of the lm was measured using a micrometer (Foil Dial Thickness Gauge F 1101/30). Films samples were cut according to the standard methods for the measurement of functional properties. For each lm sample, ve thickness measurements were randomly taken at different locations. The average value was taken to calculate the water barrier and mechanical properties. Films density was calculated by dividing the lm weight by the lm volume, where the lm volume was calculated by multiplying the lm area by the thickness. Water Vapor Permeability Films used for WVP measurement were rst conditioned at 58% RH at room temperature (25 ^ 0.58C) for . 48 h. A modication of the ASTM E96-80 (ASTM, 1989) gravimetric method was used to measure the WVP of the lms. Three replicates of each lm type were tested, and the result was reported as the mean value in mol m/m2 sec Pa. Mechanical Properties The mechanical properties of the lms were determined with a texture analyzer TA-XT2i (Microstable System, Godalming, England). Prior to the measurements, lm samples were conditioned in the same environment at 258C and 58% RH for at least 2 days. All properties measurements were performed immediately after removing lm specimens from the chamber to minimize moisture variances of these natural hydrophilic lms. The ASTM D882-83 method (ASTM, 1983) was used for measuring tensile strength (TS) and percent elongation at the break (EL). The TS value was reported as measured maximum load (N) divided by lm cross-sectional area (mm2) with units of Pa. El values were obtained by recording elongation at the break divided by the initial length of the specimen and multiplying by 100. The puncture test was realized as described previously by Gontard et al. (1992). To avoid any thickness variation, the puncture strength values were divided by the thickness of the lm. Three replicates of each lm type were tested. Differential Scanning Calorimetry (DSC) The measurements were performed on a DSC Mettler Toledo (DSC822e/400). About 10 mg of each lm-forming component or lm conditioned at 258C and 58% RH for 2 days previously was sealed in a standard aluminum pan. The rst scan, from 20 to 1508C was applied to remove any thermal history effects. This rst scan was stopped before the material melted, and samples

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were then cooled down quickly to 2 608C. The second scan from 2 40 to 2608C was performed to establish the glass-transition temperature (Tg). The DSC curve was obtained in a temperature range of 2 40 to 2608C at a heating rate of 58C/min. The Tg values quoted correspond to the midpoint of the transition. Statistical Analysis All experiments were replicated three times in a randomized complete block experiment. Each analysis was done in duplicate. Statistical analysis was conducted using STATGRAPHIC PLUS (Statistical Graphics Corp.).

Results and Discussion

Water Vapor Permeability WVP of SC-based lms incorporated with nutraceuticals is shown in Figure 28.1. The lm thickness was controlled in the range of 70 to 90 mm for all the lms. The water vapor property of the lms was signicantly improved by incorporation of mineral or vitamin C in the lm matrix (P , 0.05). By adding a high concentration of mineral salts into the lm matrix, counterion interactions increase among adjacent molecular structures and small mineral ions act as llers, thus resulting in a decrease in diffusivity of water vapor through a lm matrix and a decrease in hydrophilic tendency of SC (Park and Zhao, 2004). Ionic calcium is known
Water Vapor Permeability

7.00E-13 6.00E-13 WVP (mol m /m 2*s*Pa) 5.00E-13 4.00E-13 3.00E-13 2.00E-13 1.00E-13 0.00E+00 10

20 % Glycerol
5% GC 10% GC 5 GC-VC

30
10 GC-VC

CONTROL

FIGURE 28.1 Water vapor permeability (WVP) of sodium caseinate lms with nutraceutical substances. Film thickness was controlled in a range of 70 to 90 mm for all lms tested.

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to form strong molecular crosslinking in caseinate lms, which would improve protein network formation and stability (Park et al., 2001), reducing the moisture diffusion rate. Ionic calcium crosslinking also reduced protein solubility in water and led to decreased WVP through the protein matrix (Avena-Bustillos and Krochta, 1993). Park et al. (Park et al., 2001) indicated that calcium sulfate improved water barrier properties of soy protein isolate (SPI) lm by reacting with protein constituents to yield insoluble proteinates and altered lm consistency. From this study associated with a high concentration of calcium incorporation into lm-forming solution, it might be concluded that calcium improves the WVP of SC lms through its capacity to promote aggregation, thus reducing the solubility of protein. Incorporating a high concentration of glycerol increase the WVP of the SC lms signicantly (P . 0.05). McHugh et al. (1993) suggested that as lm thickness increased, the lm provided an increased resistance to mass transfer across it, but in this study, lm thickness varied among the lms used for WVP determination (data not shown) and this might affect the accuracy of WVP calculation. Future research must have better control of lm thickness to fully understand the WVP of lms. Mechanical Properties The tensile strength of SC lm was signicantly affected by the addition of glycerol and nutraceuticals substances (P . 0.05). At increased concentration of glycerol in the lm matrix, the tensile strength is lowered, but adding VC increased the value markedly. The percent elongation of the lms decreased with increasing GC concentration (Table 28.1). The puncture strength was signicantly decreased with the incorporation of glycerol and decreased by adding GC and VC (Figure 28.2). The thickness for the test specimen was also important. Tensile strength is the measurement of maximum strength a lm can withstand against applied tensile stress, and percent elongation represents the ability of lms to stretch. While the puncture strength is the measurement of hardness of a lm under the stress applied at right angles to its surface that generates multidirectional forces. Puncture deformation is the measurement of lm elasticity under vertically loaded stress. Both tensile and puncture strength values can be used as a measurement of hardness of lms. The Youngs modulus is the measurement of the elastic properties of the materials, and can be regarded as a measurement of the rigidity. (Table 28.1) The addition of glycerol was signicantly in this study (P . 0.05). Park and Zhao (2004) showed a similar effect with chitosan-based lms. Differential Scanning Calorimetry (DSC) The Tg values of SC lms are shown in Figure 28.3. Increasing plasticizer concentration led to a signicant reduction in Tg values in all SC lms, the addition of GC decreased the Tg and the VC increased lightly the Tg.

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TABLE 28.1 Mechanical Properties of Sodium Caseinate Films with Glycerola

Tensile Strength (Pa) Treatments 10G 20G 30G 5GC10G 5GC20G 5GC30G 10GC10G 10GC20G 10GC30G 5GC10GV 5GC20GV 5GC30GV 10GC10GV 10GC20GV 10GC30GV
a

% Elongation Average 2.99 7.31 52.69 4.33911765 5.64 14.6935294 5.60156863 6.49735294 15.0313725 3.40568627 8.42164706 48.3591176 2.95 8.16729412 44.4615126 SD 0.37 1.01 3.58 1.82 1.31 2.70 0.07 2.21 3.12 0.31 1.84 5.56 0.07 1.87 1.34

Youngs Module (Pa) Average 1273.11 492.57 33.99 1178.34 488.95 130.75 644.95 906.95 151.68 936.39 272.56 16.50 104.52 321.66 29.00 SD 434.74 47.67 10.74 629.50 88.18 16.11 17.03 314.90 28.13 130.93 54.26 1.76 11.34 76.19 19.18

Average 277545.80 158520.00 76456.83 182484.23 128406.1967 100342.4201 230261.9197 145909.8158 96900.9394 821465.02 671683.1505 711665.3339 725000.00 663294.8142 628574.8432

SD 83158.46 16577.86 5297.73 47533.72 10917.01 17224.34 29165.21 24964.26 11827.92 54390.07 35887.36 2510.47 5349.21 11130.02 35251.11

Means of ve measurements.

Puncture Strength 200000 180000 Strength / Thickness (N/m) 160000 140000 120000 100000 80000 60000 40000 20000 0 0 10 20 % Glycerol
5 gC 5 GC-V 10 GC

30

CONTROL

10 GC-V

FIGURE 28.2 Effect puncture strength in sodium caseinate lms. The puncture strength was calculated considering the thickness.

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Differential Scanning Calorimetry 120 100 Tg (C) 80 60 40 20 0 G10
CONTROL 5 GC

G20 % Glycerol
5 GC-VC 10 GC

G30
10 VC-C

FIGURE 28.3 Tg values of sodium caseinate lms incorporated with mineral and vitamin C.

Changes in the thermal properties of SC lms were correlated with effects on the mechanical properties of lm, i.e., reduced lm strength and increased lm elongation. Plasticizations disrupt intermolecular interactions between polymer molecules with the effect of decreasing brittleness and increasing lm exibility (Sears and Darby, 1982). Lillie and Gosline (1993) pointed out that the Tg of proteins, especially at a low water content, may occur over a wide temperature range. However, few Tg data are available for proteins (Roos, 1995).

Conclusions
This study quantied the properties of SC lms containing different types and concentrations of mineral or vitamin. The intended use of the coating or lms would dictate the requirements for water barrier and mechanical properties. The GC and VC affect the evaluation of lm functionality. The mechanical properties of the lms were inuenced by the glycerol concentration and nutraceuticals substances; the addition of plasticizers to the lm formulations generally decreased the puncture strength values. The WVP was inuenced by the addition of GC in the lm matrix. Moisture content of the lms correlated well with glass-transition temperatures. The specic formulation of such lms depends strongly on their intended applications, in which the concentration of mineral or vitamin C incorporated into the lms needs to be carefully selected to meet the required water barrier, mechanical, and thermal properties of the lms. The feasibility and stability of these coatings applied to fresh fruits for enhancing storability and nutritional values are under evaluation now. To understand their effects fully, future research must study the gas

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permeability and microstructure of the lms at different levels of glycerol incorporation.

References
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Park, S.K., Rhee, C.O., Bae, D.H., and Hettiarachechy, N.S. Mechanical properties and water-vapor permeability of soy-protein lms affected by calcium salts and glucono-d-lactone, J. Agric. Food Chem., 49, 2308, 2001. Park, S. and Zhao, Y. Incorporation of a high concentration of mineral or vitamin into chitosan-based lms, J. Agric. Food Chem., 52, 1933, 2004. Pszezola, D.E. The ABCs of nutraceutical ingredients, Food Technol., 52, 30, 1998. Roos, Y.H. Phase Transitions in Foods, Academic Press, London, pp. 360, 1995. Sears, J.K. and Darby, J.R. The Technology of Plasticizers, Wiley, New York, 1982.

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