K. COUGHLAN, N.B. SHAW, J.F. KERRY, AND J.P. KERRY ABSTRACT:
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1 JFS E: Food Engineering and Physical Properties Combined Effects of Proteins and Polysaccharides on Physical Properties of Whey Protein Concentrate based Edible Films K. COUGHLAN, N.B. SHAW, J.F. KERRY, AND J.P. KERRY ABSTRACT: The water-vapor permeability (WVP) and mechanical properties of edible films formed from dry blends or co-dried preparations of protein-polysaccharide powders prepared from whey protein concentrate (WPC)-45 and alginate, pectin, carrageenan, or konjac flour (WPC-45-to-polysaccharide ratio of 95:5 w/w) were investigated. Films were prepared from 8% WPC using WPC-45 (45% protein powder), consisting of g of WPC-45 in g of water per 100 g solution to give 8% protein w/w. Films formed from co-dried powders had lower WVP and higher tensile strength (TS), elastic modulus (EM) (P < 0.05), and elongation (EL) than equivalent films formed from the dry blended powders. Films containing alginate had lower WVP and higher TS, EM, and EL than films containing pectin, carrageenan, or konjac flour. There is potential to alter the physical properties of hydrophilic films by combining whey protein and polysaccharide components. Keywords: WPC, alginate, pectin, physical properties, edible films Introduction Edible films are generally formed from hydrophilic components such as proteins or polysaccharides. Films based on proteins have poor water-vapor barrier properties but are more resistant to oxygen transfer than are films prepared from polysaccharides (Kester and Fennema 1986). The properties of edible films formed from hydrocolloids have been reviewed by a number of authors, including Kester and Fennema (1986) and, more recently, Cuq and others (1998). Formation of flexible films from hydrocolloids normally requires 2 essential ingredients: a film-forming polymeric material that provides the structural matrix and a plasticizer, such as a low-molecular-weight polyol, which imparts flexibility. In the absence of sufficient amounts of plasticizer, a brittle inflexible film is formed and is difficult to peel from the casting surface (McHugh and others 1993). Biopolymer films, which contain both protein and polysaccharide ingredients, may advantageously use the distinct functional characteristics of each film-forming ingredient. Some workers have indicated that incorporation of polysaccharides into globular protein matrices may extend the functional properties of these ingredients (Zaleska and others 2000; Turgeon and Beaulieu 2001). Schmitt and others (1999) have shown that protein-polysaccharide complexes gel more effectively than polysaccharides and proteins in isolation. These effects may be attributed to the simultaneous presence of the 2 biopolymers, as well as to the structure of the complexes. According to Turgeon and Beaulieu (2001), heat treatment of a protein-polysaccharide complex weakens the low-energy bonds responsible for co-solubilization of the protein and polysaccharide. After breaking of this equilibrium, a competition occurs between phase separation and protein gelation in the system. MS Submitted 4/28/03, Revised 7/18/03, Accepted 3/31/04. Authors Coughlan, Shaw, J.F. Kerry, and J.P. Kerry are with the Dept. of Food and Nutritional Sciences, Univ. College Cork-Natl. Univ. of Ireland, Cork, Ireland. Direct inquiries to author J.P. Kerry ( joe.kerry@ucc). When protein gelation is favored by the experimental conditions, a continuous protein network is formed with polysaccharide inclusions, thereby strengthening the complex. Turgeon and Beaulieu (2001) strengthened the structure of a whey protein gel by incorporating -carrageenan into the gel. From subsequent rheological investigation, they reported that gelation of the 2 components were independent, where the protein gel formed 1st and the -carrageenan gel formed during the cooling phase. Zaleska and others (2000) formed gels from pectin and whey protein isolate, and they suggested that anionic interactions may occur between the protein and polysaccharide components if they are electrically compatible. It may therefore be possible to manipulate combinations of polysaccharide and protein component edible films to adjust water-vapor resistance or structural strength. Little information exists in the literature on the film-forming ability of such combinations of protein and polysaccharide components. Parris and others (1995) formed films from whey proteins and alginate or pectin and reported that the films formed from protein-polysaccharide blends had lower water-vapor permeability (WVP) than those formed from protein alone. They also suggested that future studies should be directed toward improving the physical properties of films through covalent bonding of proteins and polysaccharides using processing techniques such as spray-drying. Turgeon and Beaulieu (2001) and Zaleska and others (2000) formed gels from separate solutions of polysaccharide or protein powders, often called dry blending, whereby the individual protein and polysaccharide materials are blended together in powder form. The protein and polysaccharide materials may also be blended by spray-drying solutions of the combined ingredients to form a co-dried, biopolymer powder. This process, known as codrying, is a novel process; it is believed that combining the powders in such a way may promote increased interaction between the protein and polysaccharide constituents. Schmitt and others (1999) suggested that protein-polysaccharide complexes were formed with a polyelectrolyte chain bound by several protein 2004 Institute of Food Technologists Further reproduction without permission is prohibited Vol. 69, Nr. 6, 2004 JOURNAL OF FOOD SCIENCE E271 Published on Web 7/27/2004
2 molecules. These primary complexes may aggregate during spraydrying to form inter-biopolymer complexes with a structure consisting of several interacting polymer chains. To our knowledge, little exists in the literature on blending ingredients using such codrying techniques. The objectives of this study were to investigate the effect of both biopolymer combination and sample preparation technique on the physical properties of protein-polysaccharide based edible films. Materials and Methods Reagents Whey protein concentrate, containing 45% protein (WPC-45), was obtained from Dairygold Ltd. (Mitchelstown, Ireland). Alginate was obtained from Kelco Intl. Ltd. (Surrey, U.K.). Pectin and carrageenan were obtained from FMC Co. (Vallenstaek Strand, Copenhagen, Denmark). Konjac flour (Nutricol) was obtained from the FMC Corp. (Philadelphia, Pa., U.S.A.). Glycerol was obtained from Sigma Aldrich Ltd. (Poole, Dorset, U.K.). All chemicals were of reagent grade. Water-vapor permeability Circular test cups made of perspex were manufactured to the specifications outlined by McHugh and others (1993). Distilled water (4 ml) was placed in each test cup, and a film sample was mounted across the cup opening. The cups were further sealed using silicon sealant. The cups were stored in a temperature- and humidity-controlled room (50% ± 5% RH, 23 ± 2 C), and a fan set at an air velocity of 152 m/min was placed over the cups to ensure uniform movement of air across the WVP test cells. Within 2 h, steady-state conditions were assumed to have occurred (McHugh and others 1993). The weight loss of the cups was monitored over a 24-h period with intervals of greater than 2 h between readings. At least 3 film samples were prepared for each film type, and WVP was calculated according to the WVP correction method of McHugh and others (1993). This is a modification of the ASTM E-96 Standard Method (ASTM 1980) for determining WVP of synthetic packaging, which has adapted the calculations to consider the more hydrophilic nature of edible films. The WVP correction method was developed to account for the water-vapor partial-pressure gradient in the stagnant air layer of the test cup (McHugh and others 1993). Co-dried powder formation After initial screening of WPC at varying protein levels (35%, 45%, 75%) by industrial partners Dairygold Ltd., it was decided to proceed with WPC-45 because it combined optimum gel strength with cost effectiveness. Co-dried powders were manufactured in Univ. College Cork in conjunction with Dairygold Ltd. Aqueous solutions of WPC-45 (19% w/w) at 20 C and alginate, carrageenan, pectin, or konjac flour (1% w/w, heated to 60 C for complete miscibility) were fed into a single-stage spray-dryer (NIRO Atomiser-L55432, Copenhagen, Denmark) with inlet and outlet temperatures set at 200 C and 95 C, respectively. The solutions were dried at a flow rate of 18 kg/h through an atomizer, 2-mm dia, to form a co-dried powder containing WPC-45 and the desired polysaccharide at a protein-to-polysaccharide ratio of 95:5 w/w. Carrageenan required the addition of salt at 0.5% w/w to lower the viscosity of the solution. Film formation Films were prepared from 8% protein w/w solutions of WPC-45 and dry-blended or co-dried WPC-45/polysaccharide powders (8% w/w protein). These solutions were stirred continuously at room temperature for 2 h. It is well documented that the optimal temperature-time profile for whey protein denaturation is 80 C to 90 C for approximately 0.5 h (McHugh and others 1993; Mate and Krochta 1996; Shaw 2001). The solutions in the current study were heated in a water bath to 85 C and held at this temperature for 0.5 h. Glycerol was added as a plasticizer (10% w/w) to the cooled heat-denatured solutions at a glycerol-to-protein ratio of 0.35 w/w (Shaw 2001). A control film was prepared from an 8% solution of WPC-45. For each film, 30 ± 0.5 g of film-forming solution was poured onto level, circular, teflon-coated perspex plates machined at the physics department workshop in Univ. College Cork. They were dried for approximately 24 h to constant weight at controlled conditions of 50% ± 5% RH and 23 ± 2 C. Many researchers have described film casting and peeling similar to the methods used in the current study (McHugh and others 1993; Mate and Krochta 1996; Shaw 2001). Films were peeled immediately before testing. To obtain sufficient samples for WVP and mechanical testing, at least 12 films were cast from the film-forming solution of each film type. Film thickness was measured using a 0- to 25-mm micrometer screw gauge (Mitutoyo Corp., Kawasaki, Kanagawa, Japan), and overall thickness was expressed as an average of 10 readings taken randomly on each film. All films were cast in triplicate. Mechanical tests Film samples were preconditioned and tested under conditions of controlled temperature and humidity (50% ± 5% RH, 23 ± 2 C). The films were cut into strips 25.4 mm wide and 130 mm long using a scalpel. The ends of the strips were mounted between cardboard grips (26 /times/ 70 mm) using double-sided adhesive tape, and the final film area exposed was 100 /times/ 25.4 mm. An Instron Universal testing instrument (Instron Model 4301, Instron Corp. Canton, Mass., U.S.A.) was used to measure the tensile properties of the films according to the static weighing, constant rate-of-grip separation standard testing method D882 (ASTM 1985). The Instron was set to tensile mode and the mounted film samples were clamped into the screw-action grip and jaw faces of the tensile rig. Initial grip separation was set at 100 mm and crosshead speed at 100 mm/min. At least 16 strips of each film type were analyzed, and percent elongation at break (% EL), tensile strength at break (TS), and elastic modulus (EM) were calculated as outlined in ASTM D882 (ASTM 1985). Statistical analysis At least 10 films were cast from each of the film-forming solutions of each of the blends being evaluated. Data was analyzed using SPSS 10.0 for Windows (SPSS, Chicago, Ill., U.S.A.) software package. A 2-way statistical analysis of variance, least significant difference multiple comparison tests and regression analyses were performed. All experiments were conducted 3 times. Results and Discussion Film formation All films prepared were peelable from the casting surface. Average values for film thickness were generally in the range of to mm and were not significantly different (P > 0.05). Water-vapor permeability Overall trends indicated that films formed from co-dried powders had lower WVP than the control film (Figure 1). The films formed from dry blended powders had higher WVP values (P < 0.05) than the control film (WPC-45). Overall, the WVP values for films formed from co-dried preparations were lower than equivalent films formed from dry blended powders (P < 0.05). Regardless of blending, it was shown that alginate-containing films had lower WVP E272 JOURNAL OF FOOD SCIENCE Vol. 69, Nr. 6, 2004 URLs and addresses are active links at
3 values than films containing other polysaccharides (indicated by trends), and WVP of the films could be arranged in the following increasing order: WPC-45/alginate < WPC-45/pectin < WPC-45/ carrageenan < WPC-45/konjac flour. Mechanical properties Trends indicated that (Figure 2) that films formed from co-dried preparations were more extensible than the control film (WPC-45) or equivalent films formed from dry blends of the protein and polysaccharide materials. Film extensibility was greatest in films prepared from co-dried WPC-45/alginate powders (indicated by trends). Films formed from co-dried preparations (Figure 3) had greater tensile strength than equivalent films formed from dry blends (P < 0.05). Tensile strength was greatest in the films prepared from co-dried WPC-45/alginate and WPC-45/pectin powders (P < 0.05). The elastic modulus values (Figure 4) obtained for films containing co-dried blends were greater than those obtained for the equivalent films formed from dry blends (P < 0.05). Overall, elastic modulus was affected more by the sample preparation method than the polysaccharide included in the protein-polysaccharide complexes. Discussion It is generally regarded that protein-polysaccharide complexes Figure 1 Water-vapor permeability (WVP, g mm/d/m 2 /kpa) of control film ( ) (WPC-45), and co-dried ( ) and dry blended ( ) edible films prepared from WPC-45 and alginate, pectin, carrageenan, or konjac flour. a,b,c refers to test of significance where means bearing different superscripts are significantly different (P < 0.05). Figure 3 Tensile strength (TS, MPa) of control film ( ) (WPC-45), and co-dried ( ) and dry blended ( ) edible films prepared from WPC-45 and alginate, pectin, carrageenan, or konjac flour. a,b,c,d,e refer to test of significance where means bearing different superscripts are significantly different (P < 0.05). Figure 2 Percentage elongation (% EL) of control film ( ) (WPC-45), and co-dried ( ) and dry blended ( ) edible films prepared from WPC-45 and alginate, pectin, carrageenan, or konjac flour. a,b,c refer to test of significance where means bearing different superscripts are significantly different (P < 0.05). Figure 4 Elastic modulus (EM, MPa) of control film ( ) (WPC-45), and co-dried ( ) and dry blended ( ) edible films prepared from WPC-45 and alginate, pectin, carrageenan, or konjac flour. a,b refer to test of significance where means bearing different superscripts are significantly different (P < 0.05). URLs and addresses are active links at Vol. 69, Nr. 6, 2004 JOURNAL OF FOOD SCIENCE E273
4 exhibit more effective functional properties (for example, gelation and aggregation) than proteins or polysaccharides alone (Schmitt and others 1999; Zaleska and others 2000). This may explain why films containing co-dried powders had slightly lower WVP than films formed from WPC-45 exclusively (Figure 1). Parris and others (1995) formed films from combinations of whey protein and alginate or pectin and, similar to the current study, reported lower WVP values than films formed from proteins in isolation. As suggested by Zaleska and others (2000), the effects reported in both studies may be attributed to an electrical incompatibility between the protein and polysaccharide components. Such incompatibility may lead to the formation of 2 separate protein and polysaccharide gels, which may in turn lead to a decrease in intermolecular interaction between the components. Furthermore, the lower WVP values reported for films containing co-dried powders, rather than equivalent dry blended-based films, may be attributed to an increase in electrical compatibility between the protein and polysaccharides arising from the spray-drying process, which may have promoted more intermolecular interactions in the co-dried powders, leading to more effective moisture barrier properties. Bangs and Reineccius (1988) and Trubiano and Lacourse (1988) reported that encapsulating properties of protein-containing powders increased with the addition of polysaccharide material during spray-drying. The polysaccharide material led to changes in the surface active properties of the powder, which subsequently led to improvements in the functional properties of the powders. Overall, it was shown that alginate-containing films generally had lower WVP values than films containing other polysaccharides and that the WVP of films could approximately be arranged in the following increasing order: WPC-45/alginate < WPC-45/pectin < WPC-45/carrageenan < WPC-45/konjac flour. The differences in WVP reported between films formed from blends of WPC and polysaccharides may be attributed to differences in gel formation and interactions between WPC and each of the polysaccharide components. As suggested by Ustunol and others (1992) and Zasypkin and others (1997), the complex gel network, which is set up between protein and alginate, may be because of hydrogen bonding and electrostatic interactions between oppositely charged polyelectrolytes. Zaleska and others (2000) formed gels from blends of pectin and WPI and indicated that attractive forces between the protein and anionic polysaccharide were responsible for the formation of the structural matrix. Parris and others (1995) formed edible films from combinations of whey protein and, in agreement with the current study, alginate or pectin and reported lower WVP in films containing alginate than for films containing pectin. They attributed this effect to an increase in intermolecular hydrogen bonding between polymer molecules in the alginate-containing films when compared with the pectin-containing films. As a result, spacing between the macromolecules may be decreased, leading to a reduction in WVP (Parris and others 1995). Many workers have shown that protein-carrageenan complexes form 2 distinct gel phases, which occur at different time-temperature intervals (Turgeon and Beaulieu 2001; Euston and others 2002) and may explain why the WVP values of films incorporating carrageenan were higher than those reported for films containing alginate or pectin. Drohan and others (1997) suggested that the gelation of carrageenan occurs in 2 stages: helix formation followed by aggregation. When heated in solution, carrageenan undergoes a structural transition somewhat analogous to protein denaturation, adopting a helical form in solution that is disrupted to a random coil during heating. On cooling, the random coils reassociate into double helices. Consequently, the gelation of carrageenan is usually achieved by lowering the temperature, whereas denaturation of globular proteins such as WPC-45 occurs during heating (Turgeon and Beaulieu 2001; Euston and others 2002). In the case of konjac flour, it was suggested by Maekaji (1978) that the crosslinks forming the junction zones within the gel structure were formed by weak noncovalent bonds. Nishinari and others (1992) suggested that these cross-links may be formed by hydrogen bonding between konjac mannan deficient in acetyl groups. Little exists in the literature on the physical properties of films formed from blends of proteins and polysaccharides; however, Parris and others (1995) reported that films formed from proteins and polysaccharides in combination had greater tensile properties than films formed from proteins or polysaccharides in isolation. Consistent with the current study, many authors have indicated that protein-polysaccharide complexes exhibit more effective functional properties than systems containing proteins or polysaccharides in isolation (Parris and others 1995; Schmitt and others 1999; Zaleska and others 2000; Turgeon and Beaulieu 2001). The higher elastic modulus values (Figure 4) obtained for films containing codried preparation than equivalent films formed from dry blends suggests that there were a greater number of intermolecular crosslinks within the matrices of the films formed from co-dried preparations because elastic modulus is regarded as an indication of the number of intermolecular cross-links within the film matrix (Doolittle 1965). Overall, it was shown that alginate-containing films generally had slightly higher tensile strength, elongation, and elastic modulus than films containing other polysaccharides. These results were consistent with those of Parris and others (1995), who reported that whey protein/alginate biopolymer films were stronger and more extensible than equivalent films formed from whey protein and pectin dry blends. This effect may be attributed to differences in gel formation and in the nature of their protein-pectin interactions, as suggested by Zaleska and others (2000). To our knowledge, it has been reported that the strongest effect on the physical properties of mixed gel systems is the difference in electrical charge of the different components (Zaleska and others 2000). Within the confines of this study, it was difficult to determine exactly how the different protein-polysaccharide mixtures affected WVP. The results reported provide an indication only of the differences between the different protein-polysaccharide systems, but they cannot explain fully why these differences exist. A more detailed study of the powders on a molecular level may be required to provide these answers. This study only served to highlight the potential film-forming ability of protein-polysaccharide blends formed from the novel co-drying technique. Conclusions There is potential to improve the physical properties of hydrophilic films by incorporating a hydrocolloid component into the film system. Protein-polysaccharide films formed from co-dried blends were more effective moisture barriers and had higher tensile properties than films formed from protein in isolation. There are advantages to combining proteins and polysaccharides by the process known as co-drying, such as lower WVP and higher tensile properties than equivalent films formed from the dry blended powders. Future work in this area should focus on improving the physical properties of co-dried whey protein-polysaccharide edible films. Acknowledgments This research was co-funded by the European Regional Development Fund and Enterprise Ireland Strategic Research Grants Scheme. We also acknowledge the assistance of Dairygold Ltd. and Kerry Algae. E274 JOURNAL OF FOOD SCIENCE Vol. 69, Nr. 6, 2004 URLs and addresses are active links at
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