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Adsorption Behaviour of Whey Proteins Measured by Two Different Methods
PII : S0958-6946(98)00032-6
Int. Dairy Journal 8 (1998) 79—81 ( 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$19.00#0.00
Adsorption Behaviour of Whey Proteins Measured by Two Different Methods Elisabeth Eugster a*, Susanne E. Taylor b, Zdenko Puhan c and Hans Eyer a a Federal Dairy Research Institute, Schwarzenburgstr. 161, Bern, Switzerland b Biozentrum of the University of Basel, Klingelbergstr. 70, Basel, Switzerland c Swiss Federal Institute of Technology, Department of Food Science, Zu¨ rich, Switzerland (Received for publication 2 April 1998) ABSTRACT The adsorption behaviour of the commercially available whey protein isolate, PROTARMOR, at the air—water and oil—water interface has been investigated by two different methods. The adsorption rate at a constant interface area (‘isochorous mode’) as a function of time was measured by a method using a ‘bursting membrane’ device. After 10 min the slope of increase in film pressure reaches higher values at the air—water surface compared to adsorption at the oil—water interface. The slope of increase in film pressure at a constant interfacial area is strongly dependent on the protein concentration in the aqueous phase. For pressure—area curves (‘isothermal mode’) a rectangular Teflon trough was used with the surface layer being compressed by moving a Teflon barrier. From these measurements it can be concluded that the adsorption of Protarmor at the the air—water surface is reproducible with this method. There is evidence that the adsorption of this protein mixture at the air—water surface is reversible. ( 1998 Elsevier Science Ltd. All rights reserved Keywords: monolayer; b-lactoglobulin; a-lactalbumin; interfacial tension
INTRODUCTION
used as solvent for the protein solution. Concentrations of the whey protein in the subphase are indicated in wt%. The purification of the vegetable oil used for the measurements at the oil—water interface has been described by Gaonkar (1989). To remove interfacially active contaminants the commercial vegetable oil has been mixed with Silica gel (60 g L~1). The mixture was stirred (20 h) and then centrifuged. The supernatant oil was used for measurements at the oil—water interface. The adsorption rate at a constant interface area (‘isochorous mode’) as a function of time has been investigated by a method based on a ‘bursting membrane’. After release of the emulsifier solution the interfacial tension was measured (Anbarci and Armbruster, 1987). The measurement device (made by our workshop) is shown in Fig. 1. The surface area of the trough was 20 cm2, the volume of the aqueous phase was 118 mL. Twentyfive mL of purified vegetable oil has been poured on top of the water. This method and applications are described in detail in Stang et al. (1994). For pressure—area curves (‘isothermal mode’) a rectangular Teflon trough (NIMA Technology Ltd., Coventry, UK) equipped with three tiny Teflon stirrers was used. The surface layer was compressed between 495 and 90 cm2 by moving a Teflon barrier. The trough was filled with 350 mL pure water and the Protarmor stock solution was injected into the subphase with a syringe (Hamilton, Switzerland). The preparation and cleaning of the trough was described elsewhere (Schwarz and Taylor, 1995). In both cases the surface tension was measured by the Wilhelmy plate method. Film pressure n has
Milk proteins are well known for their functional properties in forming and in stabilizing emulsions. They facilitate the disruption of droplets by reducing the interfacial tension, and stabilize the emulsion by immediately occupying the newly formed interfaces after disruption and, thereby, inducing repulsive forces between droplets. The adsorption behaviour of protein molecules at the interface is crucial for the short-term stability of an emulsion. If the rate of adsorption at the interface is too low, the newly formed droplets might coalesce during the emulsification process (Stang et al., 1994, Karbstein and Schubert, 1995). Milk proteins, especially whey proteins, also act as stabilizers by forming a cohesive, viscous film via intermolecular interactions at the interface (Dickinson et al., 1989; Walstra and de Roos, 1993). In this study the adsorption behaviour of a commercially available whey protein isolate at the air—water and oil—water interface has been investigated. MATERIALS AND METHODS The whey protein powder was of food grade and used without further purification. Protarmor 906 (ARMOR PROTEINES S.A.S., France) is a bovine whey protein isolate (87 wt% protein) consisting of b-lactoglobulin and a-lactalbumin mainly. Purified water (Millipore) was * Corresponding author. 79
80
E. Eugster et al.
Fig. 2. Time dependence (‘isochorous mode’) of film pressure n at the air—water interface for Protarmor at different subphase concentrations at 25°C. The concentrations are from top to bottom: 3, 1, 10~1, 10~2, 10~3 and 10~4 wt%. Fig. 1. Device for measuring the adsorption kinetics of watersoluble surfactants at the air—water surface and oil—water interface. The surface tension is recorded as force F divided by perimeter º of the Wilhelmy platelet (Stang et al., 1994).
been calculated by using Antonow’s equation (Birdi, 1989) n"c !c 0 where n represents the film pressure, c the interfacial 0 tension of purified water and c the interfacial tension with adsorbed molecules.
RESULTS AND DISCUSSION In Figs 2 and 3 the adsorption rate of different concentrations of whey protein isolate at a constant interface area of 20 cm2 is shown. The film pressure is plotted as a function of time elapsed between t"0 (free from surfactant) and the newly formed interface with attached whey proteins. By comparison of these isochores, representing the mean value of a series of measurements, it can be concluded that the general characteristics of adsorption are similar at the oil—water and at the air—water interface. After 10 min higher film pressures are reached at the air—water interface, where the adsorbed protein molecules might be denatured to a smaller extent than those at the oil—water interface. Graham and Phillips (1979) found similar results with the adsorption isotherms of globular proteins (lysozyme, bovine serum albumin) at the air—water and oil—water interface. The apparent-equilibrium interfacial tension was not for any of the protein concentrations after 30 min reached, which has been reported by De Feijter and Benjamins (1987). Several hours are needed to reach an apparent equilibrium, as preliminary results indicated. It also can be seen that for a constant interfacial area the slope of the increase in film pressure is very much dependent on the protein concentration in the aqueous solution, which has to be considered while formulating emulsions including whey proteins. Figure 4 shows three isotherms of the whey protein solution obtained compressing the adsorbed surface layer by moving a Teflon barrier with a speed of v "10 cm2 min~1. The concentration of the protein "!33*%3
Fig. 3. Isochorous mode of measurement at the oil—water interface for different subphase concentrations of Protarmor at 25°C. The concentrations are from top to bottom: 1, 10~1, 10~2 and 10~3 wt%.
Fig. 4. Surface pressure—area isotherms of Protarmor at 25°C (2]10~4 wt%, v "10 cm2 min~1). "!33*%3
solution was 2]10~4 wt% each. Since the three isotherms are more or less identical, it can be assumed that the adsorption of Protarmor is reproducible with this method. There is evidence that the adsorption of this protein mixture at the air—water interface is reversible when the starting surface concentration is low. Similar
Adsorption behaviour of whey proteins
results were found by MacRitchie (1985) for b-lactoglobulin, and by Relkin et al. (1995) for a-lactalbumin, b-lactoglobulin and bovine serum albumin (BSA). Since a hysteresis between compression and expansion of the film is observed (data not shown), no apparent equilibrium is reached during the movement of the barriers. CONCLUSIONS The bursting membrane device has been found to be a useful apparatus for measuring the adsorption rate of whey proteins at different interfaces. For this method it was found that the film pressure for a constant interfacial area after 10 min is higher at the air—water surface compared to adsorption at an oil—water interface. This is an indication for the occurrence of different adsorption behaviour depending on the hydrophobic phase. At such a constant interfacial area the incorporation rate of the proteins strongly depends on the concentration in the subphase. With variable interfacial area (‘isothermal mode’) it has been found that the adsorption of whey proteins at the air-water interface is reversible when starting the compression at low surface amounts of Protarmor. ACKNOWLEDGEMENTS Thanks are due to ARMOR PROTEINES S.A.S., France, for providing the whey protein isolate. REFERENCES Anbarci, A. and Armbruster, H. (1987) Bestimmung der Grenzfla¨chenbesetzungskinetik. ¹enside Surfactants Detergents 24, 111—115.
81
Birdi, K. S. (1989) Thermodynamics of liquid surfaces. In ¸ipid and Biopolymer Monolayers at ¸iquid Interfaces, Chap. 2. Plenum Press, New York, pp. 16—17. Dickinson, E., Rolfe, S. E. and Dalgleish, D. G. (1989) Competitive adsorption in oil-in-water emulsions containing a-lactalbumin and b-lactoglobulin. Food Hydrocolloids 3(3), 193—203. De Feijter, J. A. and Benjamins, J. (1987) Adsorption kinetics of proteins at the air—water interface. In Food Emulsions and Foams, ed. E. Dickinson. The Royal Society of Chemistry, Cambridge, pp. 72—85. Gaonkar, A. G. (1989) Interfacial tensions of vegetable oil/ water systems: effect of oil purification. Journal of American Oil Chemical Society 66(8), 1090—1092. Graham, D. E. and Phillips, M. C. (1979) Proteins at liquid interfaces. II. Adsorption isotherms. Journal of Colloid and Interface Science 79(3), 415—426. Karbstein, H. and Schubert, H. (1995) Development in the continuous mechanical production of oil in water macroemulsions. Chemical Engineering and Processing 34, 205—211. MacRitchie, F. (1985) Desorption of proteins from the air/water interface. Journal of Colloid and Interface Science 105(1), 119—123. Relkin, P., Muller, A. and Launay, B. (1995) Conformational stability of globular proteins: a differential scanning calorimetry study of whey proteins. In Food Macromolecules and Colloids, eds E. Dickinson and D. Lorient. The Royal Society of Chemistry, Cambridge, pp. 167—170. Schwarz, G. and Taylor, S. E. (1995) Thermodynamic analysis of the surface activity exhibited by a largely hydrophobic peptide. ¸angmuir 11, 4341—4346. Stang, M., Karbstein, H. and Schubert, H. (1994) Adsorption kinetics of emulsifiers at oil—water interfaces and their effect on mechanical emulsification. Chemical Engineering and Processing 33, 307—311. Walstra, P. and de Roos, A. L. (1993) Proteins at air—water and oil—water interfaces: static and dynamic aspects. Food Reviews International 9(4), 503—525.
Int. Dairy Journal 8 (1998) 79—81 ( 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$19.00#0.00
Adsorption Behaviour of Whey Proteins Measured by Two Different Methods Elisabeth Eugster a*, Susanne E. Taylor b, Zdenko Puhan c and Hans Eyer a a Federal Dairy Research Institute, Schwarzenburgstr. 161, Bern, Switzerland b Biozentrum of the University of Basel, Klingelbergstr. 70, Basel, Switzerland c Swiss Federal Institute of Technology, Department of Food Science, Zu¨ rich, Switzerland (Received for publication 2 April 1998) ABSTRACT The adsorption behaviour of the commercially available whey protein isolate, PROTARMOR, at the air—water and oil—water interface has been investigated by two different methods. The adsorption rate at a constant interface area (‘isochorous mode’) as a function of time was measured by a method using a ‘bursting membrane’ device. After 10 min the slope of increase in film pressure reaches higher values at the air—water surface compared to adsorption at the oil—water interface. The slope of increase in film pressure at a constant interfacial area is strongly dependent on the protein concentration in the aqueous phase. For pressure—area curves (‘isothermal mode’) a rectangular Teflon trough was used with the surface layer being compressed by moving a Teflon barrier. From these measurements it can be concluded that the adsorption of Protarmor at the the air—water surface is reproducible with this method. There is evidence that the adsorption of this protein mixture at the air—water surface is reversible. ( 1998 Elsevier Science Ltd. All rights reserved Keywords: monolayer; b-lactoglobulin; a-lactalbumin; interfacial tension
INTRODUCTION
used as solvent for the protein solution. Concentrations of the whey protein in the subphase are indicated in wt%. The purification of the vegetable oil used for the measurements at the oil—water interface has been described by Gaonkar (1989). To remove interfacially active contaminants the commercial vegetable oil has been mixed with Silica gel (60 g L~1). The mixture was stirred (20 h) and then centrifuged. The supernatant oil was used for measurements at the oil—water interface. The adsorption rate at a constant interface area (‘isochorous mode’) as a function of time has been investigated by a method based on a ‘bursting membrane’. After release of the emulsifier solution the interfacial tension was measured (Anbarci and Armbruster, 1987). The measurement device (made by our workshop) is shown in Fig. 1. The surface area of the trough was 20 cm2, the volume of the aqueous phase was 118 mL. Twentyfive mL of purified vegetable oil has been poured on top of the water. This method and applications are described in detail in Stang et al. (1994). For pressure—area curves (‘isothermal mode’) a rectangular Teflon trough (NIMA Technology Ltd., Coventry, UK) equipped with three tiny Teflon stirrers was used. The surface layer was compressed between 495 and 90 cm2 by moving a Teflon barrier. The trough was filled with 350 mL pure water and the Protarmor stock solution was injected into the subphase with a syringe (Hamilton, Switzerland). The preparation and cleaning of the trough was described elsewhere (Schwarz and Taylor, 1995). In both cases the surface tension was measured by the Wilhelmy plate method. Film pressure n has
Milk proteins are well known for their functional properties in forming and in stabilizing emulsions. They facilitate the disruption of droplets by reducing the interfacial tension, and stabilize the emulsion by immediately occupying the newly formed interfaces after disruption and, thereby, inducing repulsive forces between droplets. The adsorption behaviour of protein molecules at the interface is crucial for the short-term stability of an emulsion. If the rate of adsorption at the interface is too low, the newly formed droplets might coalesce during the emulsification process (Stang et al., 1994, Karbstein and Schubert, 1995). Milk proteins, especially whey proteins, also act as stabilizers by forming a cohesive, viscous film via intermolecular interactions at the interface (Dickinson et al., 1989; Walstra and de Roos, 1993). In this study the adsorption behaviour of a commercially available whey protein isolate at the air—water and oil—water interface has been investigated. MATERIALS AND METHODS The whey protein powder was of food grade and used without further purification. Protarmor 906 (ARMOR PROTEINES S.A.S., France) is a bovine whey protein isolate (87 wt% protein) consisting of b-lactoglobulin and a-lactalbumin mainly. Purified water (Millipore) was * Corresponding author. 79
80
E. Eugster et al.
Fig. 2. Time dependence (‘isochorous mode’) of film pressure n at the air—water interface for Protarmor at different subphase concentrations at 25°C. The concentrations are from top to bottom: 3, 1, 10~1, 10~2, 10~3 and 10~4 wt%. Fig. 1. Device for measuring the adsorption kinetics of watersoluble surfactants at the air—water surface and oil—water interface. The surface tension is recorded as force F divided by perimeter º of the Wilhelmy platelet (Stang et al., 1994).
been calculated by using Antonow’s equation (Birdi, 1989) n"c !c 0 where n represents the film pressure, c the interfacial 0 tension of purified water and c the interfacial tension with adsorbed molecules.
RESULTS AND DISCUSSION In Figs 2 and 3 the adsorption rate of different concentrations of whey protein isolate at a constant interface area of 20 cm2 is shown. The film pressure is plotted as a function of time elapsed between t"0 (free from surfactant) and the newly formed interface with attached whey proteins. By comparison of these isochores, representing the mean value of a series of measurements, it can be concluded that the general characteristics of adsorption are similar at the oil—water and at the air—water interface. After 10 min higher film pressures are reached at the air—water interface, where the adsorbed protein molecules might be denatured to a smaller extent than those at the oil—water interface. Graham and Phillips (1979) found similar results with the adsorption isotherms of globular proteins (lysozyme, bovine serum albumin) at the air—water and oil—water interface. The apparent-equilibrium interfacial tension was not for any of the protein concentrations after 30 min reached, which has been reported by De Feijter and Benjamins (1987). Several hours are needed to reach an apparent equilibrium, as preliminary results indicated. It also can be seen that for a constant interfacial area the slope of the increase in film pressure is very much dependent on the protein concentration in the aqueous solution, which has to be considered while formulating emulsions including whey proteins. Figure 4 shows three isotherms of the whey protein solution obtained compressing the adsorbed surface layer by moving a Teflon barrier with a speed of v "10 cm2 min~1. The concentration of the protein "!33*%3
Fig. 3. Isochorous mode of measurement at the oil—water interface for different subphase concentrations of Protarmor at 25°C. The concentrations are from top to bottom: 1, 10~1, 10~2 and 10~3 wt%.
Fig. 4. Surface pressure—area isotherms of Protarmor at 25°C (2]10~4 wt%, v "10 cm2 min~1). "!33*%3
solution was 2]10~4 wt% each. Since the three isotherms are more or less identical, it can be assumed that the adsorption of Protarmor is reproducible with this method. There is evidence that the adsorption of this protein mixture at the air—water interface is reversible when the starting surface concentration is low. Similar
Adsorption behaviour of whey proteins
results were found by MacRitchie (1985) for b-lactoglobulin, and by Relkin et al. (1995) for a-lactalbumin, b-lactoglobulin and bovine serum albumin (BSA). Since a hysteresis between compression and expansion of the film is observed (data not shown), no apparent equilibrium is reached during the movement of the barriers. CONCLUSIONS The bursting membrane device has been found to be a useful apparatus for measuring the adsorption rate of whey proteins at different interfaces. For this method it was found that the film pressure for a constant interfacial area after 10 min is higher at the air—water surface compared to adsorption at an oil—water interface. This is an indication for the occurrence of different adsorption behaviour depending on the hydrophobic phase. At such a constant interfacial area the incorporation rate of the proteins strongly depends on the concentration in the subphase. With variable interfacial area (‘isothermal mode’) it has been found that the adsorption of whey proteins at the air-water interface is reversible when starting the compression at low surface amounts of Protarmor. ACKNOWLEDGEMENTS Thanks are due to ARMOR PROTEINES S.A.S., France, for providing the whey protein isolate. REFERENCES Anbarci, A. and Armbruster, H. (1987) Bestimmung der Grenzfla¨chenbesetzungskinetik. ¹enside Surfactants Detergents 24, 111—115.
81
Birdi, K. S. (1989) Thermodynamics of liquid surfaces. In ¸ipid and Biopolymer Monolayers at ¸iquid Interfaces, Chap. 2. Plenum Press, New York, pp. 16—17. Dickinson, E., Rolfe, S. E. and Dalgleish, D. G. (1989) Competitive adsorption in oil-in-water emulsions containing a-lactalbumin and b-lactoglobulin. Food Hydrocolloids 3(3), 193—203. De Feijter, J. A. and Benjamins, J. (1987) Adsorption kinetics of proteins at the air—water interface. In Food Emulsions and Foams, ed. E. Dickinson. The Royal Society of Chemistry, Cambridge, pp. 72—85. Gaonkar, A. G. (1989) Interfacial tensions of vegetable oil/ water systems: effect of oil purification. Journal of American Oil Chemical Society 66(8), 1090—1092. Graham, D. E. and Phillips, M. C. (1979) Proteins at liquid interfaces. II. Adsorption isotherms. Journal of Colloid and Interface Science 79(3), 415—426. Karbstein, H. and Schubert, H. (1995) Development in the continuous mechanical production of oil in water macroemulsions. Chemical Engineering and Processing 34, 205—211. MacRitchie, F. (1985) Desorption of proteins from the air/water interface. Journal of Colloid and Interface Science 105(1), 119—123. Relkin, P., Muller, A. and Launay, B. (1995) Conformational stability of globular proteins: a differential scanning calorimetry study of whey proteins. In Food Macromolecules and Colloids, eds E. Dickinson and D. Lorient. The Royal Society of Chemistry, Cambridge, pp. 167—170. Schwarz, G. and Taylor, S. E. (1995) Thermodynamic analysis of the surface activity exhibited by a largely hydrophobic peptide. ¸angmuir 11, 4341—4346. Stang, M., Karbstein, H. and Schubert, H. (1994) Adsorption kinetics of emulsifiers at oil—water interfaces and their effect on mechanical emulsification. Chemical Engineering and Processing 33, 307—311. Walstra, P. and de Roos, A. L. (1993) Proteins at air—water and oil—water interfaces: static and dynamic aspects. Food Reviews International 9(4), 503—525.