- Email: [email protected]
Foam formation and stabilisation by pre-denatured ovalbumin
Colloids and Surfaces B: Biointerfaces 12 (1999) 409 – 416
Foam formation and stabilisation by pre-denatured ovalbumin P. Relkin *, N. Hagolle, D.G. Dalgleish 1, B. Launay Department Science de l’Aliment, Laboratoire de Biophysique des Mate´riaux Alimentaires, Ecole Nationale Supe´rieure des Industries Alimentaires, 1 A6enue des Olympiades, 91 744 Massy, France
Abstract Various mild heat-treatments of ovalbumin solutions were applied to produce molecular species with different conformational states, and having different kinetics of adsorption to the air/water interface and different foaming properties. Molecular species with a higher degree of shear-induced deformation and a low degree of thermal conformational stability showed a slight enhancement of the rate of decrease of surface tension, 5 min after the creation of the fresh interface, and decreasing long-term values of surface tension. Solutions of ovalbumin molecular species exhibiting such initial structural patterns were shown to have enhanced foam capacity and stability against liquid drainage. Ovalbumin molecules with some degree of secondary and tertiary structural changes and increased viscosity, before adsorption at the air/water interface, were shown to be relevant to produce more or less hydrated foams with more or less stability against liquid drainage. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Ovalbumin; Denaturation; Aggregation; Adsorption at air/water interface; Foaming properties
1. Introduction A number of studies [1 – 3] have focussed on how globular proteins accomodate to various surfaces. It is well established that due to their amphiphilic (polar/non polar) nature and marginal conformational stability, globular proteins may adsorb from aqueous solutions to solid surfaces and fluid/fluid interfaces and act as surfactants by reducing the interfacial tension and forming a cohesive film. By using mild treatments * Corresponding author.. E-mail: [email protected]. 1 Present address: Danone, CIRDC, 15 Av. Galille´e, 92350 Le Plessis Robinson -France.
of protein solutions such as heating or change of physicochemical environmental conditions to cause denaturation, it has been shown that globular proteins may undergo a conformational transition from the native structure to a native-like secondary one without residual tertiary structure [4]. More recent studies have indicated that heatinduced aggregation of partially unfolded ovalbumin molecules [5] and emulsification of a-lactalbumin at acidic pH or in the apo-form involved the so-called ‘molten globule’ state [6]. Dynamic surface tension measurements have been carried out [7,8] on globular proteins (high-order structured) in comparison with b-casein (randomcoiled) and in relation to their difference in hydro-
0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 8 ) 0 0 0 9 5 - 2
410
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
phobicity and foaming properties [7]. Some studies have been made which consider the effects of both controlled heating/cooling treatments and pH on the effects of the structural changes of whey proteins on their foam formation and stability [9] or of ovalbumin on its emulsifying properties [10]. In recent studies [11,12] we have shown that stable heat-denatured ovalbumin molecules may be formed by various heating/cooling treatments of their aqueous solutions at pH 3 and 7, where they may be oppositely charged (pI 5.2). Characteristic transition temperatures of conformational changes [11], as determined by circular dichroism (CD), differential scanning calorimetry (DSC) and rheology have been closely related to temperatures corresponding to both increased intrinsic viscosity and appearance [12] of non covalently bound aggregates (pH 3) and mixture of non covalently and covalently bound aggregates (pH 7). We also showed that pre-heated solutions, in acidic and neutral pH conditions, exhibited a long-term convergence of the steady state surface pressure values, depending on the ovalbumin structural patterns before adsorption to the air/ water interface [12]. In the present work, we extend the study to show how mild heat-treatment can be applied to enhance foaming properties of ovalbumin molecules which are considered, when in their ‘native’ conformational state, as ‘hard’ spheres.
2. Experimental
2.1. Protein solutions Chicken egg white ovalbumin was purchased from Sigma (grade V, lot 101H7155) and used without further purification. Samples were dispersed in distilled water, dialysed against 0.1 M NaCl for 24 h and pH was adjusted to 3 or 7 with 1 M HCl or NaOH. Solutions were gently stirred for 30 min then centrifuged for 30 min at 15 000× g. Concentrations were determined by the biuret method and then adjusted to values required by each methodology. All the experimental results reported in this study are mean values
obtained from experiments.
at
least
two
independent
2.2. Shearing effects and intrinsic 6iscosity Viscosimetric properties of protein solutions (5% concentration) at pH 7 and 3, were monitored with a Contrave LS-30 viscosimeter to determine time-dependent viscosity values (0.11 s − 1 rate) and intrinsic viscosity values (51.2 s − 1 rate) of ovalbumin molecules from unheated or previously heated solutions up to various temperatures. The methodology used to determine the intrinsic viscosity was described elsewhere [11].
2.3. Heat-induced structural changes in solution The methodology used to determine characteristic temperatures of transitions in the high-ordered secondary structure (far UV-CD), overall conformational stability (DSC) and intrinsic viscosity (rheology) have been described elsewhere [11,12]. The CD measurements were performed (Jasco J-600) on solutions (0.015 wt.%) which were heated from 25 to 90°C at 1°C min − 1 and critical temperatures (Ta and Tb ) of secondary structure changes were determined from the change in slope of the mean residue values of ellipticity, [U] at 222 nm (a-helix) and 213 nm (b-sheet), as functions of heating temperature. For the DSC study (Perkin-Elmer DSC7), solutions (5 wt.%) were heated from 20 to 110°C at 5°C min − 1 and the calorimetric parameters of protein heat conformational stability were deduced from the DSC signal [13]. Ti, the onset of heat-induced conformational changes was defined as the temperature of the first increase in DSC heat flow, Tp was the peak temperature and DHapp the apparent enthalpy change of heat transition that was determined from the area under the peak using a straight baseline. In our previous studies [13] we have shown that in our experimental conditions (relatively high protein concentration and presence of salt ions), where the structure of water molecules may change and protein-protein electrostatic interactions may be enhanced, the use of a straight baseline instead of a sigmoidal baseline is a good approximation. The experimen-
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
tal uncertainties were less than 0.5°C (temperature) and less than 0.8 J g − 1 (enthalpy).
2.4. Tensiometry Protein solutions (0.5%) were pre-heated from 20°C to various temperatures and then quenched in an ice bath before surface tension measurements. Kinetics of surface tension decay were measured continuously by using a processor tensiometer (Kruss K12) equipped with a Wilhelmy plate. The new interface was created by pouring out the solution, then allowing it to equilibrate for 2 min before measuring for up to 3 h. Each value of measurement was automatically determined and all experiments were performed twice. Adsorption kinetics were expressed in term of surface pressure, p:
drainage time, t1/2 (s) was determined from the time needed to drain half the liquid volume, 1/2 (Vmax + Vmin) during the duration of the analysis. Vmax is the volume of liquid incorporated during the air sparging and Vmin is the volume which remained in the foam at the end of the experiment (20 min). The foam stability against liquid drainage was also determined by the use of the capillary method of Torikata et al. [16]. Accordingly to this method, experimental values of Dh (change in height of the foam liquid boundary) as a function of time were fitted using the equation: Dh = t/(a 2 + abt)
(1)
where a and b are adjustable parameters (s cm)−1/2 which are related to T (s), a characteristic time of liquid drainage, by the following expressions:
p(t)=go − g(t)
a= (T 1/2L − 1/2)
where go (72 mN90.5 mN) was the surface tension of the solvent and g(t), that of the protein solution at time t. Measurement of protein concentration at the end of the surface tension experiments indicated no significant change, in comparison with initial concentration.
where L (cm) is the height of the foam.
2.5. Foaming properties The foaming properties of protein solutions (0.5% protein concentration, 0.1 M NaCl) at pH 3 and 7, were characterised through their foam formation and stability. We used a foam analyser based on conductimetric measurements of the liquid drained during foam destabilisation [14,15]. The foam was generated by sparging air (15 ml min − 1) into an initial volume of protein solution (8 ml). The foaming ability was evaluated by the sparging time needed to reach a certain total foam volume (35 ml) that was detected by an on-line camera, while the volume of liquid under the foam was measured by conductimetry and compared to the initial one. The foam formation and liquid drainage were analysed over 20 min time period. The foam density at the end of sparging was determined from the maximum volume of foam and the volume of air incorporated. The half
411
b=(TL) − 1/2
(2)
3. Results and discussion The curves shown in Fig. 1 were obtained with unheated solutions that were submitted to a low shear-rate deformation (0.11 s − 1). It is apparent that their viscosity value was time-dependent, but
Fig. 1. Variation of the apparent viscosity of ovalbumin molecules obtained with unheated solutions (5% protein concentration) at pH 3 () and pH 7 (), as a function of time (0.11 s − 1 shear-rate).
412
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
Fig. 2. Effects of pH of ovalbumin solutions on differential scanning calorimetric signals and on variations of mean residue values [U] at 213 and 222 nm, as functions of temperature treatments. pH 3 (a); pH 7 (b); [U]213 () and [U]222 ().
solutions at pH 3 seemed to undergo a steeper jump in the viscosity to a higher plateau value, relative to solutions at pH 7. This suggests that orthokinetic aggregation seems to be more favoured at pH 3 than at pH 7 in solutions which had not been heated previously. The corresponding intrinsic viscosity values, determined from apparent viscosity measurements at a high shear rate (55.1 s − 1), were higher for ovalbumin molecules at pH 3 (7.2 ml g − 1) than at pH 7 (5.4 ml g − 1). The intrinsic viscosity value measured at pH 7 is in fair agreement with published data, but it is much lower than that (40 ml g − 1) of chemically denatured ovalbumin molecules [17]. The higher value of intrinsic viscosity, found at pH 3 compared with pH 7 may be due to more expanded molecular forms and explained on the basis of other studies [10,17,18]. Those studies have shown higher flexibility and surface hydrophobicity of ovalbumin in acidic conditions. The existence of aggregates of ovalbumin molecules in a wide range of protein concentration [19], resulting in a higher average state of association number in pH 3 solutions, could also be responsible for the higher intrinsic viscosity at pH 3. The mean residue values of ellipticity measured at 222 and 213 nm, as functions of temperatures of treatments are shown in Fig. 2(a) and (b). They indicated that when protein solutions were heated at temperatures between 25 and 90°C, the trend at 222 mm (a-helix) and 213 nm (b-forms) showed an apparent cooperative decrease of the overall
secondary structure for pH 7 (Fig. 2(b)) but a formation of newly created b-structure above T60°C for pH 3 (Fig. 2(a)). Although we used protein solutions at a much higher protein concentration for the DSC measurements, the examples of DSC signals reported in the same figures showed that the temperatures corresponding to the onset of changes in the secondary structure a-forms (222 nm) and b (213 nm) are close to Ti (temperature of first increase in heat flow) but lower than Tp (temperature of maximum rate of heat conformational transition). The calorimetric parameters of protein conformational transition, as determined by DSC are lower at pH 3 (Ti = 62°C; Tp = 76°C and DHapp = 5.8 J g − 1) than at pH 7 (Ti = 68°C; Tp = 88°C and DHapp = 17.6 J g − 1). These values are in fair agreement with previous studies [20]. The lower value of the heat transition observed at pH 3, relative to that observed at pH 7 cannot be attributed to the methodology used for its determination (straight baseline instead of sigmoidal one) but it could rather indicate either a partial unfolding of the globular state (the so-called molten globule state) at acidic pH [4,5,10,17,18] or a possible exothermic contribution to the overall calorimetric heat of reaction due to destruction under heating of the hydrophobic contacts between protein molecules formed at pH 3 or both. Recently [21] we found a close relationship between the decreasing values of residual enthalpy change (DSC) and increasing values of surface hydrophobicity
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
413
Fig. 3. Effects of pH and temperature of prior treatments of ovalbumin solutions (0.5% protein concentration) on the kinetics of their surface pressure. pH 3 (a) and pH 7 (b).
(fluorimetry-dye binding) of b-lactoglobulin of solutions that were pre-heated to a preset temperature and in previous studies we have shown that ovalbumin concentrations between 0.25 and 5% have not a significant effect on Tp [11], whereas DHapp decreases with increased concentrations, as well as for solutions of b-lactoglobulin, in conditions favouring protein-protein interactions [22]. Thus, it seems clearly that ovalbumin molecules at pH 3, having a higher degree of deformability (time-dependent viscosity) and a higher intrinsic viscosity, have a lower heat-conformational stability (secondary and tertiary structure changes) than at pH 7. The ability of ovalbumin molecules to adsorb to the air/water interface was evaluated through kinetics of surface pressure (p(t)) or surface tension (g(t)), as functions of pre-heating treatments. It is well established that globular proteins can be transported to an interface where they can attach (reversibly or irreversibly) to the surface with further structural rearrangements [1 – 3]. The curves in Fig. 3(a) and (b) are characterised by a rapid rate of increase (or decrease) in p (or g) values at both pH 3 and 7. The corresponding steady-state value (48 mN m − 1) of the surface tension observed with unheated protein solutions is close to that observed by dynamic surface tension measurements performed on lysozyme [7] and b-lactoglobulin [8] at 0.1% and pH 7, but lower than the value (53 mN m − 1) observed by
de Feijter and Benjamin [23] and Tripp et al. [7] for ovalbumin solution (0.1% and pH 6.4) and bovin serum albumin (0.1% and pH 7), respectively. Both bulk protein concentration, pH and ionic strength are well known to affect the surface adsorption properties of globular proteins. Furthermore, previous studies have demonstrated that in experimental conditions which favour protein-protein interactions, a lateral surface compression of adsorbed BSA molecules leads to a negative contribution to steady state values of surface pressure [24]. It might be suggested that in such conditions, ovalbumin molecules and BSA behave similarly, when they are at a high bulk protein concentration and in presence of salt ions. When the protein solutions were pre-heated, the rate of decrease in surface tension, determined 5 min after the creation of fresh interface seems to be only slightly dependent on temperature of preheating (Fig. 4). Although there is a large experimental uncertainty, the values reported in Fig. 4 might suggest that molecular species which are initially present in the bulk medium exhibit the highest rate of adsorption at the air/water interface after heat-treatment at T 62°C (pH 3) and T68°C (pH 7). Again, these temperatures are close to the onset temperatures of tertiary structure changes (Fig. 2(a) and (b)). In the long time region (Fig. 3(a) and (b)), we observed a slight decrease in the surface pressure, when protein solutions were pre-heated at a tem-
414
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
Fig. 4. Variation of the values of rate of decrease of surface tension of ovalbumin solutions at pH 3 ( ) and pH 7 (), determined at 5 min after the creation of the interface, as functions of pre-heating temperatures.
perature related to the development of b-structures for pH 3, and to secondary and tertiary structure changes for pH 7 (Fig. 2(a) and (b)), and also to increasing intrinsic viscosity values as functions of temperatures (shown in Fig. 5). These intrinsic viscosity values (taken from [11]), determined from protein solutions that were pre-heated up to various temperatures, indicated a steep increase from : 55°C at pH 3 instead of 75°C at pH 7. Previous studies indicated that soluble ovalbumin aggregates are formed by hydrophobic in-
Fig. 5. Effects of pH and temperature of prior treatments on the intrinsic viscosity of ovalbumin molecules (see [11] for the methodology) and corresponding foam density. pH 7 (filled symbols) and pH 3 (open symbols); intrinsic viscosity (circles); foam density (triangles).
teraction [10,17,18]. On the basis of the last hypothesis, combined with our previous results [12] observed from analysis of PAGE electrophoresis band patterns of solutions which have been heated at temperatures between 20 and 65°C (pH 3) and 20 and 80°C (pH 7), it seems that these increases in intrinsic viscosity could be due to heat-induced non covalently bound aggregates at pH 3 (up to 65°C) but also to covalently bound ones at pH 7 and T\ 75°C. It might be possible that close-packing of molecular species associated by non covalent bonds, including intermolecular b-sheet forms (pH 3) or other interaction forces (pH 7), in addition to slowing down their diffusion rate to the interface might explain the slight decrease in surface pressure relative to unheated solutions. The results reported in Figs. 2–4, indicate also that molecular species with different conformational states, that may have different initial flexibility and/or different surface hydrophobicity [10,17,18] and also different mass transport properties, may display different kinetics of surface tension as measured by the Wilhelmy balance. Thus, in unstirred conditions, the initial adsorption rate of the protein molecules at the air/water interface may be supposed to be controlled both by their flexibility, surface hydrophobicity and mass transport properties. This hypothesis could be investigated or confirmed by further measurements with sensitive techniques such as ellipsometry and interfacial rheology which could provide information on the thickness and elasticity of the protein adsorbed layer. Proteins are used in food technology to stabilise foams. To be a good foaming agent, proteins must be able to entrap the gas bubbles in a continuous liquid lamellar phase. In the bubbling tests, it has been shown that the higher the foam density (larger amount of incorporated liquid), the higher was the foam expansion [25]. When compared to protein solutions at pH 7, we observed [26] that unheated ovalbumin solutions at pH 3 required a lower bubbling time to attain a foam volume of 35 ml, and the amount of liquid incorporated in the corresponding foams and therefore the foam density (Fig. 5) are higher. Furthermore, their liquid drainage characteristics (Fig. 6(a) and (b)) are very different. At pH 3,
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
415
Fig. 6. Effects of pH and temperature of prior treatments of ovalbumin solutions. Half-drainage time of liquid, t1/2 (a) and characteristic time of liquid drainage, T (s) (b); pH 7 (open symbols; pH 3 (filled symbols).
where the protein molecules in solution are more sensitive to orthokinetic aggregation (Fig. 1) and less resistant to thermal denaturation (Fig. 2), the foaming capacity and stability are much higher than at pH 7, where the protein molecules may be considered as more rigid. The original heat-treatment has a wide effect on the intrinsic viscosity but only a slight effect on the foam density at pH 3, whereas at pH 7 the bubbling time decreased [26] and the foam density and the intrinsic viscosity increased when solutions were pre-heated at temperatures higher than 68°C (Fig. 5). This temperature is close to that of the transition of secondary and tertiary structure changes (Fig. 2(b)) and it also corresponds to that of a slightly higher rate of decrease of protein surface tension (from pure solvent value), relative to unheated solutions (Fig. 4). The half-drainage time, t1/2 (Fig. 6(a)) and the time constant value, T(s), determined by the capillary method (Fig. 6(b)), are shown to have increased values (more than 100%) for temperature of pre-heating ranging from 75 to 80°C at pH 7, and decreased values (more than 30%) for solutions at pH 3 and T\ 50°C. The value of t1/2 obtained at T=50°C and pH 3 is not significantly changed relative to that obtained with unheated solutions, whereas T (s) values decreases. These results indicate that mild-heat treatments which denature the protein conformation in solution, without formation of a large percentage of
covalently bound aggregates as assumed in our previous study [12], can be used to enhance foaming properties of globular proteins.
4. Conclusion In this work, we have shown that heat-treatment of ovalbumin solutions leading to molecular species with mild-denatured structures can be used to improve the foaming properties of globular proteins that have an initially ‘rigid’ conformational state. At pH 3, heat-induced b-sheet structures are shown to be newly created before the occurrence of the DSC peak transition and in parallel with increasing viscosity. At pH 7 no significant development of b-sheets was detected, but a rather large decrease in the overall secondary and tertiary structures accompanied increased viscosity. When such stable molecular species are submitted to adsorption at the air/water interface, the long-term surface pressure values decreased with the temperature of pre-heating, while the rate of decrease of the surface tension from bulk solvent seemed to be slightly higher for pre-denatured proteins, as long as non covalently bound aggregates were assumed not to form, as demonstrated in a previous study [12]. Finally we showed that it is possible to stabilise foams with ovalbumin molecules in such heat
416
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
denatured forms. Although we have not investigated by direct techniques, the re-conformation of adsorbed proteins nor their surface rheological properties, this study could contribute to more understanding of a possible relationship between the conformational state of adsorbing proteins and their resulting foaming properties.
Acknowledgements The financial supports of the Ministe`re de l’Agriculture et de la Peˆche (programme ‘Aliment demain’, R 94/51) and Danone (centre Jean The`ves) are gratefully acknowledged. Thanks also to Dr M-N. Bellon-Fontaine (INRA-Massy) and Dr Y. Popineau (INRA-Nantes) for their material and scientific help.
References [1] F. MacRitchie, A.E. Alexander, J. Colloid Interface Sci. 18 (1963) 458. [2] D.E. Graham, M.C. Phillips, J. Colloid Interface Sci. 76 (1980) 227. [3] E. Dickinson, in: E.J. Wilson (Ed.), Foams: Physics, Chemistry and Structure, Springer, Berlin, 1989, p. 39. [4] K. Kuwajima, Y. Hiraoka, M. Ikeguchi, S. Sugai, Biochemistry 24 (1985) 874. [5] H. Hirose, Trends Food Sci. Technol. 4 (1993) 48. [6] E. Dickinson, Y. Matsumura, Colloids Surf. B: Biointerfaces 3 (1994) 1.
.
[7] C.B. Tripp, J.J. Magda, J.D. Andrade, J. Colloid Interface Sci. 173 (1995) 16. [8] R. Wu¨stneck, J. Kra¨gel, R. Miller, V.B. Fainerman, P.J. Wilde, D.K. Sarker, D.C. Clark, Food Hydrocolloids 10 (1996) 395. [9] R.D. Waniska, J.E. Kinsella, J. Agric. Food Chem. 33 (1985) 1143. [10] Y. Mine, T. Noutomi, N. Haga, J. Agric. Food Chem. 38 (1990) 2122. [11] N. Hagolle, P. Relkin, D.G. Dalgleish, B. Launay, Food Hydrocolloids 11 (1997) 311. [12] N. Hagolle, B. Launay, P. Relkin, Colloids Surf. B. Biointerfaces 10 (1997) 191. [13] P. Relkin, Thermochim. Acta 246 (1994) 371. [14] W. Loisel W, European Patent, PCT/FR92/00509. [15] A. Baniel, A. Fains, Y. Popineau, J. Food Sci. 62 (1997) 377. [16] Y. Torikata, M. Kato, H. Kumagai, T. Yano, Agric. Biol. Chem. 55 (1991) 1307. [17] T. Koseki, N. Kitabatake, E. Doi, J. Biochem. 103 (1988) 425. [18] A. Kato, Y. Nagase, N. Matsudomi, K. Kobayasi, Agric. Biol. Chem. 47 (1983) 1829. [19] T. Matsumoto, J. Chiba, H. Inoue, Colloid Polym. Sci. 270 (1992) 687. [20] P.O. Hegg, H. Martens, B. Lofqvist, J. Sci. Food Agric. 30 (1979) 981. [21] P. Relkin, J. Int. Biol. Macromol. 22 (1998) 59. [22] P. Relkin, B. Launay, Food Hydrocolloids 4 (1990) 19. [23] I.A De Feijter, J. Benjamins, in: E. Dickinson (Ed.), Food Emulsions and Foams, Royal Society of Chemistry, London, 1986, p. 72. [24] A. Dussaud, G.B. Han, L. ter Minassian-Saraga, M. Vignes-Adler, J. Colloid Interfacial Sci. 167 (1994) 247. [25] P.J. Halling, CRC Crit. Rev. Food Sci. Nutr. 15 (1981) 155. [26] N. Hagolle, PhD Thesis, ENSIA, France, 1997.
Foam formation and stabilisation by pre-denatured ovalbumin P. Relkin *, N. Hagolle, D.G. Dalgleish 1, B. Launay Department Science de l’Aliment, Laboratoire de Biophysique des Mate´riaux Alimentaires, Ecole Nationale Supe´rieure des Industries Alimentaires, 1 A6enue des Olympiades, 91 744 Massy, France
Abstract Various mild heat-treatments of ovalbumin solutions were applied to produce molecular species with different conformational states, and having different kinetics of adsorption to the air/water interface and different foaming properties. Molecular species with a higher degree of shear-induced deformation and a low degree of thermal conformational stability showed a slight enhancement of the rate of decrease of surface tension, 5 min after the creation of the fresh interface, and decreasing long-term values of surface tension. Solutions of ovalbumin molecular species exhibiting such initial structural patterns were shown to have enhanced foam capacity and stability against liquid drainage. Ovalbumin molecules with some degree of secondary and tertiary structural changes and increased viscosity, before adsorption at the air/water interface, were shown to be relevant to produce more or less hydrated foams with more or less stability against liquid drainage. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Ovalbumin; Denaturation; Aggregation; Adsorption at air/water interface; Foaming properties
1. Introduction A number of studies [1 – 3] have focussed on how globular proteins accomodate to various surfaces. It is well established that due to their amphiphilic (polar/non polar) nature and marginal conformational stability, globular proteins may adsorb from aqueous solutions to solid surfaces and fluid/fluid interfaces and act as surfactants by reducing the interfacial tension and forming a cohesive film. By using mild treatments * Corresponding author.. E-mail: [email protected]. 1 Present address: Danone, CIRDC, 15 Av. Galille´e, 92350 Le Plessis Robinson -France.
of protein solutions such as heating or change of physicochemical environmental conditions to cause denaturation, it has been shown that globular proteins may undergo a conformational transition from the native structure to a native-like secondary one without residual tertiary structure [4]. More recent studies have indicated that heatinduced aggregation of partially unfolded ovalbumin molecules [5] and emulsification of a-lactalbumin at acidic pH or in the apo-form involved the so-called ‘molten globule’ state [6]. Dynamic surface tension measurements have been carried out [7,8] on globular proteins (high-order structured) in comparison with b-casein (randomcoiled) and in relation to their difference in hydro-
0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 8 ) 0 0 0 9 5 - 2
410
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
phobicity and foaming properties [7]. Some studies have been made which consider the effects of both controlled heating/cooling treatments and pH on the effects of the structural changes of whey proteins on their foam formation and stability [9] or of ovalbumin on its emulsifying properties [10]. In recent studies [11,12] we have shown that stable heat-denatured ovalbumin molecules may be formed by various heating/cooling treatments of their aqueous solutions at pH 3 and 7, where they may be oppositely charged (pI 5.2). Characteristic transition temperatures of conformational changes [11], as determined by circular dichroism (CD), differential scanning calorimetry (DSC) and rheology have been closely related to temperatures corresponding to both increased intrinsic viscosity and appearance [12] of non covalently bound aggregates (pH 3) and mixture of non covalently and covalently bound aggregates (pH 7). We also showed that pre-heated solutions, in acidic and neutral pH conditions, exhibited a long-term convergence of the steady state surface pressure values, depending on the ovalbumin structural patterns before adsorption to the air/ water interface [12]. In the present work, we extend the study to show how mild heat-treatment can be applied to enhance foaming properties of ovalbumin molecules which are considered, when in their ‘native’ conformational state, as ‘hard’ spheres.
2. Experimental
2.1. Protein solutions Chicken egg white ovalbumin was purchased from Sigma (grade V, lot 101H7155) and used without further purification. Samples were dispersed in distilled water, dialysed against 0.1 M NaCl for 24 h and pH was adjusted to 3 or 7 with 1 M HCl or NaOH. Solutions were gently stirred for 30 min then centrifuged for 30 min at 15 000× g. Concentrations were determined by the biuret method and then adjusted to values required by each methodology. All the experimental results reported in this study are mean values
obtained from experiments.
at
least
two
independent
2.2. Shearing effects and intrinsic 6iscosity Viscosimetric properties of protein solutions (5% concentration) at pH 7 and 3, were monitored with a Contrave LS-30 viscosimeter to determine time-dependent viscosity values (0.11 s − 1 rate) and intrinsic viscosity values (51.2 s − 1 rate) of ovalbumin molecules from unheated or previously heated solutions up to various temperatures. The methodology used to determine the intrinsic viscosity was described elsewhere [11].
2.3. Heat-induced structural changes in solution The methodology used to determine characteristic temperatures of transitions in the high-ordered secondary structure (far UV-CD), overall conformational stability (DSC) and intrinsic viscosity (rheology) have been described elsewhere [11,12]. The CD measurements were performed (Jasco J-600) on solutions (0.015 wt.%) which were heated from 25 to 90°C at 1°C min − 1 and critical temperatures (Ta and Tb ) of secondary structure changes were determined from the change in slope of the mean residue values of ellipticity, [U] at 222 nm (a-helix) and 213 nm (b-sheet), as functions of heating temperature. For the DSC study (Perkin-Elmer DSC7), solutions (5 wt.%) were heated from 20 to 110°C at 5°C min − 1 and the calorimetric parameters of protein heat conformational stability were deduced from the DSC signal [13]. Ti, the onset of heat-induced conformational changes was defined as the temperature of the first increase in DSC heat flow, Tp was the peak temperature and DHapp the apparent enthalpy change of heat transition that was determined from the area under the peak using a straight baseline. In our previous studies [13] we have shown that in our experimental conditions (relatively high protein concentration and presence of salt ions), where the structure of water molecules may change and protein-protein electrostatic interactions may be enhanced, the use of a straight baseline instead of a sigmoidal baseline is a good approximation. The experimen-
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
tal uncertainties were less than 0.5°C (temperature) and less than 0.8 J g − 1 (enthalpy).
2.4. Tensiometry Protein solutions (0.5%) were pre-heated from 20°C to various temperatures and then quenched in an ice bath before surface tension measurements. Kinetics of surface tension decay were measured continuously by using a processor tensiometer (Kruss K12) equipped with a Wilhelmy plate. The new interface was created by pouring out the solution, then allowing it to equilibrate for 2 min before measuring for up to 3 h. Each value of measurement was automatically determined and all experiments were performed twice. Adsorption kinetics were expressed in term of surface pressure, p:
drainage time, t1/2 (s) was determined from the time needed to drain half the liquid volume, 1/2 (Vmax + Vmin) during the duration of the analysis. Vmax is the volume of liquid incorporated during the air sparging and Vmin is the volume which remained in the foam at the end of the experiment (20 min). The foam stability against liquid drainage was also determined by the use of the capillary method of Torikata et al. [16]. Accordingly to this method, experimental values of Dh (change in height of the foam liquid boundary) as a function of time were fitted using the equation: Dh = t/(a 2 + abt)
(1)
where a and b are adjustable parameters (s cm)−1/2 which are related to T (s), a characteristic time of liquid drainage, by the following expressions:
p(t)=go − g(t)
a= (T 1/2L − 1/2)
where go (72 mN90.5 mN) was the surface tension of the solvent and g(t), that of the protein solution at time t. Measurement of protein concentration at the end of the surface tension experiments indicated no significant change, in comparison with initial concentration.
where L (cm) is the height of the foam.
2.5. Foaming properties The foaming properties of protein solutions (0.5% protein concentration, 0.1 M NaCl) at pH 3 and 7, were characterised through their foam formation and stability. We used a foam analyser based on conductimetric measurements of the liquid drained during foam destabilisation [14,15]. The foam was generated by sparging air (15 ml min − 1) into an initial volume of protein solution (8 ml). The foaming ability was evaluated by the sparging time needed to reach a certain total foam volume (35 ml) that was detected by an on-line camera, while the volume of liquid under the foam was measured by conductimetry and compared to the initial one. The foam formation and liquid drainage were analysed over 20 min time period. The foam density at the end of sparging was determined from the maximum volume of foam and the volume of air incorporated. The half
411
b=(TL) − 1/2
(2)
3. Results and discussion The curves shown in Fig. 1 were obtained with unheated solutions that were submitted to a low shear-rate deformation (0.11 s − 1). It is apparent that their viscosity value was time-dependent, but
Fig. 1. Variation of the apparent viscosity of ovalbumin molecules obtained with unheated solutions (5% protein concentration) at pH 3 () and pH 7 (), as a function of time (0.11 s − 1 shear-rate).
412
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
Fig. 2. Effects of pH of ovalbumin solutions on differential scanning calorimetric signals and on variations of mean residue values [U] at 213 and 222 nm, as functions of temperature treatments. pH 3 (a); pH 7 (b); [U]213 () and [U]222 ().
solutions at pH 3 seemed to undergo a steeper jump in the viscosity to a higher plateau value, relative to solutions at pH 7. This suggests that orthokinetic aggregation seems to be more favoured at pH 3 than at pH 7 in solutions which had not been heated previously. The corresponding intrinsic viscosity values, determined from apparent viscosity measurements at a high shear rate (55.1 s − 1), were higher for ovalbumin molecules at pH 3 (7.2 ml g − 1) than at pH 7 (5.4 ml g − 1). The intrinsic viscosity value measured at pH 7 is in fair agreement with published data, but it is much lower than that (40 ml g − 1) of chemically denatured ovalbumin molecules [17]. The higher value of intrinsic viscosity, found at pH 3 compared with pH 7 may be due to more expanded molecular forms and explained on the basis of other studies [10,17,18]. Those studies have shown higher flexibility and surface hydrophobicity of ovalbumin in acidic conditions. The existence of aggregates of ovalbumin molecules in a wide range of protein concentration [19], resulting in a higher average state of association number in pH 3 solutions, could also be responsible for the higher intrinsic viscosity at pH 3. The mean residue values of ellipticity measured at 222 and 213 nm, as functions of temperatures of treatments are shown in Fig. 2(a) and (b). They indicated that when protein solutions were heated at temperatures between 25 and 90°C, the trend at 222 mm (a-helix) and 213 nm (b-forms) showed an apparent cooperative decrease of the overall
secondary structure for pH 7 (Fig. 2(b)) but a formation of newly created b-structure above T60°C for pH 3 (Fig. 2(a)). Although we used protein solutions at a much higher protein concentration for the DSC measurements, the examples of DSC signals reported in the same figures showed that the temperatures corresponding to the onset of changes in the secondary structure a-forms (222 nm) and b (213 nm) are close to Ti (temperature of first increase in heat flow) but lower than Tp (temperature of maximum rate of heat conformational transition). The calorimetric parameters of protein conformational transition, as determined by DSC are lower at pH 3 (Ti = 62°C; Tp = 76°C and DHapp = 5.8 J g − 1) than at pH 7 (Ti = 68°C; Tp = 88°C and DHapp = 17.6 J g − 1). These values are in fair agreement with previous studies [20]. The lower value of the heat transition observed at pH 3, relative to that observed at pH 7 cannot be attributed to the methodology used for its determination (straight baseline instead of sigmoidal one) but it could rather indicate either a partial unfolding of the globular state (the so-called molten globule state) at acidic pH [4,5,10,17,18] or a possible exothermic contribution to the overall calorimetric heat of reaction due to destruction under heating of the hydrophobic contacts between protein molecules formed at pH 3 or both. Recently [21] we found a close relationship between the decreasing values of residual enthalpy change (DSC) and increasing values of surface hydrophobicity
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
413
Fig. 3. Effects of pH and temperature of prior treatments of ovalbumin solutions (0.5% protein concentration) on the kinetics of their surface pressure. pH 3 (a) and pH 7 (b).
(fluorimetry-dye binding) of b-lactoglobulin of solutions that were pre-heated to a preset temperature and in previous studies we have shown that ovalbumin concentrations between 0.25 and 5% have not a significant effect on Tp [11], whereas DHapp decreases with increased concentrations, as well as for solutions of b-lactoglobulin, in conditions favouring protein-protein interactions [22]. Thus, it seems clearly that ovalbumin molecules at pH 3, having a higher degree of deformability (time-dependent viscosity) and a higher intrinsic viscosity, have a lower heat-conformational stability (secondary and tertiary structure changes) than at pH 7. The ability of ovalbumin molecules to adsorb to the air/water interface was evaluated through kinetics of surface pressure (p(t)) or surface tension (g(t)), as functions of pre-heating treatments. It is well established that globular proteins can be transported to an interface where they can attach (reversibly or irreversibly) to the surface with further structural rearrangements [1 – 3]. The curves in Fig. 3(a) and (b) are characterised by a rapid rate of increase (or decrease) in p (or g) values at both pH 3 and 7. The corresponding steady-state value (48 mN m − 1) of the surface tension observed with unheated protein solutions is close to that observed by dynamic surface tension measurements performed on lysozyme [7] and b-lactoglobulin [8] at 0.1% and pH 7, but lower than the value (53 mN m − 1) observed by
de Feijter and Benjamin [23] and Tripp et al. [7] for ovalbumin solution (0.1% and pH 6.4) and bovin serum albumin (0.1% and pH 7), respectively. Both bulk protein concentration, pH and ionic strength are well known to affect the surface adsorption properties of globular proteins. Furthermore, previous studies have demonstrated that in experimental conditions which favour protein-protein interactions, a lateral surface compression of adsorbed BSA molecules leads to a negative contribution to steady state values of surface pressure [24]. It might be suggested that in such conditions, ovalbumin molecules and BSA behave similarly, when they are at a high bulk protein concentration and in presence of salt ions. When the protein solutions were pre-heated, the rate of decrease in surface tension, determined 5 min after the creation of fresh interface seems to be only slightly dependent on temperature of preheating (Fig. 4). Although there is a large experimental uncertainty, the values reported in Fig. 4 might suggest that molecular species which are initially present in the bulk medium exhibit the highest rate of adsorption at the air/water interface after heat-treatment at T 62°C (pH 3) and T68°C (pH 7). Again, these temperatures are close to the onset temperatures of tertiary structure changes (Fig. 2(a) and (b)). In the long time region (Fig. 3(a) and (b)), we observed a slight decrease in the surface pressure, when protein solutions were pre-heated at a tem-
414
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
Fig. 4. Variation of the values of rate of decrease of surface tension of ovalbumin solutions at pH 3 ( ) and pH 7 (), determined at 5 min after the creation of the interface, as functions of pre-heating temperatures.
perature related to the development of b-structures for pH 3, and to secondary and tertiary structure changes for pH 7 (Fig. 2(a) and (b)), and also to increasing intrinsic viscosity values as functions of temperatures (shown in Fig. 5). These intrinsic viscosity values (taken from [11]), determined from protein solutions that were pre-heated up to various temperatures, indicated a steep increase from : 55°C at pH 3 instead of 75°C at pH 7. Previous studies indicated that soluble ovalbumin aggregates are formed by hydrophobic in-
Fig. 5. Effects of pH and temperature of prior treatments on the intrinsic viscosity of ovalbumin molecules (see [11] for the methodology) and corresponding foam density. pH 7 (filled symbols) and pH 3 (open symbols); intrinsic viscosity (circles); foam density (triangles).
teraction [10,17,18]. On the basis of the last hypothesis, combined with our previous results [12] observed from analysis of PAGE electrophoresis band patterns of solutions which have been heated at temperatures between 20 and 65°C (pH 3) and 20 and 80°C (pH 7), it seems that these increases in intrinsic viscosity could be due to heat-induced non covalently bound aggregates at pH 3 (up to 65°C) but also to covalently bound ones at pH 7 and T\ 75°C. It might be possible that close-packing of molecular species associated by non covalent bonds, including intermolecular b-sheet forms (pH 3) or other interaction forces (pH 7), in addition to slowing down their diffusion rate to the interface might explain the slight decrease in surface pressure relative to unheated solutions. The results reported in Figs. 2–4, indicate also that molecular species with different conformational states, that may have different initial flexibility and/or different surface hydrophobicity [10,17,18] and also different mass transport properties, may display different kinetics of surface tension as measured by the Wilhelmy balance. Thus, in unstirred conditions, the initial adsorption rate of the protein molecules at the air/water interface may be supposed to be controlled both by their flexibility, surface hydrophobicity and mass transport properties. This hypothesis could be investigated or confirmed by further measurements with sensitive techniques such as ellipsometry and interfacial rheology which could provide information on the thickness and elasticity of the protein adsorbed layer. Proteins are used in food technology to stabilise foams. To be a good foaming agent, proteins must be able to entrap the gas bubbles in a continuous liquid lamellar phase. In the bubbling tests, it has been shown that the higher the foam density (larger amount of incorporated liquid), the higher was the foam expansion [25]. When compared to protein solutions at pH 7, we observed [26] that unheated ovalbumin solutions at pH 3 required a lower bubbling time to attain a foam volume of 35 ml, and the amount of liquid incorporated in the corresponding foams and therefore the foam density (Fig. 5) are higher. Furthermore, their liquid drainage characteristics (Fig. 6(a) and (b)) are very different. At pH 3,
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
415
Fig. 6. Effects of pH and temperature of prior treatments of ovalbumin solutions. Half-drainage time of liquid, t1/2 (a) and characteristic time of liquid drainage, T (s) (b); pH 7 (open symbols; pH 3 (filled symbols).
where the protein molecules in solution are more sensitive to orthokinetic aggregation (Fig. 1) and less resistant to thermal denaturation (Fig. 2), the foaming capacity and stability are much higher than at pH 7, where the protein molecules may be considered as more rigid. The original heat-treatment has a wide effect on the intrinsic viscosity but only a slight effect on the foam density at pH 3, whereas at pH 7 the bubbling time decreased [26] and the foam density and the intrinsic viscosity increased when solutions were pre-heated at temperatures higher than 68°C (Fig. 5). This temperature is close to that of the transition of secondary and tertiary structure changes (Fig. 2(b)) and it also corresponds to that of a slightly higher rate of decrease of protein surface tension (from pure solvent value), relative to unheated solutions (Fig. 4). The half-drainage time, t1/2 (Fig. 6(a)) and the time constant value, T(s), determined by the capillary method (Fig. 6(b)), are shown to have increased values (more than 100%) for temperature of pre-heating ranging from 75 to 80°C at pH 7, and decreased values (more than 30%) for solutions at pH 3 and T\ 50°C. The value of t1/2 obtained at T=50°C and pH 3 is not significantly changed relative to that obtained with unheated solutions, whereas T (s) values decreases. These results indicate that mild-heat treatments which denature the protein conformation in solution, without formation of a large percentage of
covalently bound aggregates as assumed in our previous study [12], can be used to enhance foaming properties of globular proteins.
4. Conclusion In this work, we have shown that heat-treatment of ovalbumin solutions leading to molecular species with mild-denatured structures can be used to improve the foaming properties of globular proteins that have an initially ‘rigid’ conformational state. At pH 3, heat-induced b-sheet structures are shown to be newly created before the occurrence of the DSC peak transition and in parallel with increasing viscosity. At pH 7 no significant development of b-sheets was detected, but a rather large decrease in the overall secondary and tertiary structures accompanied increased viscosity. When such stable molecular species are submitted to adsorption at the air/water interface, the long-term surface pressure values decreased with the temperature of pre-heating, while the rate of decrease of the surface tension from bulk solvent seemed to be slightly higher for pre-denatured proteins, as long as non covalently bound aggregates were assumed not to form, as demonstrated in a previous study [12]. Finally we showed that it is possible to stabilise foams with ovalbumin molecules in such heat
416
P. Relkin et al. / Colloids and Surfaces B: Biointerfaces 12 (1999) 409–416
denatured forms. Although we have not investigated by direct techniques, the re-conformation of adsorbed proteins nor their surface rheological properties, this study could contribute to more understanding of a possible relationship between the conformational state of adsorbing proteins and their resulting foaming properties.
Acknowledgements The financial supports of the Ministe`re de l’Agriculture et de la Peˆche (programme ‘Aliment demain’, R 94/51) and Danone (centre Jean The`ves) are gratefully acknowledged. Thanks also to Dr M-N. Bellon-Fontaine (INRA-Massy) and Dr Y. Popineau (INRA-Nantes) for their material and scientific help.
References [1] F. MacRitchie, A.E. Alexander, J. Colloid Interface Sci. 18 (1963) 458. [2] D.E. Graham, M.C. Phillips, J. Colloid Interface Sci. 76 (1980) 227. [3] E. Dickinson, in: E.J. Wilson (Ed.), Foams: Physics, Chemistry and Structure, Springer, Berlin, 1989, p. 39. [4] K. Kuwajima, Y. Hiraoka, M. Ikeguchi, S. Sugai, Biochemistry 24 (1985) 874. [5] H. Hirose, Trends Food Sci. Technol. 4 (1993) 48. [6] E. Dickinson, Y. Matsumura, Colloids Surf. B: Biointerfaces 3 (1994) 1.
.
[7] C.B. Tripp, J.J. Magda, J.D. Andrade, J. Colloid Interface Sci. 173 (1995) 16. [8] R. Wu¨stneck, J. Kra¨gel, R. Miller, V.B. Fainerman, P.J. Wilde, D.K. Sarker, D.C. Clark, Food Hydrocolloids 10 (1996) 395. [9] R.D. Waniska, J.E. Kinsella, J. Agric. Food Chem. 33 (1985) 1143. [10] Y. Mine, T. Noutomi, N. Haga, J. Agric. Food Chem. 38 (1990) 2122. [11] N. Hagolle, P. Relkin, D.G. Dalgleish, B. Launay, Food Hydrocolloids 11 (1997) 311. [12] N. Hagolle, B. Launay, P. Relkin, Colloids Surf. B. Biointerfaces 10 (1997) 191. [13] P. Relkin, Thermochim. Acta 246 (1994) 371. [14] W. Loisel W, European Patent, PCT/FR92/00509. [15] A. Baniel, A. Fains, Y. Popineau, J. Food Sci. 62 (1997) 377. [16] Y. Torikata, M. Kato, H. Kumagai, T. Yano, Agric. Biol. Chem. 55 (1991) 1307. [17] T. Koseki, N. Kitabatake, E. Doi, J. Biochem. 103 (1988) 425. [18] A. Kato, Y. Nagase, N. Matsudomi, K. Kobayasi, Agric. Biol. Chem. 47 (1983) 1829. [19] T. Matsumoto, J. Chiba, H. Inoue, Colloid Polym. Sci. 270 (1992) 687. [20] P.O. Hegg, H. Martens, B. Lofqvist, J. Sci. Food Agric. 30 (1979) 981. [21] P. Relkin, J. Int. Biol. Macromol. 22 (1998) 59. [22] P. Relkin, B. Launay, Food Hydrocolloids 4 (1990) 19. [23] I.A De Feijter, J. Benjamins, in: E. Dickinson (Ed.), Food Emulsions and Foams, Royal Society of Chemistry, London, 1986, p. 72. [24] A. Dussaud, G.B. Han, L. ter Minassian-Saraga, M. Vignes-Adler, J. Colloid Interfacial Sci. 167 (1994) 247. [25] P.J. Halling, CRC Crit. Rev. Food Sci. Nutr. 15 (1981) 155. [26] N. Hagolle, PhD Thesis, ENSIA, France, 1997.