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Temperature-dependent complexation between sodium caseinate and gum arabic
Food Hydrocolloids 26 (2012) 82e88
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Temperature-dependent complexation between sodium caseinate and gum arabic Aiqian Ye a, *, Patrick J.B. Edwards b, Janiene Gilliland a, Geoffrey B. Jameson a, b, Harjinder Singh a a b
Riddet Institute, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 June 2010 Accepted 11 April 2011
Complex formation between sodium caseinate and gum arabic as a function of temperature was investigated using dynamic light scattering, fluorescence and NMR. At neutral pH, the turbidity and the particle size increased when sodium caseinate and gum arabic mixtures were heated in situ at temperatures above a critical temperature. The increases in turbidity and particle size were reversible. This effect was considered to be due to hydrophobic interactions, leading to the formation of a complex between sodium caseinate and gum arabic. 1H NMR spectroscopy showed that ANS, which bound to caseinate at low temperatures in caseinate solution or a caseinateegum arabic mixture, was released at high temperatures upon formation of a caseinate or caseinateegum arabic complex. This supported changes observed in the fluorescence of 8-anilino-1-naphthalene sulfonate upon binding to caseinate, which decreased at high temperatures for caseinate alone or when sodium caseinate was mixed with gum arabic. Light-scattering (turbidity) and dynamic light-scattering studies show that the temperaturedependent complexation between sodium caseinate and gum arabic was sensitive to the mass ratio of protein to gum arabic (greater complexation at a 1:5 ratio than a 1:1 ratio) and the pH (maximum complexation at pH 6.5). Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Complexation Sodium caseinate Gum arabic Hydrophobic interaction Temperature
1. Introduction Proteins and polysaccharides are often used simultaneously in the food industry (Tolstoguzov, 1997), and knowledge of their interactions is of great importance in controlling the structure and texture of manufactured foods (Dickinson, 1995; McClements, 2002; Tolstoguzov, 1997). The overall interaction between two biopolymers is an average of the large number of different intermolecular forces arising between the various segments and side chains on the two macromolecules (Tolstoguzov, 1997). Depending on the aqueous environmental conditions and the distribution of the different types of groups (charged, hydrophobic etc.), the overall proteinepolysaccharide interaction may be net attractive or net repulsive. Attractive interactions vary widely in strength and specificity. A number of researchers have studied in vitro the complexation of proteins with synthetic and natural polyelectrolytes; this involves Coulombic (electrostatic) interactions, which depend on the pH of the solution, the pI of the proteins and the nature of the polysaccharides (Schmitt, Sanchez, Thomas, & Hardy, 1999; Weinbreck,
* Corresponding author. Tel.: þ64 6 350 5072; fax: þ64 6 350 5655. E-mail address: [email protected] (A. Ye). 0268-005X/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2011.04.004
de Vries, Schrooyen, & de Kruif, 2003; Ye, 2008; Ye, Flanagan, & Singh, 2006). Schmitt et al. (1999) and Weinbreck et al. (2003) studied the complex coacervation of whey proteins with gum arabic (GA). Ye et al. (2006) reported that the electrostatic interactions between sodium caseinate and GA under certain pH conditions give rise to complex nanoparticles in the size range from 100 to 200 nm. However, little work on another important attractive interaction, hydrophobic interaction, between proteins and polysaccharides has been reported. The relationship between electrostatic and hydrophobic interactions and the influence of temperature on proteine polysaccharide systems are yet to be fully elucidated. In foods, the nutritional quality of casein products is obviously an important property. However, caseinates are in particular demand as functional food ingredients, in which their surface properties predominate (Mulvihill & Fox, 1989). Sodium caseinate, produced through the addition of sodium hydroxide to acid casein, is a heterogeneous mixture of caseins, of which as1-, as2-, b- and k-casein represent approximately 38, 10, 36 and 12% respectively of whole casein (Fox & McSweeney, 2003). The caseins have a molecular mass of 20e25 kDa and contain high levels of proline. The presence of a high level of proline limits the formation of a-helices, b-sheets and b-turns. The caseins are also phosphorylated, but the degree of phosphorylation varies among the individual caseins. The
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phosphorylated residues are often found clustered in groups of two, three or four, and they provide considerable negative charge around neutral pH. The casein molecules are distinctly hydrophobic. Because of their lack of stable secondary and tertiary structures, most of their hydrophobic residues are exposed and consequently caseins are prone to aggregation. Sodium caseinate dissolves readily in water to give a translucent solution. This solution contains the protein itself and Naþ ions only. The latter tend to be localised close to the anionic regions of the protein (Ho & Waugh, 1965) and repulsive forces between the protein molecules dominate over the hydrophobic forces that tend to draw the proteins into associates. Thus pH, which affects the number of negative charges along the protein chain, and salt content, which affects the shielding of these ionic charges, can have substantial overall effects on associates. GA is a complex polysaccharide that is exuded from the African tree Acacia senegal (Anderson, Millar, & Weiping, 1991). It is an arabinogalactan-type polysaccharide that is composed of six carbohydrate moieties and a protein fraction. It has been suggested that this polysaccharide has a “wattle blossom”-type structure with a number of polysaccharide units linked to a common polypeptide chain (Connolly, Fenyo, & Vandeve1de, 1988; Dickinson, Elverson, & Murray, 1989; Wang, Burchard, Cui, Huang, & Phillips, 2008). This polysaccharide presents good emulsifying properties and a remarkably low viscosity (Osman, Menzies, Williams, Phillips, & Baldwin, 1993). GA is a weak polyelectrolyte that carries carboxyl groups, and microelectrophoretic measurements have shown that GA is negatively charged above pH 2.2. The charge density of the polyelectrolyte can be estimated from the molecular structure of GA to be one carboxylic group per 5 nm (Connolly, Fenyo, & Vandevelde, 1987). This peculiar composition confers to GA a charge density that is six times greater than that of a linear polysaccharide having the same composition (Vandevelde & Fenyo, 1987) and a good cold solubility because of the presence of residual charged groups and peptidic fragments (Phillips, Takigami, & Takigami, 1996). GA is a widely used polysaccharide in microencapsulation procedures, film formation and emulsion stabilisation (Islam, Phillips, Sljivo, Snowden, & Williams, 1997; Ray, Bird, Iacobucci, & Clark, 1995; Stephen & Churms, 1995). In this work, the temperature-dependent complexation between sodium caseinate and GA as a function of the mass ratio of protein to gum and the pH was examined in situ over a temperature range from 20 to 80 C and from pH 7 to pH 2. Increase in the turbidity and hydrodynamic size of particles in mixtures of sodium caseinate and GA induced by increasing temperature was reported at neutral pH (where both casein and GA are negatively charged). 2. Materials and methods 2.1. Materials Sodium caseinate (ALANATE 180) was supplied by Fonterra Cooperative Group Ltd, Auckland, New Zealand. This product contained w96% dry matter, of which about 94% was protein, 1.38% was sodium and 0.06% was calcium. GA was obtained from Bronson and Jacobs Ltd, Auckland, New Zealand. All the chemicals used were of analytical grade and were obtained from either BDH Chemicals (BDH Ltd, Poole, England) or Sigma Chemical Co. (St. Louis, MO, USA) unless specified otherwise. 2.2. Preparation of sodium caseinate and GA mixtures Sodium caseinate (1 wt%) and GA (10 wt%) powders were dispersed in Milli-Q water under gentle stirring at room temperature for 5 h and the dispersions were stored at 4 C overnight to
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allow complete hydration. Various concentrations of sodium caseinate and GA mixtures were obtained by diluting the stock dispersions in Milli-Q water. The concentration of the protein varied from 0.1 to 0.5%, and the concentration of GA was set to be from 0.01 to 5%. The initial pH of the sodium caseinate/GA mixture was adjusted to pH 7.0 with 0.1 M NaOH and 0.1 M HCl. The pH of the mixture was acidified by the addition of 1 M and 0.1 M HCl at room temperature. 2.3. Turbidity measurements Turbidity measurements were carried out with a 160A UVeVis spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at a wavelength of 480 nm. The samples were put in a cuvette of 1 cm pathlength, and their turbidity was measured as a function of temperature from 20 to 80 C. The measurements were repeated at least twice at each condition. 2.4. Particle size analysis Particle size (average hydrodynamic diameter) was measured by dynamic light scattering using a Nanosizer ZS (Malvern Instruments Ltd, Malvern, Worcestershire, England) with a He/Ne laser emitting at 633 nm and a 4.0 mW power source. The instrument used a backscattering configuration with detection at a scattering angle of 173 using an avalanche photodiode. The temperature of the cell was maintained at 20 0.5 C for the duration of the experiments. Average diffusion coefficients were determined by the method of cumulants and were translated into average hydrodynamic diameters using the StokeseEinstein relationship for spheres. The hydrodynamic diameter was calculated assuming that the diffusing particles were monodisperse spheres and was the result of the average of 13 measurements. 2.5. Fluorescence measurements Fluorescence was measured with a luminescence spectrophotometer Fluoro Max-4 (Horiba, Kyoto, Japan). Fluorescence emission spectra of 8-anilino-1-naphthalene sulfonate (bis-ANS, as 1 H NMR spectroscopy showed unequivocally that the material was dimerized) were measured as a function of temperature from 25 to 75 C. The samples were diluted threefold and were placed in 10 mm square quartz cells that were held at different temperatures in a water-jacketed cell holder attached to a temperaturecontrolled water bath. Each spectrum was determined after the mixture had attained temperature equilibrium. The excitation wavelength was 370 nm, and the emission spectrum was scanned from 385 to 650 nm using emission slits of 3 nm respectively at interval time of 1 s. Bis-ANS was dissolved (1 mg/ml) in degassed ethanol and were stored in the dark. Bis-ANS solutions were added to samples at 1:10 w/w. 2.6. NMR measurements Samples of sodium caseinate and/or gum arabic were made up in deuterium oxide or 10 mM phosphate buffer with 5% deuterium oxide both containing w1 mM 4,4-dimethyl-4-silapentane-1sulfonic acid (DSS) as an internal reference. Just prior to measurement 100 ml of 1% 4,40 -bis(1-anilinonaphthalene 8-sulfonate) (bisANS) in d4-methanol was added to 1 ml of each sample. After mixing, w0.6 ml of this solution was transferred to a standard 5 mm NMR tube. 1H NMR spectra for the bis-ANS/CAS/GA mixtures were recorded using a Bruker Avance 500 MHz NMR spectrometer equipped with a 5 mm probe. Spectra were recorded at 25 C before subsequently heating the samples and re-recording the spectra at
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elevated temperature. Temperature was regulated with a standard Bruker gas flow variable temperature unit. Excitation sculpting water suppression was used for samples in phosphate buffer. 3. Results and discussion 3.1. Change in turbidity The turbidity profiles (at 480 nm) of mixtures containing 0.1% wt sodium caseinate and different concentrations of GA (0e1.0%) at pH 7.0 as a function of temperature in situ during heating and cooling are shown in Fig. 1. Sodium caseinate solution without added GA displayed low turbidity at temperatures from 20 to w62 C, with a slight increase in turbidity at temperatures higher than 62 C. In addition, the turbidity of GA solutions did not change at all with change in the temperature (data not shown). Similar to sodium caseinate solution, the turbidity of the mixtures was very low until the temperature reached around 50 C but then increased markedly when the temperature was increased further (Fig. 1A). The increase in the turbidity with increasing temperature was more dramatic in the mixtures containing higher concentrations of GA.
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For example, the turbidity values of a mixture containing 1.0% GA were greater than those of mixtures containing 0.5% GA at high temperatures. Similar changes in turbidity were observed in mixtures of 0.5% sodium caseinate with different ratios of GA as a function of temperature during heating and cooling (Fig. 2). The changes were dependent mainly on the ratio of sodium caseinate to GA in the mixtures; for example, the mixture containing 0.5% sodium caseinate and 0.5% GA (1:1 in ratio) had a similar profile to the mixture containing 0.1% sodium caseinate and 0.1% GA (1:1), which showed a slight increase in turbidity at temperatures greater than 60 C. However, marked increases in turbidity were observed in the mixture with a 1:5 ratio of sodium caseinate to GA in both mixtures containing 0.1% caseinate and 0.5% caseinate when the temperature was greater than 60 C (Figs. 1A and 2A). In addition, the high turbidity of the mixtures at high temperatures decreased when the temperature was decreased and the
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turbidity became similar to the initial turbidity when the samples were cooled to ambient temperature (Figs. 1B and 2B). 3.2. Temperature dependence of particle size The particle sizes of mixtures of sodium caseinate and GA were determined by dynamic light scattering as a function of temperature (Fig. 3). The average hydrodynamic diameter of particles in the 0.1% sodium caseinate solution was about 90 nm, which remained constant at temperatures lower than 60 C and then increased to about 120 nm from 60 to 80 C. Compared with sodium caseinate solution, the particle size for sodium caseinate and GA mixtures started to increase at a lower temperature and increased to a larger size when the temperature was increased to 80 C. For the mixture containing 0.1% sodium caseinate and 0.1% GA (protein:GA ¼ 1:1), the average diameter was about 150 nm at 80 C. For the mixture containing 0.1% sodium caseinate and 0.5% GA (protein:GA ¼ 1:5), the particle size increased more dramatically. Fig. 3B shows the increase in the hydrodynamic diameters of particles in the mixture containing 0.5% sodium caseinate and GA with an increase in the temperature, indicating the mixtures containing higher concentrations of protein and GA formed very much
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larger particles at high temperatures (as well as larger particles at 25 C). These trends in particle size were consistent with the change in the turbidity of the samples (Figs. 1 and 2). The results indicate that complexation between sodium caseinate and GA occurred when the temperature of the mixtures increased to a critical point. This temperature-induced complex formation may be attributed to the hydrophobic interactions between sodium caseinate and GA. Because of their limited secondary structure, and the high content of hydrophobic amino acids, the hydrophobic bonding is the principal interactive force that is responsible for the endothermic self-assembly of b-casein, and thus the self-assembly is markedly temperature dependent (O’Connell, Grinberg, & de Kruif, 1997; Pearce, 1975). The amphipathic nature of casein, with distinct hydrophilic (N-terminal domain) and hydrophobic (C-terminal domain) parts, allows it to associate with other molecules with hydrophobic non-polar residues. At other side, GA, containing w2% protein (Islam et al., 1997), consists of, among other subunits, a glycoprotein and an arabinogalactan protein, which consist of a hydrophobic polypeptide chain (Whistler & BeMiller, 1993). Therefore, it might be suggested that the hydrophobic polypeptide chains of GA would bind with the hydrophobic non-polar residues of caseins at high temperatures to form complex composite particles. In mixture solutions of sodium caseinate and GA, at temperatures below about 50 C, strong electrostatic repulsions predominate over hydrophobic attractions, and hence no attractive interactions between sodium caseinate and GA take place. Increasing the temperature is known to increase the strength of hydrophobic interactions, and is likely to shift the balance of electrostatic repulsion and hydrophobic attraction in favour of hydrophobic attraction, leading to complex formation between sodium caseinate and GA. Therefore, the hydrophobic attraction between the non-polar segments of GA and protein provides the driving force for aggregation. In contrast, the repulsion forces provided by electrostatic or steric forces between the polar or charged fragments inhibit the formation of complexes and limit the increase in the size of the complexes. The greater extent of complexation in the mixtures containing high concentrations of protein and GA at high temperature can be attributed to the more hydrophobic non-polar segments being involved in the interaction. 3.3. Effect of pH
Fig. 3. Average size of mixtures of 0.1% (A) and 0.5% (B) sodium caseinate (NaCas) and different concentrations of gum arabic (GA) as a function of temperature at pH 7.0.
Turbidity profiles of mixture solutions (0.1% sodium caseinate and 0.5% GA) as a function of temperature are shown in Fig. 4, in which the mixtures were adjusted to different pH values. The changes in the turbidity of the mixtures with temperature were apparently affected by pH. At pH 6.0, the mixture displayed the largest increase in turbidity with increasing temperature and a reversible decrease in turbidity during cooling. This increase in turbidity with temperature was less when the pH of the samples was below 6.0. However, it is worth noting that the increase in turbidity of the mixture at pH 6.5 was less than that at pH 6.0. At pH 4.2, the turbidity of the mixture did not change with an increase in temperature, although the turbidity at 25 C was higher than that for mixtures of the same composition subjected to higher values of pH (Fig. 4A). Very little change in turbidity with temperature was observed at pH 2.0. Moreover, it is interesting to note that the turbidity of the sample at pH 5.0 increased when the temperature was higher than 50 C, but the increased turbidity did not return to the original value when the sample was cooled (Fig. 4B), indicating that irreversible complexation occurred at this pH. Stronger interaction and the formation of complexes at pH 6.0 and pH 5.6 support and confirm the hydrophobic interaction between sodium caseinate and GA when the temperature is increased. The
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To explore further the effect of temperature on the interaction between sodium caseinate and GA, the binding of bis-ANS to sodium caseinate was monitored as a function of temperature. Bis-ANS preferentially occupies solvent-accessible hydrophobic pockets or patches of proteins, producing a marked increase in the emission intensity and a large blue shift of the emission maximum, when compared with its emission wavelength in water (Hawe, Sutter, & Jiskoot, 2008). The fluorescent probe (ANS) provides information on the number and the affinity of binding sites (Considine, Patel, Singh, & Creamer, 2005). A plot of the maximum bis-ANS fluorescence intensity of the mixtures of sodium caseinate and GA versus the heating temperature is shown in Fig. 5. The fluorescence quantum yield of ANS in 10 mM phosphate buffer/64 mM NaCl solution (control) was unchanged with increasing temperature from 25 to 65 C and showed a very low fluorescence quantum yield and no change in the wavelength of the fluorescence maximum. Meanwhile, GA in 10 mM phosphate buffer with added bis-ANS also displayed no change in fluorescence intensity (nor in the wavelength of the fluorescence maximum) with increasing temperature. However, sodium caseinate with added bis-ANS showed strong fluorescence and a 25 nm blue shift at pH 7 and 25 C. Upon heating, the bis-ANS fluorescence intensity decreased, along with a red-shift towards but even at 75 C not equal to the wavelength of the fluorescent maximum of the controls, suggesting a change in the association of bis-ANS with caseinate upon the increasing micellisation of sodium caseinate with temperature shown by turbidity and DLS measurements. In the micellisation of sodium caseinate, N-terminal polar domains extend into the aqueous phase whereas long hydrophobic tails are oriented towards the hydrophobic micellar core; the hydrophobic peptide regions in the N-terminal polar domain may not make a significant contribution to the bis-ANS binding.
Fig. 4. Turbidity of mixtures of 0.1% sodium caseinate and 0.5% gum arabic as a function of temperature at different pHs. (A) Heating; (B) cooling.
electrostatic repulsion was lower at pH 6.0 and pH 5.6, which are close to the pI of the caseinate, than at higher pH (pH 6.5 and pH 7.0). Thus, the hydrophobic attractive force dominates over repulsive forces and leads to complexation between sodium caseinate and GA at lower temperatures. However, when the pH is close to or lower than the pI of casein proteins (about pH 5), the casein proteins carry more and more positive charges. It has been shown that electrostatic interaction between milk protein and GA occurs in this situation, leading to the formation of an electrostatic complex and thus resulting in high turbidity of the mixture (Ye et al., 2006). Electrostatic complexation is not a temperature-dependent interaction. However, the turbidity increased at temperatures > w45 C at pH 5.0, indicating that the complexation is enhanced at high temperature. This might be attributed to further associations between GA and protein molecules via hydrophobic interactions. Furthermore, the high turbidity observed at high temperature did not decrease when the temperature was decreased, suggesting that electrostatic interactions dominate complex formation under these conditions and that these interactions are not affected by temperature.
Fig. 5. Maximum ANS fluorescence intensity of samples as a function of temperature. ANS in 10 mM phosphate buffer/64 mM NaCl (-), 0.5% GA in 10 mM phosphate buffer (,), 0.1% sodium caseinate (NaCas) in 10 mM phosphate buffer (:) and mixtures of 0.1% sodium caseinate (NaCas) and 0.5% gum arabic (GA) (B) at pH 7.0.
A. Ye et al. / Food Hydrocolloids 26 (2012) 82e88
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Fig. 6. 1H NMR spectra of bis-ANS/CAS/GA mixtures. (a) 1% Sodium caseinate with 0.1% bis-ANS in unbuffered deuterium oxide, (b) 1% sodium caseinate with 0.1% bis-ANS in 10 mM phosphate buffer, (c) 5% gum arabic with 0.1% bis-ANS in 10 mM phosphate buffer, (d) 1% sodium caseinate with 5% gum arabic and 0.1% ANS in 10 mM phosphate buffer.
Mixing sodium caseinate with GA weakens slightly the fluorescence intensity at each temperature. With increasing temperature the ANS fluorescence intensity decreased steadily and the wavelength of maximum emission red-shifted, both changing at the same rate as for the caseinate solution alone (Fig. 5).
expelled from the caseinate and caseinateeGA aggregates at elevated temperature, consistent with a strong hydrophobic interaction of caseinate with itself and with the gum arabic.
3.5. NMR spectroscopy
Above a critical temperature, complexation through hydrophobic attraction occurred between sodium caseinate and GA. The temperature-dependent complexation was largely reversible (although not in case of pH 5.0) when the mixture was cooled, confirming non-covalent association of the caseinate and caseinateeGA particles. The ratio of protein to GA in the mixtures, pH and the concentrations of protein and GA influenced the temperature required for complexation and the particle size of the complexes. At pHs close to the pI of the protein, the formation of complexes between caseins and GA may be driven by both electrostatic and hydrophobic interactions at high temperatures. The findings from this work could be used to form novel, complex, composite particles that change with temperature. The particles could potentially be used to form complex surface layers of emulsion droplets.
Spectra from bis-ANS/caseinate/GA mixtures are shown in Fig. 6. In all samples containing caseinate (Fig. 6a, b, and d), the signals from the aromatic protons of bis-ANS (in the region w6.5e8.5 ppm) are significantly broadened at 25 C. Since line widths tend to increase with the correlation time of molecular tumbling, this indicates that the bis-ANS is interacting with the much larger caseinate molecules at low temperature. In contrast, the signals from the DSS reference at 0 ppm (another small molecule) remain sharp indicating that this molecule is tumbling freely in solution. At 75 C, the signals from the bis-ANS have narrowed significantly, in contrast to those from the caseinate, which remain relatively broad. This is consistent with bisANS no longer being bound to the caseinate aggregates. In the mixture of GA with bis-ANS (Fig. 6c) the aromatic signals from bisANS remain sharp at all temperatures indicating that the tumbling of the bis-ANS molecules is not affected significantly in the absence of caseinate, and that bis-ANS is not interacting to a significant extent with GA. These results augment the ANS fluorescence measurements, indicating that at elevated temperatures bis-ANS is
4. Conclusions
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Temperature-dependent complexation between sodium caseinate and gum arabic Aiqian Ye a, *, Patrick J.B. Edwards b, Janiene Gilliland a, Geoffrey B. Jameson a, b, Harjinder Singh a a b
Riddet Institute, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 June 2010 Accepted 11 April 2011
Complex formation between sodium caseinate and gum arabic as a function of temperature was investigated using dynamic light scattering, fluorescence and NMR. At neutral pH, the turbidity and the particle size increased when sodium caseinate and gum arabic mixtures were heated in situ at temperatures above a critical temperature. The increases in turbidity and particle size were reversible. This effect was considered to be due to hydrophobic interactions, leading to the formation of a complex between sodium caseinate and gum arabic. 1H NMR spectroscopy showed that ANS, which bound to caseinate at low temperatures in caseinate solution or a caseinateegum arabic mixture, was released at high temperatures upon formation of a caseinate or caseinateegum arabic complex. This supported changes observed in the fluorescence of 8-anilino-1-naphthalene sulfonate upon binding to caseinate, which decreased at high temperatures for caseinate alone or when sodium caseinate was mixed with gum arabic. Light-scattering (turbidity) and dynamic light-scattering studies show that the temperaturedependent complexation between sodium caseinate and gum arabic was sensitive to the mass ratio of protein to gum arabic (greater complexation at a 1:5 ratio than a 1:1 ratio) and the pH (maximum complexation at pH 6.5). Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Complexation Sodium caseinate Gum arabic Hydrophobic interaction Temperature
1. Introduction Proteins and polysaccharides are often used simultaneously in the food industry (Tolstoguzov, 1997), and knowledge of their interactions is of great importance in controlling the structure and texture of manufactured foods (Dickinson, 1995; McClements, 2002; Tolstoguzov, 1997). The overall interaction between two biopolymers is an average of the large number of different intermolecular forces arising between the various segments and side chains on the two macromolecules (Tolstoguzov, 1997). Depending on the aqueous environmental conditions and the distribution of the different types of groups (charged, hydrophobic etc.), the overall proteinepolysaccharide interaction may be net attractive or net repulsive. Attractive interactions vary widely in strength and specificity. A number of researchers have studied in vitro the complexation of proteins with synthetic and natural polyelectrolytes; this involves Coulombic (electrostatic) interactions, which depend on the pH of the solution, the pI of the proteins and the nature of the polysaccharides (Schmitt, Sanchez, Thomas, & Hardy, 1999; Weinbreck,
* Corresponding author. Tel.: þ64 6 350 5072; fax: þ64 6 350 5655. E-mail address: [email protected] (A. Ye). 0268-005X/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2011.04.004
de Vries, Schrooyen, & de Kruif, 2003; Ye, 2008; Ye, Flanagan, & Singh, 2006). Schmitt et al. (1999) and Weinbreck et al. (2003) studied the complex coacervation of whey proteins with gum arabic (GA). Ye et al. (2006) reported that the electrostatic interactions between sodium caseinate and GA under certain pH conditions give rise to complex nanoparticles in the size range from 100 to 200 nm. However, little work on another important attractive interaction, hydrophobic interaction, between proteins and polysaccharides has been reported. The relationship between electrostatic and hydrophobic interactions and the influence of temperature on proteine polysaccharide systems are yet to be fully elucidated. In foods, the nutritional quality of casein products is obviously an important property. However, caseinates are in particular demand as functional food ingredients, in which their surface properties predominate (Mulvihill & Fox, 1989). Sodium caseinate, produced through the addition of sodium hydroxide to acid casein, is a heterogeneous mixture of caseins, of which as1-, as2-, b- and k-casein represent approximately 38, 10, 36 and 12% respectively of whole casein (Fox & McSweeney, 2003). The caseins have a molecular mass of 20e25 kDa and contain high levels of proline. The presence of a high level of proline limits the formation of a-helices, b-sheets and b-turns. The caseins are also phosphorylated, but the degree of phosphorylation varies among the individual caseins. The
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phosphorylated residues are often found clustered in groups of two, three or four, and they provide considerable negative charge around neutral pH. The casein molecules are distinctly hydrophobic. Because of their lack of stable secondary and tertiary structures, most of their hydrophobic residues are exposed and consequently caseins are prone to aggregation. Sodium caseinate dissolves readily in water to give a translucent solution. This solution contains the protein itself and Naþ ions only. The latter tend to be localised close to the anionic regions of the protein (Ho & Waugh, 1965) and repulsive forces between the protein molecules dominate over the hydrophobic forces that tend to draw the proteins into associates. Thus pH, which affects the number of negative charges along the protein chain, and salt content, which affects the shielding of these ionic charges, can have substantial overall effects on associates. GA is a complex polysaccharide that is exuded from the African tree Acacia senegal (Anderson, Millar, & Weiping, 1991). It is an arabinogalactan-type polysaccharide that is composed of six carbohydrate moieties and a protein fraction. It has been suggested that this polysaccharide has a “wattle blossom”-type structure with a number of polysaccharide units linked to a common polypeptide chain (Connolly, Fenyo, & Vandeve1de, 1988; Dickinson, Elverson, & Murray, 1989; Wang, Burchard, Cui, Huang, & Phillips, 2008). This polysaccharide presents good emulsifying properties and a remarkably low viscosity (Osman, Menzies, Williams, Phillips, & Baldwin, 1993). GA is a weak polyelectrolyte that carries carboxyl groups, and microelectrophoretic measurements have shown that GA is negatively charged above pH 2.2. The charge density of the polyelectrolyte can be estimated from the molecular structure of GA to be one carboxylic group per 5 nm (Connolly, Fenyo, & Vandevelde, 1987). This peculiar composition confers to GA a charge density that is six times greater than that of a linear polysaccharide having the same composition (Vandevelde & Fenyo, 1987) and a good cold solubility because of the presence of residual charged groups and peptidic fragments (Phillips, Takigami, & Takigami, 1996). GA is a widely used polysaccharide in microencapsulation procedures, film formation and emulsion stabilisation (Islam, Phillips, Sljivo, Snowden, & Williams, 1997; Ray, Bird, Iacobucci, & Clark, 1995; Stephen & Churms, 1995). In this work, the temperature-dependent complexation between sodium caseinate and GA as a function of the mass ratio of protein to gum and the pH was examined in situ over a temperature range from 20 to 80 C and from pH 7 to pH 2. Increase in the turbidity and hydrodynamic size of particles in mixtures of sodium caseinate and GA induced by increasing temperature was reported at neutral pH (where both casein and GA are negatively charged). 2. Materials and methods 2.1. Materials Sodium caseinate (ALANATE 180) was supplied by Fonterra Cooperative Group Ltd, Auckland, New Zealand. This product contained w96% dry matter, of which about 94% was protein, 1.38% was sodium and 0.06% was calcium. GA was obtained from Bronson and Jacobs Ltd, Auckland, New Zealand. All the chemicals used were of analytical grade and were obtained from either BDH Chemicals (BDH Ltd, Poole, England) or Sigma Chemical Co. (St. Louis, MO, USA) unless specified otherwise. 2.2. Preparation of sodium caseinate and GA mixtures Sodium caseinate (1 wt%) and GA (10 wt%) powders were dispersed in Milli-Q water under gentle stirring at room temperature for 5 h and the dispersions were stored at 4 C overnight to
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allow complete hydration. Various concentrations of sodium caseinate and GA mixtures were obtained by diluting the stock dispersions in Milli-Q water. The concentration of the protein varied from 0.1 to 0.5%, and the concentration of GA was set to be from 0.01 to 5%. The initial pH of the sodium caseinate/GA mixture was adjusted to pH 7.0 with 0.1 M NaOH and 0.1 M HCl. The pH of the mixture was acidified by the addition of 1 M and 0.1 M HCl at room temperature. 2.3. Turbidity measurements Turbidity measurements were carried out with a 160A UVeVis spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at a wavelength of 480 nm. The samples were put in a cuvette of 1 cm pathlength, and their turbidity was measured as a function of temperature from 20 to 80 C. The measurements were repeated at least twice at each condition. 2.4. Particle size analysis Particle size (average hydrodynamic diameter) was measured by dynamic light scattering using a Nanosizer ZS (Malvern Instruments Ltd, Malvern, Worcestershire, England) with a He/Ne laser emitting at 633 nm and a 4.0 mW power source. The instrument used a backscattering configuration with detection at a scattering angle of 173 using an avalanche photodiode. The temperature of the cell was maintained at 20 0.5 C for the duration of the experiments. Average diffusion coefficients were determined by the method of cumulants and were translated into average hydrodynamic diameters using the StokeseEinstein relationship for spheres. The hydrodynamic diameter was calculated assuming that the diffusing particles were monodisperse spheres and was the result of the average of 13 measurements. 2.5. Fluorescence measurements Fluorescence was measured with a luminescence spectrophotometer Fluoro Max-4 (Horiba, Kyoto, Japan). Fluorescence emission spectra of 8-anilino-1-naphthalene sulfonate (bis-ANS, as 1 H NMR spectroscopy showed unequivocally that the material was dimerized) were measured as a function of temperature from 25 to 75 C. The samples were diluted threefold and were placed in 10 mm square quartz cells that were held at different temperatures in a water-jacketed cell holder attached to a temperaturecontrolled water bath. Each spectrum was determined after the mixture had attained temperature equilibrium. The excitation wavelength was 370 nm, and the emission spectrum was scanned from 385 to 650 nm using emission slits of 3 nm respectively at interval time of 1 s. Bis-ANS was dissolved (1 mg/ml) in degassed ethanol and were stored in the dark. Bis-ANS solutions were added to samples at 1:10 w/w. 2.6. NMR measurements Samples of sodium caseinate and/or gum arabic were made up in deuterium oxide or 10 mM phosphate buffer with 5% deuterium oxide both containing w1 mM 4,4-dimethyl-4-silapentane-1sulfonic acid (DSS) as an internal reference. Just prior to measurement 100 ml of 1% 4,40 -bis(1-anilinonaphthalene 8-sulfonate) (bisANS) in d4-methanol was added to 1 ml of each sample. After mixing, w0.6 ml of this solution was transferred to a standard 5 mm NMR tube. 1H NMR spectra for the bis-ANS/CAS/GA mixtures were recorded using a Bruker Avance 500 MHz NMR spectrometer equipped with a 5 mm probe. Spectra were recorded at 25 C before subsequently heating the samples and re-recording the spectra at
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elevated temperature. Temperature was regulated with a standard Bruker gas flow variable temperature unit. Excitation sculpting water suppression was used for samples in phosphate buffer. 3. Results and discussion 3.1. Change in turbidity The turbidity profiles (at 480 nm) of mixtures containing 0.1% wt sodium caseinate and different concentrations of GA (0e1.0%) at pH 7.0 as a function of temperature in situ during heating and cooling are shown in Fig. 1. Sodium caseinate solution without added GA displayed low turbidity at temperatures from 20 to w62 C, with a slight increase in turbidity at temperatures higher than 62 C. In addition, the turbidity of GA solutions did not change at all with change in the temperature (data not shown). Similar to sodium caseinate solution, the turbidity of the mixtures was very low until the temperature reached around 50 C but then increased markedly when the temperature was increased further (Fig. 1A). The increase in the turbidity with increasing temperature was more dramatic in the mixtures containing higher concentrations of GA.
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For example, the turbidity values of a mixture containing 1.0% GA were greater than those of mixtures containing 0.5% GA at high temperatures. Similar changes in turbidity were observed in mixtures of 0.5% sodium caseinate with different ratios of GA as a function of temperature during heating and cooling (Fig. 2). The changes were dependent mainly on the ratio of sodium caseinate to GA in the mixtures; for example, the mixture containing 0.5% sodium caseinate and 0.5% GA (1:1 in ratio) had a similar profile to the mixture containing 0.1% sodium caseinate and 0.1% GA (1:1), which showed a slight increase in turbidity at temperatures greater than 60 C. However, marked increases in turbidity were observed in the mixture with a 1:5 ratio of sodium caseinate to GA in both mixtures containing 0.1% caseinate and 0.5% caseinate when the temperature was greater than 60 C (Figs. 1A and 2A). In addition, the high turbidity of the mixtures at high temperatures decreased when the temperature was decreased and the
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turbidity became similar to the initial turbidity when the samples were cooled to ambient temperature (Figs. 1B and 2B). 3.2. Temperature dependence of particle size The particle sizes of mixtures of sodium caseinate and GA were determined by dynamic light scattering as a function of temperature (Fig. 3). The average hydrodynamic diameter of particles in the 0.1% sodium caseinate solution was about 90 nm, which remained constant at temperatures lower than 60 C and then increased to about 120 nm from 60 to 80 C. Compared with sodium caseinate solution, the particle size for sodium caseinate and GA mixtures started to increase at a lower temperature and increased to a larger size when the temperature was increased to 80 C. For the mixture containing 0.1% sodium caseinate and 0.1% GA (protein:GA ¼ 1:1), the average diameter was about 150 nm at 80 C. For the mixture containing 0.1% sodium caseinate and 0.5% GA (protein:GA ¼ 1:5), the particle size increased more dramatically. Fig. 3B shows the increase in the hydrodynamic diameters of particles in the mixture containing 0.5% sodium caseinate and GA with an increase in the temperature, indicating the mixtures containing higher concentrations of protein and GA formed very much
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larger particles at high temperatures (as well as larger particles at 25 C). These trends in particle size were consistent with the change in the turbidity of the samples (Figs. 1 and 2). The results indicate that complexation between sodium caseinate and GA occurred when the temperature of the mixtures increased to a critical point. This temperature-induced complex formation may be attributed to the hydrophobic interactions between sodium caseinate and GA. Because of their limited secondary structure, and the high content of hydrophobic amino acids, the hydrophobic bonding is the principal interactive force that is responsible for the endothermic self-assembly of b-casein, and thus the self-assembly is markedly temperature dependent (O’Connell, Grinberg, & de Kruif, 1997; Pearce, 1975). The amphipathic nature of casein, with distinct hydrophilic (N-terminal domain) and hydrophobic (C-terminal domain) parts, allows it to associate with other molecules with hydrophobic non-polar residues. At other side, GA, containing w2% protein (Islam et al., 1997), consists of, among other subunits, a glycoprotein and an arabinogalactan protein, which consist of a hydrophobic polypeptide chain (Whistler & BeMiller, 1993). Therefore, it might be suggested that the hydrophobic polypeptide chains of GA would bind with the hydrophobic non-polar residues of caseins at high temperatures to form complex composite particles. In mixture solutions of sodium caseinate and GA, at temperatures below about 50 C, strong electrostatic repulsions predominate over hydrophobic attractions, and hence no attractive interactions between sodium caseinate and GA take place. Increasing the temperature is known to increase the strength of hydrophobic interactions, and is likely to shift the balance of electrostatic repulsion and hydrophobic attraction in favour of hydrophobic attraction, leading to complex formation between sodium caseinate and GA. Therefore, the hydrophobic attraction between the non-polar segments of GA and protein provides the driving force for aggregation. In contrast, the repulsion forces provided by electrostatic or steric forces between the polar or charged fragments inhibit the formation of complexes and limit the increase in the size of the complexes. The greater extent of complexation in the mixtures containing high concentrations of protein and GA at high temperature can be attributed to the more hydrophobic non-polar segments being involved in the interaction. 3.3. Effect of pH
Fig. 3. Average size of mixtures of 0.1% (A) and 0.5% (B) sodium caseinate (NaCas) and different concentrations of gum arabic (GA) as a function of temperature at pH 7.0.
Turbidity profiles of mixture solutions (0.1% sodium caseinate and 0.5% GA) as a function of temperature are shown in Fig. 4, in which the mixtures were adjusted to different pH values. The changes in the turbidity of the mixtures with temperature were apparently affected by pH. At pH 6.0, the mixture displayed the largest increase in turbidity with increasing temperature and a reversible decrease in turbidity during cooling. This increase in turbidity with temperature was less when the pH of the samples was below 6.0. However, it is worth noting that the increase in turbidity of the mixture at pH 6.5 was less than that at pH 6.0. At pH 4.2, the turbidity of the mixture did not change with an increase in temperature, although the turbidity at 25 C was higher than that for mixtures of the same composition subjected to higher values of pH (Fig. 4A). Very little change in turbidity with temperature was observed at pH 2.0. Moreover, it is interesting to note that the turbidity of the sample at pH 5.0 increased when the temperature was higher than 50 C, but the increased turbidity did not return to the original value when the sample was cooled (Fig. 4B), indicating that irreversible complexation occurred at this pH. Stronger interaction and the formation of complexes at pH 6.0 and pH 5.6 support and confirm the hydrophobic interaction between sodium caseinate and GA when the temperature is increased. The
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To explore further the effect of temperature on the interaction between sodium caseinate and GA, the binding of bis-ANS to sodium caseinate was monitored as a function of temperature. Bis-ANS preferentially occupies solvent-accessible hydrophobic pockets or patches of proteins, producing a marked increase in the emission intensity and a large blue shift of the emission maximum, when compared with its emission wavelength in water (Hawe, Sutter, & Jiskoot, 2008). The fluorescent probe (ANS) provides information on the number and the affinity of binding sites (Considine, Patel, Singh, & Creamer, 2005). A plot of the maximum bis-ANS fluorescence intensity of the mixtures of sodium caseinate and GA versus the heating temperature is shown in Fig. 5. The fluorescence quantum yield of ANS in 10 mM phosphate buffer/64 mM NaCl solution (control) was unchanged with increasing temperature from 25 to 65 C and showed a very low fluorescence quantum yield and no change in the wavelength of the fluorescence maximum. Meanwhile, GA in 10 mM phosphate buffer with added bis-ANS also displayed no change in fluorescence intensity (nor in the wavelength of the fluorescence maximum) with increasing temperature. However, sodium caseinate with added bis-ANS showed strong fluorescence and a 25 nm blue shift at pH 7 and 25 C. Upon heating, the bis-ANS fluorescence intensity decreased, along with a red-shift towards but even at 75 C not equal to the wavelength of the fluorescent maximum of the controls, suggesting a change in the association of bis-ANS with caseinate upon the increasing micellisation of sodium caseinate with temperature shown by turbidity and DLS measurements. In the micellisation of sodium caseinate, N-terminal polar domains extend into the aqueous phase whereas long hydrophobic tails are oriented towards the hydrophobic micellar core; the hydrophobic peptide regions in the N-terminal polar domain may not make a significant contribution to the bis-ANS binding.
Fig. 4. Turbidity of mixtures of 0.1% sodium caseinate and 0.5% gum arabic as a function of temperature at different pHs. (A) Heating; (B) cooling.
electrostatic repulsion was lower at pH 6.0 and pH 5.6, which are close to the pI of the caseinate, than at higher pH (pH 6.5 and pH 7.0). Thus, the hydrophobic attractive force dominates over repulsive forces and leads to complexation between sodium caseinate and GA at lower temperatures. However, when the pH is close to or lower than the pI of casein proteins (about pH 5), the casein proteins carry more and more positive charges. It has been shown that electrostatic interaction between milk protein and GA occurs in this situation, leading to the formation of an electrostatic complex and thus resulting in high turbidity of the mixture (Ye et al., 2006). Electrostatic complexation is not a temperature-dependent interaction. However, the turbidity increased at temperatures > w45 C at pH 5.0, indicating that the complexation is enhanced at high temperature. This might be attributed to further associations between GA and protein molecules via hydrophobic interactions. Furthermore, the high turbidity observed at high temperature did not decrease when the temperature was decreased, suggesting that electrostatic interactions dominate complex formation under these conditions and that these interactions are not affected by temperature.
Fig. 5. Maximum ANS fluorescence intensity of samples as a function of temperature. ANS in 10 mM phosphate buffer/64 mM NaCl (-), 0.5% GA in 10 mM phosphate buffer (,), 0.1% sodium caseinate (NaCas) in 10 mM phosphate buffer (:) and mixtures of 0.1% sodium caseinate (NaCas) and 0.5% gum arabic (GA) (B) at pH 7.0.
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Fig. 6. 1H NMR spectra of bis-ANS/CAS/GA mixtures. (a) 1% Sodium caseinate with 0.1% bis-ANS in unbuffered deuterium oxide, (b) 1% sodium caseinate with 0.1% bis-ANS in 10 mM phosphate buffer, (c) 5% gum arabic with 0.1% bis-ANS in 10 mM phosphate buffer, (d) 1% sodium caseinate with 5% gum arabic and 0.1% ANS in 10 mM phosphate buffer.
Mixing sodium caseinate with GA weakens slightly the fluorescence intensity at each temperature. With increasing temperature the ANS fluorescence intensity decreased steadily and the wavelength of maximum emission red-shifted, both changing at the same rate as for the caseinate solution alone (Fig. 5).
expelled from the caseinate and caseinateeGA aggregates at elevated temperature, consistent with a strong hydrophobic interaction of caseinate with itself and with the gum arabic.
3.5. NMR spectroscopy
Above a critical temperature, complexation through hydrophobic attraction occurred between sodium caseinate and GA. The temperature-dependent complexation was largely reversible (although not in case of pH 5.0) when the mixture was cooled, confirming non-covalent association of the caseinate and caseinateeGA particles. The ratio of protein to GA in the mixtures, pH and the concentrations of protein and GA influenced the temperature required for complexation and the particle size of the complexes. At pHs close to the pI of the protein, the formation of complexes between caseins and GA may be driven by both electrostatic and hydrophobic interactions at high temperatures. The findings from this work could be used to form novel, complex, composite particles that change with temperature. The particles could potentially be used to form complex surface layers of emulsion droplets.
Spectra from bis-ANS/caseinate/GA mixtures are shown in Fig. 6. In all samples containing caseinate (Fig. 6a, b, and d), the signals from the aromatic protons of bis-ANS (in the region w6.5e8.5 ppm) are significantly broadened at 25 C. Since line widths tend to increase with the correlation time of molecular tumbling, this indicates that the bis-ANS is interacting with the much larger caseinate molecules at low temperature. In contrast, the signals from the DSS reference at 0 ppm (another small molecule) remain sharp indicating that this molecule is tumbling freely in solution. At 75 C, the signals from the bis-ANS have narrowed significantly, in contrast to those from the caseinate, which remain relatively broad. This is consistent with bisANS no longer being bound to the caseinate aggregates. In the mixture of GA with bis-ANS (Fig. 6c) the aromatic signals from bisANS remain sharp at all temperatures indicating that the tumbling of the bis-ANS molecules is not affected significantly in the absence of caseinate, and that bis-ANS is not interacting to a significant extent with GA. These results augment the ANS fluorescence measurements, indicating that at elevated temperatures bis-ANS is
4. Conclusions
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