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The effect of surfactants on the solubility, zeta potential, and viscosity of soy protein isolates
Food Hydrocolloids 18 (2004) 101–108 www.elsevier.com/locate/foodhyd
The effect of surfactants on the solubility, zeta potential, and viscosity of soy protein isolates Anupam Malhotra, John N. Coupland* Department of Food Science, The Pennsylvania State University, 103, Borland Lab., University Park, PA 16802, USA Received 27 April 2002; revised 6 October 2002; accepted 30 January 2003
Abstract The interactions of soy protein isolate (SPI, 5 wt%) with an anionic (sodium dodecyl sulfate, SDS) and a nonionic (polyoxyethylene sorbitan monolaurate, Tween 20) surfactant were studied as a function of pH. The solubility of the Tween 20 and surfactant free samples reached a minimum close to the isoelectric point of the protein (pH 4 – 5) whereas the SDS-containing sample was highly soluble over the whole range studied. The viscosity of the Tween 20-containing and surfactant-free samples were low (,3 mPa s) and largely pH independent while the SDS-containing samples were much more viscous, particularly at high pH (. pI) and high [SDS] (.critical micellar concentration (CMC)). The effect of the surfactants and pH on the z-potential of more dilute protein suspensions was measured by phase analysis light scattering. The charge on the SDS samples was high and negative at all pH values while the Tween 20 and surfactant-free samples showed a typical but distinct sigmoidal changes from positive to negative with increasing pH and a neutral point corresponding to the isoelectric point and the point of minimum solubility. The z-potential changed linearly with added SDS from a pH-determined value at 0 mM to approximately 2 50 mV at 7.5 mM. These observations are discussed in terms of the changing colloidal interactions of soy protein particles. q 2003 Elsevier Ltd. All rights reserved. Keywords: Soy protein; Solubility; Zeta-potential; Surfactants; Rheology
1. Introduction Soybean (Glycine Max) is a native crop of eastern Asia where it has served as an important part of the diet for centuries while soybeans produced in the US are used predominantly as animal food with only a small percentage used for human consumption (Wolf & Cowan, 1977). Until recently, soy was mainly eaten in Western cultures by vegetarians and various ethnic populations as foods such as tofu, tempeh, miso, and soy sauce. However, recent research has shown the anti-cancer and other health benefits of soy (Messina & Barnes, 1991), which has spurred research into better ways to use soy extracts as food ingredients. Soybeans contain around 40% protein, 35% carbohydrate, 20% lipids and 5% ash. Oil is the highest value soy product, but after extraction the defatted soy contains 50% protein, of which 90% is globular storage protein (Bravo, 1987) with significant potential as a food ingredient. * Corresponding author. Tel.: þ 1-814-865-2636; fax: þ1-814-863-6132. E-mail address: [email protected] (J.N. Coupland). 0268-005X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0268-005X(03)00047-X
Soy protein can be fractionated into four main groups on the basis of sedimentation velocity as 2S, 7S, 11S, and 15S (Rhee, 1994). The most abundant soy proteins (, 70%) are the globulins, which are largely glycinin (11S) and conglycinin (globulin portion of 7S). Soy proteins are extracted from the beans, partially purified and concentrated as various soy protein flours (40 – 54% protein), concentrate ($ 70% protein), and isolates ($ 90% protein) with differing functional properties and commercial value. We are most concerned with soy protein isolate (SPI), which is typically manufactured by grinding the defatted beans in an alkali solution (pH , 8) to selectively solubilize a protein fraction. The soluble fraction is separated and the protein is precipitated and separated by lowering the pH to its isoelectric point (pH , 4.5) and dried (Puppo & Anon, 1998). The sample is frequently heated during this process to induce different degrees of protein denaturation. SPI can be added to foods to improve their nutritional quality (including both essential amino acids and other associated micronutrients), to act as a low cost bulking agent, and to provide desirable functional properties.
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The functional properties of proteins are those chemical and physical properties that affect food systems during processing, storage, preparation and consumption and are determined by the chemical structure of proteins and their interactions with other food components (Bravo, 1987). Functional properties include structure formation (e.g. emulsification, foam or film formation) and texture modification (e.g. gelation and thickening). Soy proteins are frequently incorporated into the product to stabilize fat emulsions, to improve viscosity, to gel and to improve water retention (Morr, 1987). Many functional attributes of proteins require their incomplete precipitation from water. For example, in order to form a gel a protein must first form a well dispersed mixture in the solvent then form limited protein– protein interactions to create a solid-like behavior. When the protein cannot adequately form a solution (that is to say when the protein– protein interactions are much stronger than combination of protein –solvent interactions and the free energy of mixing) it cannot proceed to form a strong gel (Kinsella, 1979). Frequently, the functional properties of soy proteins are limited by their relatively poor solubility, particularly close to the isoelectric point (Kinsella, 1979). Surfactants have been used to improve the solubility of poorly soluble materials including proteins. Nakai, Ho, and Tung (1980) showed that anionic surfactants were effective in solubilizing seed proteins. The dispersibility of SPI in sodium dodecyl sulfate (SDS) solution was 95 and 98% at pH 7.0 and 8.2, respectively, as compared to 71 and 82% for the controls. They postulated that there was an interaction between hydrophobic groups of SDS and hydrophobic sites of the soy protein, which led to an increase of negative charge on the protein causing an increase in interparticle electrostatic repulsion. It is well known that certain surfactants denature and precipitate proteins, which is indicative of strong surfactant –protein binding (Greener, Contestable, & Bale, 1987). However, the functional properties of the surfactant – protein complexes are less well understood. Greener and co-workers (1987) used various rheological methods to study the interactions of anionic surfactants with gelatin (pH . pI). For some systems above a critical surfactant concentration, the viscosity increased several orders of scale suggesting strong complex formation. They concluded that the extent of thickening was closely related to surfactant type, surfactant/gelatin composition and ionic strength and that the mechanism responsible for the thickening was micelle-mediated cross-links between peptide chains. Dreja, Heine, Tieke and Junkers (1996) demonstrated the importance of the effect of pH on the viscosity of gelatin– surfactant solutions. However, these workers explained their results using a different approach, arguing the viscosity changes were due to electrostatically driven changes in polymer conformation after binding highly charged micelles. Howe, Wilkins, and Goodwin (1992) found that
nonionic surfactants had little effect on the viscosity of gelatin solutions. In this work, different amounts of surfactant (anionic and uncharged) were added to a commercial sample of SPI and the viscosity and solubility of the complexes were measured over a range of pH. The z-potentials of similar, but more dilute, samples were measured to provide details of the colloidal properties of the system useful in interpreting our observations. By studying a single commercial protein isolate and two types of surfactant under a range of conditions using a number of techniques we seek to demonstrate the main interactions involved. A secondary and more long-term objective is to develop a new approach to the study of poorly soluble and poorly characterized protein powders. It is our contention that this large class of ingredients is better considered as suspensions of colloidal particles than through the traditional approaches of protein chemistry (Boulet, Britten, & Lamarche, 1998, 2000). The recently developed technique of phase analysis light scattering (PALS), applied here for the first time to the study of protein functionality, provides a valuable tool to characterize these experimentally difficult systems.
2. Materials and methods SPI was donated by the Archer Daniels Midland Company (Pro-Fam 974, Decatur, Il); SDS and polyoxyethylene sorbitan monolaurate (Tween 20) were purchased from the Sigma Chemical Company (St Louis, MO). According to the product literature, Pro-Fam 974 is 6.5% moisture (maximum), 90% protein (by Kjeldahl), 1% fat (by ether extraction) and 5% ash. It is recommended for use in milk replacers. All other reagents were purchased from Fisher Scientific Company (Fair Lawn, NJ). The SPI was dispersed (5 wt%) in SDS (0 – 5 wt%) or Tween 20 (0 – 5 wt%) solutions and stirred for 24 h to allow complete hydration. In some experiments, the pH was adjusted (1 –8) with small volumes of sodium hydroxide or hydrochloric acid. The soy formed an opaque suspension rather than a true solution in the solvents. Preliminary optical microscopy studies revealed a heterogeneous mixture of irregularly shaped particles with several in the 10– 100 mm range. There were no detectable thermal transitions on heating SPI and SPI– surfactant complexes in a differential scanning calorimeter that might have corresponded to denaturation or complex melting. From these observations we conclude that the proteins were largely denatured during the manufacturing process and that the clumps formed on drying do not readily re-disperse in aqueous solvents. 2.1. Rheology The shear viscosity measurements were carried out a controlled strain rheometer (Rheometrics Fluid Spectrometer
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II, Piscataway, NJ) operating in concentric cylinder geometry (cylinder length 13 mm, inner diameter 16.5 mm, outer diameter 17.0 mm). Samples were equilibrated at the analysis temperature (30 8C), gently mixed, and then poured into the instrument. The instrument had previously been equilibrated at 30 8C and the test was run immediately. The rate of rotation of the outer cylinder was increased from 10 to 3000 s21 and the torque on the inner cylinder was measured and used to calculate viscosity at each shear rate. Although some of the samples had a tendency to precipitate during analysis, this process was slow compared to the time required for analysis (, 100 s) and reproducible measurements could be made. 2.2. Solubility Soy protein solubility was determined using a variation of a method described by Wang and Zayas (1991). The insoluble soy protein was separated from the soluble fraction by gentle centrifugation (1000 rpm, 5 min) and the protein concentration in the original sample and the supernatant after centrifugation was determined using a Kjeldahl method (AOAC International, 1995) (Preliminary studies showed the surfactants used here did not interfere with the assay. Several colorimetric protein assays attempted did not work in the presence of SDS.). The protein solubility was calculated as Solubility ¼
Psupernatant £ 100% Ptotal
ð1Þ
where P is the protein concentration in the phase designated by the subscript. It is important to be aware that solubility measured by this method is a very empirical parameter—slight variations in the centrifugation or hydration conditions would cause a change in apparent solubility. Solubility in this work means only the proportion of protein that was not precipitated under these conditions.
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ions move with the particle and thus the potential determined is not that at the surface but rather at a short, undefined distance into the diffuse layer—the z-potential. Surface charge in protein particles is due to the partial ionization of various amino acid residues (Vanapalli & Coupland, 2000). The effective charge on a protein particle is affected by pH, ionic strength and the accumulation of ligands or surfactants at the interface. Zeta potential was measured by PALS. In this method a laser beam is split and a portion used to generate a scattering signal (at 158) from a dilute suspension of particles moving in a sinusoidally oscillating voltage. The second portion has a finite frequency modulation applied to it and it is recombined with the scattered light. Analysis of the time dependency of the signal phase is sensitive to the electrically induced rate of motion and hence electrophoretic mobility of the particles. More details on the mathematical (Miller, Schatzel, & Vincent, 1991) and practical basis (Tscharnuter, McNeil-Watson, & Fairhurst, 1996) of PALS are available in the literature. PALS has been used to measure the electrophoretic mobility (a parameter related to z-potential) of aqueous solutions of globular proteins (Vanapalli & Coupland, 2000). Protein solutions (0.05 wt%) were prepared in water or different surfactant solutions (SDS 5 wt%; Tween 20 5% wt%). The solutions were placed in a standard foursided, 1 cm polystyrene cuvette and a parallel plate electrode (0.45 cm2 square platinum plates with a 0.4 cm gap) was inserted. The cuvette was placed in a temperaturecontrolled holder (25 8C). The electrophoretic mobility was measured by PALS (ZetaPALS, Brookhaven Instruments, Holtsville, NY). Each measurement was the average of 50 (five sets of 10) measurements and the entire experiment was conducted in triplicate. The z-potential was calculated from the electrophoretic mobility using the Smoulokowski model (assuming the double layer thickness is much less than the particle size) (Hunter, 1986).
2.3. Zeta potential 3. Results Colloidal particles accumulate charge at their surface that can be expressed as a surface potential. Surface potential is an important factor for determining the magnitude of charged-based colloidal interactions of a particle, most crucially electrostatic repulsion of other like charged particles. The surface charge on a particle perturbs the ionic distribution in the medium surrounding it. First a layer of tightly bound counter-ions (i.e. of opposing charge) accumulates at the particle surface, the Stern layer, and beyond this a region of decaying excess concentration, the diffuse layer, extends a considerable distance (, nm) into the surrounding aqueous media (Hunter, 1986). Measuring the colloidal charge typically involves applying an electrical voltage to the particle and measuring the speed of movement induced. In practice, one or more layers of hydrated
3.1. Rheology Fig. 1 shows representative data for the dependency of apparent viscosity on applied shear rate for the SPI and SPI – surfactant systems (Preliminary measurements on pure surfactant solutions at these concentrations were below the sensitivity of the instrument.). In all cases, the solutions were shear thinning and the viscosity of SDS – SPI solution is much higher than SPI –Tween 20 and SPI solutions at all shear rates. For example for 5 wt% SDS (pH 6.3) the apparent viscosity decreased from 32.3 mPa s at 160 s21 to 18.9 mPa s at 2500 s21. Similarly for 5 wt% Tween 20 (pH 6.0), the viscosity decreased from 5.0 mPa s (160 s21) to 3.6 mPa s (2500 s21) and for the surfactant-free solution (pH 5.6) viscosity decreased from 6.4 mPa s (160 s21) to
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Fig. 1. Representative plots of viscosity as a function of applied shear rate for (X) 5 wt% SDS–SPI, (W) 5 wt% Tween 20– SPI and (P) surfactant-free SPI solutions at pH , 6.
3.5 mPa s (2500 s21). The shear thinning curves could not be adequately characterized by a power-law equation. The viscosity measured at high shear was more reproducible than those at lower shear (e.g. the standard error for the 5 wt% SDS sample was 5% at 16 s21, 0.7% at 1000 s21). Consequently the apparent viscosity at 1000 s21 was used henceforth to compare the rheology of the various samples. The viscosities of 5 wt% SDS, 5 wt% Tween 20, and surfactant-free samples are shown as a function of pH in Fig. 2a. The viscosity of the 5 wt% SDS sample increased with pH (e.g. at pH 7.9 viscosity was highest (28.7 mPa s) but gradually decreased to 9.6 mPa s at pH 2.4). At pH , 6 the viscosity was 22.8 mPa s for the 5 wt% SDS sample but only 1.9 mPa s for the surfactant-free or Tween 20 samples. In the surfactant-free samples the viscosity (, 3.0 mPa s) was effectively independent of pH but the Tween 20 samples were slightly more viscous under acid conditions. The effect of surfactant concentration on protein viscosity at pH , 4.4 (, pI) and pH 6.4 (. pI) is shown in Fig. 2b. The viscosities of the Tween-containing samples was low (, 3 mPa s) and largely independent of surfactant concentration, while the SDS-containing samples became more viscous with increased [SDS]. At low [SDS] the viscosity was similar to the corresponding Tween-containing samples, but at approximately 2 wt% (i.e. 8.7 mM) the viscosity of the high pH sample began to increase rapidly with further additions of surfactant. 3.2. Solubility The solubilities of soy protein and soy protein – surfactant complexes are shown as a function of pH in Fig. 3. The SDS samples were highly soluble (. 90%) at all pH values whereas the Tween 20 and surfactant-free samples showed a typical U-shaped pH – solubility profile, with a minimum of , 10% soluble at pH 3.5 – 4.5. At all pH values the solubility of the SDS samples was greatest and
Fig. 2. (a) Viscosity as a function of pH for (X) 5 wt% SDS–SPI, (W) 5 wt% Tween 20–SPI and (P) surfactant-free SPI solutions. (b) Viscosity of 5 wt% SPI suspensions as a function of (X, W) SDS or (B, A) Tween 20 concentration. Open points pH 4.4 ^ 0.4, filled points pH 6.4 ^ 0.4.
there were relatively minor differences between Tween 20 and surfactant-free samples. 3.3. Zeta potential Light scattering studies can only be performed in optically clear suspensions so it was necessary to make these measurements at much lower protein concentrations than were used in the rheological and solubility studies. To account for the changes in protein concentration we could either maintain the same surfactant concentrations and thus get unrealistic protein:surfactant ratios or we could commensurately reduce the surfactant concentrations and potentially reduce the surfactant:water ratio to close to the CMC. In either case, the results of the different methods will not be directly comparable and care must be taken in interpreting the data. The z-potentials of 5 wt% SDS –SPI, 5 wt% Tween 20– SPI, and surfactant-free samples are shown as a function of pH in Fig. 4a. The z-potential of the SDS – SPI complex
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Fig. 3. Solubility as a function of pH for (X) 5 wt% SDS– SPI, (W) 5 wt% Tween 20–SPI and (P) surfactant-free SPI solutions.
was strongly negative (approx. 2 50 mV) and largely independent of pH. Both the Tween 20 and surfactantfree samples were negative at high pH values and increased and became positive as the pH was decreased.
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The magnitude of the z-potential of the Tween 20 samples was lower than that of the surfactant-free samples at all values of pH, except for the neutrality point (pH , 4.1)— close to the established isoelectric point of soy globulins (pH 4 –5) (Smith & Circle, 1978). The charge on a protein can be estimated from the sum of the charges on the individual amino acid residues using the Henderson –Hasselbach equation. The total charge on soy protein was calculated as a function of pH using literature data for amino acid composition and the pK values of amino acid side chains listed in Table 1. Over the pH range considered, the net charge is dominated by a positive contribution from lysine and histidine and increasingly (with increasing pH) negative contributions from aspartic and glutamic acid. Other amino acids carry charge but are not present on an adequate mass basis to influence the total charge. The net charge decreased sigmoidally with increasing pH passing through zero at pH 4.3, again similar to the isoelectric point of soy globulins (pH 4 – 5) (Smith & Circle, 1978). The measured z-potential of soy proteins (using the Henderson – Hasselbach approach set out earlier) correlated well with the calculated charge: z-potential (mV) ¼ 39.1 £ net calculated charge (mol kg21) 2 2.5, r 2 ¼ 0:96: The good agreement between measured and calculated charge for the surfactant-free SPI suggests strongly that this method is indeed sensitive to the electrical properties of the complex colloids present here and is an appropriate method to track the charge-based interactions of soy protein and surfactants. However the correlation is not perfect, in particular around pH 6, which may be due to the likelihood that the scattering particles do not have the same amino acid composition as average soy protein or that the mean pK values selected are not reliable. Alternatively, some charged groups may be buried in the protein core and do not contribute to the surface charge. Fig. 4b shows the effect of surfactant concentration on the z-potential of SPI at selected values of pH close to, or significantly above, the isoelectric point. As expected, the Table 1 Calculations of the charge on soy protein Concentrationa (mol kg21 protein)
Aspartic acid Glutamic acid Histidine Lysine Totald Fig. 4. (a) z-Potential as a function of pH for (X) 5 wt% SDS–0.05 wt% SPI, (W) 5 wt% Tween 20–0.05 wt% SPI and (P) surfactant-free 0.05 wt% SPI solutions. Solid line is the calculated net charge on soy protein. (b) zPotential of 0.05 wt% SPI suspensions as a function of (X, W) Tween 20 or (B, A) SDS concentration. Filled points pH , pI ( ¼ 4.4), open points pH . pI (6.4 ^ 0.1 for Tween 20 samples and 7.9 ^ 0.1 for SDS samples).
a
0.77 1.19 0.15 0.36
pK b
4.6 4.6 7.0 10.2
Net chargec (mol kg21 protein) pH 7.9
pH 6.4
pH 4.4
20.77 21.19 0.02 0.36
20.75 21.17 0.12 0.36
20.30 20.46 0.15 0.36
21.58
21.44
20.25
US Department of Agriculture (1999). Damodaran (1996). The pK values used are those of the amino acid residues. c Calculated using the Henderson–Hasselbach equation (see text for details). d Other amino acids contributed insignificant charge to the total. b
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protein at its isoelectric point is close to neutral and the basic protein is strongly negatively charged. As SDS is added to the neutral protein, the charge decreases linearly to approximately 2 40 mV at approximately 7.5 mM surfactant (i.e. 1.5 mM SDS g21 protein). When SDS is added to the negative protein the charge increases slightly to a similar value at a similar surfactant concentration, but the magnitude of the changes are relatively small compared to the error in the measurements. Adding Tween 20 caused a very small increase in z-potential of SPI.
4. Discussion These results indicate some clear differences between the SDS and Tween 20 or surfactant-free samples. The viscosities of the SDS samples are high and pH dependent whereas the other samples are not. The solubilities and zpotentials of the SDS samples are high and pH-independent while the values for the other samples are lower and pH dependant. Viscosity is independent of low SDS concentrations but, depending on the pH, significantly increases at higher concentrations. The z-potential changes on the addition of moderate amounts of SDS to a pH-independent plateau but is largely unaffected by added Tween 20. Plotting solubility as a function of viscosity (Fig. 5) reveals trends in the data (considering only the 5 wt% surfactant data at all measured pH values). Increasing the viscosity is associated with an increase in solubility up to close to 100%. Further changes in viscosity beyond a critical point (, 7 mPa s) cause no further changes in solubility. The changes in solubility with viscosity (Fig. 5) seem to follow a single function independent of surfactant type. Both viscosity and z-potential could be expected to increase the tendency of protein particles to remain in suspension for different reasons. First, it would be harder for protein particles to sediment through a thicker solution.
Fig. 5. Solubility as a function of viscosity for (X) 5 wt% SDS–SPI, (W) 5 wt% Tween 20 –SPI and (P) surfactant-free SPI solutions. Measurements were made at a range of pH values.
Despite the fact that the continuous phase viscosity does not change, as would be required for a classic Stokes law explanation of the process, a common analogous observation is that highly flocculated emulsions are often resistant to creaming. Second, increasing the surface charge on colloidal particles increases the magnitude of inter-particle electrostatic repulsion (Walstra, 1996), which will tend to disrupt existing protein aggregates and discourage further aggregate formation. According to Stokes law the rate of sedimentation of a particle is proportional to the square of its diameter (Walstra, 1996) and so a smaller particle would sediment less rapidly. The question remains—why do surfactants and pH changes have such a large effect on the z-potential and viscosity of soy protein solutions? SDS is a charged surfactant so it can interact with a protein both electrostatically (through its sulfate group) or hydrophobically (via its alkane tail-group), while Tween 20 can only interact hydrophobically. While the absolute charge on SPI changes with pH (Fig. 4a), there are some positive charges present over the entire pH range considered (Table 1) so electrostatic interactions are possible in all cases. Damodaran and Kinsella (1986) showed that electrostatic interactions are important in forming complexes between soy proteins and lysozyme, and Utsumi and Kinsella (1985) showed that both electrostatic and hydrophobic interactions (as well as hydrogen and disulfide bonding) could all play a role in forming soy protein gels. The effects of SDS are most dramatic so we will first attempt to create a model for charged surfactant –protein interactions and then more briefly explore the different effects seen with Tween. The major experimental observations from the SDS – SPI complexes in our studies that should be incorporated into any model are: Observation 1. Solubility is high and pH independent (Fig. 3), Observation 2. z-potential is highly negative and pHindependent (Fig. 4a), Observation 3. At low pH, z-potential becomes more negative with [SDS] and reaches a plateau , 0.2 wt%. At high pH, z-potential is largely pH independent (Fig. 4b), Observation 4. Viscosity increases with [SDS] . 2 wt% if pH . pI and with pH (again if pH . pI) (Fig. 2). Observations 1 and 2 appear to be related. The surfactant binds to the protein and, because the sulfate group is not protonated over this pH range, maintains a strong pHindependent negative charge. The highly charged complex better interacts with water and is therefore soluble. The z-potential measurements that form the basis of Observation 3 relate to the binding of monomeric SDS ([SDS] p CMC). Monomeric surfactant can bind electrostatically or hydrophobically; electrostatic binding would eliminate one net positive charge from the protein and hydrophobic binding would add one net negative charge. If
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electrostatic bonding were solely responsible, the net charge on the proteins at high [SDS] would be zero, while if hydrophobic binding were responsible it would be negative. The net charge at high [SDS] seen in this work is not only negative (Fig. 4, i.e. implying hydrophobic surfactant binding), but also pH-independent (Observation 2) suggesting that there is some critical level of charge that can be carried on a single protein beyond which no further surfactant can bind. Observation 2 implies that beyond a certain (low) level of surfactant the charge on the proteins is pH-independent. However in Observation 4, we note that only for the high pH samples there is an increase in viscosity and so there must be some distinction in their behaviors. Interestingly the point of divergence in the viscosity data for the high pH system (Fig. 2b) is at approximately 2 wt% or 8.7 mM SDS—reasonably close to the aqueous CMC of SDS ( ¼ 8.1 mM) so it seems likely that the micelles are responsible for the viscosity increase. A micelle has a high charge density, so when a protein binds a micelle there would be an electrostatic pressure to physically disperse the charge as widely as possible. Although we have argued that the charges are similar and pH independent before micelle binding, the negative charges on the anionic protein are more due to amino acid charges than those on the cationic protein which are more due to bound monomeric SDS. Therefore the anionic protein can only disperse its charge by expanding its structure while the cationic protein can displace some bound monomers. Consequently, the anionic protein would have an increased hydrodynamic radius, and hence viscosity, while the cationic protein would be unchanged. This reasoning is a variation of the ‘conformational change’ model proposed by Dreja and co-workers (1996) (although we are unable to say if the micelle is electrostatically or hydrophobically bound). However it would be possible to make a similar argument based on the ‘cross-linking model’ proposed by Greener and co-workers (1987). In this case, the observed changes in viscosity could be due to the acidic or basic protein binding micellar surfactant via its localized positive groups. The basic SPI has relatively few positive charges, so the same micelle could simultaneously bind several peptide chains and serve as inter-molecular links and thus lead to an increase in viscosity (fig. 13d in Greener et al., 1987). The acidic protein has more positive charges and therefore each protein would tend to wrap around any bound micelles acting as intra- rather than inter-molecular links (fig. 13b in Greener et al., 1987) and therefore not increase the viscosity. In reality it seems probable that a combination of the ‘conformational change’ and ‘cross-linking’ are responsible for the effects of SDS on SPI rheology. The Tween 20 samples have properties distinct from both the SDS and surfactant-free samples. While the viscosity of the soy was not significantly affected by addition of Tween 20 (Fig. 2), the z-potential was lower than that of the surfactant-free samples at all pH values (Fig. 4). Tween 20
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is a non-ionic surfactant and so can onlt interact hydrophobically with a protein. The amount of Tween 20 bound to the protein surface should therefore be independent of pH and would have little impact on protein conformation nor could it lead to protein association and there would be no increase in viscosity. However, binding surfactant would shield the hydrophobic regions of the protein from the solvent and therefore increase solubility. Bound Tween 20 would not in itself affect the absolute charge on the protein, but during a z-potential measurement could move with the protein particle in the applied electrical field. The charges present on the protein would then be further away from the plane of shear, so the measured charge, the z-potential, would be reduced. Even though the Tween 20 is to some extent reducing the effective charge on the protein, it is providing a much more hydrophilic surface and there is little loss in solubility.
5. Conclusion One of the most important limitations of soy protein as a food ingredient is its poor solubility at pH , pI. This is because the charge on the particles is insufficient to prevent extensive aggregation (presumably driven by a hydrophobic mechanism) and the aggregates rapidly sediment. Charged surfactants are capable of maintaining a pH-independent, negative charge on the proteins so their complexes are highly soluble even at the isoelectric point. In practice it is impossible to add sufficient levels of SDS to have this effect in a real food systems but there are several other food molecules with the right characteristics, i.e. a localized, pHindependent charge and a tendency to form hydrophobically driven, self assembled structures (e.g. lecithin and other phospholipids or lactic or acetic acid esters of mono-and diglycerides). The ideal solubilizing molecule will also have a bland taste, be permissible in foods, hopefully have an acceptable label declaration, and be reasonably cheap. Soy protein is representative of a group of food ingredients that are polymeric, chemically complex and poorly soluble. Prominent in this group are the protein concentrates and flours extracted from various sources. The behavior of these ingredients is difficult to study in that classical protein chemistry is most often concerned with polymers in solution In this work we have instead taken the approach of Boulet and co-workers (1998, 2000) and considered functionality in terms of the soy proteins as colloidal particles. These workers argue that in many real food systems the quaternary aggregates of proteins are functional particles and are a more useful way to represent the properties of the system than studies on dilute monomers. In our case some of the particles in the commercial preparation are even larger than those described by Boulet and others (1998, 2000) and thus presumably more readily described in terms of their particle-like behavior.
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These large particles could not be well characterized by the photon correlation spectroscopy technique used by Boulet et al. (1998, 2000) but their effective charge could be readily measured by PALS. Large particles scattering light more efficiently than smaller ones so the PALS technique will be most sensitive to them and the Smoulokowski approach used to convert the mobilities to charges is insensitive to absolute size. In the context of this work it is likely the large particles that will sediment the most rapidly and be responsible for insolubility defects, so it is with the properties of these particles we are most concerned. Measured charge on surfactant-free samples agrees well with the calculated charge so we can reasonably infer the measurements are also representative of systems where we cannot make the calculations. The measurement of zpotential is therefore a way to probe a characteristic colloidal property in a complex mixture of particles and is a useful technique to explain their behavior. The phase analysis approach is particularly appropriate as it allows measurement of very small changes in z-potential indistinguishable by conventional light scattering methodologies (Vanapalli & Coupland, 2000). This study is the first to apply PALS to understanding the functional properties of protein sols.
Acknowledgements We are grateful to the Pennsylvania Soybean Promotion Board for funding this research.
References AOAC International (1995). Official methods of analysis. Gaithersburg: AOAC International. Bravo, E. N. D. (1987). Effect of blending on the structure and functional properties of soybean major storage proteins. PhD Thesis. University Park: Department of Food Science. Pennsylvania State University Boulet, M., Britten, M., & Lamarche, F. (1998). Volumosity of some food proteins in aqueous dispersions at various pH and ionic strengths. Food Hydrocolloids, 12(4), 433 –441. Boulet, M., Britten, M., & Lamarche, F. (2000). Aggregation of some food proteins in aqueous dispersions: effects of concentration, pH and ionic strength. Food Hydrocolloids, 14(2), 135–144. Damodaran, S., Kinsella, J. E. (1986). Role of electrostatic forces in the interaction of soy proteins with lysozyme. Cereal Chemistry, 63(5), 381– 383. Damodaran, S. (1996). Amino acids, peptides, and proteins. In O. R. Fennema (Ed.), Food chemistry (pp. 321–430). New York: Marcel Dekker.
Dreja, M., Heine, K., Tieke, B., & Junkers, G. (1996). Rheological study of the pH-dependence of interactions between gelatin and anionic surfactants: flow behavior and gelation. Colloid and Polymer Science, 274(11), 1044–1053. Greener, J., Contestable, B. A., & Bale, M. D. (1987). Interaction of anionic surfactants with gelatin—viscosity effects. Macromolecules, 20(10), 2490–2498. Howe, A. M., Wilkins, A. G., & Goodwin, J. W. (1992). The interactions between gelatin and surfactants—a rheological study. Journal of Photographic Science, 40(5/6), 234 –243. Hunter, R. J. (1986). Foundations of colloidal science. Oxford: Oxford University Press. Kinsella, J. E. (1979). Functional properties of soy proteins. Journal of the American Oil Chemists Society, 56, 242–258. Messina, M., & Barnes, S. (1991). The role of soy products in reducing risk of cancer. Journal of the National Cancer Institute, 83(8), 541–546. Miller, J. F., Schatzel, K., & Vincent, B. (1991). The determination of very small electrophoretic mobilities in polar and nonpolar colloidal dispersions using phase analysis light scattering. Journal of Colloidal and Interface Science, 143(2), 532– 554. Morr, C. V. (1987). Current status of soy protein functionality in food systems. Journal of the American Oil Chemists Society, 64(7), 265. Nakai, S., Ho, L., & Tung, M. A. (1980). Solubilization of rapeseed, soy and sunflower protein isolates by surfactants and proteinase treatments. Canadian Institute of Food Science and Technology Journal, 13, 14 –22. Puppo, M. C., & Anon, M. C. (1998). Structural properties of heat-induced soy protein gels as affected by ionic strength and pH. Journal of Agricultural and Food Chemistry, 46(9), 3583–3589. Rhee, K. C. (1994). Functionality of soy proteins. In N. S. Hettiarachy, & G. R. Ziegler (Eds.), Protein functionality in food systems. New York: Marcel Dekker. Smith, A. K., & Circle, S. J. (1978). Soybean: Chemistry and technology. Westport: AVI Publishing. Tscharnuter, W. W., McNeil-Watson, F., & Fairhurst, D. (1996). A new instrument for the measurement of very small electrophoretic mobilities using phase analysis light scattering. In T. Provder (Ed.), Particle size distribution 3, assessment and characterization (Vol. 693). ACS Symposium Series, Washington, DC: ACS Press. US Department of Agriculture, Agricultural Research Service (1999). USDA nutrient database for standard reference, release 13. Nutrient Data Laboratory home page, http://www.nal.usda.gov/fnic/foodcomp Utsumi, S., & Kinsella, J. E. (1985). Forces involved in soy protein gelation: effects of various reagents on the formation, hardness and solubility of heat-induced gels made from 7S, 11S and soy isolate. Journal of Food Science, 50(5), 1278–1282. Vanapalli, S., & Coupland, J. N. (2000). Characterization of food colloids by phase analysis light scattering. Food Hydrocolloids, 14(4), 315 –317. Walstra, P. (1996). Dispersed systems: Basic considerations. In O. R. Fennema (Ed.), Food chemistry. New York: Marcel Dekker. Wang, C. R., & Zayas, J. F. (1991). Water retention and solubility of soy proteins and corn germ proteins in a model system. Journal of Food Science, 56(2), 455 –458. Wolf, W. J., & Cowan, J. C. (1977). Soybean as food source. Cleveland: CRC Press.
The effect of surfactants on the solubility, zeta potential, and viscosity of soy protein isolates Anupam Malhotra, John N. Coupland* Department of Food Science, The Pennsylvania State University, 103, Borland Lab., University Park, PA 16802, USA Received 27 April 2002; revised 6 October 2002; accepted 30 January 2003
Abstract The interactions of soy protein isolate (SPI, 5 wt%) with an anionic (sodium dodecyl sulfate, SDS) and a nonionic (polyoxyethylene sorbitan monolaurate, Tween 20) surfactant were studied as a function of pH. The solubility of the Tween 20 and surfactant free samples reached a minimum close to the isoelectric point of the protein (pH 4 – 5) whereas the SDS-containing sample was highly soluble over the whole range studied. The viscosity of the Tween 20-containing and surfactant-free samples were low (,3 mPa s) and largely pH independent while the SDS-containing samples were much more viscous, particularly at high pH (. pI) and high [SDS] (.critical micellar concentration (CMC)). The effect of the surfactants and pH on the z-potential of more dilute protein suspensions was measured by phase analysis light scattering. The charge on the SDS samples was high and negative at all pH values while the Tween 20 and surfactant-free samples showed a typical but distinct sigmoidal changes from positive to negative with increasing pH and a neutral point corresponding to the isoelectric point and the point of minimum solubility. The z-potential changed linearly with added SDS from a pH-determined value at 0 mM to approximately 2 50 mV at 7.5 mM. These observations are discussed in terms of the changing colloidal interactions of soy protein particles. q 2003 Elsevier Ltd. All rights reserved. Keywords: Soy protein; Solubility; Zeta-potential; Surfactants; Rheology
1. Introduction Soybean (Glycine Max) is a native crop of eastern Asia where it has served as an important part of the diet for centuries while soybeans produced in the US are used predominantly as animal food with only a small percentage used for human consumption (Wolf & Cowan, 1977). Until recently, soy was mainly eaten in Western cultures by vegetarians and various ethnic populations as foods such as tofu, tempeh, miso, and soy sauce. However, recent research has shown the anti-cancer and other health benefits of soy (Messina & Barnes, 1991), which has spurred research into better ways to use soy extracts as food ingredients. Soybeans contain around 40% protein, 35% carbohydrate, 20% lipids and 5% ash. Oil is the highest value soy product, but after extraction the defatted soy contains 50% protein, of which 90% is globular storage protein (Bravo, 1987) with significant potential as a food ingredient. * Corresponding author. Tel.: þ 1-814-865-2636; fax: þ1-814-863-6132. E-mail address: [email protected] (J.N. Coupland). 0268-005X/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0268-005X(03)00047-X
Soy protein can be fractionated into four main groups on the basis of sedimentation velocity as 2S, 7S, 11S, and 15S (Rhee, 1994). The most abundant soy proteins (, 70%) are the globulins, which are largely glycinin (11S) and conglycinin (globulin portion of 7S). Soy proteins are extracted from the beans, partially purified and concentrated as various soy protein flours (40 – 54% protein), concentrate ($ 70% protein), and isolates ($ 90% protein) with differing functional properties and commercial value. We are most concerned with soy protein isolate (SPI), which is typically manufactured by grinding the defatted beans in an alkali solution (pH , 8) to selectively solubilize a protein fraction. The soluble fraction is separated and the protein is precipitated and separated by lowering the pH to its isoelectric point (pH , 4.5) and dried (Puppo & Anon, 1998). The sample is frequently heated during this process to induce different degrees of protein denaturation. SPI can be added to foods to improve their nutritional quality (including both essential amino acids and other associated micronutrients), to act as a low cost bulking agent, and to provide desirable functional properties.
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The functional properties of proteins are those chemical and physical properties that affect food systems during processing, storage, preparation and consumption and are determined by the chemical structure of proteins and their interactions with other food components (Bravo, 1987). Functional properties include structure formation (e.g. emulsification, foam or film formation) and texture modification (e.g. gelation and thickening). Soy proteins are frequently incorporated into the product to stabilize fat emulsions, to improve viscosity, to gel and to improve water retention (Morr, 1987). Many functional attributes of proteins require their incomplete precipitation from water. For example, in order to form a gel a protein must first form a well dispersed mixture in the solvent then form limited protein– protein interactions to create a solid-like behavior. When the protein cannot adequately form a solution (that is to say when the protein– protein interactions are much stronger than combination of protein –solvent interactions and the free energy of mixing) it cannot proceed to form a strong gel (Kinsella, 1979). Frequently, the functional properties of soy proteins are limited by their relatively poor solubility, particularly close to the isoelectric point (Kinsella, 1979). Surfactants have been used to improve the solubility of poorly soluble materials including proteins. Nakai, Ho, and Tung (1980) showed that anionic surfactants were effective in solubilizing seed proteins. The dispersibility of SPI in sodium dodecyl sulfate (SDS) solution was 95 and 98% at pH 7.0 and 8.2, respectively, as compared to 71 and 82% for the controls. They postulated that there was an interaction between hydrophobic groups of SDS and hydrophobic sites of the soy protein, which led to an increase of negative charge on the protein causing an increase in interparticle electrostatic repulsion. It is well known that certain surfactants denature and precipitate proteins, which is indicative of strong surfactant –protein binding (Greener, Contestable, & Bale, 1987). However, the functional properties of the surfactant – protein complexes are less well understood. Greener and co-workers (1987) used various rheological methods to study the interactions of anionic surfactants with gelatin (pH . pI). For some systems above a critical surfactant concentration, the viscosity increased several orders of scale suggesting strong complex formation. They concluded that the extent of thickening was closely related to surfactant type, surfactant/gelatin composition and ionic strength and that the mechanism responsible for the thickening was micelle-mediated cross-links between peptide chains. Dreja, Heine, Tieke and Junkers (1996) demonstrated the importance of the effect of pH on the viscosity of gelatin– surfactant solutions. However, these workers explained their results using a different approach, arguing the viscosity changes were due to electrostatically driven changes in polymer conformation after binding highly charged micelles. Howe, Wilkins, and Goodwin (1992) found that
nonionic surfactants had little effect on the viscosity of gelatin solutions. In this work, different amounts of surfactant (anionic and uncharged) were added to a commercial sample of SPI and the viscosity and solubility of the complexes were measured over a range of pH. The z-potentials of similar, but more dilute, samples were measured to provide details of the colloidal properties of the system useful in interpreting our observations. By studying a single commercial protein isolate and two types of surfactant under a range of conditions using a number of techniques we seek to demonstrate the main interactions involved. A secondary and more long-term objective is to develop a new approach to the study of poorly soluble and poorly characterized protein powders. It is our contention that this large class of ingredients is better considered as suspensions of colloidal particles than through the traditional approaches of protein chemistry (Boulet, Britten, & Lamarche, 1998, 2000). The recently developed technique of phase analysis light scattering (PALS), applied here for the first time to the study of protein functionality, provides a valuable tool to characterize these experimentally difficult systems.
2. Materials and methods SPI was donated by the Archer Daniels Midland Company (Pro-Fam 974, Decatur, Il); SDS and polyoxyethylene sorbitan monolaurate (Tween 20) were purchased from the Sigma Chemical Company (St Louis, MO). According to the product literature, Pro-Fam 974 is 6.5% moisture (maximum), 90% protein (by Kjeldahl), 1% fat (by ether extraction) and 5% ash. It is recommended for use in milk replacers. All other reagents were purchased from Fisher Scientific Company (Fair Lawn, NJ). The SPI was dispersed (5 wt%) in SDS (0 – 5 wt%) or Tween 20 (0 – 5 wt%) solutions and stirred for 24 h to allow complete hydration. In some experiments, the pH was adjusted (1 –8) with small volumes of sodium hydroxide or hydrochloric acid. The soy formed an opaque suspension rather than a true solution in the solvents. Preliminary optical microscopy studies revealed a heterogeneous mixture of irregularly shaped particles with several in the 10– 100 mm range. There were no detectable thermal transitions on heating SPI and SPI– surfactant complexes in a differential scanning calorimeter that might have corresponded to denaturation or complex melting. From these observations we conclude that the proteins were largely denatured during the manufacturing process and that the clumps formed on drying do not readily re-disperse in aqueous solvents. 2.1. Rheology The shear viscosity measurements were carried out a controlled strain rheometer (Rheometrics Fluid Spectrometer
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II, Piscataway, NJ) operating in concentric cylinder geometry (cylinder length 13 mm, inner diameter 16.5 mm, outer diameter 17.0 mm). Samples were equilibrated at the analysis temperature (30 8C), gently mixed, and then poured into the instrument. The instrument had previously been equilibrated at 30 8C and the test was run immediately. The rate of rotation of the outer cylinder was increased from 10 to 3000 s21 and the torque on the inner cylinder was measured and used to calculate viscosity at each shear rate. Although some of the samples had a tendency to precipitate during analysis, this process was slow compared to the time required for analysis (, 100 s) and reproducible measurements could be made. 2.2. Solubility Soy protein solubility was determined using a variation of a method described by Wang and Zayas (1991). The insoluble soy protein was separated from the soluble fraction by gentle centrifugation (1000 rpm, 5 min) and the protein concentration in the original sample and the supernatant after centrifugation was determined using a Kjeldahl method (AOAC International, 1995) (Preliminary studies showed the surfactants used here did not interfere with the assay. Several colorimetric protein assays attempted did not work in the presence of SDS.). The protein solubility was calculated as Solubility ¼
Psupernatant £ 100% Ptotal
ð1Þ
where P is the protein concentration in the phase designated by the subscript. It is important to be aware that solubility measured by this method is a very empirical parameter—slight variations in the centrifugation or hydration conditions would cause a change in apparent solubility. Solubility in this work means only the proportion of protein that was not precipitated under these conditions.
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ions move with the particle and thus the potential determined is not that at the surface but rather at a short, undefined distance into the diffuse layer—the z-potential. Surface charge in protein particles is due to the partial ionization of various amino acid residues (Vanapalli & Coupland, 2000). The effective charge on a protein particle is affected by pH, ionic strength and the accumulation of ligands or surfactants at the interface. Zeta potential was measured by PALS. In this method a laser beam is split and a portion used to generate a scattering signal (at 158) from a dilute suspension of particles moving in a sinusoidally oscillating voltage. The second portion has a finite frequency modulation applied to it and it is recombined with the scattered light. Analysis of the time dependency of the signal phase is sensitive to the electrically induced rate of motion and hence electrophoretic mobility of the particles. More details on the mathematical (Miller, Schatzel, & Vincent, 1991) and practical basis (Tscharnuter, McNeil-Watson, & Fairhurst, 1996) of PALS are available in the literature. PALS has been used to measure the electrophoretic mobility (a parameter related to z-potential) of aqueous solutions of globular proteins (Vanapalli & Coupland, 2000). Protein solutions (0.05 wt%) were prepared in water or different surfactant solutions (SDS 5 wt%; Tween 20 5% wt%). The solutions were placed in a standard foursided, 1 cm polystyrene cuvette and a parallel plate electrode (0.45 cm2 square platinum plates with a 0.4 cm gap) was inserted. The cuvette was placed in a temperaturecontrolled holder (25 8C). The electrophoretic mobility was measured by PALS (ZetaPALS, Brookhaven Instruments, Holtsville, NY). Each measurement was the average of 50 (five sets of 10) measurements and the entire experiment was conducted in triplicate. The z-potential was calculated from the electrophoretic mobility using the Smoulokowski model (assuming the double layer thickness is much less than the particle size) (Hunter, 1986).
2.3. Zeta potential 3. Results Colloidal particles accumulate charge at their surface that can be expressed as a surface potential. Surface potential is an important factor for determining the magnitude of charged-based colloidal interactions of a particle, most crucially electrostatic repulsion of other like charged particles. The surface charge on a particle perturbs the ionic distribution in the medium surrounding it. First a layer of tightly bound counter-ions (i.e. of opposing charge) accumulates at the particle surface, the Stern layer, and beyond this a region of decaying excess concentration, the diffuse layer, extends a considerable distance (, nm) into the surrounding aqueous media (Hunter, 1986). Measuring the colloidal charge typically involves applying an electrical voltage to the particle and measuring the speed of movement induced. In practice, one or more layers of hydrated
3.1. Rheology Fig. 1 shows representative data for the dependency of apparent viscosity on applied shear rate for the SPI and SPI – surfactant systems (Preliminary measurements on pure surfactant solutions at these concentrations were below the sensitivity of the instrument.). In all cases, the solutions were shear thinning and the viscosity of SDS – SPI solution is much higher than SPI –Tween 20 and SPI solutions at all shear rates. For example for 5 wt% SDS (pH 6.3) the apparent viscosity decreased from 32.3 mPa s at 160 s21 to 18.9 mPa s at 2500 s21. Similarly for 5 wt% Tween 20 (pH 6.0), the viscosity decreased from 5.0 mPa s (160 s21) to 3.6 mPa s (2500 s21) and for the surfactant-free solution (pH 5.6) viscosity decreased from 6.4 mPa s (160 s21) to
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Fig. 1. Representative plots of viscosity as a function of applied shear rate for (X) 5 wt% SDS–SPI, (W) 5 wt% Tween 20– SPI and (P) surfactant-free SPI solutions at pH , 6.
3.5 mPa s (2500 s21). The shear thinning curves could not be adequately characterized by a power-law equation. The viscosity measured at high shear was more reproducible than those at lower shear (e.g. the standard error for the 5 wt% SDS sample was 5% at 16 s21, 0.7% at 1000 s21). Consequently the apparent viscosity at 1000 s21 was used henceforth to compare the rheology of the various samples. The viscosities of 5 wt% SDS, 5 wt% Tween 20, and surfactant-free samples are shown as a function of pH in Fig. 2a. The viscosity of the 5 wt% SDS sample increased with pH (e.g. at pH 7.9 viscosity was highest (28.7 mPa s) but gradually decreased to 9.6 mPa s at pH 2.4). At pH , 6 the viscosity was 22.8 mPa s for the 5 wt% SDS sample but only 1.9 mPa s for the surfactant-free or Tween 20 samples. In the surfactant-free samples the viscosity (, 3.0 mPa s) was effectively independent of pH but the Tween 20 samples were slightly more viscous under acid conditions. The effect of surfactant concentration on protein viscosity at pH , 4.4 (, pI) and pH 6.4 (. pI) is shown in Fig. 2b. The viscosities of the Tween-containing samples was low (, 3 mPa s) and largely independent of surfactant concentration, while the SDS-containing samples became more viscous with increased [SDS]. At low [SDS] the viscosity was similar to the corresponding Tween-containing samples, but at approximately 2 wt% (i.e. 8.7 mM) the viscosity of the high pH sample began to increase rapidly with further additions of surfactant. 3.2. Solubility The solubilities of soy protein and soy protein – surfactant complexes are shown as a function of pH in Fig. 3. The SDS samples were highly soluble (. 90%) at all pH values whereas the Tween 20 and surfactant-free samples showed a typical U-shaped pH – solubility profile, with a minimum of , 10% soluble at pH 3.5 – 4.5. At all pH values the solubility of the SDS samples was greatest and
Fig. 2. (a) Viscosity as a function of pH for (X) 5 wt% SDS–SPI, (W) 5 wt% Tween 20–SPI and (P) surfactant-free SPI solutions. (b) Viscosity of 5 wt% SPI suspensions as a function of (X, W) SDS or (B, A) Tween 20 concentration. Open points pH 4.4 ^ 0.4, filled points pH 6.4 ^ 0.4.
there were relatively minor differences between Tween 20 and surfactant-free samples. 3.3. Zeta potential Light scattering studies can only be performed in optically clear suspensions so it was necessary to make these measurements at much lower protein concentrations than were used in the rheological and solubility studies. To account for the changes in protein concentration we could either maintain the same surfactant concentrations and thus get unrealistic protein:surfactant ratios or we could commensurately reduce the surfactant concentrations and potentially reduce the surfactant:water ratio to close to the CMC. In either case, the results of the different methods will not be directly comparable and care must be taken in interpreting the data. The z-potentials of 5 wt% SDS –SPI, 5 wt% Tween 20– SPI, and surfactant-free samples are shown as a function of pH in Fig. 4a. The z-potential of the SDS – SPI complex
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Fig. 3. Solubility as a function of pH for (X) 5 wt% SDS– SPI, (W) 5 wt% Tween 20–SPI and (P) surfactant-free SPI solutions.
was strongly negative (approx. 2 50 mV) and largely independent of pH. Both the Tween 20 and surfactantfree samples were negative at high pH values and increased and became positive as the pH was decreased.
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The magnitude of the z-potential of the Tween 20 samples was lower than that of the surfactant-free samples at all values of pH, except for the neutrality point (pH , 4.1)— close to the established isoelectric point of soy globulins (pH 4 –5) (Smith & Circle, 1978). The charge on a protein can be estimated from the sum of the charges on the individual amino acid residues using the Henderson –Hasselbach equation. The total charge on soy protein was calculated as a function of pH using literature data for amino acid composition and the pK values of amino acid side chains listed in Table 1. Over the pH range considered, the net charge is dominated by a positive contribution from lysine and histidine and increasingly (with increasing pH) negative contributions from aspartic and glutamic acid. Other amino acids carry charge but are not present on an adequate mass basis to influence the total charge. The net charge decreased sigmoidally with increasing pH passing through zero at pH 4.3, again similar to the isoelectric point of soy globulins (pH 4 – 5) (Smith & Circle, 1978). The measured z-potential of soy proteins (using the Henderson – Hasselbach approach set out earlier) correlated well with the calculated charge: z-potential (mV) ¼ 39.1 £ net calculated charge (mol kg21) 2 2.5, r 2 ¼ 0:96: The good agreement between measured and calculated charge for the surfactant-free SPI suggests strongly that this method is indeed sensitive to the electrical properties of the complex colloids present here and is an appropriate method to track the charge-based interactions of soy protein and surfactants. However the correlation is not perfect, in particular around pH 6, which may be due to the likelihood that the scattering particles do not have the same amino acid composition as average soy protein or that the mean pK values selected are not reliable. Alternatively, some charged groups may be buried in the protein core and do not contribute to the surface charge. Fig. 4b shows the effect of surfactant concentration on the z-potential of SPI at selected values of pH close to, or significantly above, the isoelectric point. As expected, the Table 1 Calculations of the charge on soy protein Concentrationa (mol kg21 protein)
Aspartic acid Glutamic acid Histidine Lysine Totald Fig. 4. (a) z-Potential as a function of pH for (X) 5 wt% SDS–0.05 wt% SPI, (W) 5 wt% Tween 20–0.05 wt% SPI and (P) surfactant-free 0.05 wt% SPI solutions. Solid line is the calculated net charge on soy protein. (b) zPotential of 0.05 wt% SPI suspensions as a function of (X, W) Tween 20 or (B, A) SDS concentration. Filled points pH , pI ( ¼ 4.4), open points pH . pI (6.4 ^ 0.1 for Tween 20 samples and 7.9 ^ 0.1 for SDS samples).
a
0.77 1.19 0.15 0.36
pK b
4.6 4.6 7.0 10.2
Net chargec (mol kg21 protein) pH 7.9
pH 6.4
pH 4.4
20.77 21.19 0.02 0.36
20.75 21.17 0.12 0.36
20.30 20.46 0.15 0.36
21.58
21.44
20.25
US Department of Agriculture (1999). Damodaran (1996). The pK values used are those of the amino acid residues. c Calculated using the Henderson–Hasselbach equation (see text for details). d Other amino acids contributed insignificant charge to the total. b
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protein at its isoelectric point is close to neutral and the basic protein is strongly negatively charged. As SDS is added to the neutral protein, the charge decreases linearly to approximately 2 40 mV at approximately 7.5 mM surfactant (i.e. 1.5 mM SDS g21 protein). When SDS is added to the negative protein the charge increases slightly to a similar value at a similar surfactant concentration, but the magnitude of the changes are relatively small compared to the error in the measurements. Adding Tween 20 caused a very small increase in z-potential of SPI.
4. Discussion These results indicate some clear differences between the SDS and Tween 20 or surfactant-free samples. The viscosities of the SDS samples are high and pH dependent whereas the other samples are not. The solubilities and zpotentials of the SDS samples are high and pH-independent while the values for the other samples are lower and pH dependant. Viscosity is independent of low SDS concentrations but, depending on the pH, significantly increases at higher concentrations. The z-potential changes on the addition of moderate amounts of SDS to a pH-independent plateau but is largely unaffected by added Tween 20. Plotting solubility as a function of viscosity (Fig. 5) reveals trends in the data (considering only the 5 wt% surfactant data at all measured pH values). Increasing the viscosity is associated with an increase in solubility up to close to 100%. Further changes in viscosity beyond a critical point (, 7 mPa s) cause no further changes in solubility. The changes in solubility with viscosity (Fig. 5) seem to follow a single function independent of surfactant type. Both viscosity and z-potential could be expected to increase the tendency of protein particles to remain in suspension for different reasons. First, it would be harder for protein particles to sediment through a thicker solution.
Fig. 5. Solubility as a function of viscosity for (X) 5 wt% SDS–SPI, (W) 5 wt% Tween 20 –SPI and (P) surfactant-free SPI solutions. Measurements were made at a range of pH values.
Despite the fact that the continuous phase viscosity does not change, as would be required for a classic Stokes law explanation of the process, a common analogous observation is that highly flocculated emulsions are often resistant to creaming. Second, increasing the surface charge on colloidal particles increases the magnitude of inter-particle electrostatic repulsion (Walstra, 1996), which will tend to disrupt existing protein aggregates and discourage further aggregate formation. According to Stokes law the rate of sedimentation of a particle is proportional to the square of its diameter (Walstra, 1996) and so a smaller particle would sediment less rapidly. The question remains—why do surfactants and pH changes have such a large effect on the z-potential and viscosity of soy protein solutions? SDS is a charged surfactant so it can interact with a protein both electrostatically (through its sulfate group) or hydrophobically (via its alkane tail-group), while Tween 20 can only interact hydrophobically. While the absolute charge on SPI changes with pH (Fig. 4a), there are some positive charges present over the entire pH range considered (Table 1) so electrostatic interactions are possible in all cases. Damodaran and Kinsella (1986) showed that electrostatic interactions are important in forming complexes between soy proteins and lysozyme, and Utsumi and Kinsella (1985) showed that both electrostatic and hydrophobic interactions (as well as hydrogen and disulfide bonding) could all play a role in forming soy protein gels. The effects of SDS are most dramatic so we will first attempt to create a model for charged surfactant –protein interactions and then more briefly explore the different effects seen with Tween. The major experimental observations from the SDS – SPI complexes in our studies that should be incorporated into any model are: Observation 1. Solubility is high and pH independent (Fig. 3), Observation 2. z-potential is highly negative and pHindependent (Fig. 4a), Observation 3. At low pH, z-potential becomes more negative with [SDS] and reaches a plateau , 0.2 wt%. At high pH, z-potential is largely pH independent (Fig. 4b), Observation 4. Viscosity increases with [SDS] . 2 wt% if pH . pI and with pH (again if pH . pI) (Fig. 2). Observations 1 and 2 appear to be related. The surfactant binds to the protein and, because the sulfate group is not protonated over this pH range, maintains a strong pHindependent negative charge. The highly charged complex better interacts with water and is therefore soluble. The z-potential measurements that form the basis of Observation 3 relate to the binding of monomeric SDS ([SDS] p CMC). Monomeric surfactant can bind electrostatically or hydrophobically; electrostatic binding would eliminate one net positive charge from the protein and hydrophobic binding would add one net negative charge. If
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electrostatic bonding were solely responsible, the net charge on the proteins at high [SDS] would be zero, while if hydrophobic binding were responsible it would be negative. The net charge at high [SDS] seen in this work is not only negative (Fig. 4, i.e. implying hydrophobic surfactant binding), but also pH-independent (Observation 2) suggesting that there is some critical level of charge that can be carried on a single protein beyond which no further surfactant can bind. Observation 2 implies that beyond a certain (low) level of surfactant the charge on the proteins is pH-independent. However in Observation 4, we note that only for the high pH samples there is an increase in viscosity and so there must be some distinction in their behaviors. Interestingly the point of divergence in the viscosity data for the high pH system (Fig. 2b) is at approximately 2 wt% or 8.7 mM SDS—reasonably close to the aqueous CMC of SDS ( ¼ 8.1 mM) so it seems likely that the micelles are responsible for the viscosity increase. A micelle has a high charge density, so when a protein binds a micelle there would be an electrostatic pressure to physically disperse the charge as widely as possible. Although we have argued that the charges are similar and pH independent before micelle binding, the negative charges on the anionic protein are more due to amino acid charges than those on the cationic protein which are more due to bound monomeric SDS. Therefore the anionic protein can only disperse its charge by expanding its structure while the cationic protein can displace some bound monomers. Consequently, the anionic protein would have an increased hydrodynamic radius, and hence viscosity, while the cationic protein would be unchanged. This reasoning is a variation of the ‘conformational change’ model proposed by Dreja and co-workers (1996) (although we are unable to say if the micelle is electrostatically or hydrophobically bound). However it would be possible to make a similar argument based on the ‘cross-linking model’ proposed by Greener and co-workers (1987). In this case, the observed changes in viscosity could be due to the acidic or basic protein binding micellar surfactant via its localized positive groups. The basic SPI has relatively few positive charges, so the same micelle could simultaneously bind several peptide chains and serve as inter-molecular links and thus lead to an increase in viscosity (fig. 13d in Greener et al., 1987). The acidic protein has more positive charges and therefore each protein would tend to wrap around any bound micelles acting as intra- rather than inter-molecular links (fig. 13b in Greener et al., 1987) and therefore not increase the viscosity. In reality it seems probable that a combination of the ‘conformational change’ and ‘cross-linking’ are responsible for the effects of SDS on SPI rheology. The Tween 20 samples have properties distinct from both the SDS and surfactant-free samples. While the viscosity of the soy was not significantly affected by addition of Tween 20 (Fig. 2), the z-potential was lower than that of the surfactant-free samples at all pH values (Fig. 4). Tween 20
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is a non-ionic surfactant and so can onlt interact hydrophobically with a protein. The amount of Tween 20 bound to the protein surface should therefore be independent of pH and would have little impact on protein conformation nor could it lead to protein association and there would be no increase in viscosity. However, binding surfactant would shield the hydrophobic regions of the protein from the solvent and therefore increase solubility. Bound Tween 20 would not in itself affect the absolute charge on the protein, but during a z-potential measurement could move with the protein particle in the applied electrical field. The charges present on the protein would then be further away from the plane of shear, so the measured charge, the z-potential, would be reduced. Even though the Tween 20 is to some extent reducing the effective charge on the protein, it is providing a much more hydrophilic surface and there is little loss in solubility.
5. Conclusion One of the most important limitations of soy protein as a food ingredient is its poor solubility at pH , pI. This is because the charge on the particles is insufficient to prevent extensive aggregation (presumably driven by a hydrophobic mechanism) and the aggregates rapidly sediment. Charged surfactants are capable of maintaining a pH-independent, negative charge on the proteins so their complexes are highly soluble even at the isoelectric point. In practice it is impossible to add sufficient levels of SDS to have this effect in a real food systems but there are several other food molecules with the right characteristics, i.e. a localized, pHindependent charge and a tendency to form hydrophobically driven, self assembled structures (e.g. lecithin and other phospholipids or lactic or acetic acid esters of mono-and diglycerides). The ideal solubilizing molecule will also have a bland taste, be permissible in foods, hopefully have an acceptable label declaration, and be reasonably cheap. Soy protein is representative of a group of food ingredients that are polymeric, chemically complex and poorly soluble. Prominent in this group are the protein concentrates and flours extracted from various sources. The behavior of these ingredients is difficult to study in that classical protein chemistry is most often concerned with polymers in solution In this work we have instead taken the approach of Boulet and co-workers (1998, 2000) and considered functionality in terms of the soy proteins as colloidal particles. These workers argue that in many real food systems the quaternary aggregates of proteins are functional particles and are a more useful way to represent the properties of the system than studies on dilute monomers. In our case some of the particles in the commercial preparation are even larger than those described by Boulet and others (1998, 2000) and thus presumably more readily described in terms of their particle-like behavior.
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These large particles could not be well characterized by the photon correlation spectroscopy technique used by Boulet et al. (1998, 2000) but their effective charge could be readily measured by PALS. Large particles scattering light more efficiently than smaller ones so the PALS technique will be most sensitive to them and the Smoulokowski approach used to convert the mobilities to charges is insensitive to absolute size. In the context of this work it is likely the large particles that will sediment the most rapidly and be responsible for insolubility defects, so it is with the properties of these particles we are most concerned. Measured charge on surfactant-free samples agrees well with the calculated charge so we can reasonably infer the measurements are also representative of systems where we cannot make the calculations. The measurement of zpotential is therefore a way to probe a characteristic colloidal property in a complex mixture of particles and is a useful technique to explain their behavior. The phase analysis approach is particularly appropriate as it allows measurement of very small changes in z-potential indistinguishable by conventional light scattering methodologies (Vanapalli & Coupland, 2000). This study is the first to apply PALS to understanding the functional properties of protein sols.
Acknowledgements We are grateful to the Pennsylvania Soybean Promotion Board for funding this research.
References AOAC International (1995). Official methods of analysis. Gaithersburg: AOAC International. Bravo, E. N. D. (1987). Effect of blending on the structure and functional properties of soybean major storage proteins. PhD Thesis. University Park: Department of Food Science. Pennsylvania State University Boulet, M., Britten, M., & Lamarche, F. (1998). Volumosity of some food proteins in aqueous dispersions at various pH and ionic strengths. Food Hydrocolloids, 12(4), 433 –441. Boulet, M., Britten, M., & Lamarche, F. (2000). Aggregation of some food proteins in aqueous dispersions: effects of concentration, pH and ionic strength. Food Hydrocolloids, 14(2), 135–144. Damodaran, S., Kinsella, J. E. (1986). Role of electrostatic forces in the interaction of soy proteins with lysozyme. Cereal Chemistry, 63(5), 381– 383. Damodaran, S. (1996). Amino acids, peptides, and proteins. In O. R. Fennema (Ed.), Food chemistry (pp. 321–430). New York: Marcel Dekker.
Dreja, M., Heine, K., Tieke, B., & Junkers, G. (1996). Rheological study of the pH-dependence of interactions between gelatin and anionic surfactants: flow behavior and gelation. Colloid and Polymer Science, 274(11), 1044–1053. Greener, J., Contestable, B. A., & Bale, M. D. (1987). Interaction of anionic surfactants with gelatin—viscosity effects. Macromolecules, 20(10), 2490–2498. Howe, A. M., Wilkins, A. G., & Goodwin, J. W. (1992). The interactions between gelatin and surfactants—a rheological study. Journal of Photographic Science, 40(5/6), 234 –243. Hunter, R. J. (1986). Foundations of colloidal science. Oxford: Oxford University Press. Kinsella, J. E. (1979). Functional properties of soy proteins. Journal of the American Oil Chemists Society, 56, 242–258. Messina, M., & Barnes, S. (1991). The role of soy products in reducing risk of cancer. Journal of the National Cancer Institute, 83(8), 541–546. Miller, J. F., Schatzel, K., & Vincent, B. (1991). The determination of very small electrophoretic mobilities in polar and nonpolar colloidal dispersions using phase analysis light scattering. Journal of Colloidal and Interface Science, 143(2), 532– 554. Morr, C. V. (1987). Current status of soy protein functionality in food systems. Journal of the American Oil Chemists Society, 64(7), 265. Nakai, S., Ho, L., & Tung, M. A. (1980). Solubilization of rapeseed, soy and sunflower protein isolates by surfactants and proteinase treatments. Canadian Institute of Food Science and Technology Journal, 13, 14 –22. Puppo, M. C., & Anon, M. C. (1998). Structural properties of heat-induced soy protein gels as affected by ionic strength and pH. Journal of Agricultural and Food Chemistry, 46(9), 3583–3589. Rhee, K. C. (1994). Functionality of soy proteins. In N. S. Hettiarachy, & G. R. Ziegler (Eds.), Protein functionality in food systems. New York: Marcel Dekker. Smith, A. K., & Circle, S. J. (1978). Soybean: Chemistry and technology. Westport: AVI Publishing. Tscharnuter, W. W., McNeil-Watson, F., & Fairhurst, D. (1996). A new instrument for the measurement of very small electrophoretic mobilities using phase analysis light scattering. In T. Provder (Ed.), Particle size distribution 3, assessment and characterization (Vol. 693). ACS Symposium Series, Washington, DC: ACS Press. US Department of Agriculture, Agricultural Research Service (1999). USDA nutrient database for standard reference, release 13. Nutrient Data Laboratory home page, http://www.nal.usda.gov/fnic/foodcomp Utsumi, S., & Kinsella, J. E. (1985). Forces involved in soy protein gelation: effects of various reagents on the formation, hardness and solubility of heat-induced gels made from 7S, 11S and soy isolate. Journal of Food Science, 50(5), 1278–1282. Vanapalli, S., & Coupland, J. N. (2000). Characterization of food colloids by phase analysis light scattering. Food Hydrocolloids, 14(4), 315 –317. Walstra, P. (1996). Dispersed systems: Basic considerations. In O. R. Fennema (Ed.), Food chemistry. New York: Marcel Dekker. Wang, C. R., & Zayas, J. F. (1991). Water retention and solubility of soy proteins and corn germ proteins in a model system. Journal of Food Science, 56(2), 455 –458. Wolf, W. J., & Cowan, J. C. (1977). Soybean as food source. Cleveland: CRC Press.