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Solubility and density of egg white proteins: Effect of pH and saline concentration
ARTICLE IN PRESS
LWT 40 (2007) 1304–1307 www.elsevier.com/locate/lwt
Research Note
Solubility and density of egg white proteins: Effect of pH and saline concentration Fla´via Ferreira Machadoa, Jane S.R. Coimbraa,, Edwin E. Garcia Rojasb, Luis A. Minima, Fabı´ ola C. Oliveiraa, Rita de Ca´ssia S. Sousaa a
Departamento de Tecnologia de Alimentos, Universidade Federal de Vic- osa, Av. P.H. Rolfs s/n, 36570-000, Vic- osa, MG, Brazil b Departamento de Engenharia de Agronegocios e Administrac- a˜o, Universidade Federal Fluminense, Av. dos Trabalhadores 420, 27225-250, Volta Redonda, RJ, Brazil Received 30 January 2006; received in revised form 26 August 2006; accepted 31 August 2006
Abstract Influence of different pH values (3.0, 4.6, 6.0, 8.0 and 9.0) and salt concentrations (0.05, 0.1, 0.2, 0.35 and 0.5 mol/l) for three types of salt (NaCl, Na2SO4 and (NH4)2SO4) on the solubility of egg white protein and density of egg white, at room temperature (25 1C) was studied. The results showed that the solubility of egg white protein was influenced by pH as well as concentration and type of salt present in the medium. Protein solubility increased with increase in pH, with higher solubility showed at pH 9.0 and lower solubility at pH 4.6. This behavior was verified for all the salts analysed. At acid pH (pH 3.0), it was observed the tendency of solubility elevation on the increase of the saline concentration, due to the salting-in effect. The density increased with increase in the salt concentration. r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Density; Egg white; Functional properties; Protein; Solubility
1. Introduction Egg white is a natural source of proteins of recognized nutritional, biological, and technological potential interest (Awade, Moreau, Molle, Brule, & Maubois, 1994; Croguennec, Francoise, Stephane, & Gerard, 2000). Three particularly important proteins present in egg white are lysozyme, ovotransferrin and ovoalbumin. Lysozyme (3.5 g/100 g of the total egg white protein) has antibactericidal properties and is widely used in food preservation and pharmaceutical industry. Ovotransferrin (13 g/100 g of the total egg white protein) is a glycoprotein with an important role in the iron transport and with wide antimicrobial activity. Ovoalbumin, the major egg white component, corresponding to 54 g/100 g of the total egg white protein, is a glycoprotein with coagulation and gelation properties (Vachier, Piot, & Awade, 1995). Corresponding author. Tel.: +55 31 38991618/1804; fax: +55 31 38992208. E-mail address: [email protected] (J.S.R. Coimbra).
Due to unique functional properties of proteins, such as gel and foam formation, hen egg white proteins have been extensively used as ingredients in processed foods, besides being desirable ingredients in many foods, such as bakery products, pies, cookies, and meat by-products (Mine & Bergougnoux, 1998; Wong, Herald, & Hachmeister, 1996). Additionally, the commercialization of egg-derived products has gained great importance in the international market (Stadelman & Cotterill, 1995). The way proteins interact with a solvent is a manifestation of the physicochemical properties of proteins under given conditions. A wide range of protein–protein and protein–solvent interactions are involved. Many important functional food properties, including solubility, may be described through these interactions (Kakalis & Regenstein, 1986). The primary importance of protein solubility is that it influences other functional properties, such as gelation, emulsification and foam formation, so the main factor for proteins to exhibit gelatinization, foaming and emulsifying characteristics it is solubility in a food matrix (Nakai & Chan,
0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2006.08.020
ARTICLE IN PRESS F. Ferreira Machado et al. / LWT 40 (2007) 1304–1307
1985; Wit, 1989). Besides, solubility data are applied to determine the optimum conditions of protein extraction and purification from natural sources (Fennema, 1993). Solubility is affected by many factors such as amino acid composition, protein structure (native or denatured) and medium factors, such as pH, temperature, pressure, type and content of salts and protein concentration. Overall, proteins remain in solution until reaching a maximum quantity, after which they precipitate (Vojdani, 1996); pH affects the nature and distribution charges of a protein. In general, proteins are more soluble in low (acid) or high (alkaline) pH values, due to excess of charges of the same signal, producing repulsion among the molecules and, consequently, contributing to their higher solubility (Pelegrine & Gasparetto, 2004). Proteins at isolectric point (pI) generally present minimum solubility. The protein– protein interaction increases, at pI, because the electrostatic forces between the protein molecules and water molecules are minimum, which reduces the number of water molecules that interact with protein molecules. This is a favorable condition for the approximation and aggregation of protein molecules, leading to their precipitation (Vojdani, 1996). Salts can also affect the electrostatic interactions among the macromolecules, contributing through the ionic force (Fennema, 1993). The effect of salts on protein solubility in aqueous solutions is a function of the ionic species present (Arakawa & Timasheff, 1982). Kinsella (1982) showed that a salt concentration of 0.15 mol/l is sufficient to change the structure of the water and conformation of the proteins. However, this will depend on the content and type of salt present in the medium. Thus, the objective of this work was to evaluate the influence of pH and saline concentration on solubility and density of egg white protein for three types of salt (NaCl, Na2SO4 and (NH4)2SO4) at room temperature. 2. Materials and methods
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immediately mixed in magnetic stirrer until complete dissolution. The solution was quantitatively transferred to a volumetric flask of 10 ml, completed with 1 mol equivalent/l NaOH, manually agitated, and the protein content quantified. 2.3. Solubility determination Solubility data were obtained according to Kakalis and Regenstein (1986), by adding 200 mg of dehydrated egg white into 30 ml of a buffer solution. The buffers used were glycine–HCl, citrate–citric acid, phosphate–citrate, phosphate and Tris–HCl leading to the pH values of 3.0; 4.6; 6.0; 8 and 9.0, respectively. Each buffer solution was previously added by the salt to be evaluated (NaCl, Na2SO4 or (NH4)2SO4) at pre-determined concentrations (0.05; 0.01; 0.2; 0.35 and 0.5 mol/l). The mixture was agitated for 1 h, at room temperature (25 1C), using a device to simulate an agitated tank (Ferreira, 2001) and immediately centrifuged at 20 000 g for 30 min at approximately 4 1C (Beckman centrifuge, model J2-MC, USA). After centrifugation, the supernatant was collected for analyses. The solubility (S, g protein in the supernatant/100 g total amount protein in sample before centrifugation) was calculated by PS S¼ 100, (1) PA where PS and PA are the protein amounts (g) in the supernatant and sample, respectively. Each experiment was performed in duplicate, with the soluble protein content being the average of the values of two repetitions. 2.4. Density data Density data were determined by applying the Pycnometric Method (Constenla, Lozano, & Crapiste, 1989), using a 10 ml pycnometer. The pycnometer was calibrated with deionized water at room temperature. Corrections for temperature were shown to be negligible.
2.1. Materials 3. Results and discussion Samples of standard dehydrated egg white were a kindly gift from Sohovos Industrial Ltda (Brazil), packed in polyethylene bags. Deionized water and analytical grade reagents were used in the experiments. 2.2. Protein quantification Protein determinations were based on the Biureto reaction (Gornall, Bardawill, & David, 1949), using an analytical curve built by varying ovoalbumin (Sigma Chemicals, USA) concentration in aqueous solutions from 0.5 to 8.0 mg/ml. Absorbance was measured at 540 nm using a Cary 50 spectrophotometer (Varian, Australia). For protein quantification of dehydrated egg white protein, 100 mg of the material was carefully weighed in a beaker of 25 ml, added 6 ml of 1 mol equivalent/l NaOH, and
3.1. Effect of pH, type and concentration of salt on protein solubility It is observed in Table 1 the solubility increase with the pH increase, for all tested salts. Therefore, extraction yield and solubility are higher in alkaline pH than in acid pH. In fact, the number of negatively charged ions at pH4pI is larger than the number of positively charged ions at pHopI (Fennema, 1993). At pH 3 it was verified a tendency of solubility increase with increasing concentrations of the salts Na2SO4 and (NH4)2SO4. Some type of salts may increase protein solubility, at concentrations ranging from 0.1 to 1.0 mol/l, due to the salting-in phenomenon by which saline ions interact with groups of opposite protein charges to form a
ARTICLE IN PRESS F. Ferreira Machado et al. / LWT 40 (2007) 1304–1307
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Table 1 Solubility of egg white protein (g/100 g) as a function of pH and salt concentrations pH 3.0
4.6
6.0
8.0
9.0
NaCl (mol/l) 0.05 0.10 0.20 0.35 0.50
34.7571.48 55.8872.52 55.0571.55 38.5074.33 39.8372.46
47.8675.60 47.4670.56 38.8072.61 56.1870.09 61.9271.58
63.6270.74 66.1272.87 78.9770.38 68.6870.19 72.3372.05
73.9472.50 72.5771.44 73.5570.80 71.9371.85 76.8371.75
79.2270.12 70.0772.95 62.2371.85 68.8572.01 59.2770.87
Na2SO4 (mol/l) 0.05 0.10 0.20 0.35 0.50
57.4970.26 72.2070.57 62.1971.74 65.8876.51 68.4473.62
65.3773.48 62.5871.74 68.1275.28 65.9171.13 63.7372.10
81.9473.53 84.7371.60 65.6670.46 84.0070.30 75.8570.61
81.0372.91 84.2773.41 83.8771.50 74.6670.27 72.0676.78
91.9470.60 90.1570.93 93.2470.64 81.0271.11 84.1172.77
(NH4)2SO4 (mol/l) 0.05 0.10 0.20 0.35 0.50
60.3270.14 60.6970.88 62.4270.88 77.2876.58 63.6370.02
56.3371.40 66.1072.78 56.5671.19 67.1873.93 65.1871.72
79.2871.33 83.3270.34 72.9170.67 73.1073.94 73.0771.56
85.0971.09 87.1573.71 81.3070.12 78.8171.23 72.4371.20
90.6170.28 78.9372.76 86.5170.38 81.4670.60 81.4670.48
double layer of ionic groups, than the electrostatic interaction among the protein molecules reduces, increasing their solvatation (Vojdani, 1996). A lower solubility was observed around pH 4.5, which is the isoelectric point of ovoalbumin, the major egg white component (54 g/100 g of the egg white protein). At the isoelectric point, the protein molecule has zero liquid charge, exhibiting the maximum of electrostatic interactions among the proteins charged groups. Since the charged groups offer minimum availability to interact with water molecules, the amount of bound water decreases minimally, thus reducing solubility (Wong, 1995). Proteins solubility at pH 8.0, for NaCl was practically constant. In the presence of Na2SO4 and (NH4)2SO4, a reduction tendency of egg white protein solubility on the salt content increase was observed. Such behavior is also consequence of the salting-out effect due to the presence of the SO2 4 anions. Egg white proteins presented the higher solubility data at alkaline pH values (8.0 and 9.0). According to Fennema (1993), at alkaline pH, protein molecules have negative net charges (COO) on their surfaces and repulsion between them increases, decreasing the protein–protein interactions and increasing the protein–water interactions. At these pH values, the increase of salt content reduces the solubility (salting-out effect), due to the competition between the protein and saline ions for water molecules. Consequently the water in the protein neighborhood will be removed increasing the protein–protein interaction, thus leading to aggregation of the protein molecules, followed by precipitation (Fennema, 1993). Solubility was higher for Na2SO4 than for (NH4)2SO4 following the Hofmeister series where alkaline metal
cations are more effective in protein precipitation than ammonium cations (Leberman, 1991). Solubility increased in the order of Na2SO44(NH4)2 SO44NaCl, as observed in Table 1. Egg white protein solubility varied according to type and content of salt present in the medium for a respective pH. Similar results were reported by Arakawa and Timasheff (1982, 1984), for protein solubility of bovine serum albumin, b-lactoglobulin and lysozyme at different types of salt. These authors related a differential interaction of each protein with each type of salt present in the studied medium. Thus, the preferential salt–protein interaction is an important parameter for understanding salt efficiency in maintaining or not the protein structure stability, besides evaluating salt behavior as a promoting agent of salting-in or salting-out.
3.2. Density of egg white solution as a function of the pH and salt concentration Table 2 shows the density of egg white protein solutions as a function of the pH and salt concentration. It is observed in Table 2, for the three types of salts evaluated, the density elevation on the salt concentration increase at the pH range tested. So the addition of salt increased the protein solution density. Also, only a smooth change of density was observed on the pH variation for the studied salts. Addition of saline ions to water alters the hydrogen bond length and the chemical structure of the liquid. Changes in the salt concentration produce a gradient in hydrogen bond strength as a function of salt concentration that varies from salt to salt (Dougherty, 2001).
ARTICLE IN PRESS F. Ferreira Machado et al. / LWT 40 (2007) 1304–1307 Table 2 Density values (g/cm3) of egg white solutions as a function of pH and salt concentrationsa pH 3.0
4.6
6.0
8.0
9.0
NaCl (mol/l) 0.05 0.10 0.20 0.35 0.50
1.002 1.107 1.010 1.015 1.022
1.007 1.009 1.013 1.020 1.026
1.010 1.012 1.016 1.022 1.028
1.007 1.009 1.013 1.019 1.025
1.002 1.005 1.008 1.015 1.020
Na2SO4 (mol/l) 0.05 0.10 0.20 0.35 0.50
1.006 1.018 1.025 1.043 1.060
1.012 1.019 1.031 1.048 1.071
1.015 1.021 1.038 1.051 1.069
1.011 1.018 1.030 1.048 1.065
1.002 1.006 1.014 1.024 1.035
(NH4)2SO4 (mol/l) 0.05 1.003 0.10 1.007 0.20 1.014 0.35 1.030 0.50 1.036
1.008 1.017 1.019 1.030 1.041
1.010 1.015 1.022 1.033 1.043
1.008 1.012 1.018 1.030 1.040
1.002 1.006 1.014 1.024 1.035
a
The standard deviation was of 0.001 for all measurements.
4. Conclusion Egg white protein solubility was influenced by changes in both values of pH and salt concentrations, presenting a specific behavior for each type of salt tested. Minimum solubility values were obtained at pH 4.6 and maximum at pH 9.0, for any type of salt. At acid pH (pH 3.0) it was observed a tendency of solubility elevation on the saline concentrations increase (salting-in effect). At basic pH (pH 9.0) a solubility decrease was verified with the increase of salt content (salting-out effect). The density of the solution containing the proteins presented a direct relationship with the salt concentration. Acknowledgment The authors thank CNPq and FAPEMIG for the financial support of this work. References Arakawa, T., & Timasheff, S. N. (1982). Preferential interactions of proteins with salts in concentrated solutions. Biochemistry, 21, 6545–6552.
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Arakawa, T., & Timasheff, S. N. (1984). Mechanism of protein in saltingin and salting-out by divalent cation salts: balance between hydration and salt binding. Biochemistry, 23, 5912–5923. Awade, A. C., Moreau, S., Molle, D., Brule, G., & Maubois, S. J. L. (1994). Two-step chromatographic procedure for the purification of hen egg white ovomucin, ovotransferrin and ovalbumin and characterization of purified proteins. Journal of Chromatography A, 677, 279–288. Constenla, D. T., Lozano, J. E., & Crapiste, G. H. (1989). Thermophysical properties of clarified apple juice as a function of concentration and temperature. Journal of Food Science, 54, 663–668. Croguennec, T., Francoise, N., Stephane, P., & Gerard, B. (2000). Simple rapid procedure for preparation of large quantities of ovalbumin. Journal of Agricultural and Food Chemistry, 46, 4883–4889. Dougherty, R. C. (2001). Density of salt solutions: Effect of ions on the apparent density of water. Journal Physical Chemistry B, 105, 4514–4519. Fennema, O. R. (1993). Food chemistry (2nd ed.). New York: Marcel Dekker Inc. Ferreira, R. C. (2001). Separac- a˜o de a-lactoalbumina e b-lactoglobulina de proteı´ nas do soro de queijo por adsorc- a˜o em colunas de leito fixo (81pp). Master Science, Universidade Federal de Vic- osa, MG, Brasil. Gornall, A. G., Bardawill, C. J., & David, M. M. (1949). Determination of serum proteins by means of the biuret reaction. Journal of Biological Chemistry, 177–751. Kakalis, L. T., & Regenstein, J. M. (1986). Effect of pH and salts on the solubility of egg white protein. Journal of Food Science, 51(6), 1445–1447. Kinsella, J. E. (1982). Structure and functional properties of food proteins. In: P. P. Fox, & J. J. Condon (Eds.), Food proteins (pp. 72–85). Applied Science Published. Leberman, R. (1991). The Hofmeister series and ionic strength. Federation of European Biochemical Societies, 284(2), 293–294. Mine, Y., & Bergougnoux (1998). Adsorption properties of cholesterol reduced egg yolk low-density liprotein at oil-in-water interfaces. Journal of Agricultural and Food Chemistry, 46, 2152–2158. Nakai, S., & Chan, L. (1985). Structure modification and functionality of whey proteins: Quantitative structure—activity relationship approach. Journal of Dairy Science, 68(10), 2763–2772. Pelegrine, D. H. G., & Gasparetto, C. A. (2004). Whey proteins solubility as a function of temperature and pH. LWT—Food Science and Technology, 38, 77–80. Stadelman, W. J., & Cotterill, O. J. (1995). Egg science and technology (4th ed.). Wesport: AVI Publisher Company. Vachier, M. C., Piot, M., & Awade, A. C. (1995). Isolation of hen egg white lysozyme, ovotransferrin and ovalbumin, using a quaternary ammonium bound to a highly crosslinked agarose matrix. Journal of Chromatography B, 664, 201–210. Vojdani, F. (1996). Solubility. In G. M. Hall (Ed.), Methods of testing protein functionality (pp. 11–46). London: Blackie Academic & Professional. Wit, J. N. (1989). Functional properties of whey proteins. In P. F. Fox (Ed.), Developments in dairy chemistry (Vol.4) (pp. 285–321). London: Elsevier Applied Science. Wong, D. W. S. (1995). Mechanism and theory in food chemistry. New York: Van Nostrand Reinhold. Wong, Y. C., Herald, T. J., & Hachmeister, K. A. (1996). Comparison between irradiated and thermally pasteurised liquid egg white on functional, physical and microbiological properties. Poultry Science, 75(6), 803–808.
LWT 40 (2007) 1304–1307 www.elsevier.com/locate/lwt
Research Note
Solubility and density of egg white proteins: Effect of pH and saline concentration Fla´via Ferreira Machadoa, Jane S.R. Coimbraa,, Edwin E. Garcia Rojasb, Luis A. Minima, Fabı´ ola C. Oliveiraa, Rita de Ca´ssia S. Sousaa a
Departamento de Tecnologia de Alimentos, Universidade Federal de Vic- osa, Av. P.H. Rolfs s/n, 36570-000, Vic- osa, MG, Brazil b Departamento de Engenharia de Agronegocios e Administrac- a˜o, Universidade Federal Fluminense, Av. dos Trabalhadores 420, 27225-250, Volta Redonda, RJ, Brazil Received 30 January 2006; received in revised form 26 August 2006; accepted 31 August 2006
Abstract Influence of different pH values (3.0, 4.6, 6.0, 8.0 and 9.0) and salt concentrations (0.05, 0.1, 0.2, 0.35 and 0.5 mol/l) for three types of salt (NaCl, Na2SO4 and (NH4)2SO4) on the solubility of egg white protein and density of egg white, at room temperature (25 1C) was studied. The results showed that the solubility of egg white protein was influenced by pH as well as concentration and type of salt present in the medium. Protein solubility increased with increase in pH, with higher solubility showed at pH 9.0 and lower solubility at pH 4.6. This behavior was verified for all the salts analysed. At acid pH (pH 3.0), it was observed the tendency of solubility elevation on the increase of the saline concentration, due to the salting-in effect. The density increased with increase in the salt concentration. r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Density; Egg white; Functional properties; Protein; Solubility
1. Introduction Egg white is a natural source of proteins of recognized nutritional, biological, and technological potential interest (Awade, Moreau, Molle, Brule, & Maubois, 1994; Croguennec, Francoise, Stephane, & Gerard, 2000). Three particularly important proteins present in egg white are lysozyme, ovotransferrin and ovoalbumin. Lysozyme (3.5 g/100 g of the total egg white protein) has antibactericidal properties and is widely used in food preservation and pharmaceutical industry. Ovotransferrin (13 g/100 g of the total egg white protein) is a glycoprotein with an important role in the iron transport and with wide antimicrobial activity. Ovoalbumin, the major egg white component, corresponding to 54 g/100 g of the total egg white protein, is a glycoprotein with coagulation and gelation properties (Vachier, Piot, & Awade, 1995). Corresponding author. Tel.: +55 31 38991618/1804; fax: +55 31 38992208. E-mail address: [email protected] (J.S.R. Coimbra).
Due to unique functional properties of proteins, such as gel and foam formation, hen egg white proteins have been extensively used as ingredients in processed foods, besides being desirable ingredients in many foods, such as bakery products, pies, cookies, and meat by-products (Mine & Bergougnoux, 1998; Wong, Herald, & Hachmeister, 1996). Additionally, the commercialization of egg-derived products has gained great importance in the international market (Stadelman & Cotterill, 1995). The way proteins interact with a solvent is a manifestation of the physicochemical properties of proteins under given conditions. A wide range of protein–protein and protein–solvent interactions are involved. Many important functional food properties, including solubility, may be described through these interactions (Kakalis & Regenstein, 1986). The primary importance of protein solubility is that it influences other functional properties, such as gelation, emulsification and foam formation, so the main factor for proteins to exhibit gelatinization, foaming and emulsifying characteristics it is solubility in a food matrix (Nakai & Chan,
0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2006.08.020
ARTICLE IN PRESS F. Ferreira Machado et al. / LWT 40 (2007) 1304–1307
1985; Wit, 1989). Besides, solubility data are applied to determine the optimum conditions of protein extraction and purification from natural sources (Fennema, 1993). Solubility is affected by many factors such as amino acid composition, protein structure (native or denatured) and medium factors, such as pH, temperature, pressure, type and content of salts and protein concentration. Overall, proteins remain in solution until reaching a maximum quantity, after which they precipitate (Vojdani, 1996); pH affects the nature and distribution charges of a protein. In general, proteins are more soluble in low (acid) or high (alkaline) pH values, due to excess of charges of the same signal, producing repulsion among the molecules and, consequently, contributing to their higher solubility (Pelegrine & Gasparetto, 2004). Proteins at isolectric point (pI) generally present minimum solubility. The protein– protein interaction increases, at pI, because the electrostatic forces between the protein molecules and water molecules are minimum, which reduces the number of water molecules that interact with protein molecules. This is a favorable condition for the approximation and aggregation of protein molecules, leading to their precipitation (Vojdani, 1996). Salts can also affect the electrostatic interactions among the macromolecules, contributing through the ionic force (Fennema, 1993). The effect of salts on protein solubility in aqueous solutions is a function of the ionic species present (Arakawa & Timasheff, 1982). Kinsella (1982) showed that a salt concentration of 0.15 mol/l is sufficient to change the structure of the water and conformation of the proteins. However, this will depend on the content and type of salt present in the medium. Thus, the objective of this work was to evaluate the influence of pH and saline concentration on solubility and density of egg white protein for three types of salt (NaCl, Na2SO4 and (NH4)2SO4) at room temperature. 2. Materials and methods
1305
immediately mixed in magnetic stirrer until complete dissolution. The solution was quantitatively transferred to a volumetric flask of 10 ml, completed with 1 mol equivalent/l NaOH, manually agitated, and the protein content quantified. 2.3. Solubility determination Solubility data were obtained according to Kakalis and Regenstein (1986), by adding 200 mg of dehydrated egg white into 30 ml of a buffer solution. The buffers used were glycine–HCl, citrate–citric acid, phosphate–citrate, phosphate and Tris–HCl leading to the pH values of 3.0; 4.6; 6.0; 8 and 9.0, respectively. Each buffer solution was previously added by the salt to be evaluated (NaCl, Na2SO4 or (NH4)2SO4) at pre-determined concentrations (0.05; 0.01; 0.2; 0.35 and 0.5 mol/l). The mixture was agitated for 1 h, at room temperature (25 1C), using a device to simulate an agitated tank (Ferreira, 2001) and immediately centrifuged at 20 000 g for 30 min at approximately 4 1C (Beckman centrifuge, model J2-MC, USA). After centrifugation, the supernatant was collected for analyses. The solubility (S, g protein in the supernatant/100 g total amount protein in sample before centrifugation) was calculated by PS S¼ 100, (1) PA where PS and PA are the protein amounts (g) in the supernatant and sample, respectively. Each experiment was performed in duplicate, with the soluble protein content being the average of the values of two repetitions. 2.4. Density data Density data were determined by applying the Pycnometric Method (Constenla, Lozano, & Crapiste, 1989), using a 10 ml pycnometer. The pycnometer was calibrated with deionized water at room temperature. Corrections for temperature were shown to be negligible.
2.1. Materials 3. Results and discussion Samples of standard dehydrated egg white were a kindly gift from Sohovos Industrial Ltda (Brazil), packed in polyethylene bags. Deionized water and analytical grade reagents were used in the experiments. 2.2. Protein quantification Protein determinations were based on the Biureto reaction (Gornall, Bardawill, & David, 1949), using an analytical curve built by varying ovoalbumin (Sigma Chemicals, USA) concentration in aqueous solutions from 0.5 to 8.0 mg/ml. Absorbance was measured at 540 nm using a Cary 50 spectrophotometer (Varian, Australia). For protein quantification of dehydrated egg white protein, 100 mg of the material was carefully weighed in a beaker of 25 ml, added 6 ml of 1 mol equivalent/l NaOH, and
3.1. Effect of pH, type and concentration of salt on protein solubility It is observed in Table 1 the solubility increase with the pH increase, for all tested salts. Therefore, extraction yield and solubility are higher in alkaline pH than in acid pH. In fact, the number of negatively charged ions at pH4pI is larger than the number of positively charged ions at pHopI (Fennema, 1993). At pH 3 it was verified a tendency of solubility increase with increasing concentrations of the salts Na2SO4 and (NH4)2SO4. Some type of salts may increase protein solubility, at concentrations ranging from 0.1 to 1.0 mol/l, due to the salting-in phenomenon by which saline ions interact with groups of opposite protein charges to form a
ARTICLE IN PRESS F. Ferreira Machado et al. / LWT 40 (2007) 1304–1307
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Table 1 Solubility of egg white protein (g/100 g) as a function of pH and salt concentrations pH 3.0
4.6
6.0
8.0
9.0
NaCl (mol/l) 0.05 0.10 0.20 0.35 0.50
34.7571.48 55.8872.52 55.0571.55 38.5074.33 39.8372.46
47.8675.60 47.4670.56 38.8072.61 56.1870.09 61.9271.58
63.6270.74 66.1272.87 78.9770.38 68.6870.19 72.3372.05
73.9472.50 72.5771.44 73.5570.80 71.9371.85 76.8371.75
79.2270.12 70.0772.95 62.2371.85 68.8572.01 59.2770.87
Na2SO4 (mol/l) 0.05 0.10 0.20 0.35 0.50
57.4970.26 72.2070.57 62.1971.74 65.8876.51 68.4473.62
65.3773.48 62.5871.74 68.1275.28 65.9171.13 63.7372.10
81.9473.53 84.7371.60 65.6670.46 84.0070.30 75.8570.61
81.0372.91 84.2773.41 83.8771.50 74.6670.27 72.0676.78
91.9470.60 90.1570.93 93.2470.64 81.0271.11 84.1172.77
(NH4)2SO4 (mol/l) 0.05 0.10 0.20 0.35 0.50
60.3270.14 60.6970.88 62.4270.88 77.2876.58 63.6370.02
56.3371.40 66.1072.78 56.5671.19 67.1873.93 65.1871.72
79.2871.33 83.3270.34 72.9170.67 73.1073.94 73.0771.56
85.0971.09 87.1573.71 81.3070.12 78.8171.23 72.4371.20
90.6170.28 78.9372.76 86.5170.38 81.4670.60 81.4670.48
double layer of ionic groups, than the electrostatic interaction among the protein molecules reduces, increasing their solvatation (Vojdani, 1996). A lower solubility was observed around pH 4.5, which is the isoelectric point of ovoalbumin, the major egg white component (54 g/100 g of the egg white protein). At the isoelectric point, the protein molecule has zero liquid charge, exhibiting the maximum of electrostatic interactions among the proteins charged groups. Since the charged groups offer minimum availability to interact with water molecules, the amount of bound water decreases minimally, thus reducing solubility (Wong, 1995). Proteins solubility at pH 8.0, for NaCl was practically constant. In the presence of Na2SO4 and (NH4)2SO4, a reduction tendency of egg white protein solubility on the salt content increase was observed. Such behavior is also consequence of the salting-out effect due to the presence of the SO2 4 anions. Egg white proteins presented the higher solubility data at alkaline pH values (8.0 and 9.0). According to Fennema (1993), at alkaline pH, protein molecules have negative net charges (COO) on their surfaces and repulsion between them increases, decreasing the protein–protein interactions and increasing the protein–water interactions. At these pH values, the increase of salt content reduces the solubility (salting-out effect), due to the competition between the protein and saline ions for water molecules. Consequently the water in the protein neighborhood will be removed increasing the protein–protein interaction, thus leading to aggregation of the protein molecules, followed by precipitation (Fennema, 1993). Solubility was higher for Na2SO4 than for (NH4)2SO4 following the Hofmeister series where alkaline metal
cations are more effective in protein precipitation than ammonium cations (Leberman, 1991). Solubility increased in the order of Na2SO44(NH4)2 SO44NaCl, as observed in Table 1. Egg white protein solubility varied according to type and content of salt present in the medium for a respective pH. Similar results were reported by Arakawa and Timasheff (1982, 1984), for protein solubility of bovine serum albumin, b-lactoglobulin and lysozyme at different types of salt. These authors related a differential interaction of each protein with each type of salt present in the studied medium. Thus, the preferential salt–protein interaction is an important parameter for understanding salt efficiency in maintaining or not the protein structure stability, besides evaluating salt behavior as a promoting agent of salting-in or salting-out.
3.2. Density of egg white solution as a function of the pH and salt concentration Table 2 shows the density of egg white protein solutions as a function of the pH and salt concentration. It is observed in Table 2, for the three types of salts evaluated, the density elevation on the salt concentration increase at the pH range tested. So the addition of salt increased the protein solution density. Also, only a smooth change of density was observed on the pH variation for the studied salts. Addition of saline ions to water alters the hydrogen bond length and the chemical structure of the liquid. Changes in the salt concentration produce a gradient in hydrogen bond strength as a function of salt concentration that varies from salt to salt (Dougherty, 2001).
ARTICLE IN PRESS F. Ferreira Machado et al. / LWT 40 (2007) 1304–1307 Table 2 Density values (g/cm3) of egg white solutions as a function of pH and salt concentrationsa pH 3.0
4.6
6.0
8.0
9.0
NaCl (mol/l) 0.05 0.10 0.20 0.35 0.50
1.002 1.107 1.010 1.015 1.022
1.007 1.009 1.013 1.020 1.026
1.010 1.012 1.016 1.022 1.028
1.007 1.009 1.013 1.019 1.025
1.002 1.005 1.008 1.015 1.020
Na2SO4 (mol/l) 0.05 0.10 0.20 0.35 0.50
1.006 1.018 1.025 1.043 1.060
1.012 1.019 1.031 1.048 1.071
1.015 1.021 1.038 1.051 1.069
1.011 1.018 1.030 1.048 1.065
1.002 1.006 1.014 1.024 1.035
(NH4)2SO4 (mol/l) 0.05 1.003 0.10 1.007 0.20 1.014 0.35 1.030 0.50 1.036
1.008 1.017 1.019 1.030 1.041
1.010 1.015 1.022 1.033 1.043
1.008 1.012 1.018 1.030 1.040
1.002 1.006 1.014 1.024 1.035
a
The standard deviation was of 0.001 for all measurements.
4. Conclusion Egg white protein solubility was influenced by changes in both values of pH and salt concentrations, presenting a specific behavior for each type of salt tested. Minimum solubility values were obtained at pH 4.6 and maximum at pH 9.0, for any type of salt. At acid pH (pH 3.0) it was observed a tendency of solubility elevation on the saline concentrations increase (salting-in effect). At basic pH (pH 9.0) a solubility decrease was verified with the increase of salt content (salting-out effect). The density of the solution containing the proteins presented a direct relationship with the salt concentration. Acknowledgment The authors thank CNPq and FAPEMIG for the financial support of this work. References Arakawa, T., & Timasheff, S. N. (1982). Preferential interactions of proteins with salts in concentrated solutions. Biochemistry, 21, 6545–6552.
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