- Email: [email protected]
Rheological properties of acidic soybean protein gels: Salt addition effect
Food Hydrocolloids 13 (1999) 167–176
Rheological properties of acidic soybean protein gels: Salt addition effect Marı´a Cecilia Puppo*, Marı´a Cristina An˜o´n Centro de Investigacio´n y Desarrollo en Criotecnologı´a de Alimentos (CIDCA), Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), 47 Y 116 (1900) La Plata, Argentina Received 17 March 1998; received in revised form 10 July 1998; accepted 30 September 1998
Abstract The objective of this work was to study rheological properties of acidic soybean protein gels in the presence of sodium and calcium chloride. The viscoelastic properties of acidic gels were affected by the amount and type of the assay salt. The pH 2.75 dispersions prepared with 0.1–0.25 M NaCl behaved as diluted macromolecular or entanglement solutions and were more viscous than elastic. At high ionic strength (2.0 M NaCl), samples were more elastic with a gel-like behavior. A semidiluted macromolecular solutions was observed for dispersions at pH 3.50 with 0.1 M NaCl, while at high salt concentration (0.25–2.0 M) gels were obtained. At pH 2.75 gelation kinetics was different at several NaCl concentrations, while at pH 3.50, first order kinetics was observed. At pH 2.75 and 0.1 M CaCl2, the dispersions behaved as semidiluted macromolecular solutions, while at 0.2 M CaCl2 diluted macromolecular solutions were obtained. A semidiluted solution dynamic spectra for pH 3.50 samples (0.1–0.2 M CaCl2) was observed. Both pH 2.75 and 3.50 gels prepared with several NaCl concentrations were more fragile, hardest and resistant rupture than those obtained with CaCl2. The textural properties of acidic gels were less modified by CaCl2. 䉷 1999 Elsevier Science Ltd. All rights reserved. Keywords: Soybean protein gelation; Gelation in acidic conditions; Heat-induced gelation; Salt effect; Rheological properties
1. Introduction Soybean proteins play an important role in many foodstuffs because they have high nutritional value and contribute significally to food texture. Studies on the use of these proteins as functional ingredients in foods must be carried out to optimize food formulation. Previous authors investigated the effect of heat treatment, pH, protein concentration, ions (sodium and calcium chloride) in protein dispersions (Hermansson, 1978; Van Kleef, 1986). It was found that heat treatment induces denaturation and aggregation of soy protein molecules and that at high protein concentration (more than 7% w/w in soy proteins), the aggregates formed produce a self-supporting gel. Soy protein isolate-based gels contain a variety of food ingredients such as sodium chloride to enhance flavor (Yao et al., 1990) or a nutrient mineral used as a coagulant agent such as calcium chloride (Lee and Rha, 1977). To understand the effect of salt addition on the gelation properties of proteins, food protein gels should be described in physicochemical and rheological terms, according to Clark and LeeTuffnell (1986). Gel characteristics are straightforwardly related to their texture (Kinsella, 1979; Hermansson, * Corresponding author. Tel.: ⫹ 54 21 254853; e-mail: mca@nahuel. biol.unlp.edu.ar
1985; Kohyama and Nishinari, 1993) and also to their viscoelasticity and both properties features are determined in turn by the molecular properties of the constituent protein. Since globulins form gels upon heating (Utsumi et al., 1982; Nakamura et al., 1984; Mori etal., 1986), thorough studies at neutral pH have been carried out on the effect of sodium (Catsimpoolas and Meyer, 1970; Hermansson, 1978, 1986) and calcium (Scilingo and An˜o´n, 1996) cations on the behavior of the major soybean protein globulins. NaCl and CaCl2 salts are extensively used as additives along with soy protein to enhance emulsification, gelation and water and lipid retention in meat products, allowing the desired texture to be achieved (Visser and Thomas, 1987; McMindes, 1991). The preparation of acidic soy proteinbased foods require an exhaustive analysis of the gelation properties of soybean proteins at pH values below the pI, and for different ionic strengths obtained with either NaCl or CaCl2 addition. Structural properties of heat-induced acidic soy-protein gels under different conditions of pH and ionic strength have been studied (Puppo and An˜o´n, 1998a). Hydration properties such as water holding capacity, the type of bonds that stabilize gel structure and the protein species that make up and/or stabilize gel structure were also analyzed (Puppo and An˜o´n, 1998a). The WHC of gels prepared with NaCl and CaCl2 decreased with
0268-005X/99/$ - see front matter 䉷 1999 Elsevier Science Ltd. All rights reserved. PII: S0268-005 X( 98)00 079-4
168
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
increasing salt concentration. This fact suggested, as was corroborated by gel SEM, that at high ionic strength a more open matrix was formed (Puppo and An˜o´n, 1998a). The structure of acidic gels, stabilized by non-covalent bonds, changed with the addition of 2 M NaCl. Both 7S and 11S globulin subunits participated via hydrophobic interactions on the stabilization of pH 2.75 gel structure. At pH 3.50 the gel matrix was stabilized by hydrophobic interactions among b -conglycinin subunits, whereas the AB-11S subunit and the AB-11S polymers, linked by disulfide bonds, would be soluble in the matrix interior owing to the glycinin fraction that remains native after thermal treatment (Puppo and An˜o´n, 1998a). The objective of the present work was to study the influence of pH and ionic strength on viscoelastic and textural properties of acidic soybean protein gels. 2. Materials and methods 2.1. Preparation of soy protein isolate Soy proteins isolates were prepared from defatted flour produced by Santista Alimentos S.A. (Brasil). Proteins were obtained by alkaline extraction (pH 8.0) of the flour and subsequent precipitation at the isoelectric point (pI 4.5), as described by Puppo et al. (1995). The isoelectric precipitate was dispersed in distilled water and brought to pH 3.25 with 2 N HCl. To obtain the acidic isolate, the resulting dispersion was freeze-dried. The pH 3.25 isolate thus obtained, contained 95.22 ^ 0.15% protein as determined by Kjeldahl method (N × 6.25). 2.2. Preparation of soy protein dispersions 10% w/w protein suspensions were prepared by hydrating the pH 3.25 isolate in distilled water, NaCl (0.1–2.0 M) or CaCl2 (0.1 and 0.2 M) solutions under constant magnetic stirring at room temperature during 20 min. Dispersions were adjusted to pH 2.75 and 3.50 with 1 N HCl or 1 N NaOH, respectively. 2.3. Dynamic tests: viscoelasticity determinations Rheological measurements were carried out in a Haake CV20 Rheometer. The sample dispersion (0.5 ml) was placed between parallel plates (d 27.83 mm) and the gap between the two plates was set to 1 mm. The lower plate was held at 90⬚C. Low viscosity silicone was added around the plate edges to prevent dehydration and to permit a total contact between sample and silicone. The equipment was driven through the Haake software osc. 2.0. Complex modulus (G*), storage modulus (G 0 ), loss modulus (G 00 ), tan d (G 00 /G 0 ) and complex viscosity (h *), were recorded as a function of time and frequency of oscillation. The scans were performed by duplicate. The linear viscoelasticity range was determined in all
dispersions measuring G* as a function of deformation at a shear oscillation of 1 Hz. In all tests, a deformation of 10% was determined within the linear viscoelasticity range. The variation of G 0 , G 00 and h * (10% deformation) as a function of dynamic frequency was determined after heating the sample for 30 min at 90⬚C. Sample rheological behavior was studied comparing the dependence of G 0 and G 00 with frequency (Giboreau et al., 1994; Ross-Murphy, 1956, 1987, 1995). The variation of G 0 , G 00 and tan d (f 1 Hz) with sample pH and ionic strength was analyzed. Storage modulus, G 0 , of pH 2.75 and 3.50 (10% w/w) dispersions, prepared with NaCl (0.1–2.0 M) and CaCl2 (0.1–0.2 M) solutions, was recorded as a function of time (1 Hz, 10% deformation, 90⬚C). Based on previous work of other researchers (Nishinari et al., 1991; Yoshida et al., 1992; Kokini and Dickie, 1981), different non-linear models were adapted to interpret experimental data. The data corresponding to dispersions prepared with NaCl (0.1 M, pH 2.75; pH 3.50) and with CaCl2 were represented by Eq. (1) (Nishinari et al., 1991; Yoshida et al., 1992): G 0 t G 0sat 1 ⫺ exp ⫺kt
1
where G 0sat is the saturation storage modulus, k the thermal treatment rate constant and t the time. The values corresponding to dispersions of pH 2.75 prepared with 0.5 and 2.0 M NaCl were represented by Eq. (2): G 0 t G 0sat 1 ⫹ at ⫺ 1exp ⫺k 0 t
2
were G 0sat represents the saturation storage modulus and, a and k 0 are constants. Eq. (2) is analogous to the Bird–Leider equation used by Kokini and Dickie (1981), a and k 0 are related to the G 0 increment at short times and the G 0 decrease at saturation times, respectively. A non-linear regression (SYSTAT, V5.0) was used. 2.4. Compression tests 2.4.1. Preparation of gels The dispersions prepared in distilled water, NaCl (0.1– 2.0 M) and CaCl2 (0.1–0.2 M) solutions were partially deaerated at 15⬚C by centrifugation at 1000g for 1 min, and resuspended using a magnetic stirrer. The samples were placed in glass tubes (length, 6 cm; internal diameter, 2.2 cm) hermetically sealed in both ends with rubber plugs. Gelation was carried out by heating the tubes at 90⬚C for 30 min followed by immediate cooling, by using a water bath set at 15⬚C. The gels so formed were kept at 4⬚C for 24–48 h to ensure complete gelation (Puppo et al., 1995). 2.4.2. Determination of texture Gels were compressed at room temperature (20⬚C) to 80% of their original height (3 cm) (Lee and Batt, 1993) by means of 1101 model Instron press, using a 50 N cell with a cross head speed of 100 mm/min. In each
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
169
Fig. 1. Variation of Storage modulus, G 0 (X) (Pa), Loss modulus, G 00 (O) (Pa) and Complex viscosity, h * (B) (Pa.s) with frequency for (10% w/w) acidic soybean protein dispersions: pH 2.75 (a, b, c, d) and 3.50 (e, f, g, h). NaCl: (a, e) 0.1 M; (b, f) 0.25 M; (c, g) 0.5 M; (d, h) 2.0 M.
determination, a minimum of 4 gels were used. A one-cycle uniaxial compression test was carried out using parallel plates to determine rupture strength or fracturability (F) parameters and hardness (H) (Andersson et al., 1973). The curves obtained allowed the deformability modulus (ED) to be calculated from the initial slope along with the relative deformation at rupture (Dr) (Peleg, 1977; Nussinovitch et al., 1989, 1990). The data were statistically analyzed by a two-way analysis of variance (ANOVA). Sources of variation were pH (2.75 and 3.50) and salt (NaCl or CaCl2) concentration. The significance of differences among means of the several treatments was determined by Tukey’s test at P ⬍ 0.05. Difference between means (D0.05) for each texture parameter was calculated.
3. Results and discussion 3.1. Effect of sodium chloride: viscoelasticity of dispersions The dispersions of 10% w/w acidic soy protein isolates in NaCl were heated for 90⬚C during 30 min. The temperature was maintained at 90⬚C and the storage and loss moduli were recorded at 10% strain as a function of the oscillation frequency (Fig. 1). In the presence of 0.1 M (Fig. 1(a)) and 0.25 M NaCl (Fig. 1(b)), the pH 2.75 samples behave as dilute macromolecular solutions or entanglement solutions (Giboreau et al., 1994; Ross-Murphy, 1995) where G 0 is below G 00 at low frequencies. Both moduli, G 0 and G 00 , experience a linear increase at increasing oscillation frequencies and cross-over at high frequencies (5–6 Hz).
170
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
Fig. 2. Variation of Storage modulus, G 0 (a, c), Loss modulus, G 00 (a, c) and Loss tangent, tan d (b, d) with the NaCl concentration of the gel. Gels of pH 2.75 (a, b); pH 3.50 (c, d). Error bars: standard deviation.
In this case there is no long time interactions between macromolecules. The dispersions of pH 2.75 in 0.5 M NaCl behave as semidiluated macromolecular solutions or more entangled semidiluted solutions (Giboreau et al., 1994; Ross-Murphy, 1995), G 0 increase continuously and cross-over G 00 at a frequency of 0.4 Hz (Fig. 1(c)). This type of spectrum is closer to a gel and can be described as ‘‘highly elastic solutions’’ where an association phenomenon between ordered protein chain segments has been assumed (Cuvelier and Launay, 1986). At 2 M NaCl, samples behave as a gel, G 0 remains constant and G 00 shows a minimum at about 2 Hz (Fig. 1(d)). Gel like (Giboreau et al., 1994) or strong (Ross-Murphy, 1995) materials show a prevailing elastic modulus over the complete range of frequencies. G 0 moduli is independent of frequency and can be described as an elastic plateau, characteristic of gels. In the samples whose pH is close to the pI (pH 3.50, 0.1 M NaCl) (Fig. 1(e)), the G 0 and G 00 curves cross over at low frequencies (⬇ 0:2–0.3 Hz) before reaching the plateau region. This behavior corresponds to semidiluted macromolecular dispersions. At frequencies above 0.2, the pH 3.50 dispersions prepared with 0.25, 0.5 and 2 M NaCl behave like gels (Giboreau et al., 1994; Ross-Murphy, 1987, 1995) where G 0 is independent of frequency and a minimum
of G 00 is observed (Fig. 1(f–h)). The viscoelastic behavior of dispersions can be characterized not only by their dynamic spectra but also by the variation of the dynamic viscosity (h *). In both acidic dispersions (pH 2.75 and 3.50), h * decreases with increasing frequency (Fig 1(a–h)). Adding NaCl to the samples induce changes in the viscoelastic moduli and the magnitude of these depends on pH. Both G 0 and G 00 values of pH 2.75 samples prepared with 0.1 M NaCl were higher than those observed in acidic soy protein isolate dispersed in distilled water, particularly the viscous modulus. By contrast, higher salt concentrations ( ⬎ 0.25 M) decreases both parameters (Fig. 2(a)). After salt addition, the tan d increases and so does the viscous component in dispersions prepared with 0.1 and 0.25 M NaCl. A higher NaCl content increases elasticity in pH 2.75 dispersions (Fig. 2(b)). All pH 3.50 gels are more elastic than viscous (tan d ⬍ 1) regardless of gel ionic strength (Fig. 2(d)). Both the elasticity and the viscous moduli decrease for increasing ionic strengths (Fig. 2(c)), and they do so to an extent that gel elasticity becomes unaffected by saline concentration, as demonstrated by the constancy of tan d (Fig. 2d). The decrease of tan d indicates that the system tends to a more solid-like state with the formation of gel networks (Kohyama and Nishinari, 1992; Nagano et al., 1994).
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
171
3.2. Gelation kinetics
Fig. 3. Variation of Storage modulus, G 0 with time of heating of gels (10% w/w) of pH 2.75 (a) and 3.50 (b) prepared with NaCl 0.1 M (O), 0.5 (D) and 2.0 M (A); (—) empirical model (r ⬎ 0.997).
Typical gelation curves for acidic dispersions (10% w/w) containing different amount of NaCl are shown in Fig. 3. For the pH 2.75 and 0.1 M NaCl dispersion, a constant G 0 value G 0sat reached within 30 min of heat treatment was obtained (Fig. 3(a)). The dispersion kinetics could be well approximated by Eq. (1) (Materials and methods, (Section 2.3)). The G 0sat and k values obtained were 1492 Pa and 1.97 × 10 ⫺3 s ⫺1, respectively. These values were higher than those observed in distilled water dispersions: 915 Pa and 4.08 × 10 ⫺4 s ⫺1, respectively (Puppo and An˜o´n, 1998b). Therefore the addition of small amounts of NaCl increased rheological parameters. For higher ionic strengths (0.5 and 2.0 M NaCl), a maximum of G 0 modulus was obtained before reaching the G 0sat value (Fig. 3(a)). Eq. (2) (Materials and methods, (Section 2.3)) better fit these kinetic data. At high ionic strength two phenomenon were observed: gelation (G 0 maximum) followed by protein aggregation at high heating times evidenced by a slight decrease in G 0 modulus. The ratio a /k 0 is related to the G 0 maximum observed at short times. The a /k 0 values, for 0.5 and 2.0 M NaCl pH 2.75 dispersions, were 0.072 and 0.123, respectively. This fact would indicate that at 2.0 M NaCl the gelation process was more pronounced than at 0.5 M MaCl. The curves of the pH 3.50 dispersions showed that G 0sat values were reached within the 15 min of heating regardless of the amount of NaCl present (Fig. 3(b)). The G 0sat values were 1930, 2079 and 367 Pa for the dispersions prepared with 0.1, 0.5 and 2.0 M NaCl, respectively. The dispersions followed the kinetics expression given by Eq. (1) (Materials and methods, (Section 2.3)) and the gelation rate constants obtained were 3.48 × 10 ⫺3 s ⫺1, 5.96 × 10 ⫺3 s ⫺1 and 1.11 × 10 ⫺3 s ⫺1 for the 0.1, 0.5 and 2.0 M NaCl, respectively. The results suggest that lower values of G 0sat and gelation rate were observed at high ionic strength. At low ionic strengths, where soy proteins are partially stabilized by the addition of NaCl (Puppo and An˜o´n, 1999, thermal treatment produced a steady gelation of both acidic pH dispersions. At high ionic strengths and pH 2.75, protein dispersion gelation was observed; followed (after 30 min heat treatment) by a decrease in the elastic component owing to protein aggregation (Fig. 3(a)). 3.3. Texture of gels
Fig. 4. Force-time curves of pH 2. 75 (a) and pH 3.50 (b) gels prepared with different NaCl solutions. NaCl concentration: (A) 0.1 M, (B) 0.25 M and (C) 0.5 M.
The gels prepared with NaCl presented a two-peak profile (Fig. 4). The first peak represents the fracturability (I) and the second, the hardness (II). In more acidic pH (2.75), the only peak observed in the absence of salt splits into two peaks (I and II), the gap between which increase with saline content (Fig. 4(a)). Acidic gels containing a large amount of NaCl produced exudate owing to protein aggregation, in turn caused by the salting out effect. In the presence of NaCl, the pH 3.50 gels maintained the two peak profile observed in the gels prepared with distilled water, where
172
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
Fig. 5. Rupture force, F (a), hardness, H (b), deformability moduli, ED (c) and relative deformability at rupture, Dr (d) of gels (10% w/w) prepared with NaCl solutions of concentration: o 0 M, g 0.1 M, n0.25 M and m 0.5 M. Error bars: standard deviation. (a) D0.05 1.23, (b) D0.05 1.97, (c) D0.05 2.84, (d) D0.05 0.10.
the fracturability peak (I) decreases with the saline content (Fig. 4(b)). After compression, the acidic gels displayed a negative peak (III) of increasing area, indicating an increase in adhesiveness with the increase of saline concentration (Fig. 4 (a,b)). Higher rupture force values of pH 2.75 gels prepared with
0.1 M NaCl were observed (Fig. 5(a)) while the hardest gel was obtained with 0.25 M NaCl (Fig. 5(b)). The pH 3.50 gels behaved differently; the rupture force decreased with addition of NaCl and reached (0.5 M NaCl) values below 1 N (Fig. 5(a)). The harder gels were those prepared with 0.1 and 0.5 M NaCl (Fig. 5(b)).
Fig. 6. Variation of Storage modulus, G 0 (X) (Pa), Loss modulus, G 00 (O) (Pa) and Complex viscosity, h * (B) (Pa.s) with frequency of 10% w/w gels of pH 2.75 (a, b) and pH 3.50 (c, d) prepared with CaCl2 0.1 M (a, c) and 0.2 M (b, d).
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
173
Fig. 7. Variation of Storage modulus, G 0 (a, c), Loss modulus, G 00 (a, c) and Loss tangent, tan d (b, d) with CaCl2 concentration. pH of the gels: 2.75 (a, b); pH 3.50 (c, d). Error bars: Standard deviation.
At acidic pH, the denaturation temperatures of soybean proteins are below the working temperature (90⬚C) even at 0.5 M NaCl concentrations (Puppo and An˜o´n, 1999). Therefore, gels formed are structurally more fragile, with decreasing water holding capacity for increasing ionic strengths (Puppo et al., 1995). At high concentrations, salt causes specific ionic effects on protein conformation: the modification of hydrophobic interactions between non-polar residues owing to charge neutralizing effect (Chronakis, 1996). Acidic gels with more resistance to deformation (higher ED) were those prepared at pH 2.75, 0.25 M NaCl and pH 3.50, 0.1 and 0.25 M NaCl. At very high saline concentrations (0.5 M), the resistance to deformation becomes similar to that of gels prepared in distilled water (Fig. 5(c)). Fig. 5(d) shows that the relative deformability at rupture (Dr) increases for the more acidic gels in 0.5 M NaCl, whereas at pH 3.50, small amounts of added salt produce gels of high Dr that becomes independent of the amount of salt added to the gel. 3.4. Effect of calcium chloride: viscoelasticity of dispersions The viscoelastic behavior of pH 2.75 dispersions prepared in 0.1 and 0.2 M CaCl2 are shown in Fig. 6(a) and (b). At 0.1 M CaCl2, G 0 and G 00 cross over at a frequency of 1 Hz while at 0.2 M, the curves increase linearly for increasing frequencies until they cross over beyond 7 Hz.
Fig. 8. Variation of Storage modulus, G 0 with heating time, for gels (10% w/w) of pH 2.75 (a) and 3.50 (b) prepared with CaCl2 0.1 M (W) and 0.2 M (K). (—) empirical model (r ⬎ 0.993).
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
174
Table 1 Effect of CaCl2 addition on the constant gelation rate (k × 10 3 s ⫺1) of acidic soybean protein dispersions (10% w/w) heated at 90⬚C r ⬎ 0.993 CaCl2 (M)
pH 2.75
pH 3.50
0.1 0.2
1.25 1.61
2.17 1.00
Rheological behavior at low ionic strength is comparable to that of semidiluted macromolecular dispersions (Giboreau et al., 1994; Ross-Murphy, 1987, 1995), and the dispersion looses elasticity at higher ionic strengths, becoming more fluid-like. The frequency scanning of pH 3.50 dispersions (Fig. 6(c) and (d)) at 0.1 and 0.2 M CaCl2 show that G 0 and G 00 crossed over at frequencies lower than 0.5 Hz and for frequencies above 0.5 Hz typical behavior of semidilute macromolecular solutions was observed. The mechanical spectrum of h * (Fig 6(a–d)) also reflected this viscoelastic behavior. Rheological behavior suggests that, at more acidic pH, a weak matrix is formed at 0.1 M CaCl2, while no structure is developed with addition of 0.2 M CaCl2. At pH close to the pI the matrix was not affected by the increment of CaCl2 concentration. Fig. 7 shows, for acidic dispersions, the variation of G 0 and G 00 moduli, and of tan d (1 Hz frequency) with CaCl2 concentration. At pH 2.75 and 0.1 M CaCl2, both the elastic and the viscous moduli increase. Although G 00 is lower than G 0 , it increases more than the elastic modulus (Fig. 7(a)); so that tan d increases (Fig. 7(b)) and the sample is less elastic. At 0.2 M CaCl2, G 00 becomes higher than G 0 and the sample gets even more viscous-like than in the absence of salt; this fact was confirmed by the large increase observed in tan d (Fig. 7(a) and (b)). Fig. 7(c,d) shows, for pH 3.50 dispersions, the change in viscoelastic parameters (1 Hz) with the CaCl2 content. The elastic modulus is higher than the viscous one regardless of the ionic strength. Besides, the same decrease in G 0 and G 00 moduli is observed as the amount of calcium added to the gel increases (Fig. 7(c)). Thus, tan d does not vary with saline content (Fig. 7(d)).
3.5. Gelation kinetics Fig. 8 shows the variation of G 0 with heating time (90⬚C) of 10% w/w acidic dispersions. After 60 min thermal treatment, the elastic moduli of pH 2.75 dispersions prepared in distilled water (graph not shown) was 915 Pa (Puppo and An˜o´n, 1998b), while the dispersions prepared with 0.1 and 0.2 M CaCl2 G 0 values were higher (Fig. 8(a)). No differences between 0.1 and 0.2 M CaCl2 heating curves were observed (Fig. 8(a)), whereas G 0 values of pH 3.50 dispersions were higher at 0.2 M CaCl2 (Fig. 8(b)) with a G 0sat value of 3678 Pa. Gelation kinetic of dispersions prepared with CaCl2 can be represented by the gelation rate constants shown in Table 1. These constants were obtained by fitting experimental data to the model represented by Eq. (1). At pH 2.75, increased CaCl2 concentrations increase the gelation rate, whereas close to the pI an opposite behavior is observed. 3.6. Texture of gels Fig. 9 displays the texture profiles of acidic gels prepared with 0.1 M CaCl2 (m 0.3). As in the case of NaCl, both gels containing CaCl2 show two peaks, the hardness peak is lower than the rupture one. The gels prepared with the divalent salt are more resistant to rupture than the gels prepared with NaCl. The profile of the acidic gels prepared with 0.2 M CaCl2 (m 0.6) is similar to that observed at the lower ionic strength, but the time observed between strength and hardness peaks is longer than at m 0.3 (graph not shown). Gels of pH 2.75 prepared with 0.1 and 0.2 M CaCl2 presented high rupture strength, while gel hardness did not change with salt addition (Fig. 10 (a) and (b)). Gels of pH 2.75 prepared with CaCl2 (m 0.3) presented higher gel strength than those prepared with NaCl (m 0.25). The electrostatic bond linking Ca 2⫹ to the protein is stronger than that of a noncovalent cation (e.g., Na ⫹), leading to high viscosity soy protein dispersions [37]. The texture of pH 3.50 gels was unaffected by the presence of CaCl2 (Fig. 10(a) and (b)). The resistance to deformation of pH 2.75 gels is higher when adding 0.2 M CaCl2 whereas, in pH 3.50 gels, the highest value is obtained for 0.1 M CaCl2 (Fig. 10(c)). However, the relative deformability neither changes with the pH nor with the CaCl2 content (Fig. 10(d)). 4. Conclusions
Fig. 9. Force–time curves of gels (10% w/w) of pH 2.75 (A) and pH 3.50 (B) prepared with CaCl2 0.1 M solution.
Acidic dispersions prepared with sodium and calcium salts showed different rheological behavior at different ionic strengths. An increase of ionic strength from 0.25– 0.5 of pH 2.75 dispersions prepared with NaCl favored molecular entanglement; meanwhile dispersions prepared with CaCl2 were more fluid. At pH 3.50, in the same range of ionic strength, the addition of sodium produced dispersions with a gel like behavior; whereas in the presence
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
175
Fig. 10. Rupture force, F (a), hardness, H (b), deformability moduli, ED (c) and relative deformability at rupture, Dr (d) of acidic gels (10% w/w) prepared with CaCl2: o 0 mM; g 0.1 M; n 0.2 M. Error bars: standard deviation. (a) D0.05 1.60, (b) D0.05 1.48, (c) D0.05 1.19, (d) D0.05 0.14.
of calcium, macromolecular semidiluted dispersions were formed. Both pH 2.75 and 3.50 gels (m 0.25–0.5) prepared with NaCl were more fragile but more harder than those prepared with CaCl2. At 0.25 ionic strength, gels with much more resistance to deformation and of greater relative deformability at rupture were obtained with NaCl than with CaCl2. Acknowledgements This investigation was supported by the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET). Argentina. M.C.P. is a Research Fellow of the Comisio´n Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET) and M.C.A. is Member of the Researcher Career of the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET). This work was also supported by grants from BID-SECYT. References Andersson, Y., Drake, B., Granquist, A., Halldin, L., Johansson, B., Pangborn, R. M., & Akesson, C. (1973). Fracture force, hardness and brittleness in crisp bread, with a generalized regression analysis approach to instrumental-sensory comparisons. J. Text. Stud., 4, 119. Catsimpoolas, N., & Meyer, E. W. (1970). Gelation phenomena of soybean globulins. I. Protein–protein interactions. Cereal Chem., 47, 559–570. Chen, W. S., & Soucie, W. G. (1986). The ionic modification of the surface charge and isoelectric point of soy protein. J. Am. Oil Chem. Soc., 63, 1346–1350. Chronakis, I. S. (1996). Network formation and viscoelastic properties of commercial soy protein dispersions: effect of heat treatment pH and calcium ions. Food Res. Intern., 29, 123–134.
Clark, H. A., & Lee-Tuffnell, C. D. (1986). In J. R. Mitchell & D. A. Ledward (Eds.), Functional properties of food macromolecules, 5, (pp. 203). Barking, Essex, England: Applied Science. Cuvelier, G., & Launay, B. (1986). Concentration regimes in xanthan gum solutions deducted from flow and viscoelastic properties. Carbohydr. Polym., 6, 321–333. Giboreau, A., Cuvelier, G., & Launay, B. (1994). Rheological behaviour of three biopolymer/water systems, with emphasis on yield stress and viscoelastic properties. J. Text. Stud., 25, 119–137. Hermansson, A. M. (1978). Physico-chemical aspects of soy proteins structure formation. J. Texture. Stud., 9, 33–58. Hermansson, A. M. (1985). Structure of soya glycinin and conglycinin gels. J. Sci. Food Agric., 36, 822–832. Hermansson, A. M. (1986). Soy protein gelation. J. Am. Oil Chem. Soc., 63, 658–666. Kinsella, J. E. (1979). Functional properties of soy proteins. J. Am. Oil Chem. Soc., 56, 242–258. Kohyama, K., & Nishinari, K. (1992). The effect of glucono-d-lactone on the gelation time of soybean 11S protein: concentration dependence. Food Hydrocolloids, 6, 263–274. Kohyama, K., & Nishinari, K. (1993). Rheological studies on the gelation process of soybean 7S and 11S proteins in the presence of glucono-dlactone. J. Agric. Food Chem., 41, 8–14. Kokini, J. L., & Dickie, A. (1981). An attempt to identify and model transient viscoelastic flow in foods. J. Text. Stud., 12, 539–557. Lee, S. P., & Batt, C. A. (1993). A micro-scale method for measuring the hardness of heat-induced protein gels. J. Text. Stud., 24, 73–79. Lee, C. H., & Rha, C. K. (1977). Thichenning of soy protein suspensions with calcium. J. Text. Stud., 7, 441–449. Mc Mindes, M. K. (1991). Applications of isolated soy protein in low-fat meat products. Food Technol., 61–64. Mori, T., Nakamura, T., & Utsumi, S. (1986). Behavior of intermolecular bond formation in the late stage of heat-induced gelation of glycinin. J. Agric. Food Chem., 34, 33–36. Nagano, T., Mori, H., & Nishinari, K. (1994). Effect of heating and cooling on the gelation kinetics of 7S globulin from soybeans. J. Agric. Food Chem., 42, 1415–1419. Nakamura, T., Utsumi, S., Kitamura, K., Harada, K., & Mori, T. (1984).
176
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
Cultivar differences in gelling characteristics of soybean glycinin. J. Agric. Food Chem., 32, 647–651. Nishinari, K., Kohyama, K., Zhang, Y., Kitamura, K., Sugimoto, T., Saio, K., & Kawamura, Y. (1991). Rheological study on the effect of the A5 subunit on the gelation characteristics of soybean proteins. Agric. Biol. Chem., 55, 351–355. Nussinovitch, A., Peleg, M., & Normand, M. D. (1989). A modified Maxwell and a non-exponential model for the characterization of the stress relaxation of agar and alginate gels. J. Food Sci., 54, 1013–1016. Nussinovitch, A., Ak, M. M., Normand, M. D., & Peleg, M. (1990). Characterization of gellan gels by uniaxial compression, stress relaxation and creep. J. Text. Stud., 21, 37–49. Peleg, M. (1977). Operational conditions and the stress–strain relationship of solid foods-Theoretical evaluation. J. Text. Stud., 8, 283–295. Puppo, M. C., & An˜o´n, M. C. (1998). Structural properties of heat-induced soy-protein gels as affected by ionic strength and pH. J. Agric. Food Chem, 46, 3583–3589. Puppo, M. C., & An˜on, M. C. (1998). Effect of pH and protein concentration on rheological behavior of acidic soybean protein gels. J. Agric. Food. Chem., 46, 3039–3046. Puppo, M. C., & An˜on, M. C. (1999). Soybean protein dispersions at acidic pH. Thermal and rheological properties. J. Food Sci. (In press). Puppo, M. C., Lupano, C. E., & An˜o´n, M. C. (1995). Gelation of soybean protein isolates in acidic conditions. Effect of pH and protein concentration. J. Agric. Food Chem., 43, 2353–2361.
Ross-Murphy, S. B. (1956). In E. Dickinson (Ed.), New Physico-chemical techniques for the characterization of complex food systems, 6, (pp. 130). Glasgow, UK: Blackie Academic and Professional. Ross-Murphy, S. B. (1987). Physical gelation of biopolymers. Food Hydrocoll., 1, 485–495. Ross-Murphy, S. B. (1995). Rheological characterisation of gels. J. Text Stud., 26, 391–400. Scilingo, A. A., & An˜o´n, M. C. (1996). Calorimetric study of soybean protein isolates: effect of calcium and thermal treatments. J. Agric. Food Chem., 44, 3751–3756. Utsumi, S., Nakamura, T., & Mori, T. (1982). A. micro-method for the measurement of gel properties of soyban 11S globulin. Agric. Biol. Chem., 46, 1923–1924. Visser, A., & Thomas, A. (1987). Review: Soya protein products-their processing, functionality and application aspects. Food Rev. Int., 3, 1–32. Van Kleef, F. S. M. (1986). Thermally induced protein gelation: gelation and rheological characterization of highly concentrated ovalbumin and soybean protein gels. Biopolym., 25, 31–59. Yao, J. J., Tanteeratarm, K., & Wei, L. S. (1990). Effects of maturation and storage on solubility, emulsion stability and gelation properties of isolated soy proteins. J. Am. Oil Chem. Soc., 67, 974–979. Yoshida, M., Kohyama, K., & Nishinari, K. (1992). Gelation properties of soymilk and soybean 11S globulin from Japanese-grown soybeans. Biosci. Biotech. Biochem., 56, 725–728.
Rheological properties of acidic soybean protein gels: Salt addition effect Marı´a Cecilia Puppo*, Marı´a Cristina An˜o´n Centro de Investigacio´n y Desarrollo en Criotecnologı´a de Alimentos (CIDCA), Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), 47 Y 116 (1900) La Plata, Argentina Received 17 March 1998; received in revised form 10 July 1998; accepted 30 September 1998
Abstract The objective of this work was to study rheological properties of acidic soybean protein gels in the presence of sodium and calcium chloride. The viscoelastic properties of acidic gels were affected by the amount and type of the assay salt. The pH 2.75 dispersions prepared with 0.1–0.25 M NaCl behaved as diluted macromolecular or entanglement solutions and were more viscous than elastic. At high ionic strength (2.0 M NaCl), samples were more elastic with a gel-like behavior. A semidiluted macromolecular solutions was observed for dispersions at pH 3.50 with 0.1 M NaCl, while at high salt concentration (0.25–2.0 M) gels were obtained. At pH 2.75 gelation kinetics was different at several NaCl concentrations, while at pH 3.50, first order kinetics was observed. At pH 2.75 and 0.1 M CaCl2, the dispersions behaved as semidiluted macromolecular solutions, while at 0.2 M CaCl2 diluted macromolecular solutions were obtained. A semidiluted solution dynamic spectra for pH 3.50 samples (0.1–0.2 M CaCl2) was observed. Both pH 2.75 and 3.50 gels prepared with several NaCl concentrations were more fragile, hardest and resistant rupture than those obtained with CaCl2. The textural properties of acidic gels were less modified by CaCl2. 䉷 1999 Elsevier Science Ltd. All rights reserved. Keywords: Soybean protein gelation; Gelation in acidic conditions; Heat-induced gelation; Salt effect; Rheological properties
1. Introduction Soybean proteins play an important role in many foodstuffs because they have high nutritional value and contribute significally to food texture. Studies on the use of these proteins as functional ingredients in foods must be carried out to optimize food formulation. Previous authors investigated the effect of heat treatment, pH, protein concentration, ions (sodium and calcium chloride) in protein dispersions (Hermansson, 1978; Van Kleef, 1986). It was found that heat treatment induces denaturation and aggregation of soy protein molecules and that at high protein concentration (more than 7% w/w in soy proteins), the aggregates formed produce a self-supporting gel. Soy protein isolate-based gels contain a variety of food ingredients such as sodium chloride to enhance flavor (Yao et al., 1990) or a nutrient mineral used as a coagulant agent such as calcium chloride (Lee and Rha, 1977). To understand the effect of salt addition on the gelation properties of proteins, food protein gels should be described in physicochemical and rheological terms, according to Clark and LeeTuffnell (1986). Gel characteristics are straightforwardly related to their texture (Kinsella, 1979; Hermansson, * Corresponding author. Tel.: ⫹ 54 21 254853; e-mail: mca@nahuel. biol.unlp.edu.ar
1985; Kohyama and Nishinari, 1993) and also to their viscoelasticity and both properties features are determined in turn by the molecular properties of the constituent protein. Since globulins form gels upon heating (Utsumi et al., 1982; Nakamura et al., 1984; Mori etal., 1986), thorough studies at neutral pH have been carried out on the effect of sodium (Catsimpoolas and Meyer, 1970; Hermansson, 1978, 1986) and calcium (Scilingo and An˜o´n, 1996) cations on the behavior of the major soybean protein globulins. NaCl and CaCl2 salts are extensively used as additives along with soy protein to enhance emulsification, gelation and water and lipid retention in meat products, allowing the desired texture to be achieved (Visser and Thomas, 1987; McMindes, 1991). The preparation of acidic soy proteinbased foods require an exhaustive analysis of the gelation properties of soybean proteins at pH values below the pI, and for different ionic strengths obtained with either NaCl or CaCl2 addition. Structural properties of heat-induced acidic soy-protein gels under different conditions of pH and ionic strength have been studied (Puppo and An˜o´n, 1998a). Hydration properties such as water holding capacity, the type of bonds that stabilize gel structure and the protein species that make up and/or stabilize gel structure were also analyzed (Puppo and An˜o´n, 1998a). The WHC of gels prepared with NaCl and CaCl2 decreased with
0268-005X/99/$ - see front matter 䉷 1999 Elsevier Science Ltd. All rights reserved. PII: S0268-005 X( 98)00 079-4
168
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
increasing salt concentration. This fact suggested, as was corroborated by gel SEM, that at high ionic strength a more open matrix was formed (Puppo and An˜o´n, 1998a). The structure of acidic gels, stabilized by non-covalent bonds, changed with the addition of 2 M NaCl. Both 7S and 11S globulin subunits participated via hydrophobic interactions on the stabilization of pH 2.75 gel structure. At pH 3.50 the gel matrix was stabilized by hydrophobic interactions among b -conglycinin subunits, whereas the AB-11S subunit and the AB-11S polymers, linked by disulfide bonds, would be soluble in the matrix interior owing to the glycinin fraction that remains native after thermal treatment (Puppo and An˜o´n, 1998a). The objective of the present work was to study the influence of pH and ionic strength on viscoelastic and textural properties of acidic soybean protein gels. 2. Materials and methods 2.1. Preparation of soy protein isolate Soy proteins isolates were prepared from defatted flour produced by Santista Alimentos S.A. (Brasil). Proteins were obtained by alkaline extraction (pH 8.0) of the flour and subsequent precipitation at the isoelectric point (pI 4.5), as described by Puppo et al. (1995). The isoelectric precipitate was dispersed in distilled water and brought to pH 3.25 with 2 N HCl. To obtain the acidic isolate, the resulting dispersion was freeze-dried. The pH 3.25 isolate thus obtained, contained 95.22 ^ 0.15% protein as determined by Kjeldahl method (N × 6.25). 2.2. Preparation of soy protein dispersions 10% w/w protein suspensions were prepared by hydrating the pH 3.25 isolate in distilled water, NaCl (0.1–2.0 M) or CaCl2 (0.1 and 0.2 M) solutions under constant magnetic stirring at room temperature during 20 min. Dispersions were adjusted to pH 2.75 and 3.50 with 1 N HCl or 1 N NaOH, respectively. 2.3. Dynamic tests: viscoelasticity determinations Rheological measurements were carried out in a Haake CV20 Rheometer. The sample dispersion (0.5 ml) was placed between parallel plates (d 27.83 mm) and the gap between the two plates was set to 1 mm. The lower plate was held at 90⬚C. Low viscosity silicone was added around the plate edges to prevent dehydration and to permit a total contact between sample and silicone. The equipment was driven through the Haake software osc. 2.0. Complex modulus (G*), storage modulus (G 0 ), loss modulus (G 00 ), tan d (G 00 /G 0 ) and complex viscosity (h *), were recorded as a function of time and frequency of oscillation. The scans were performed by duplicate. The linear viscoelasticity range was determined in all
dispersions measuring G* as a function of deformation at a shear oscillation of 1 Hz. In all tests, a deformation of 10% was determined within the linear viscoelasticity range. The variation of G 0 , G 00 and h * (10% deformation) as a function of dynamic frequency was determined after heating the sample for 30 min at 90⬚C. Sample rheological behavior was studied comparing the dependence of G 0 and G 00 with frequency (Giboreau et al., 1994; Ross-Murphy, 1956, 1987, 1995). The variation of G 0 , G 00 and tan d (f 1 Hz) with sample pH and ionic strength was analyzed. Storage modulus, G 0 , of pH 2.75 and 3.50 (10% w/w) dispersions, prepared with NaCl (0.1–2.0 M) and CaCl2 (0.1–0.2 M) solutions, was recorded as a function of time (1 Hz, 10% deformation, 90⬚C). Based on previous work of other researchers (Nishinari et al., 1991; Yoshida et al., 1992; Kokini and Dickie, 1981), different non-linear models were adapted to interpret experimental data. The data corresponding to dispersions prepared with NaCl (0.1 M, pH 2.75; pH 3.50) and with CaCl2 were represented by Eq. (1) (Nishinari et al., 1991; Yoshida et al., 1992): G 0 t G 0sat 1 ⫺ exp ⫺kt
1
where G 0sat is the saturation storage modulus, k the thermal treatment rate constant and t the time. The values corresponding to dispersions of pH 2.75 prepared with 0.5 and 2.0 M NaCl were represented by Eq. (2): G 0 t G 0sat 1 ⫹ at ⫺ 1exp ⫺k 0 t
2
were G 0sat represents the saturation storage modulus and, a and k 0 are constants. Eq. (2) is analogous to the Bird–Leider equation used by Kokini and Dickie (1981), a and k 0 are related to the G 0 increment at short times and the G 0 decrease at saturation times, respectively. A non-linear regression (SYSTAT, V5.0) was used. 2.4. Compression tests 2.4.1. Preparation of gels The dispersions prepared in distilled water, NaCl (0.1– 2.0 M) and CaCl2 (0.1–0.2 M) solutions were partially deaerated at 15⬚C by centrifugation at 1000g for 1 min, and resuspended using a magnetic stirrer. The samples were placed in glass tubes (length, 6 cm; internal diameter, 2.2 cm) hermetically sealed in both ends with rubber plugs. Gelation was carried out by heating the tubes at 90⬚C for 30 min followed by immediate cooling, by using a water bath set at 15⬚C. The gels so formed were kept at 4⬚C for 24–48 h to ensure complete gelation (Puppo et al., 1995). 2.4.2. Determination of texture Gels were compressed at room temperature (20⬚C) to 80% of their original height (3 cm) (Lee and Batt, 1993) by means of 1101 model Instron press, using a 50 N cell with a cross head speed of 100 mm/min. In each
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
169
Fig. 1. Variation of Storage modulus, G 0 (X) (Pa), Loss modulus, G 00 (O) (Pa) and Complex viscosity, h * (B) (Pa.s) with frequency for (10% w/w) acidic soybean protein dispersions: pH 2.75 (a, b, c, d) and 3.50 (e, f, g, h). NaCl: (a, e) 0.1 M; (b, f) 0.25 M; (c, g) 0.5 M; (d, h) 2.0 M.
determination, a minimum of 4 gels were used. A one-cycle uniaxial compression test was carried out using parallel plates to determine rupture strength or fracturability (F) parameters and hardness (H) (Andersson et al., 1973). The curves obtained allowed the deformability modulus (ED) to be calculated from the initial slope along with the relative deformation at rupture (Dr) (Peleg, 1977; Nussinovitch et al., 1989, 1990). The data were statistically analyzed by a two-way analysis of variance (ANOVA). Sources of variation were pH (2.75 and 3.50) and salt (NaCl or CaCl2) concentration. The significance of differences among means of the several treatments was determined by Tukey’s test at P ⬍ 0.05. Difference between means (D0.05) for each texture parameter was calculated.
3. Results and discussion 3.1. Effect of sodium chloride: viscoelasticity of dispersions The dispersions of 10% w/w acidic soy protein isolates in NaCl were heated for 90⬚C during 30 min. The temperature was maintained at 90⬚C and the storage and loss moduli were recorded at 10% strain as a function of the oscillation frequency (Fig. 1). In the presence of 0.1 M (Fig. 1(a)) and 0.25 M NaCl (Fig. 1(b)), the pH 2.75 samples behave as dilute macromolecular solutions or entanglement solutions (Giboreau et al., 1994; Ross-Murphy, 1995) where G 0 is below G 00 at low frequencies. Both moduli, G 0 and G 00 , experience a linear increase at increasing oscillation frequencies and cross-over at high frequencies (5–6 Hz).
170
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
Fig. 2. Variation of Storage modulus, G 0 (a, c), Loss modulus, G 00 (a, c) and Loss tangent, tan d (b, d) with the NaCl concentration of the gel. Gels of pH 2.75 (a, b); pH 3.50 (c, d). Error bars: standard deviation.
In this case there is no long time interactions between macromolecules. The dispersions of pH 2.75 in 0.5 M NaCl behave as semidiluated macromolecular solutions or more entangled semidiluted solutions (Giboreau et al., 1994; Ross-Murphy, 1995), G 0 increase continuously and cross-over G 00 at a frequency of 0.4 Hz (Fig. 1(c)). This type of spectrum is closer to a gel and can be described as ‘‘highly elastic solutions’’ where an association phenomenon between ordered protein chain segments has been assumed (Cuvelier and Launay, 1986). At 2 M NaCl, samples behave as a gel, G 0 remains constant and G 00 shows a minimum at about 2 Hz (Fig. 1(d)). Gel like (Giboreau et al., 1994) or strong (Ross-Murphy, 1995) materials show a prevailing elastic modulus over the complete range of frequencies. G 0 moduli is independent of frequency and can be described as an elastic plateau, characteristic of gels. In the samples whose pH is close to the pI (pH 3.50, 0.1 M NaCl) (Fig. 1(e)), the G 0 and G 00 curves cross over at low frequencies (⬇ 0:2–0.3 Hz) before reaching the plateau region. This behavior corresponds to semidiluted macromolecular dispersions. At frequencies above 0.2, the pH 3.50 dispersions prepared with 0.25, 0.5 and 2 M NaCl behave like gels (Giboreau et al., 1994; Ross-Murphy, 1987, 1995) where G 0 is independent of frequency and a minimum
of G 00 is observed (Fig. 1(f–h)). The viscoelastic behavior of dispersions can be characterized not only by their dynamic spectra but also by the variation of the dynamic viscosity (h *). In both acidic dispersions (pH 2.75 and 3.50), h * decreases with increasing frequency (Fig 1(a–h)). Adding NaCl to the samples induce changes in the viscoelastic moduli and the magnitude of these depends on pH. Both G 0 and G 00 values of pH 2.75 samples prepared with 0.1 M NaCl were higher than those observed in acidic soy protein isolate dispersed in distilled water, particularly the viscous modulus. By contrast, higher salt concentrations ( ⬎ 0.25 M) decreases both parameters (Fig. 2(a)). After salt addition, the tan d increases and so does the viscous component in dispersions prepared with 0.1 and 0.25 M NaCl. A higher NaCl content increases elasticity in pH 2.75 dispersions (Fig. 2(b)). All pH 3.50 gels are more elastic than viscous (tan d ⬍ 1) regardless of gel ionic strength (Fig. 2(d)). Both the elasticity and the viscous moduli decrease for increasing ionic strengths (Fig. 2(c)), and they do so to an extent that gel elasticity becomes unaffected by saline concentration, as demonstrated by the constancy of tan d (Fig. 2d). The decrease of tan d indicates that the system tends to a more solid-like state with the formation of gel networks (Kohyama and Nishinari, 1992; Nagano et al., 1994).
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
171
3.2. Gelation kinetics
Fig. 3. Variation of Storage modulus, G 0 with time of heating of gels (10% w/w) of pH 2.75 (a) and 3.50 (b) prepared with NaCl 0.1 M (O), 0.5 (D) and 2.0 M (A); (—) empirical model (r ⬎ 0.997).
Typical gelation curves for acidic dispersions (10% w/w) containing different amount of NaCl are shown in Fig. 3. For the pH 2.75 and 0.1 M NaCl dispersion, a constant G 0 value G 0sat reached within 30 min of heat treatment was obtained (Fig. 3(a)). The dispersion kinetics could be well approximated by Eq. (1) (Materials and methods, (Section 2.3)). The G 0sat and k values obtained were 1492 Pa and 1.97 × 10 ⫺3 s ⫺1, respectively. These values were higher than those observed in distilled water dispersions: 915 Pa and 4.08 × 10 ⫺4 s ⫺1, respectively (Puppo and An˜o´n, 1998b). Therefore the addition of small amounts of NaCl increased rheological parameters. For higher ionic strengths (0.5 and 2.0 M NaCl), a maximum of G 0 modulus was obtained before reaching the G 0sat value (Fig. 3(a)). Eq. (2) (Materials and methods, (Section 2.3)) better fit these kinetic data. At high ionic strength two phenomenon were observed: gelation (G 0 maximum) followed by protein aggregation at high heating times evidenced by a slight decrease in G 0 modulus. The ratio a /k 0 is related to the G 0 maximum observed at short times. The a /k 0 values, for 0.5 and 2.0 M NaCl pH 2.75 dispersions, were 0.072 and 0.123, respectively. This fact would indicate that at 2.0 M NaCl the gelation process was more pronounced than at 0.5 M MaCl. The curves of the pH 3.50 dispersions showed that G 0sat values were reached within the 15 min of heating regardless of the amount of NaCl present (Fig. 3(b)). The G 0sat values were 1930, 2079 and 367 Pa for the dispersions prepared with 0.1, 0.5 and 2.0 M NaCl, respectively. The dispersions followed the kinetics expression given by Eq. (1) (Materials and methods, (Section 2.3)) and the gelation rate constants obtained were 3.48 × 10 ⫺3 s ⫺1, 5.96 × 10 ⫺3 s ⫺1 and 1.11 × 10 ⫺3 s ⫺1 for the 0.1, 0.5 and 2.0 M NaCl, respectively. The results suggest that lower values of G 0sat and gelation rate were observed at high ionic strength. At low ionic strengths, where soy proteins are partially stabilized by the addition of NaCl (Puppo and An˜o´n, 1999, thermal treatment produced a steady gelation of both acidic pH dispersions. At high ionic strengths and pH 2.75, protein dispersion gelation was observed; followed (after 30 min heat treatment) by a decrease in the elastic component owing to protein aggregation (Fig. 3(a)). 3.3. Texture of gels
Fig. 4. Force-time curves of pH 2. 75 (a) and pH 3.50 (b) gels prepared with different NaCl solutions. NaCl concentration: (A) 0.1 M, (B) 0.25 M and (C) 0.5 M.
The gels prepared with NaCl presented a two-peak profile (Fig. 4). The first peak represents the fracturability (I) and the second, the hardness (II). In more acidic pH (2.75), the only peak observed in the absence of salt splits into two peaks (I and II), the gap between which increase with saline content (Fig. 4(a)). Acidic gels containing a large amount of NaCl produced exudate owing to protein aggregation, in turn caused by the salting out effect. In the presence of NaCl, the pH 3.50 gels maintained the two peak profile observed in the gels prepared with distilled water, where
172
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
Fig. 5. Rupture force, F (a), hardness, H (b), deformability moduli, ED (c) and relative deformability at rupture, Dr (d) of gels (10% w/w) prepared with NaCl solutions of concentration: o 0 M, g 0.1 M, n0.25 M and m 0.5 M. Error bars: standard deviation. (a) D0.05 1.23, (b) D0.05 1.97, (c) D0.05 2.84, (d) D0.05 0.10.
the fracturability peak (I) decreases with the saline content (Fig. 4(b)). After compression, the acidic gels displayed a negative peak (III) of increasing area, indicating an increase in adhesiveness with the increase of saline concentration (Fig. 4 (a,b)). Higher rupture force values of pH 2.75 gels prepared with
0.1 M NaCl were observed (Fig. 5(a)) while the hardest gel was obtained with 0.25 M NaCl (Fig. 5(b)). The pH 3.50 gels behaved differently; the rupture force decreased with addition of NaCl and reached (0.5 M NaCl) values below 1 N (Fig. 5(a)). The harder gels were those prepared with 0.1 and 0.5 M NaCl (Fig. 5(b)).
Fig. 6. Variation of Storage modulus, G 0 (X) (Pa), Loss modulus, G 00 (O) (Pa) and Complex viscosity, h * (B) (Pa.s) with frequency of 10% w/w gels of pH 2.75 (a, b) and pH 3.50 (c, d) prepared with CaCl2 0.1 M (a, c) and 0.2 M (b, d).
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
173
Fig. 7. Variation of Storage modulus, G 0 (a, c), Loss modulus, G 00 (a, c) and Loss tangent, tan d (b, d) with CaCl2 concentration. pH of the gels: 2.75 (a, b); pH 3.50 (c, d). Error bars: Standard deviation.
At acidic pH, the denaturation temperatures of soybean proteins are below the working temperature (90⬚C) even at 0.5 M NaCl concentrations (Puppo and An˜o´n, 1999). Therefore, gels formed are structurally more fragile, with decreasing water holding capacity for increasing ionic strengths (Puppo et al., 1995). At high concentrations, salt causes specific ionic effects on protein conformation: the modification of hydrophobic interactions between non-polar residues owing to charge neutralizing effect (Chronakis, 1996). Acidic gels with more resistance to deformation (higher ED) were those prepared at pH 2.75, 0.25 M NaCl and pH 3.50, 0.1 and 0.25 M NaCl. At very high saline concentrations (0.5 M), the resistance to deformation becomes similar to that of gels prepared in distilled water (Fig. 5(c)). Fig. 5(d) shows that the relative deformability at rupture (Dr) increases for the more acidic gels in 0.5 M NaCl, whereas at pH 3.50, small amounts of added salt produce gels of high Dr that becomes independent of the amount of salt added to the gel. 3.4. Effect of calcium chloride: viscoelasticity of dispersions The viscoelastic behavior of pH 2.75 dispersions prepared in 0.1 and 0.2 M CaCl2 are shown in Fig. 6(a) and (b). At 0.1 M CaCl2, G 0 and G 00 cross over at a frequency of 1 Hz while at 0.2 M, the curves increase linearly for increasing frequencies until they cross over beyond 7 Hz.
Fig. 8. Variation of Storage modulus, G 0 with heating time, for gels (10% w/w) of pH 2.75 (a) and 3.50 (b) prepared with CaCl2 0.1 M (W) and 0.2 M (K). (—) empirical model (r ⬎ 0.993).
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
174
Table 1 Effect of CaCl2 addition on the constant gelation rate (k × 10 3 s ⫺1) of acidic soybean protein dispersions (10% w/w) heated at 90⬚C r ⬎ 0.993 CaCl2 (M)
pH 2.75
pH 3.50
0.1 0.2
1.25 1.61
2.17 1.00
Rheological behavior at low ionic strength is comparable to that of semidiluted macromolecular dispersions (Giboreau et al., 1994; Ross-Murphy, 1987, 1995), and the dispersion looses elasticity at higher ionic strengths, becoming more fluid-like. The frequency scanning of pH 3.50 dispersions (Fig. 6(c) and (d)) at 0.1 and 0.2 M CaCl2 show that G 0 and G 00 crossed over at frequencies lower than 0.5 Hz and for frequencies above 0.5 Hz typical behavior of semidilute macromolecular solutions was observed. The mechanical spectrum of h * (Fig 6(a–d)) also reflected this viscoelastic behavior. Rheological behavior suggests that, at more acidic pH, a weak matrix is formed at 0.1 M CaCl2, while no structure is developed with addition of 0.2 M CaCl2. At pH close to the pI the matrix was not affected by the increment of CaCl2 concentration. Fig. 7 shows, for acidic dispersions, the variation of G 0 and G 00 moduli, and of tan d (1 Hz frequency) with CaCl2 concentration. At pH 2.75 and 0.1 M CaCl2, both the elastic and the viscous moduli increase. Although G 00 is lower than G 0 , it increases more than the elastic modulus (Fig. 7(a)); so that tan d increases (Fig. 7(b)) and the sample is less elastic. At 0.2 M CaCl2, G 00 becomes higher than G 0 and the sample gets even more viscous-like than in the absence of salt; this fact was confirmed by the large increase observed in tan d (Fig. 7(a) and (b)). Fig. 7(c,d) shows, for pH 3.50 dispersions, the change in viscoelastic parameters (1 Hz) with the CaCl2 content. The elastic modulus is higher than the viscous one regardless of the ionic strength. Besides, the same decrease in G 0 and G 00 moduli is observed as the amount of calcium added to the gel increases (Fig. 7(c)). Thus, tan d does not vary with saline content (Fig. 7(d)).
3.5. Gelation kinetics Fig. 8 shows the variation of G 0 with heating time (90⬚C) of 10% w/w acidic dispersions. After 60 min thermal treatment, the elastic moduli of pH 2.75 dispersions prepared in distilled water (graph not shown) was 915 Pa (Puppo and An˜o´n, 1998b), while the dispersions prepared with 0.1 and 0.2 M CaCl2 G 0 values were higher (Fig. 8(a)). No differences between 0.1 and 0.2 M CaCl2 heating curves were observed (Fig. 8(a)), whereas G 0 values of pH 3.50 dispersions were higher at 0.2 M CaCl2 (Fig. 8(b)) with a G 0sat value of 3678 Pa. Gelation kinetic of dispersions prepared with CaCl2 can be represented by the gelation rate constants shown in Table 1. These constants were obtained by fitting experimental data to the model represented by Eq. (1). At pH 2.75, increased CaCl2 concentrations increase the gelation rate, whereas close to the pI an opposite behavior is observed. 3.6. Texture of gels Fig. 9 displays the texture profiles of acidic gels prepared with 0.1 M CaCl2 (m 0.3). As in the case of NaCl, both gels containing CaCl2 show two peaks, the hardness peak is lower than the rupture one. The gels prepared with the divalent salt are more resistant to rupture than the gels prepared with NaCl. The profile of the acidic gels prepared with 0.2 M CaCl2 (m 0.6) is similar to that observed at the lower ionic strength, but the time observed between strength and hardness peaks is longer than at m 0.3 (graph not shown). Gels of pH 2.75 prepared with 0.1 and 0.2 M CaCl2 presented high rupture strength, while gel hardness did not change with salt addition (Fig. 10 (a) and (b)). Gels of pH 2.75 prepared with CaCl2 (m 0.3) presented higher gel strength than those prepared with NaCl (m 0.25). The electrostatic bond linking Ca 2⫹ to the protein is stronger than that of a noncovalent cation (e.g., Na ⫹), leading to high viscosity soy protein dispersions [37]. The texture of pH 3.50 gels was unaffected by the presence of CaCl2 (Fig. 10(a) and (b)). The resistance to deformation of pH 2.75 gels is higher when adding 0.2 M CaCl2 whereas, in pH 3.50 gels, the highest value is obtained for 0.1 M CaCl2 (Fig. 10(c)). However, the relative deformability neither changes with the pH nor with the CaCl2 content (Fig. 10(d)). 4. Conclusions
Fig. 9. Force–time curves of gels (10% w/w) of pH 2.75 (A) and pH 3.50 (B) prepared with CaCl2 0.1 M solution.
Acidic dispersions prepared with sodium and calcium salts showed different rheological behavior at different ionic strengths. An increase of ionic strength from 0.25– 0.5 of pH 2.75 dispersions prepared with NaCl favored molecular entanglement; meanwhile dispersions prepared with CaCl2 were more fluid. At pH 3.50, in the same range of ionic strength, the addition of sodium produced dispersions with a gel like behavior; whereas in the presence
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
175
Fig. 10. Rupture force, F (a), hardness, H (b), deformability moduli, ED (c) and relative deformability at rupture, Dr (d) of acidic gels (10% w/w) prepared with CaCl2: o 0 mM; g 0.1 M; n 0.2 M. Error bars: standard deviation. (a) D0.05 1.60, (b) D0.05 1.48, (c) D0.05 1.19, (d) D0.05 0.14.
of calcium, macromolecular semidiluted dispersions were formed. Both pH 2.75 and 3.50 gels (m 0.25–0.5) prepared with NaCl were more fragile but more harder than those prepared with CaCl2. At 0.25 ionic strength, gels with much more resistance to deformation and of greater relative deformability at rupture were obtained with NaCl than with CaCl2. Acknowledgements This investigation was supported by the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET). Argentina. M.C.P. is a Research Fellow of the Comisio´n Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET) and M.C.A. is Member of the Researcher Career of the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET). This work was also supported by grants from BID-SECYT. References Andersson, Y., Drake, B., Granquist, A., Halldin, L., Johansson, B., Pangborn, R. M., & Akesson, C. (1973). Fracture force, hardness and brittleness in crisp bread, with a generalized regression analysis approach to instrumental-sensory comparisons. J. Text. Stud., 4, 119. Catsimpoolas, N., & Meyer, E. W. (1970). Gelation phenomena of soybean globulins. I. Protein–protein interactions. Cereal Chem., 47, 559–570. Chen, W. S., & Soucie, W. G. (1986). The ionic modification of the surface charge and isoelectric point of soy protein. J. Am. Oil Chem. Soc., 63, 1346–1350. Chronakis, I. S. (1996). Network formation and viscoelastic properties of commercial soy protein dispersions: effect of heat treatment pH and calcium ions. Food Res. Intern., 29, 123–134.
Clark, H. A., & Lee-Tuffnell, C. D. (1986). In J. R. Mitchell & D. A. Ledward (Eds.), Functional properties of food macromolecules, 5, (pp. 203). Barking, Essex, England: Applied Science. Cuvelier, G., & Launay, B. (1986). Concentration regimes in xanthan gum solutions deducted from flow and viscoelastic properties. Carbohydr. Polym., 6, 321–333. Giboreau, A., Cuvelier, G., & Launay, B. (1994). Rheological behaviour of three biopolymer/water systems, with emphasis on yield stress and viscoelastic properties. J. Text. Stud., 25, 119–137. Hermansson, A. M. (1978). Physico-chemical aspects of soy proteins structure formation. J. Texture. Stud., 9, 33–58. Hermansson, A. M. (1985). Structure of soya glycinin and conglycinin gels. J. Sci. Food Agric., 36, 822–832. Hermansson, A. M. (1986). Soy protein gelation. J. Am. Oil Chem. Soc., 63, 658–666. Kinsella, J. E. (1979). Functional properties of soy proteins. J. Am. Oil Chem. Soc., 56, 242–258. Kohyama, K., & Nishinari, K. (1992). The effect of glucono-d-lactone on the gelation time of soybean 11S protein: concentration dependence. Food Hydrocolloids, 6, 263–274. Kohyama, K., & Nishinari, K. (1993). Rheological studies on the gelation process of soybean 7S and 11S proteins in the presence of glucono-dlactone. J. Agric. Food Chem., 41, 8–14. Kokini, J. L., & Dickie, A. (1981). An attempt to identify and model transient viscoelastic flow in foods. J. Text. Stud., 12, 539–557. Lee, S. P., & Batt, C. A. (1993). A micro-scale method for measuring the hardness of heat-induced protein gels. J. Text. Stud., 24, 73–79. Lee, C. H., & Rha, C. K. (1977). Thichenning of soy protein suspensions with calcium. J. Text. Stud., 7, 441–449. Mc Mindes, M. K. (1991). Applications of isolated soy protein in low-fat meat products. Food Technol., 61–64. Mori, T., Nakamura, T., & Utsumi, S. (1986). Behavior of intermolecular bond formation in the late stage of heat-induced gelation of glycinin. J. Agric. Food Chem., 34, 33–36. Nagano, T., Mori, H., & Nishinari, K. (1994). Effect of heating and cooling on the gelation kinetics of 7S globulin from soybeans. J. Agric. Food Chem., 42, 1415–1419. Nakamura, T., Utsumi, S., Kitamura, K., Harada, K., & Mori, T. (1984).
176
M.C. Puppo, M.C. An˜o´n / Food Hydrocolloids 13 (1999) 167–176
Cultivar differences in gelling characteristics of soybean glycinin. J. Agric. Food Chem., 32, 647–651. Nishinari, K., Kohyama, K., Zhang, Y., Kitamura, K., Sugimoto, T., Saio, K., & Kawamura, Y. (1991). Rheological study on the effect of the A5 subunit on the gelation characteristics of soybean proteins. Agric. Biol. Chem., 55, 351–355. Nussinovitch, A., Peleg, M., & Normand, M. D. (1989). A modified Maxwell and a non-exponential model for the characterization of the stress relaxation of agar and alginate gels. J. Food Sci., 54, 1013–1016. Nussinovitch, A., Ak, M. M., Normand, M. D., & Peleg, M. (1990). Characterization of gellan gels by uniaxial compression, stress relaxation and creep. J. Text. Stud., 21, 37–49. Peleg, M. (1977). Operational conditions and the stress–strain relationship of solid foods-Theoretical evaluation. J. Text. Stud., 8, 283–295. Puppo, M. C., & An˜o´n, M. C. (1998). Structural properties of heat-induced soy-protein gels as affected by ionic strength and pH. J. Agric. Food Chem, 46, 3583–3589. Puppo, M. C., & An˜on, M. C. (1998). Effect of pH and protein concentration on rheological behavior of acidic soybean protein gels. J. Agric. Food. Chem., 46, 3039–3046. Puppo, M. C., & An˜on, M. C. (1999). Soybean protein dispersions at acidic pH. Thermal and rheological properties. J. Food Sci. (In press). Puppo, M. C., Lupano, C. E., & An˜o´n, M. C. (1995). Gelation of soybean protein isolates in acidic conditions. Effect of pH and protein concentration. J. Agric. Food Chem., 43, 2353–2361.
Ross-Murphy, S. B. (1956). In E. Dickinson (Ed.), New Physico-chemical techniques for the characterization of complex food systems, 6, (pp. 130). Glasgow, UK: Blackie Academic and Professional. Ross-Murphy, S. B. (1987). Physical gelation of biopolymers. Food Hydrocoll., 1, 485–495. Ross-Murphy, S. B. (1995). Rheological characterisation of gels. J. Text Stud., 26, 391–400. Scilingo, A. A., & An˜o´n, M. C. (1996). Calorimetric study of soybean protein isolates: effect of calcium and thermal treatments. J. Agric. Food Chem., 44, 3751–3756. Utsumi, S., Nakamura, T., & Mori, T. (1982). A. micro-method for the measurement of gel properties of soyban 11S globulin. Agric. Biol. Chem., 46, 1923–1924. Visser, A., & Thomas, A. (1987). Review: Soya protein products-their processing, functionality and application aspects. Food Rev. Int., 3, 1–32. Van Kleef, F. S. M. (1986). Thermally induced protein gelation: gelation and rheological characterization of highly concentrated ovalbumin and soybean protein gels. Biopolym., 25, 31–59. Yao, J. J., Tanteeratarm, K., & Wei, L. S. (1990). Effects of maturation and storage on solubility, emulsion stability and gelation properties of isolated soy proteins. J. Am. Oil Chem. Soc., 67, 974–979. Yoshida, M., Kohyama, K., & Nishinari, K. (1992). Gelation properties of soymilk and soybean 11S globulin from Japanese-grown soybeans. Biosci. Biotech. Biochem., 56, 725–728.