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Effects of Emulsifying Salts on the Turbidity and Calcium-Phosphate–Protein Interactions in Casein Micelles
J. Dairy Sci. 88:3070–3078 © American Dairy Science Association, 2005.
Effects of Emulsifying Salts on the Turbidity and CalciumPhosphate–Protein Interactions in Casein Micelles R. Mizuno1,2 and J. A. Lucey2 1
Food Research and Development Laboratory, Morinaga Milk Industry Co., Ltd., Zama, Kanagawa 228-8583 Japan Department of Food Science, University of Wisconsin–Madison, Madison 53706
2
ABSTRACT Influence of emulsifying salts (ES) on some physical properties of casein micelles was investigated. A reconstituted milk protein concentrate (MPC) solution (5% wt/wt) was used as the protein source and the effects of ES [0 to 2.0% (wt/wt)] were estimated by measuring turbidity, acid-base titration curves and amount of casein-bound Ca and inorganic P (Pi). Various ES, trisodium citrate (TSC), or sodium phosphates (ortho-, pyro-, or hexameta-) were added to MPC solution, and all samples were adjusted to pH 5.8. Acid-base buffering curves were used to observe changes in the amount and type of insoluble Ca phosphates. An increase in the concentration of TSC added to MPC solution decreased turbidity, buffering at pH ∼5 (contributed by colloidal Ca phosphate), and amount of casein-bound Ca and Pi. Addition of up to 0.7% disodium orthophosphate (DSP) did not significantly influence turbidity, buffering curves, or amount of casein-bound Ca and Pi. When higher concentrations (i.e., ≥1.0%) of DSP were added, there was a slow decrease in turbidity. With increasing concentration of added tetrasodium pyrophosphate (TSPP), turbidity and buffering at pH ∼5 decreased, and amount of casein-bound Ca and Pi increased. When small concentrations (i.e., 0.1%) of sodium hexametaphosphate were added, effects were similar to those when TSPP were added but when higher concentrations (i.e., ≥0.5%) were added, the buffering peak shifted to a higher pH value, and amount of casein-bound Ca and Pi decreased. These results suggested that each type of ES influenced casein micelles by different mechanisms. (Key words: emulsifying salt, casein, colloidal Ca phosphate) Abbreviation key: CCP = colloidal Ca phosphate, DSP = disodium orthophosphate, ES = emulsifying salts, MPC = milk protein concentrate, Pi = inorganic phosphorus, SHMP = sodium hexametaphosphate,
Received February 28, 2005. Accepted April 18, 2005. Corresponding author: J. A. Lucey; e-mail: [email protected].
TSC = trisodium citrate, TSPP = tetrasodium pyrophosphate. INTRODUCTION Emulsifying salts (ES), such as trisodium citrate (TSC) and sodium phosphates, are widely used for processed cheese manufacture to control melting, texture, and free oil formation (Meyer, 1973; Berger et al., 1998; Fox et al., 2000). In the strict sense, these compounds are not real emulsifiers, such as mono- or diglycerides, because they are not surface-active substances. The essential role of ES during processing is to improve the emulsifying capacity of cheese proteins (Caric et al., 1985). It is well known that ES are also critical in hydrating rennet caseins (Ennis et al., 1998). There have been many studies concerning the effects of ES on processed cheese functionality (e.g., Taneya et al., 1980; Gupta et al., 1984; Savello et al., 1989; Cavalier-Salou and Cheftel, 1991; Awad et al., 2002), although many of the reported results are conflicting. These differences could be due to the usage of different types and ages of cheese, pH values of cheese, or different emulsifying conditions (e.g., type of cookers, temperatures, and holding times). In commercial practice, the selections of an adequate amount and type(s) of ES are essential factors in obtaining processed cheese with the desired functionalities, depending on the type, age, and pH of the natural cheese and emulsifying conditions employed. Moreover, when ≥2 types of ES are used, it becomes more complicated to draw firm conclusions about the action of each type of individual ES. Another complication is that it is not completely understood how each ES interacts with caseins and Ca phosphate or why different types of ES can provide different functionalities for processed cheese. Textural properties of processed cheese have been investigated by sensory and physical analyses when different types of ES were used (Gupta et al., 1984). In general, the addition of phosphate, especially polyphosphate, to processed cheese during manufacturing produces firm, low-melting cheeses (Templeton and Sommer, 1936; Gupta et al., 1984; Anonymous, 2004). Conversely, citrate is known
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to make processed cheese with good melting properties (Gupta et al., 1984; Anonymous, 2004). The use of a simplified milk protein system, instead of a more complex cheese system (i.e., where there are often differences in pH, cheese types, or extent of proteolysis) can be an effective approach in clarifying these issues. It can also provide a basis for understanding and predicting the chemical behavior of ES when they are used in cheese. Some interactions between ES and milk proteins in solution (e.g., skim milk) have been studied, and some properties of ES have been reported (Odagiri and Nickerson, 1964b, 1965; Morr, 1967; Vujicic et al., 1968; Zittle, 1970; Nakajima et al., 1975; Mohammad and Fox, 1983). In our studies, a reconstituted milk protein concentrate (MPC) solution was used. This MPC powder was produced by ultrafiltration/diafiltration and evaporation of milk to reduce the lactose content and increase the protein content prior to spray drying (Novak, 1992). Commonly available MPC powders have protein contents between 56 and 80%. The casein in MPC powder, produced by these processing techniques, is highly soluble, although the micelles may not be identical to those in fresh milk because of the diafiltration and drying processes. The pH of our reconstituted MPC solutions was adjusted to 5.8, which also alters the properties of caseins so this model system did not have micelles that were identical to those in fresh unheated milk. The objective of our studies was to clarify the mode of action of each type of ES and to determine the effect of various concentrations of added ES on caseins and the state of insoluble Ca phosphate using MPC solution as a model system. MATERIALS AND METHODS
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azide was added to prevent bacterial growth, and the pH was adjusted to 5.8 with 1 N HCl. Various concentrations [0 to 2.0% (wt/wt)] of the 4 types of ES were added to MPC solutions and stirred until the ES was completely dissolved. Only a single type of ES was added to MPC solutions (i.e., no blends). When the addition of ES altered the pH of the solutions, 1 N HCl was added to restore the pH to 5.8. Solutions were left for ∼1 h at 25°C until the pH was constant before starting experiments. A 5% (wt/wt) MPC solution contains 2.1% total protein and 1.7% casein. Emulsifying salt solutions for acid-base titrations were prepared by dissolving 0.5% (wt/wt) ES to milliQ water and adjusting pH to 5.8 with 1 N HCl. Emulsifying salt solution with Ca was made by adding 0.2% (wt/wt) CaCl2ⴢ2H2O to the ES solution. Titrations were performed within 1 h at 25°C. Turbidity Measurements Turbidity measurements were made at 700 nm on a Beckman DU 520 UV/Vis Spectrophotometer (Beckman Coulter, Fullerton, CA) using a cell with a 1-mm light path. Experiments were performed in triplicate. Titration Methods Acid-base titrations were performed as described by Lucey et al. (1993) and Lucey and Fox (1993). Samples were titrated using a Mettler Toledo DL50 Autotitrator (Mettler Toledo, Greifensee, Switzerland). Buffering indices (dB/dpH) were calculated according to Van Slyke (1922) as follows: dB ml of acid or base added × normality of acid or base = . dpH volume of sample × pH change produced
Materials Milk protein concentrate powder (ALAPRO 4560) was supplied by New Zealand Milk Products (Santa Rosa, CA). Trisodium citrate dihydrate was obtained from Sigma-Aldrich (St. Louis, MO). Sodium phosphates [disodium orthophosphate (DSP), tetrasodium pyrophosphate (TSPP), and sodium hexametaphosphate (SHMP)] were obtained from Astaris (St. Louis, MO). For SHMP, an average P chain length of 5.6 was determined by the method described by Odagiri and Nickerson (1964a). Preparation of the MPC and ES Solutions Milk protein concentrate was reconstituted in milliQ water and rehydrated at 50°C for 30 min to obtain 5% (wt/wt) milk solids suspension. The solution was then cooled to 25°C in water bath, 0.02% (wt/wt) sodium
Ultrafiltration A Prep/Scale-TFF membrane (Millipore, Billerica, MA), which was made from regenerated cellulose, was used to obtain permeates. The molecular weight cutoff of this membrane was 10 kDa. Experiments were performed at 25°C. Analysis of Ca and P The amount of Ca and P in MCP solutions and permeates was determined by standard methods (IDF, 1992, 1990, respectively). Casein-bound Ca and inorganic P (Pi) were calculated based on the method of White and Davies (1958) using the following equations: Casein-bound Ca = total Ca – Ca in UF permeate, and Casein-bound Pi = total Pi – P in UF permeate. Journal of Dairy Science Vol. 88, No. 9, 2005
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Figure 1. Influence of addition of trisodium citrate (䊉), disodium orthophosphate (䊊), tetrasodium pyrophosphate (▲), or sodium hexametaphosphate (䉭) on turbidity of milk protein concentrate solution. Turbidity was measured as absorbance (arbitrary units) at 700 nm 1 h after emulsifying salts (ES) were added. Results are the means of triplicates with error bars for standard deviations.
When SHMP solution was ultrafiltered, ∼20.5% (wt/wt) of added SHMP did not go through the UF membrane. This nondiffusible P content was subtracted from total Pi content when casein-bound Pi was calculated for SHMP. RESULTS Turbidity Measurements Addition of TSC, TSPP, and SHMP to MPC solution resulted in a similar decrease in the turbidity of these solutions (Figure 1). When these 3 types of ES were added, absorbance decreased almost linearly until it reached a plateau at ∼0.5% addition. The final absorbance value for solutions with TSPP added at a level ≥ 0.5% was slightly higher than the values for solutions with TSC and SHMP added at similar concentrations. The decrease in turbidity when DSP was added was smaller than when other ES were added. Although the absorbance value gradually decreased with increasing concentration of DSP, it was still high (0.43) even with the addition of 2.0% DSP. Turbidity measurements indicated that the ability of ES to cause protein dispersion varied with the types of ES, and SHMP had the highest ability to disperse casein. Disodium orthophosphate had the lowest ability. Acid-Base Buffering Curves of ES Solutions Before MPC solutions were titrated, ES solutions were titrated to understand how ES alters buffering curves of MPC solution. Because it is known that pKa Journal of Dairy Science Vol. 88, No. 9, 2005
values of citrate and phosphate are altered in the presence of Ca (Walstra and Jenness, 1984), acid-base buffering curves were performed for 0.5% ES solutions with and without 0.2% (wt/wt) CaCl2ⴢ2H2O. All of the buffering curves obtained from the ES solutions alone were quite different from those from ES solutions in the presence of Ca. When TSC solution was titrated, there was no clear buffering peak, although a buffering region was observed at pH ≤ 6 (Figure 2a). Conversely, broad buffering with a peak at pH 4.2 was observed during both acid and base titration in TSC solution with added Ca (Figure 2b). These buffering peaks were probably due to the solubilization and formation of Ca citrate, which may be Ca hydrogen citrate. When DSP solution was titrated, a buffering peak at pH 6.7 was observed during base titration (Figure 2c). With the addition of Ca to DSP solution, a peak occurred at pH values between 4 and 5 during acid titration, and a strong buffering peak at pH 6.0 was also observed (Figure 2d). These peaks were probably due to the solubilization and formation of Ca phosphate, respectively. Tetrasodium pyrophosphate solution had broad buffering with peaks at pH 6.0 and 8.5 during base titration (Figure 2e). The TSPP solution with Ca had sharp buffering peaks in both acid and base titration at pH ∼4.5 (Figure 2f), which were not observed in the buffering curve of solutions of TSPP alone (Figure 2e). These peaks were probably due to the solubilization and formation of Ca pyrophosphate, respectively. The SHMP solutions did not have high buffering with or without Ca (Figure 2g, h). However, the addition of Ca shifted a buffering peak from pH 6.7 to 5.0. Acid-Base Buffering Curves of MPC Solutions The acid-base buffering curves of MPC solutions as a function of TSC concentration (0 to 0.7%) are shown in Figure 3. Figure 3a is an acid-base buffering curve of a control MPC sample (i.e., without TSC). When MPC solution at pH 5.8 was titrated with HCl, there was a peak in the buffering curve at pH 5.0. This buffering at pH 5.0 was due to the solubilization of colloidal Ca phosphate (CCP) (Lucey et al., 1993). When an acidified sample was back-titrated with NaOH, the buffering at pH 5.0 was low, because CCP was already solublilized, but a buffering peak occurred at pH 6.0, because of the formation of insoluble Ca phosphate (Lucey et al., 1993). The area between pH 5.8 and 4.1 can be used to estimate the amount of CCP in milk (Lucey et al., 1993). The area of this CCP peak decreased with an increase in the concentration of added TSC. When 0.5% TSC was added, most of the CCP peak area had disappeared, although there was a small gap between the acid and base titration buffering curves. It is possible that this
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Figure 2. Acid-base titration curves for 0.5% emulsifying salt solution with or without 0.2% CaCl2ⴢ2H2O. a) Trisodium citrate (TSC), b) TSC + Ca, c) disodium orthophosphate (DSP), d) DSP + Ca, e) tetrasodium pyrophosphate (TSPP), f) TSPP + Ca, g) sodium-hexametaphosphate (SHMP), and h) SHMP + Ca.
hysteresis was caused by changes in casein structure because of acidification. Buffering at pH 4.2 increased with increasing concentration of TSC (this peak was present in both the acid and base buffering curves). This peak, at pH 4.2, was probably due to the buffering by Ca citrate, which has a pKa ∼4.1 (Walstra and Jenness, 1984). This peak was also observed in TSC solutions with Ca (Figure 2b). During the back titration with NaOH, the buffering peak at pH 6.0 decreased, possibly because of a decrease in the concentration of Ca ions available for the formation of Ca phosphate attributable to the presence of Ca citrate. When DSP was added to the MPC solution, the shape of the CCP peak area between pH 5.8 and 4.1 and the position of the peak at pH 5.0 did not change significantly (Figure 4a,b,c) until the addition of 0.5 and 0.7%
DSP slightly decreased the height of this peak (Figure 4d,e). This suggests that the amount and composition of CCP did not appear to be affected by the addition of up to 0.7% DSP. The buffering peak at pH 6.0 during base titration steadily increased with the addition of DSP because of the increased concentration of phosphate, which has a pKa ∼5.8 (Walstra and Jenness, 1984). This peak was also observed in the buffering curves of DSP solution with Ca (Figure 2d). The acid-base titration curves for the addition of TSPP are shown in Figure 5. The addition of 0.1% TSPP resulted in a slight decrease in the pH value of the buffering peak during acid titration (Figure 5b). During back titration with NaOH, its buffering peak occurred at pH values between 4 and 5, instead of pH 6.0 in control MPC solutions (Figure 5a). The addition of 0.3% Journal of Dairy Science Vol. 88, No. 9, 2005
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Figure 3. Acid-base titration curves for 5% MPC solution with various concentrations of trisodium citrate (TSC): a) control, b) 0.1% TSC addition, c) 0.3% TSC addition, d) 0.5% TSC addition, and e) 0.7% TSC addition.
Figure 4. Acid-base titration curves for 5% milk protein concentrate solution with various concentrations of disodium orthophosphate (DSP): a) control, b) 0.1% DSP addition, c) 0.3% DSP addition, d) 0.5% DSP addition, and e) 0.7% DSP addition.
TSPP resulted in the near disappearance of buffering peak at pH 5.0, as this peak shifted to a lower pH (between pH 4.0 and 4.5) (Figure 5c). When > 0.3% TSPP was added, this shifted buffering peak became very high in both the acid and base titration curves (Figure 5d, e). These new peaks are probably due to the buffering of Ca pyrophosphate because these were similar to those that were observed in TSPP solution with Ca (Figure 2f). The buffering peak at pH 6.0 during base titration had almost disappeared. These observations could be due to CCP, which is converted into a new type of Ca phosphate salt involving pyrophosphate. The buffering curves for the addition of SHMP are shown in Figure 6. When 0.1% SHMP was added to this MPC solution, the peak at pH 5.0 shifted to pH 4.2 (Figure 6b), although the area of this peak was approximately the same as the control. This peak at pH 4.2 was not observed in the buffering curve of SHMP
solution with Ca (Figure 2h). During back titration with NaOH, the addition of ≥0.1% SHMP eliminated the peak at pH 6.0. However, during acidification, higher concentrations of SHMP (≥0.5%) appeared to cause the buffering peak at pH 4.2 to shift back to pH ∼5 (Figure 6d, e). The pH value of this peak was similar to that in SHMP solution with Ca (Figure 2h). This could suggest that low concentrations SHMP might convert the original form of CCP to a new type of Ca phosphate salt, and higher concentrations of SHMP might cause another, different type of Ca phosphate salt to be formed.
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Casein-Bound Ca and Pi The amount of Ca and Pi bound (associated) to casein as a function of the concentration of added ES was calculated (Figure 7). The control MPC sample (i.e., 0% ES) had 1.29 and 0.44 mmol of bound Ca and Pi,
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Figure 5. Acid-base titration curves for 5% milk protein concentrate solution with various concentrations of tetrasodium pyrophosphate (TSPP): a) control, b) 0.1% TSPP addition, c) 0.3% TSPP addition, d) 0.5% TSPP addition, and e) 0.7% TSPP addition.
Figure 6. Acid-base titration curves for 5% milk protein concentrate solution with various concentrations of sodium hexametaphosphate (SHMP): a) control, b) 0.1% SHMP addition, c) 0.3% SHMP addition, d) 0.5% SHMP addition, and e) 0.7% SHMP addition.
respectively. When TSC was added to MPC solution, both bound Ca and Pi decreased, presumably because of the release of both of these ions from casein micelles. (There was no added phosphate in the TSC.) The calculated slope was 2.21 mmol of Ca/mmol of Pi, and the intercept was ∼0.38 mmol of Ca/100 g. These values were different from those of Holt (1982), who used EDTA depletion and those of Van Hooydonk et al. (1986), who used acidification of milk. This difference may be due to the use of a pH 5.8 MPC solution instead of the skim milk previously used. It appeared that TSC was acting as Ca chelator, similar to EDTA, as previously reported by Morr (1967) and Horne (1982). As a result of Ca depletion, CCP was disrupted, and casein micelles were dispersed (i.e., turbidity was decreased). Casein-bound Ca and Pi did not change significantly with the addition of ≤0.7% DSP, in agreement with the lack of change in the CCP buffering peak at pH 5.0 during acid titration (Figure 4).
When TSPP was added to MPC solution, both caseinbound Ca and Pi increased. When high concentrations of TSPP (≥0.3%) were added and the concentration of available Ca ions was decreased, more Pi combined with casein without additional Ca ions being involved. With the addition of 0.7% TSPP to MPC solution, ∼88% of total Ca was bound to casein. It was likely that pyrophosphate anions from TSPP combined with casein micelles together with soluble Ca ions, forming a new type of caseinate-Ca phosphate complex (i.e., with different acid-base buffering properties than CCP). The increased charge resulting from the binding of Ca, and especially phosphate, increased charge repulsion between casein molecules and resulted in increased casein dispersion. The addition of low concentrations (0.1%) of SHMP resulted in an increase in both casein-bound Ca and Pi. This trend was similar to that observed with the addiJournal of Dairy Science Vol. 88, No. 9, 2005
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Figure 7. Relationship between casein-bound Ca and inorganic P (Pi) with the addition of different concentrations of trisodium citrate (䊉), disodium orthophosphate (䊊), tetrasodium pyrophosphate (▲), or sodium hexametaposphate (䉭). Concentration of emulsifying salts added is indicated next to each symbol. Results are the means of triplicates with vertical and horizontal error bars for standard deviations.
tion of TSPP. However, with the addition of 0.3% SHMP, casein-bound Ca and Pi were not changed significantly compared with 0.1% SHMP, and with the addition of ≥0.5% SHMP, both casein-bound Ca and Pi decreased. This result suggested that casein-bound Ca and Pi started to be released from casein micelles at high levels of SHMP. This observation was consistent with the study of Odagiri and Nickerson (1965). Moreover, this change in behavior at high concentrations was also observed in the buffering curves (Figure 6), where the pH of the buffering peak during the acid titration increased at high concentrations of SHMP after initially decreasing at lower SHMP levels. DISCUSSION The results of the acid-base buffering curves and the amount of casein-bound Ca and Pi indicated that phosphate anions from TSPP and 0.1% SHMP were able to combine with casein together with Ca to form a new type of insoluble Ca phosphate. This interaction resulted in a decrease in the turbidity of MPC solution (i.e., greater protein dispersion). However, in a more complicated cheese system, phosphates that are associated with casein might act as a cross-linking agent by bridging within or between casein molecules (Lucey et Journal of Dairy Science Vol. 88, No. 9, 2005
al., 2003), which could make processed cheese less meltable. Under certain conditions, casein can be precipitated or aggregated by pyrophosphate to form viscous gels, even in dilute protein solutions (Lauck and Tucker, 1962; Zittle, 1966). Zittle (1966) suggested that negatively charged pyrophosphate anions became bound to positively charged residues on casein. Leviton (1964) also suggested that polyphosphate and pyrophosphate combined with milk protein and Ca, altering the colloidal caseinate particles as it bound Ca, resulting in a more stable linkage in the caseinate-Ca phosphate complex. Panouille´ et al. (2004) observed heat-induced aggregation and gelation of casein submicelles by polyphosphate, depending on the casein concentration and pH value. Moreover, when 5% MPC solution with 0.2% TSPP at pH 5.8 was held at 25°C overnight, a thick gel was formed, suggesting that the dispersed casein molecules that were associated with pyrophosphate anions slowly interacted with each other to form a gel (R. Mizuno and J. A. Lucey, unpublished results, 2005). Therefore, it appears that, under certain conditions, some types of phosphates can induce gelation or aggregation by the formation of some types of caseinate-Ca phosphate complexes. It is possible that the interac-
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tions responsible for this gelation/aggregation could also contribute to the functionality of processed cheese. Tatsumi et al. (1991) studied the relationship between meltability and water-insoluble casein content, which was determined by centrifugation, of model cheese dispersions, which consisted of sodium caseinate, butter fat, and water. Those researchers observed the formation of insoluble casein after model cheese was heated at 80°C. They suggested that the increase in insoluble casein was related to a decrease in meltability in processed cheese. Although ES was not used in their model system, the results of Tatsumi et al. (1991) suggested that heat-induced aggregation of casein could contribute to a decrease in meltability of processed cheese. Citrates do not appear to work as a cross-linking agent. After chelating Ca from CCP, citrates formed soluble complexes. The loss of CCP from casein micelles resulted in increased dispersion of casein micelles. When CCP is partly chelated by TSC, attractive interactions decrease because of fewer CCP cross-links and electrostatic repulsion increases because of the exposure of phosphoserine residues, resulting in an increase in meltability of natural cheese (Lucey et al., 2003). Savello et al. (1989) reported that a model processed cheese with citrate showed better melting quality than those with DSP, TSPP, and sodium-aluminum phosphate because of the greater dispersion of casein. However, the mechanism as to how TSC alters the functionalities of processed cheese is still unclear because it has been reported recently that the meltability of pasteurized processed Cheddar cheese decreased with increasing concentrations of added TSC (Shirashoji et al., 2004). This may be because, in processed cheese, ES greatly increase casein dispersion during heating and shearing, which may result in a greater number of possible casein-casein interactions in the (cooled) cheese than in milk or natural cheese. In the phosphate-based ES, there may be the additional casein-ES interaction, reducing melt and increasing hardness. Presumably, there are no citrate-casein bonds, so its melting would be better than phosphate-based ES. When high concentrations of SHMP (e.g., ≥0.5%) were added to MPC solutions, a unique phenomenon occurred where the pH of the buffering peak during the acid titration increased after it had initially decreased at low SHMP levels. Although further studies are needed to explain this phenomenon, it could be related to an alteration in the type of Ca phosphate formed with increasing SHMP. Shirashoji et al. (2004) obtained titration curves of pasteurized processed Cheddar with SHMP with a degree of polymerization of 9 to 15. Although emulsification was performed with 2.75% SHMP and a 20-min holding time, a shift of the buff-
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ering peak to higher pH was not observed. This difference might reflect an alteration in concentrations of total Ca, soluble Ca, SHMP, and casein in a processed cheese environment. It is also possible that in processed cheese, SHMP may be hydrolyzed during processing or shortly afterward (Roesler, 1966). In our experiments, DSP did not exhibit any significant effects on milk proteins at low concentrations (≤0.7%). When high concentrations (≥1.0%) of DSP were added, a decrease in turbidity of MPC solution was observed, although this effect was smaller than when the other types of ES were added. Because DSP has been commonly used as ES in processed cheese manufacture, it is likely that it interacts in some manner with casein or CCP as well as increasing cheese pH. Some studies have reported that the addition of DSP affected the viscosity and the amount of soluble Ca in a dilute protein system (e.g., casein solution or skim milk) (Vujicic et al., 1968; Zittle, 1970; Udabage et al., 2000), although the extent of these changes were smaller compared with other types of ES. Fox et al. (1965) found that the addition of small quantities (1 to 15 mM) of orthophosphates to milk caused little change in the viscosity of milk, although the addition of 140 to 225 mM orthophosphates resulted in gelation; the rate of gelation was directly proportional to the level of phosphate. Disodium orthophosphate appears to have a weaker ability to react with Ca than does TSPP or SHMP. It is possible that DSP was not very efficient in our experimental conditions (i.e., at pH 5.8 without heat; concentration of ES; ≤0.7%) and that more energy (heat and shearing) and higher concentrations are required for it to perform like TSPP and SHMP. CONCLUSIONS Our results suggested that each type of ES interacted differently in this MPC cheese model system. Trisodium citrate chelated Ca from CCP, resulting in the dissolution of CCP and the increased dispersion of casein micelles. Disodium orthophosphate did not appear to have major effects on CCP and casein micelles at low concentrations (≤0.7%). When > 1.0% DSP was added, turbidity of MPC solution gradually decreased. The ability of DSP to perform as an ES appeared to be lower than other types of ES. Addition of TSPP caused the formation of caseinate-Ca phosphate complexes, resulting in dispersion of casein micelles, possibly because of an increase in charge repulsion between casein molecules. Low concentrations of SHMP (0.1%) resulted in complexes with soluble Ca and casein micelles. When higher concentrations of SHMP (≥0.5%) were added, casein-bound Ca and phosphate were released from the complexes, resulting in dispersion of casein micelles. It Journal of Dairy Science Vol. 88, No. 9, 2005
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is assumed that these different interactions between casein and ES cause ES-dependent functionality of processed cheese. ACKNOWLEDGMENTS The authors appreciate the financial support of Morinaga Milk Industry Co., Ltd. The donation of phosphate salts by Astaris LLC is also appreciated. The authors are grateful for the financial support of this program by the Wisconsin Center for Dairy Research and Dairy Management, Inc. REFERENCES Anonymous. 2004. Innovations in dairy. Controlling processed cheese functionality. Technical Bulletins, DMI, Inc. http: //www. extraordinarydairy.com/archive/innov_001_may_04.pdf. Accessed Feb. 10, 2005. Awad, R. A., L. B. Abdel-Hamid, S. A. El-Shrabrawy, and R. K. Singh. 2002. Texture and microstructure of block type processed cheese with formulated emulsifying salt mixture. Lebensm. Wiss. Technol. 35:54–61. Berger, W., H. Klostermeyer, K. Merkenich, and G. Uhlman. 1998. Processed Cheese Manufacture, A JOHA Guide. BK Giulini Chemie GmbH & Co. OHG, Ladenburg, Germany. Caric´, M., M. Gantar, and M. Kala´b. 1985. Effects of emulsifying agents on the microstructure and other characteristics of process cheese—A review. Food Microstruct. 4:297–312. Cavalier-Salou, C., and J. C. Cheftel. 1991. Emulsifying salts influence on characteristics of cheese analogs from calcium caseinate. J. Food Sci. 56:1542–1551. Ennis, M. P., M. M. O’Sullivan, and D. M. Mulvihill. 1998. The hydration behaviour of rennet caseins in calcium chelating salt solution as determined using a rheological approach. Food Hydrocoll. 12:451–457. Fox, P. F., T. P. Guinee, T. M. Cogan, and P. L. H. McSweeney. 2000. Fundamentals of Cheese Science. Aspen Publishers, Inc., Gaithersburg, MD. Fox, K. K., M. K. Harper, V. H. Holsinger, and M. J. Pallansch. 1965. Gelation of milk solids by orthophosphate. J. Dairy Sci. 48:179–185. Gupta, S. K., C. Karahadian, and R. C. Lindsay. 1984. Effect of emulsifier salts on textural and flavor properties of processed cheeses. J. Dairy Sci. 67:764–778. Holt, C. 1982. Inorganic constituents of milk. III. The colloidal calcium phosphate of cow’s milk. J. Dairy Res. 49: 29-38. Horne, D. S. 1982. Calcium-induced precipitation of a αs1-casein: Effect of inclusion of citrate or phosphate. J. Dairy Res. 49:107–118. IDF. 1990. Milk: Determination of total phosphorus content by the spectrometric method. 42B. International Dairy Federation, Brussels, Belgium. IDF. 1992. Milk and Dried Milk: Determination of calcium content by the flame atomic absorption spectrometric method. 154. International Dairy Federation, Brussels, Belgium. Lauck, R. M., and J. W. Tucker. 1962. Functional properties of calcium and protein in nonfat dry milk used in food products. Cereal Sci. Today 7:314–322. Leviton, A. 1964. Hydrolysis of polyphosphate in milk and milk concentrates. J. Dairy Sci. 47(Suppl. 1): 670. (Abstr.) Lucey, J. A., and P. F. Fox. 1993. Importance of calcium and phosphate in cheese manufacture: A review. J. Dairy Sci. 76:1714–1724. Lucey, J. A., B. Hauth, C. Gorry, and P. F. Fox. 1993. The acid-base buffering properties of milk. Milchwissenschaft 48:268–272.
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Lucey, J. A., M. E. Johnson, and D. S. Horne. 2003. Perspectives on the basis of the rheology and texture properties of cheese. J. Dairy Sci. 86:2725–2743. Meyer, A. 1973. Processed Cheese Manufacture. Food Trade Press Ltd., London, UK. Mohammad, K. S., and P. F. Fox. 1983. Influence of some polyvalent organic acids and salts on the colloidal stability of milk. J. Soc. Dairy Technol. 36:112–117. Morr, C. V. 1967. Some effects of pyrophosphate and citrate ions upon the colloidal caseinate-phosphate micelles and ultrafiltrate of raw and heated skim milk. J. Dairy Sci. 50:1038–1044. Nakajima, I., G. Kawanishi, and E. Furuichi. 1975. Reaction of melting salts upon casein micelles and their effects on calcium, phosphorus and bound water. Agric. Biol. Chem. 39:979–987. Novak, A. 1992. Milk protein concentrate. Pages 51–66 in New Applications of Membrane Processes, Special Issue 9201. International Dairy Federation, Brussels, Belgium. Odagiri, S., and T. A. Nickerson. 1964a. Chain length determination of polyphosphate. J. Dairy Sci. 47:920–921. Odagiri, S., and T. A. Nickerson. 1964b. Complexing of calcium by hexametaphosphate, oxalate, citrate, and EDTA in milk. I. Effects of complexing agents on turbidity and rennet coagulation. J. Dairy Sci. 47:1306–1309. Odagiri, S., and T. A. Nickerson. 1965. Complexing of calcium by hexametaphosphate, oxalate, citrate, and EDTA in milk. II. Dialysis of milk containing complexing agents. J. Dairy Sci. 48:19–22. Panouille´, M., T. Nicolai, and D. Durand. 2004. Heat induced aggregation and gelation of casein submicelles. Int. Dairy J. 14:297–303. Roesler, H. 1966. Verhalten der polyphosphate in schmelzka¨se. Milchwissenschaft 21:104–107. Savello, P. A., C. A. Ernstrom, and M. Kala´b. 1989. Microstructure and meltability of model process cheese made with rennet and acid casein. J. Dairy Sci. 72:1–11. Shirashoji, N., J. J. Jaeggi, and J. A. Lucey. 2004. Effect of emulsifying salts on texture of pasteurized process Cheddar cheese. J. Dairy Sci. 87(Suppl. 1):231. (Abstr.) Taneya, S., T. Kimura, T. Izutsu, and W. Buchheim. 1980. The submicroscopic structure of processed cheese with different melting properties. Milchwissenschaft 35:479–481. Tatsumi, K., T. Nishiya, K. Ido, and G. Kawanishi. 1991. Effects of heat treatment on the meltability of processed cheese. Nippon Shokuhin Kogyo Gakkaishi 38:102–106. Templeton, H. L., and N. H. Sommer. 1936. Studies on the emulsifying salts used in processed cheese. J. Dairy Sci. 19:561–572. Udabage, P., I. R. McKinnon, and M. A. Augustin. 2000. Mineral and casein equilibria in milk: Effects of added salts and calciumchelating agents. J. Dairy Res. 67:361–370. Van Hooydonk, A. C. M., H. G. Hagedoorn, and I. J. Boerrigter. 1986. pH-induced physico-chemical changes of casein micelles in milk and their effect on renneting. I. Effect of acidification on physicochemical properties. Neth. Milk Dairy J. 40:281–296. Van Slyke, D. D. 1922. On the measurement of buffer value and on the relationship of buffer value to the dissociation constant of the buffer and the concentration and reaction of the buffer solution. J. Biol. Chem. 52:525–571. Vujicic, I., J. M. deMan, and I. L. Woodrow. 1968. Interaction of polyphosphate and citrate with skim milk proteins. Can. Inst. Food Sci. Technol. J. 1:17–21. Walstra, P., and R. Jenness. 1984. Dairy Chemistry and Physics. John Wiley & Sons, Inc., New York, NY. White, J. C. D., and D. T. Davies. 1958. The relation between the chemical composition of milk and the stability of the caseinate complex. I. General introduction, description of samples, methods and chemical composition of samples. J. Dairy Res. 25:236–255. Zittle, C. A. 1966. Precipitation of casein from acidic solutions by divalent anions. J. Dairy Sci. 49:361–364. Zittle, C. A. 1970. Influence of phosphate and other factors on the rennin gel obtained with whole casein and with κ-casein in the presence of calcium salts. J. Dairy Sci. 53:1013–1017.
Effects of Emulsifying Salts on the Turbidity and CalciumPhosphate–Protein Interactions in Casein Micelles R. Mizuno1,2 and J. A. Lucey2 1
Food Research and Development Laboratory, Morinaga Milk Industry Co., Ltd., Zama, Kanagawa 228-8583 Japan Department of Food Science, University of Wisconsin–Madison, Madison 53706
2
ABSTRACT Influence of emulsifying salts (ES) on some physical properties of casein micelles was investigated. A reconstituted milk protein concentrate (MPC) solution (5% wt/wt) was used as the protein source and the effects of ES [0 to 2.0% (wt/wt)] were estimated by measuring turbidity, acid-base titration curves and amount of casein-bound Ca and inorganic P (Pi). Various ES, trisodium citrate (TSC), or sodium phosphates (ortho-, pyro-, or hexameta-) were added to MPC solution, and all samples were adjusted to pH 5.8. Acid-base buffering curves were used to observe changes in the amount and type of insoluble Ca phosphates. An increase in the concentration of TSC added to MPC solution decreased turbidity, buffering at pH ∼5 (contributed by colloidal Ca phosphate), and amount of casein-bound Ca and Pi. Addition of up to 0.7% disodium orthophosphate (DSP) did not significantly influence turbidity, buffering curves, or amount of casein-bound Ca and Pi. When higher concentrations (i.e., ≥1.0%) of DSP were added, there was a slow decrease in turbidity. With increasing concentration of added tetrasodium pyrophosphate (TSPP), turbidity and buffering at pH ∼5 decreased, and amount of casein-bound Ca and Pi increased. When small concentrations (i.e., 0.1%) of sodium hexametaphosphate were added, effects were similar to those when TSPP were added but when higher concentrations (i.e., ≥0.5%) were added, the buffering peak shifted to a higher pH value, and amount of casein-bound Ca and Pi decreased. These results suggested that each type of ES influenced casein micelles by different mechanisms. (Key words: emulsifying salt, casein, colloidal Ca phosphate) Abbreviation key: CCP = colloidal Ca phosphate, DSP = disodium orthophosphate, ES = emulsifying salts, MPC = milk protein concentrate, Pi = inorganic phosphorus, SHMP = sodium hexametaphosphate,
Received February 28, 2005. Accepted April 18, 2005. Corresponding author: J. A. Lucey; e-mail: [email protected].
TSC = trisodium citrate, TSPP = tetrasodium pyrophosphate. INTRODUCTION Emulsifying salts (ES), such as trisodium citrate (TSC) and sodium phosphates, are widely used for processed cheese manufacture to control melting, texture, and free oil formation (Meyer, 1973; Berger et al., 1998; Fox et al., 2000). In the strict sense, these compounds are not real emulsifiers, such as mono- or diglycerides, because they are not surface-active substances. The essential role of ES during processing is to improve the emulsifying capacity of cheese proteins (Caric et al., 1985). It is well known that ES are also critical in hydrating rennet caseins (Ennis et al., 1998). There have been many studies concerning the effects of ES on processed cheese functionality (e.g., Taneya et al., 1980; Gupta et al., 1984; Savello et al., 1989; Cavalier-Salou and Cheftel, 1991; Awad et al., 2002), although many of the reported results are conflicting. These differences could be due to the usage of different types and ages of cheese, pH values of cheese, or different emulsifying conditions (e.g., type of cookers, temperatures, and holding times). In commercial practice, the selections of an adequate amount and type(s) of ES are essential factors in obtaining processed cheese with the desired functionalities, depending on the type, age, and pH of the natural cheese and emulsifying conditions employed. Moreover, when ≥2 types of ES are used, it becomes more complicated to draw firm conclusions about the action of each type of individual ES. Another complication is that it is not completely understood how each ES interacts with caseins and Ca phosphate or why different types of ES can provide different functionalities for processed cheese. Textural properties of processed cheese have been investigated by sensory and physical analyses when different types of ES were used (Gupta et al., 1984). In general, the addition of phosphate, especially polyphosphate, to processed cheese during manufacturing produces firm, low-melting cheeses (Templeton and Sommer, 1936; Gupta et al., 1984; Anonymous, 2004). Conversely, citrate is known
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to make processed cheese with good melting properties (Gupta et al., 1984; Anonymous, 2004). The use of a simplified milk protein system, instead of a more complex cheese system (i.e., where there are often differences in pH, cheese types, or extent of proteolysis) can be an effective approach in clarifying these issues. It can also provide a basis for understanding and predicting the chemical behavior of ES when they are used in cheese. Some interactions between ES and milk proteins in solution (e.g., skim milk) have been studied, and some properties of ES have been reported (Odagiri and Nickerson, 1964b, 1965; Morr, 1967; Vujicic et al., 1968; Zittle, 1970; Nakajima et al., 1975; Mohammad and Fox, 1983). In our studies, a reconstituted milk protein concentrate (MPC) solution was used. This MPC powder was produced by ultrafiltration/diafiltration and evaporation of milk to reduce the lactose content and increase the protein content prior to spray drying (Novak, 1992). Commonly available MPC powders have protein contents between 56 and 80%. The casein in MPC powder, produced by these processing techniques, is highly soluble, although the micelles may not be identical to those in fresh milk because of the diafiltration and drying processes. The pH of our reconstituted MPC solutions was adjusted to 5.8, which also alters the properties of caseins so this model system did not have micelles that were identical to those in fresh unheated milk. The objective of our studies was to clarify the mode of action of each type of ES and to determine the effect of various concentrations of added ES on caseins and the state of insoluble Ca phosphate using MPC solution as a model system. MATERIALS AND METHODS
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azide was added to prevent bacterial growth, and the pH was adjusted to 5.8 with 1 N HCl. Various concentrations [0 to 2.0% (wt/wt)] of the 4 types of ES were added to MPC solutions and stirred until the ES was completely dissolved. Only a single type of ES was added to MPC solutions (i.e., no blends). When the addition of ES altered the pH of the solutions, 1 N HCl was added to restore the pH to 5.8. Solutions were left for ∼1 h at 25°C until the pH was constant before starting experiments. A 5% (wt/wt) MPC solution contains 2.1% total protein and 1.7% casein. Emulsifying salt solutions for acid-base titrations were prepared by dissolving 0.5% (wt/wt) ES to milliQ water and adjusting pH to 5.8 with 1 N HCl. Emulsifying salt solution with Ca was made by adding 0.2% (wt/wt) CaCl2ⴢ2H2O to the ES solution. Titrations were performed within 1 h at 25°C. Turbidity Measurements Turbidity measurements were made at 700 nm on a Beckman DU 520 UV/Vis Spectrophotometer (Beckman Coulter, Fullerton, CA) using a cell with a 1-mm light path. Experiments were performed in triplicate. Titration Methods Acid-base titrations were performed as described by Lucey et al. (1993) and Lucey and Fox (1993). Samples were titrated using a Mettler Toledo DL50 Autotitrator (Mettler Toledo, Greifensee, Switzerland). Buffering indices (dB/dpH) were calculated according to Van Slyke (1922) as follows: dB ml of acid or base added × normality of acid or base = . dpH volume of sample × pH change produced
Materials Milk protein concentrate powder (ALAPRO 4560) was supplied by New Zealand Milk Products (Santa Rosa, CA). Trisodium citrate dihydrate was obtained from Sigma-Aldrich (St. Louis, MO). Sodium phosphates [disodium orthophosphate (DSP), tetrasodium pyrophosphate (TSPP), and sodium hexametaphosphate (SHMP)] were obtained from Astaris (St. Louis, MO). For SHMP, an average P chain length of 5.6 was determined by the method described by Odagiri and Nickerson (1964a). Preparation of the MPC and ES Solutions Milk protein concentrate was reconstituted in milliQ water and rehydrated at 50°C for 30 min to obtain 5% (wt/wt) milk solids suspension. The solution was then cooled to 25°C in water bath, 0.02% (wt/wt) sodium
Ultrafiltration A Prep/Scale-TFF membrane (Millipore, Billerica, MA), which was made from regenerated cellulose, was used to obtain permeates. The molecular weight cutoff of this membrane was 10 kDa. Experiments were performed at 25°C. Analysis of Ca and P The amount of Ca and P in MCP solutions and permeates was determined by standard methods (IDF, 1992, 1990, respectively). Casein-bound Ca and inorganic P (Pi) were calculated based on the method of White and Davies (1958) using the following equations: Casein-bound Ca = total Ca – Ca in UF permeate, and Casein-bound Pi = total Pi – P in UF permeate. Journal of Dairy Science Vol. 88, No. 9, 2005
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Figure 1. Influence of addition of trisodium citrate (䊉), disodium orthophosphate (䊊), tetrasodium pyrophosphate (▲), or sodium hexametaphosphate (䉭) on turbidity of milk protein concentrate solution. Turbidity was measured as absorbance (arbitrary units) at 700 nm 1 h after emulsifying salts (ES) were added. Results are the means of triplicates with error bars for standard deviations.
When SHMP solution was ultrafiltered, ∼20.5% (wt/wt) of added SHMP did not go through the UF membrane. This nondiffusible P content was subtracted from total Pi content when casein-bound Pi was calculated for SHMP. RESULTS Turbidity Measurements Addition of TSC, TSPP, and SHMP to MPC solution resulted in a similar decrease in the turbidity of these solutions (Figure 1). When these 3 types of ES were added, absorbance decreased almost linearly until it reached a plateau at ∼0.5% addition. The final absorbance value for solutions with TSPP added at a level ≥ 0.5% was slightly higher than the values for solutions with TSC and SHMP added at similar concentrations. The decrease in turbidity when DSP was added was smaller than when other ES were added. Although the absorbance value gradually decreased with increasing concentration of DSP, it was still high (0.43) even with the addition of 2.0% DSP. Turbidity measurements indicated that the ability of ES to cause protein dispersion varied with the types of ES, and SHMP had the highest ability to disperse casein. Disodium orthophosphate had the lowest ability. Acid-Base Buffering Curves of ES Solutions Before MPC solutions were titrated, ES solutions were titrated to understand how ES alters buffering curves of MPC solution. Because it is known that pKa Journal of Dairy Science Vol. 88, No. 9, 2005
values of citrate and phosphate are altered in the presence of Ca (Walstra and Jenness, 1984), acid-base buffering curves were performed for 0.5% ES solutions with and without 0.2% (wt/wt) CaCl2ⴢ2H2O. All of the buffering curves obtained from the ES solutions alone were quite different from those from ES solutions in the presence of Ca. When TSC solution was titrated, there was no clear buffering peak, although a buffering region was observed at pH ≤ 6 (Figure 2a). Conversely, broad buffering with a peak at pH 4.2 was observed during both acid and base titration in TSC solution with added Ca (Figure 2b). These buffering peaks were probably due to the solubilization and formation of Ca citrate, which may be Ca hydrogen citrate. When DSP solution was titrated, a buffering peak at pH 6.7 was observed during base titration (Figure 2c). With the addition of Ca to DSP solution, a peak occurred at pH values between 4 and 5 during acid titration, and a strong buffering peak at pH 6.0 was also observed (Figure 2d). These peaks were probably due to the solubilization and formation of Ca phosphate, respectively. Tetrasodium pyrophosphate solution had broad buffering with peaks at pH 6.0 and 8.5 during base titration (Figure 2e). The TSPP solution with Ca had sharp buffering peaks in both acid and base titration at pH ∼4.5 (Figure 2f), which were not observed in the buffering curve of solutions of TSPP alone (Figure 2e). These peaks were probably due to the solubilization and formation of Ca pyrophosphate, respectively. The SHMP solutions did not have high buffering with or without Ca (Figure 2g, h). However, the addition of Ca shifted a buffering peak from pH 6.7 to 5.0. Acid-Base Buffering Curves of MPC Solutions The acid-base buffering curves of MPC solutions as a function of TSC concentration (0 to 0.7%) are shown in Figure 3. Figure 3a is an acid-base buffering curve of a control MPC sample (i.e., without TSC). When MPC solution at pH 5.8 was titrated with HCl, there was a peak in the buffering curve at pH 5.0. This buffering at pH 5.0 was due to the solubilization of colloidal Ca phosphate (CCP) (Lucey et al., 1993). When an acidified sample was back-titrated with NaOH, the buffering at pH 5.0 was low, because CCP was already solublilized, but a buffering peak occurred at pH 6.0, because of the formation of insoluble Ca phosphate (Lucey et al., 1993). The area between pH 5.8 and 4.1 can be used to estimate the amount of CCP in milk (Lucey et al., 1993). The area of this CCP peak decreased with an increase in the concentration of added TSC. When 0.5% TSC was added, most of the CCP peak area had disappeared, although there was a small gap between the acid and base titration buffering curves. It is possible that this
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Figure 2. Acid-base titration curves for 0.5% emulsifying salt solution with or without 0.2% CaCl2ⴢ2H2O. a) Trisodium citrate (TSC), b) TSC + Ca, c) disodium orthophosphate (DSP), d) DSP + Ca, e) tetrasodium pyrophosphate (TSPP), f) TSPP + Ca, g) sodium-hexametaphosphate (SHMP), and h) SHMP + Ca.
hysteresis was caused by changes in casein structure because of acidification. Buffering at pH 4.2 increased with increasing concentration of TSC (this peak was present in both the acid and base buffering curves). This peak, at pH 4.2, was probably due to the buffering by Ca citrate, which has a pKa ∼4.1 (Walstra and Jenness, 1984). This peak was also observed in TSC solutions with Ca (Figure 2b). During the back titration with NaOH, the buffering peak at pH 6.0 decreased, possibly because of a decrease in the concentration of Ca ions available for the formation of Ca phosphate attributable to the presence of Ca citrate. When DSP was added to the MPC solution, the shape of the CCP peak area between pH 5.8 and 4.1 and the position of the peak at pH 5.0 did not change significantly (Figure 4a,b,c) until the addition of 0.5 and 0.7%
DSP slightly decreased the height of this peak (Figure 4d,e). This suggests that the amount and composition of CCP did not appear to be affected by the addition of up to 0.7% DSP. The buffering peak at pH 6.0 during base titration steadily increased with the addition of DSP because of the increased concentration of phosphate, which has a pKa ∼5.8 (Walstra and Jenness, 1984). This peak was also observed in the buffering curves of DSP solution with Ca (Figure 2d). The acid-base titration curves for the addition of TSPP are shown in Figure 5. The addition of 0.1% TSPP resulted in a slight decrease in the pH value of the buffering peak during acid titration (Figure 5b). During back titration with NaOH, its buffering peak occurred at pH values between 4 and 5, instead of pH 6.0 in control MPC solutions (Figure 5a). The addition of 0.3% Journal of Dairy Science Vol. 88, No. 9, 2005
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Figure 3. Acid-base titration curves for 5% MPC solution with various concentrations of trisodium citrate (TSC): a) control, b) 0.1% TSC addition, c) 0.3% TSC addition, d) 0.5% TSC addition, and e) 0.7% TSC addition.
Figure 4. Acid-base titration curves for 5% milk protein concentrate solution with various concentrations of disodium orthophosphate (DSP): a) control, b) 0.1% DSP addition, c) 0.3% DSP addition, d) 0.5% DSP addition, and e) 0.7% DSP addition.
TSPP resulted in the near disappearance of buffering peak at pH 5.0, as this peak shifted to a lower pH (between pH 4.0 and 4.5) (Figure 5c). When > 0.3% TSPP was added, this shifted buffering peak became very high in both the acid and base titration curves (Figure 5d, e). These new peaks are probably due to the buffering of Ca pyrophosphate because these were similar to those that were observed in TSPP solution with Ca (Figure 2f). The buffering peak at pH 6.0 during base titration had almost disappeared. These observations could be due to CCP, which is converted into a new type of Ca phosphate salt involving pyrophosphate. The buffering curves for the addition of SHMP are shown in Figure 6. When 0.1% SHMP was added to this MPC solution, the peak at pH 5.0 shifted to pH 4.2 (Figure 6b), although the area of this peak was approximately the same as the control. This peak at pH 4.2 was not observed in the buffering curve of SHMP
solution with Ca (Figure 2h). During back titration with NaOH, the addition of ≥0.1% SHMP eliminated the peak at pH 6.0. However, during acidification, higher concentrations of SHMP (≥0.5%) appeared to cause the buffering peak at pH 4.2 to shift back to pH ∼5 (Figure 6d, e). The pH value of this peak was similar to that in SHMP solution with Ca (Figure 2h). This could suggest that low concentrations SHMP might convert the original form of CCP to a new type of Ca phosphate salt, and higher concentrations of SHMP might cause another, different type of Ca phosphate salt to be formed.
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Casein-Bound Ca and Pi The amount of Ca and Pi bound (associated) to casein as a function of the concentration of added ES was calculated (Figure 7). The control MPC sample (i.e., 0% ES) had 1.29 and 0.44 mmol of bound Ca and Pi,
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Figure 5. Acid-base titration curves for 5% milk protein concentrate solution with various concentrations of tetrasodium pyrophosphate (TSPP): a) control, b) 0.1% TSPP addition, c) 0.3% TSPP addition, d) 0.5% TSPP addition, and e) 0.7% TSPP addition.
Figure 6. Acid-base titration curves for 5% milk protein concentrate solution with various concentrations of sodium hexametaphosphate (SHMP): a) control, b) 0.1% SHMP addition, c) 0.3% SHMP addition, d) 0.5% SHMP addition, and e) 0.7% SHMP addition.
respectively. When TSC was added to MPC solution, both bound Ca and Pi decreased, presumably because of the release of both of these ions from casein micelles. (There was no added phosphate in the TSC.) The calculated slope was 2.21 mmol of Ca/mmol of Pi, and the intercept was ∼0.38 mmol of Ca/100 g. These values were different from those of Holt (1982), who used EDTA depletion and those of Van Hooydonk et al. (1986), who used acidification of milk. This difference may be due to the use of a pH 5.8 MPC solution instead of the skim milk previously used. It appeared that TSC was acting as Ca chelator, similar to EDTA, as previously reported by Morr (1967) and Horne (1982). As a result of Ca depletion, CCP was disrupted, and casein micelles were dispersed (i.e., turbidity was decreased). Casein-bound Ca and Pi did not change significantly with the addition of ≤0.7% DSP, in agreement with the lack of change in the CCP buffering peak at pH 5.0 during acid titration (Figure 4).
When TSPP was added to MPC solution, both caseinbound Ca and Pi increased. When high concentrations of TSPP (≥0.3%) were added and the concentration of available Ca ions was decreased, more Pi combined with casein without additional Ca ions being involved. With the addition of 0.7% TSPP to MPC solution, ∼88% of total Ca was bound to casein. It was likely that pyrophosphate anions from TSPP combined with casein micelles together with soluble Ca ions, forming a new type of caseinate-Ca phosphate complex (i.e., with different acid-base buffering properties than CCP). The increased charge resulting from the binding of Ca, and especially phosphate, increased charge repulsion between casein molecules and resulted in increased casein dispersion. The addition of low concentrations (0.1%) of SHMP resulted in an increase in both casein-bound Ca and Pi. This trend was similar to that observed with the addiJournal of Dairy Science Vol. 88, No. 9, 2005
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Figure 7. Relationship between casein-bound Ca and inorganic P (Pi) with the addition of different concentrations of trisodium citrate (䊉), disodium orthophosphate (䊊), tetrasodium pyrophosphate (▲), or sodium hexametaposphate (䉭). Concentration of emulsifying salts added is indicated next to each symbol. Results are the means of triplicates with vertical and horizontal error bars for standard deviations.
tion of TSPP. However, with the addition of 0.3% SHMP, casein-bound Ca and Pi were not changed significantly compared with 0.1% SHMP, and with the addition of ≥0.5% SHMP, both casein-bound Ca and Pi decreased. This result suggested that casein-bound Ca and Pi started to be released from casein micelles at high levels of SHMP. This observation was consistent with the study of Odagiri and Nickerson (1965). Moreover, this change in behavior at high concentrations was also observed in the buffering curves (Figure 6), where the pH of the buffering peak during the acid titration increased at high concentrations of SHMP after initially decreasing at lower SHMP levels. DISCUSSION The results of the acid-base buffering curves and the amount of casein-bound Ca and Pi indicated that phosphate anions from TSPP and 0.1% SHMP were able to combine with casein together with Ca to form a new type of insoluble Ca phosphate. This interaction resulted in a decrease in the turbidity of MPC solution (i.e., greater protein dispersion). However, in a more complicated cheese system, phosphates that are associated with casein might act as a cross-linking agent by bridging within or between casein molecules (Lucey et Journal of Dairy Science Vol. 88, No. 9, 2005
al., 2003), which could make processed cheese less meltable. Under certain conditions, casein can be precipitated or aggregated by pyrophosphate to form viscous gels, even in dilute protein solutions (Lauck and Tucker, 1962; Zittle, 1966). Zittle (1966) suggested that negatively charged pyrophosphate anions became bound to positively charged residues on casein. Leviton (1964) also suggested that polyphosphate and pyrophosphate combined with milk protein and Ca, altering the colloidal caseinate particles as it bound Ca, resulting in a more stable linkage in the caseinate-Ca phosphate complex. Panouille´ et al. (2004) observed heat-induced aggregation and gelation of casein submicelles by polyphosphate, depending on the casein concentration and pH value. Moreover, when 5% MPC solution with 0.2% TSPP at pH 5.8 was held at 25°C overnight, a thick gel was formed, suggesting that the dispersed casein molecules that were associated with pyrophosphate anions slowly interacted with each other to form a gel (R. Mizuno and J. A. Lucey, unpublished results, 2005). Therefore, it appears that, under certain conditions, some types of phosphates can induce gelation or aggregation by the formation of some types of caseinate-Ca phosphate complexes. It is possible that the interac-
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tions responsible for this gelation/aggregation could also contribute to the functionality of processed cheese. Tatsumi et al. (1991) studied the relationship between meltability and water-insoluble casein content, which was determined by centrifugation, of model cheese dispersions, which consisted of sodium caseinate, butter fat, and water. Those researchers observed the formation of insoluble casein after model cheese was heated at 80°C. They suggested that the increase in insoluble casein was related to a decrease in meltability in processed cheese. Although ES was not used in their model system, the results of Tatsumi et al. (1991) suggested that heat-induced aggregation of casein could contribute to a decrease in meltability of processed cheese. Citrates do not appear to work as a cross-linking agent. After chelating Ca from CCP, citrates formed soluble complexes. The loss of CCP from casein micelles resulted in increased dispersion of casein micelles. When CCP is partly chelated by TSC, attractive interactions decrease because of fewer CCP cross-links and electrostatic repulsion increases because of the exposure of phosphoserine residues, resulting in an increase in meltability of natural cheese (Lucey et al., 2003). Savello et al. (1989) reported that a model processed cheese with citrate showed better melting quality than those with DSP, TSPP, and sodium-aluminum phosphate because of the greater dispersion of casein. However, the mechanism as to how TSC alters the functionalities of processed cheese is still unclear because it has been reported recently that the meltability of pasteurized processed Cheddar cheese decreased with increasing concentrations of added TSC (Shirashoji et al., 2004). This may be because, in processed cheese, ES greatly increase casein dispersion during heating and shearing, which may result in a greater number of possible casein-casein interactions in the (cooled) cheese than in milk or natural cheese. In the phosphate-based ES, there may be the additional casein-ES interaction, reducing melt and increasing hardness. Presumably, there are no citrate-casein bonds, so its melting would be better than phosphate-based ES. When high concentrations of SHMP (e.g., ≥0.5%) were added to MPC solutions, a unique phenomenon occurred where the pH of the buffering peak during the acid titration increased after it had initially decreased at low SHMP levels. Although further studies are needed to explain this phenomenon, it could be related to an alteration in the type of Ca phosphate formed with increasing SHMP. Shirashoji et al. (2004) obtained titration curves of pasteurized processed Cheddar with SHMP with a degree of polymerization of 9 to 15. Although emulsification was performed with 2.75% SHMP and a 20-min holding time, a shift of the buff-
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ering peak to higher pH was not observed. This difference might reflect an alteration in concentrations of total Ca, soluble Ca, SHMP, and casein in a processed cheese environment. It is also possible that in processed cheese, SHMP may be hydrolyzed during processing or shortly afterward (Roesler, 1966). In our experiments, DSP did not exhibit any significant effects on milk proteins at low concentrations (≤0.7%). When high concentrations (≥1.0%) of DSP were added, a decrease in turbidity of MPC solution was observed, although this effect was smaller than when the other types of ES were added. Because DSP has been commonly used as ES in processed cheese manufacture, it is likely that it interacts in some manner with casein or CCP as well as increasing cheese pH. Some studies have reported that the addition of DSP affected the viscosity and the amount of soluble Ca in a dilute protein system (e.g., casein solution or skim milk) (Vujicic et al., 1968; Zittle, 1970; Udabage et al., 2000), although the extent of these changes were smaller compared with other types of ES. Fox et al. (1965) found that the addition of small quantities (1 to 15 mM) of orthophosphates to milk caused little change in the viscosity of milk, although the addition of 140 to 225 mM orthophosphates resulted in gelation; the rate of gelation was directly proportional to the level of phosphate. Disodium orthophosphate appears to have a weaker ability to react with Ca than does TSPP or SHMP. It is possible that DSP was not very efficient in our experimental conditions (i.e., at pH 5.8 without heat; concentration of ES; ≤0.7%) and that more energy (heat and shearing) and higher concentrations are required for it to perform like TSPP and SHMP. CONCLUSIONS Our results suggested that each type of ES interacted differently in this MPC cheese model system. Trisodium citrate chelated Ca from CCP, resulting in the dissolution of CCP and the increased dispersion of casein micelles. Disodium orthophosphate did not appear to have major effects on CCP and casein micelles at low concentrations (≤0.7%). When > 1.0% DSP was added, turbidity of MPC solution gradually decreased. The ability of DSP to perform as an ES appeared to be lower than other types of ES. Addition of TSPP caused the formation of caseinate-Ca phosphate complexes, resulting in dispersion of casein micelles, possibly because of an increase in charge repulsion between casein molecules. Low concentrations of SHMP (0.1%) resulted in complexes with soluble Ca and casein micelles. When higher concentrations of SHMP (≥0.5%) were added, casein-bound Ca and phosphate were released from the complexes, resulting in dispersion of casein micelles. It Journal of Dairy Science Vol. 88, No. 9, 2005
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is assumed that these different interactions between casein and ES cause ES-dependent functionality of processed cheese. ACKNOWLEDGMENTS The authors appreciate the financial support of Morinaga Milk Industry Co., Ltd. The donation of phosphate salts by Astaris LLC is also appreciated. The authors are grateful for the financial support of this program by the Wisconsin Center for Dairy Research and Dairy Management, Inc. REFERENCES Anonymous. 2004. Innovations in dairy. Controlling processed cheese functionality. Technical Bulletins, DMI, Inc. http: //www. extraordinarydairy.com/archive/innov_001_may_04.pdf. Accessed Feb. 10, 2005. Awad, R. A., L. B. Abdel-Hamid, S. A. El-Shrabrawy, and R. K. Singh. 2002. Texture and microstructure of block type processed cheese with formulated emulsifying salt mixture. Lebensm. Wiss. Technol. 35:54–61. Berger, W., H. Klostermeyer, K. Merkenich, and G. Uhlman. 1998. Processed Cheese Manufacture, A JOHA Guide. BK Giulini Chemie GmbH & Co. OHG, Ladenburg, Germany. Caric´, M., M. Gantar, and M. Kala´b. 1985. Effects of emulsifying agents on the microstructure and other characteristics of process cheese—A review. Food Microstruct. 4:297–312. Cavalier-Salou, C., and J. C. Cheftel. 1991. Emulsifying salts influence on characteristics of cheese analogs from calcium caseinate. J. Food Sci. 56:1542–1551. Ennis, M. P., M. M. O’Sullivan, and D. M. Mulvihill. 1998. The hydration behaviour of rennet caseins in calcium chelating salt solution as determined using a rheological approach. Food Hydrocoll. 12:451–457. Fox, P. F., T. P. Guinee, T. M. Cogan, and P. L. H. McSweeney. 2000. Fundamentals of Cheese Science. Aspen Publishers, Inc., Gaithersburg, MD. Fox, K. K., M. K. Harper, V. H. Holsinger, and M. J. Pallansch. 1965. Gelation of milk solids by orthophosphate. J. Dairy Sci. 48:179–185. Gupta, S. K., C. Karahadian, and R. C. Lindsay. 1984. Effect of emulsifier salts on textural and flavor properties of processed cheeses. J. Dairy Sci. 67:764–778. Holt, C. 1982. Inorganic constituents of milk. III. The colloidal calcium phosphate of cow’s milk. J. Dairy Res. 49: 29-38. Horne, D. S. 1982. Calcium-induced precipitation of a αs1-casein: Effect of inclusion of citrate or phosphate. J. Dairy Res. 49:107–118. IDF. 1990. Milk: Determination of total phosphorus content by the spectrometric method. 42B. International Dairy Federation, Brussels, Belgium. IDF. 1992. Milk and Dried Milk: Determination of calcium content by the flame atomic absorption spectrometric method. 154. International Dairy Federation, Brussels, Belgium. Lauck, R. M., and J. W. Tucker. 1962. Functional properties of calcium and protein in nonfat dry milk used in food products. Cereal Sci. Today 7:314–322. Leviton, A. 1964. Hydrolysis of polyphosphate in milk and milk concentrates. J. Dairy Sci. 47(Suppl. 1): 670. (Abstr.) Lucey, J. A., and P. F. Fox. 1993. Importance of calcium and phosphate in cheese manufacture: A review. J. Dairy Sci. 76:1714–1724. Lucey, J. A., B. Hauth, C. Gorry, and P. F. Fox. 1993. The acid-base buffering properties of milk. Milchwissenschaft 48:268–272.
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