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J. Dairy Sci. 86:3113-3121
© American Dairy Science Association, 2003.

Effect of Sodium Citrate on Structure-Function Relationships of Cheddar Cheese1

J. Pastorino, C. L. Hansen and D. J. McMahon

Western Dairy Center, Department of Nutrition and Food Sciences, Utah State University, Logan 84322

Corresponding author: D. J. McMahon; e-mail: djm{at}cc.usu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
The objective of this study was to determine the effect of sodium citrate on the structure and functionality of Cheddar cheese. The hypothesis was that citrate (sodium citrate) injection would affect cheese properties mainly through its effect on bound calcium (calculated as the difference between total calcium and the water-soluble calcium content of a cheese extract). A 9-kg block of Cheddar cheese was made, vacuum-packaged, and then stored for 2 wk at 4°C. After storage, the cheese was cut into 0.5- to 0.6-kg blocks that were vacuum-packaged and stored for 1 wk at 4°C prior to injection. Cheese blocks were then high-pressure injected with a buffer solution (pH 5.27) containing 40% (wt/wt) citric acid trisodium dihydrate and 6.25% (wt/wt) anhydrous citric acid, from zero (control) to five times (successive injections performed 24 h apart). Increased citric acid content of cheese from 0.22 (uninjected) to 1.39% (after five injections) caused phosphate solubilization. Thus, the calculated bound phosphate content of cheese decreased from 0.54 to 0.45 mmol/g of protein. However, unexpectedly, the soluble calcium content decreased from 0.34 (control) to 0.28 mmol/g of protein (after five injections), whereas the bound calcium content remained unchanged (0.42 mmol/g of protein). The decrease in soluble calcium probably resulted from the formation and concentration of crystals in the cheese surface, which was not included in samples for analysis, and from the expulsion of serum from within the cheese. Higher concentration of solutes in the water phase of cheese would increase the volume of serum, but the cheese had limited holding capacity and serum was expelled. Citrate injection increased the sodium content of cheese from 0.63 to 0.93%, but it had no effect on cheese pH (5.2). After five injections, the protein matrix expanded, occupying an increased area of cheese matrix (83 vs. 78%). Even though citrate injection had no effect on bound calcium, and thus the rate and extent of cheese flow were unaffected, increased phosphate solubilization, and possibly decreased ionic calcium content, resulted in expansion of the protein matrix and increased cheese hardness.

Key Words: calcium • high-pressure injection • phosphate • protein matrix

Abbreviation key: CCP = colloidal calcium phosphate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
The addition of citrate alters the chemical and functional properties of milk. Adding citrate to milk or casein systems results in decreased activity (Goddard and Augustin, 1995; Udabage et al., 1998, 2000) or concentration (Mohammad and Fox, 1983) of ionic calcium and increased solubilization of colloidal calcium phosphate (CCP) (Morr, 1967; Holt and Muir, 1979; Mohammad and Fox, 1983; Udabage et al., 1998). This is probably accompanied by increased hydration of casein micelles (Morr, 1967). Because of its effects on calcium, citrate is considered and normally referred to as a calcium-chelating agent.

Solubilization of CCP upon citrate addition to milk or casein suspensions leads to increased dissociation of caseins from casein micelles (Morr, 1967; Johnston and Murphy, 1992; Goddard and Augustin, 1995; Udabage et al., 2000), and casein micelles become smaller (Morr, 1967; Visser et al., 1986; Udabage et al., 2000). As a result of altered structure of casein micelles, milk viscosity increases (Mohammad and Fox, 1983), the scattering intensity of milk decreases (Johnston and Murphy, 1992; Udabage et al., 2000), and milk coagulation time increases (Mohammad and Fox, 1983). In addition, the strength of milk gels normally decreases upon citrate addition (Casiraghi and Lucisano, 1991; McMahon et al., 1991; Goddard and Augustin, 1995).

Citrate salts are normally added to the cheese blend in the manufacture of process cheese and related products. As observed in milk systems, added citrate in cheese is thought to act as a calcium-chelating agent. Thus, by promoting CCP solubilization, citrate addition should decrease the content of bound calcium in cheese. This would decrease protein-protein interactions leading to increased emulsification of fat by caseins, and it would give the product the desired body and texture (Templeton and Sommer, 1936). In addition, because of its effect on protein interactions, adding citrate could also affect the hardness and melting properties of cheese.

The ability to decrease ionic calcium activity and to solubilize colloidal calcium from casein micelles makes citrate a valuable chemical parameter whose content in cheese could be modified to study the effect of calcium on cheese properties. Previous research, aimed at determining the effect of calcium (Paulson et al., 1998a; Pastorino et al., 2003c), sodium chloride (Paulson et al., 1998b; Pastorino et al., 2003b), and pH (Pastorino et al., 2003a) on cheese properties, supports the argument that bound calcium content is the major factor controlling textural and melting properties of most cheeses (i.e., cheeses with pH greater than 5.0). By adding citrate to cheese and decreasing the content of bound calcium, while keeping the pH constant, further understanding and confirmation of the effect of calcium on cheese properties could be obtained. Therefore, the objectives of this study were to determine the effect of sodium citrate on cheese structure and to relate changes in structure to changes in functional properties of cheese.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Cheese
A 9-kg block of Cheddar cheese (pH 5.27) was made in the dairy plant at Utah State University. Pasteurized, full-fat, nonhomogenized milk was heated to 31°C and starter (0.4% wt/wt; Lactococcus lactis ssp. cremoris, DSM Food Specialties USA Inc. Millville, UT) and CaCl2 (0.12 ml/kg of milk) were added. Following, annatto (0.19 ml/kg of milk; Single Strength, DSM Food Specialties) and double-strength rennet (0.07 ml/kg of milk; Maxiren, Gist-brocades, Charlotte, NC) were added. After 30 min, the curd was cut and then allowed to heal for 5 min. Following 5 min of stirring, cheese curd was cooked by gradually raising the temperature to 39°C in 30 min with stirring, and then holding the temperature at 39°C for an additional 45 min. The whey was then drained, and cheese curd was cheddared. After cheddaring, the curd was milled and dry-salted (2.85 g/kg of milk). After salting, the curd was placed into a 9-kg mold and pressed overnight. The cheese was then vacuum-packaged and stored for 2 wk at 4°C. After storage, the cheese was cut into 0.5- to 0.6-kg blocks (approximately 6 x 9 x 6 cm) that were vacuum-packaged and stored for 1 wk at 4°C before injection.

Cheese Injection
A high-pressure injection system was used to incorporate a sodium citrate solution into cheese blocks (model HPI 3000, Uni-Foods, LLC, North Logan, UT). The pressure of injection was set at 17 MPa, and the burst duration was set to 1 s, which was observed to give an injectate penetration depth of 1 to 3 cm. Injected solution flowed at high speed through stainless steel nozzles (0.015 cm i.d.) and into the cheese. After 14 d of storage at 4°C, the cheese was high-pressure injected one, three, or five times, with a sodium citrate buffer solution (pH 5.27) containing 40% (wt/wt) citric acid trisodium dihydrate (Sigma, St. Louis, MO) and 6.25% (wt/wt) anhydrous citric acid (Nelson-Jameson, Marshfield, WI). Successive injections were performed 24 h apart and according to an injection pattern of 1 x 1 cm applied to two opposite sides of the cheese block. After injection, cheese blocks were blotted with paper towels to remove extraneous fluid and vacuum packaged, and then stored for an additional 40 d at 4°C to allow uniform distribution of injectant solution throughout the cheese, prior to analysis (Pastorino, 2002).

Chemical Composition
Fat content was determined using a modified Babcock method (Richardson, 1985) and moisture content by using the vacuum oven AOAC method 926.08 (1990). Protein content was determined by measuring total nitrogen content (Kjeldahl method) and multiplying by 6.38. Calcium, phosphorous, and sodium content were determined by inductively coupled plasma-atomic emission spectroscopy (US Environmental Protection Agency, 1992). To determine soluble calcium and phosphorous, we blended cheese samples (5 g) with 50 g of water using a hand-held, high-speed homogenizer (Omni 5000: Omni International, Gainsville, VA), and transferred them to a beaker. After standing for 20 min, the solution was filtered through Whatman # 42 filter paper. The filtrate was then analyzed for calcium and phosphate content. Bound calcium and phosphate (PO4) content were estimated as the difference between the total and water-soluble contents, most of which would be bound to proteins either directly or indirectly through the formation of insoluble complexes. Citric acid content was determined according to a modified AOAC 986.13 method (2000) (Covance Laboratories, Madison, WI). A pH meter (model 520A, Orion Research Inc., Boston, MA) with a glass probe (spear combo, Corning, Medfield, MA) was used for determining cheese pH. Cheese (15 g) was fine-grated, mixed, and massed into a ball into which the pH probe was inserted.

Scanning Electron Microscopy
After 40 d of refrigerated storage, cheese samples (approximately 1 x 1 x 10 mm) of uninjected cheese and cheese injected five times were taken and fixed in fresh 2% glutaraldehyde solution at room temperature (22°C) for at least 1 h, and then stored at 4°C. After refrigerated storage, the samples were processed according to McManus et al. (1993), but with some modifications. Samples were frozen in liquefied Freon 22 (-159°C) (Mallinckrodt Inc., Paris, KY), transferred to liquid nitrogen, cryofractured perpendicular to their long axis, and thawed in 2% glutaraldehyde. They were then dehydrated in a graded ethanol series and stored overnight in Freon 113 at 4°C. After storage, samples were rehydrated by reversing the graded ethanol series, and then washed with a 0.2 M HEPES buffer (Sigma-Aldrich, St. Louis, MO), pH 7.4. The samples were then postfixed for 2 h with a solution containing 1% OsO4 (Electron Microscopy Sciences, Fort Washington, PA) and 1.5% K4Fe(CN)6•3H2O (Fisher Scientific Co., Fair Lawn, NJ). This solution was replaced by a 2% tannic acid (Mallinckrodt Inc.) solution in HEPES buffer, and the samples were left for 3 h at room temperature. The tannic acid solution was then replaced with the mentioned solution of osmium tetroxide and potassium ferrocyanate, and samples were left for 4 h. This solution was later replaced with an aqueous solution of 1% hydroquinone (Mallinckrodt Inc.) and samples left overnight at room temperature. After postfixing, the samples were washed with distilled water, dehydrated in 2,2-Dimethyoxypropane (Electron Microscopy Sciences) acidified with 5% (vol/wt) HCl, and then air-dried (room temperature, 30% relative humidity). Samples were viewed in a field emission scanning electron microscope operated at 3 kV. Images from each sample, at 1500x magnification, from five fields were recorded on Kodak TMX 120 film, and digitally using Spectrum 2.0 software (The Dindima Group Pty. Ltd., Ringwoood, Victoria, Australia). Fields were randomly selected from areas of the sample that exhibited good quality planes of fracture.

Image Analysis
Digital images, with pixels in the gray scale 0 to 255 (from black to white) were uploaded into Adobe Photoshop 4.0, and brightness and contrast were adjusted so that images looked alike. Images with pixels in the gray scale 0 to 255 (from black to white) were then analyzed as described by Pastorino et al. (2003b). First, gray-scale images were converted into binary images by applying the threshold function of the software. A threshold level of 110 was found to provide for a satisfactory differentiation between dark and light areas as determined by visually matching the original and binary images. Thus, pixels with a gray value lower than the selected threshold level were converted into black pixels, and pixels with a gray value higher than the threshold level were converted into white pixels. In the original digital images, dark pixels corresponded to areas of the micrograph occupied by pockets that originally, mainly contained fat and/or serum, whereas light pixels corresponded to areas occupied by protein matrix. The proportions of black and white pixels, and the areas occupied by them were then determined by applying the histogram function of the software. Thus, the areas of cheese matrix occupied by fat/serum pockets (dark areas) and protein matrix (light areas) were determined.

Cheese Functionality
Cheese weight was recorded before injection and after 40 d of storage at 4°C. Also, after refrigerated storage, melting test and texture profile analysis were performed. Melting properties were analyzed using the UW Meltmeter (University of Wisconsin-Madison, WI) as described by Wang et al. (1998). Duplicate cheese samples, 3 cm in diameter and 0.7 cm in height, were tested at 60°C, with the height of cheese recorded every 0.2 s for 40 s. The initial rate of cheese flow, defined as the rate (mm/s) at which cheese height decreased at 2 and 4 s was determined. Also, the final extent of cheese flow (decrease in height) at 40 s was determined Texture profile analysis was performed as described by Pastorino et al. (2003b). Thus, a two-bite compression test was run on an Instron 5542 (Canton, MA) with a 1.2-kg static load cell (rating: ± 500N), 75% compression factor, and crosshead speed set at 20 mm/min. Samples, 20 mm long x 16 mm in diameter, were taken from the cheese immediately after removal from the refrigerator, and tested at approximately 5°C. Hardness and cohesiveness were determined by analyzing the data according to Bourne (1978).

Experimental Design and Statistical Analysis
The experiment was conducted in triplicate as a completely randomized design. Three treatments, corresponding to number of injections one, three, or five, along with a control, uninjected cheese, were considered in the experiment. Two cheese samples from each replication were analyzed for moisture content, textural properties, and melting test, and their mean was considered for analysis of variance. For scanning electron microscopy, cheese samples of uninjected cheese and cheese injected five times, from one replication, were observed with the microscope, and five fields from different samples were photographed and digitally analyzed. Thus, each field was considered a replicate for analysis. Statistical analysis (GLM and LSD) was performed using SAS (1999), and significance declared at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
White crystals were observed in cheese blocks injected three and five times, with crystals being more abundant on the surface of cheese blocks. The outer 0.5 cm of cheese blocks was thus trimmed and discarded, so that this portion of cheese was not included in samples for analysis.

Chemical Composition
The average chemical composition of the control (uninjected) cheese was 37.2% moisture, 34.0% fat, 0.69% calcium, 1.46% phosphate, 0.63% sodium, and pH 5.2.

Injecting a concentrated solution of sodium citrate significantly increased the citric acid content of cheese (P < 0.01), from 0.22% in the control cheese to 1.39% after five injections (Figure 1Go), and the sodium content of cheese (P < 0.01), from 0.63% in the control cheese to 0.93% after five injections (Figure 2Go).



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  		Figure 1. Citric acid content of Cheddar cheese injected with a buffer solution (pH 5.27) containing 40% (wt/wt) citric acid trisodium dihydrate and 6.25% (wt/wt) anhydrous citric acid, and stored for 40 d at 4°C. Successive injections performed 24 h apart.

 


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  		Figure 2. Sodium content of Cheddar cheese injected with a buffer solution (pH 5.27) containing 40% (wt/wt) citric acid trisodium dihydrate and 6.25% (wt/wt) anhydrous citric acid, and stored for 40 d at 4°C.

 
Citrate injection promoted syneresis, and after refrigerated storage there was free serum inside the package of cheese blocks injected three and five times. After each injection, the cheese had been blotted, so the serum in the package was serum expelled from the cheese rather than residual injectant not incorporated into the cheese block. Thus, citrate injection caused a slight but statistically significant decrease in the moisture content of cheese (P < 0.01), from 37.2% in the control cheese to 36.5% after five injections (Figure 3Go). As a result of syneresis and moisture losses, the weight of cheese blocks injected three and five times significantly decreased (P < 0.01) (Figure 4Go).



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  		Figure 3. Moisture content of Cheddar cheese injected with a buffer solution (pH 5.27) containing 40% (wt/wt) citric acid trisodium dihydrate and 6.25% (wt/wt) anhydrous citric acid, and stored for 40 d at 4°C.

 


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  		Figure 4. Weight of Cheddar cheese injected with a buffer solution (pH 5.27) containing 40% (wt/wt) citric acid trisodium dihydrate and 6.25% (wt/wt) anhydrous citric acid and stored for 40 d at 4°C.

 
Increased citric acid content of cheese caused significant phosphate solubilization (P < 0.01). Thus, the content of bound phosphate decreased from 0.54 to 0.45 mmol/g of protein (Figure 5Go). However, unexpectedly, the soluble calcium content of cheese significantly decreased (P < 0.01), from 0.35 mmol/g of protein in the control cheese to 0.28 mmol/g of protein after five injections, whereas the bound calcium content remained unchanged upon citrate injection (0.42 mmol/g of protein on average). The decrease in soluble calcium content probably resulted from the formation and concentration of crystal on the cheese surface, which was not included in samples for analysis, and from the expulsion of serum from within the cheese.



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  		Figure 5. Bound phosphate content of Cheddar cheese injected with a buffer solution (pH 5.27) containing 40% (wt/wt) citric acid trisodium dihydrate and 6.25% (wt/wt) anhydrous citric acid and stored for 40 d at 4°C.

 
Cheese Microstructure
The control, uninjected cheese had a structure that looked typical of a stirred/pressed-curd cheese, with protein matrix interspersed with areas that originally contained fat and/or serum (Figure 6AGo). Applying the threshold function of the software allowed us to obtain binary images of the original micrographs, and thus, fat/serum pockets (black areas) were differentiated from the protein matrix (white areas). For the control cheese, the protein matrix occupied 78% of the cheese matrix area, with fat/serum pockets occupying the remaining 22%. However, after five injections with citrate, the area of cheese occupied by protein matrix significantly increased (P < 0.05). Thus, the protein matrix expanded occupying 83% of the cheese matrix area, with fat/serum pockets occupying the remaining 17% (Figure 6BGo).



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  		Figure 6. Scanning electron micrographs of Cheddar cheese after 40 d of storage at 4°C. A: uninjected cheese (0.22% citric acid content); B: citrate-injected cheese (five injections; 1.39% citric acid content). Bar = 10 µm.

 
Cheese Functionality
The injection of a concentrated solution of sodium citrate significantly increased cheese hardness (P < 0.05), although no further statistically significant increase was observed after one injection (Figure 7Go). Cheese cohesiveness was unaffected by citrate injection.



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  		Figure 7. Hardness of Cheddar cheese injected with a buffer solution (pH 5.27) containing 40% (wt/wt) citric acid trisodium dihydrate and 6.25% (wt/wt) anhydrous citric acid, and stored for 40 d at 4°C. Dashed, regression lines for cheeses with citric acid content <= 0.5% and cheeses with citric acid content >= 0.5%. Coefficient of determination (R2) shown when greater than 0.5.

 
The effect of citrate injection on cheese hardness is in agreement with previous observations in which citrate addition resulted in increased strength of milk gels (Goddard and Augustin, 1995 [data not presented]), and increased strength of acid-set (pH 4.1) milk gels (Johnston and Murphy, 1992).

During the melting test, the initial rate of cheese flow, either at 2 or 4 s, was unaffected by citrate injection and the corresponding increase in citric acid content of cheese. Similarly, the final extent of cheese flow, as determined by the height of cheese samples after 40.0 s, was unaffected by citrate injection.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
In the present experiment, the citric acid content of cheese was modified by nonconventional means, i.e., by injecting a concentrated buffer solution of sodium citrate into cheese blocks. Thus, in contrast to the manufacture of process cheese, the cheese was not subjected to high temperature and shear, and no other salts or ingredients were added. It should also be noted that the citric acid content of cheese was increased up to 1.4%, which is lower than the amount of emulsifying salts normally added when making process cheese, 2.5 to 3.0%. Also, the addition of emulsifying salts during the manufacture of process cheese normally increases the pH of cheese, and pH reaches values much greater than 5.2, e.g., up to 6.2. These elements should be considered when comparing the results of the present experiment to those of previous studies done on the effect of citrate content on the properties of process cheese.

Chemical Composition
Moisture.
The observed loss of moisture from citrate-injected cheeses was slight in magnitude but statistically significant. As previously reported (Pastorino et al., 2003b, 2003c), the injection of a concentrated ionic solution may result in syneresis and moisture losses of cheese. In the present experiment, higher concentration of solutes in the water phase of cheese as a result of citrate injection would increase the volume of serum. However, even though the protein matrix may expand accommodating extra fluid, the cheese has limited holding capacity, and excess serum will be expelled from within the cheese. If solutes in the injected fluid are preferentially retained within the cheese, then syneresis during refrigerated storage will result in decreased moisture content of cheese, as observed in the present experiment.

Calcium.
Citric acid and/or citric acid salts are normally used as ingredients in the manufacture of process cheese. It has been normally thought that as happens in milk and casein suspensions, citrate added to cheese acts as a calcium-chelating agent, decreasing both the activity of ionic calcium and the content of CCP, which would result in decreased content of insoluble or bound calcium. In contrast, in the present experiment, increased citric acid content of cheese had no effect on the content of bound calcium. Thus, calcium-mediated protein interactions would remain basically unaffected by citrate addition.

Even though studies on the chemistry of milk and casein systems provide a basis for understanding and predicting the chemical behavior of cheese, simple extrapolations from these systems sometimes fail to accurately predict what happens in cheese. Previously, it was observed that sodium ions were unable to displace bound calcium from interacting with proteins in cheese (Pastorino et al., 2003b). In contrast, when sodium chloride is added to milk or casein suspensions, an exchange of bound calcium by sodium ions is normally observed. Similarly, in the present experiment, increased citric acid content of cheese (from 0.22 to 1.39%) failed to promote calcium solubilization and to decrease the content of bound calcium, whereas when added to milk or casein systems citrate promotes calcium solubilization. Compared with caseins in aqueous suspension, it seems that more extended and stronger interactions between caseins in the matrix of cheese make it more difficult for added ions to access sites for exchange and to displace bound ions, rendering the cheese less prone to chemical modifications.

Phosphate.
The addition of citrate to milk induces solubilization of CCP, which results in increased concentration of phosphate in solution (Morr, 1967; Holt and Muir, 1979; Mohammad and Fox, 1983; Udabage et al., 1998, 2000). Thus, interactions between caseins in milk would not only be affected by the chelating effect of citrate on calcium, but also and most certainly, by the solubilization of colloidal phosphate induced by citrate.

When added to milk or casein suspensions, phosphate promotes protein-protein interactions. Adding phosphate decreases the viscosity of whole casein suspensions (Zittle, 1970), probably because casein micelles adopt a more compact conformation, and decreases the coagulation time of a{alpha}-s1 suspensions (Horne, 1982), and of milk, when added in low levels (<0.04 M) (McMahon et al., 1984). Also, because of its effect in promoting protein-protein interactions, phosphate is required for the formation of casein renneted gels, with increased level of phosphate (from 0.42 to 102 mM) promoting increased gel firmness (Zittle, 1970). Thus, although most of the attention in explaining the effect of citrate on milk properties has been directed to the effect of citrate on calcium content, changes in phosphate content could also have a direct effect on milk properties. In addition, phosphate solubilization probably affects calcium-mediated protein interactions because of the cooperative effect that exists between phosphate and calcium ions in their interaction with caseins (Visser et al., 1986).

As observed in milk systems, adding citrate to cheese promoted phosphate solubilization that resulted in decreased content of bound phosphate. Pyne and McGann (1960) observed that adding citrate to acid milk sera led to the formation of precipitates upon neutralization. Increasing the amount of added citrate resulted in a progressive replacement of CaHPO4 by CaHCitr-, and the composition of precipitates approximated that of a citrate apatite: 3 Ca3(PO4)2, CaHCitr-. It is possible, that in the present study, a portion of the added citrate increasingly interacted with calcium in the formation of insoluble complexes, causing displacement of phosphate from within the protein matrix and into solution.

Cheese Microstructure
The decreased intensity of scattered light, increased viscosity, and decreased level of sedimentable nitrogen of milk or casein suspensions that is observed upon the addition of citrate are indications of disrupted structure of casein micelles. Thus, decreased concentration or activity of ionic calcium and decreased concentration of CCP affect protein-protein interactions between caseins. In particular, altered calcium-mediated protein interactions would result in decreased interaction between caseins that leads to disruption of the structure of casein micelles and to the presence of smaller casein particles (Morr, 1967; Visser et al., 1986; Udabage et al., 2000), with large particles still present having a more open structure (Visser et al., 1986). However, it is also possible that phosphate solubilization directly or indirectly (because of its interaction with calcium in the formation of colloidal complexes) further contributes to disrupting the structure of casein micelles.

Decreased concentration of CCP or ionic calcium results in increased electrostatic repulsion between renneted casein micelles, which locate further apart from one another (Augustin, 2000). In the present experiment, citrate injection and the corresponding increase in citric acid concentration decreased the content of bound phosphate, and most certainly, it also decreased the content of ionic calcium, which resulted in expansion of the protein matrix. Thus, altered composition and decreased content of CCP, i.e., decreased the level of bound phosphate but not of bound calcium, decreased interactions between caseins, which resulted in partial relaxation of the protein matrix. A more relaxed protein matrix would become more hydrated by absorbing free serum contained in the fat/serum pockets. At the same time, the protein matrix would expand to accommodate the additional entrapped water, and this was observed as the protein matrix occupying an increased area in the micrographs. Therefore, increased citric acid content may affect cheese properties not only because of its effect on calcium but also because of its effect on phosphate, as changes in the content of CCP and composition of these colloidal complexes significantly affect interactions between caseins in the matrix of cheese.

Cheese Functionality
Hardness.
Citrate injection not only increased the concentration of citric acid, but it also increased the content of sodium in cheese, from 0.63 to 0.93%, which would correspond to an increase in sodium chloride content from 1.6 to 2.4%. Even though increased salt content of cheese may promote expansion of the protein matrix (Paulson et al., 1998b; Pastorino et al., 2003b) and increased hardness of cheese (Pastorino et al., 2003b), no effect on hardness occurred when the salt content of cheese increased from 1.5 to 2.7%. In fact, the most significant effect of salt on cheese properties has been observed upon increasing the salt content from 0 to 0.5% (Paulson et al., 1998b; Pastorino et al., 2003b). Therefore, most probably, the increased sodium content of cheese observed upon citrate injection had no significant effect on cheese properties.

When added to milk (Goddard and Augustin, 1995), milk retentates (Casiraghi and Lucisano, 1991), or concentrated UHT milk (McMahon et al., 1991), citrate has led to the formation of weaker gels (with pH in the range of 5.5 to 6.7). In contrast, when added to milk with pH 5.2 (Goddard and Augustin, 1995), or in the making of acid-set milk gels, pH 4.1 (Johnston and Murphy, 1992), adding citrate resulted in stronger gels. Thus, the effect of citrate on gel firmness seems to be dependent on milk pH, with added citrate increasing gel firmness when pH is 5.2 or lower. Similarly, in the present experiment, adding citrate to cheese with pH 5.2 resulted in increased cheese hardness.

Cavalier-Salou and Cheftel (1991) observed decreased firmness of cheese analogs made from calcium caseinate when citrate content increased from 0.25 to 3.0%. However, increased citrate content was confounded by a concomitant increase in cheese pH (from 6.1 to 6.8) and increased casein solubilization. In contrast, an increased level of added citrate has resulted in increased hardness of process cheese (Gupta et al., 1984) and increased firmness of fat-free process cheese spreads (Swenson et al., 2000 [data not presented]). In the present experiment, no further statistically significant increase in cheese hardness was observed after one injection (0.48% citric acid content). As previously indicated for milk gels, the opposing results reported between studies on the effect of increased level of added citrate on cheese hardness may result from differences in cheese pH. Whereas in the study of Cavalier-Salou and Cheftel (1991), cheese pH increased from 6.1 to 6.8, Gupta et al. (1984) used cheese with an average pH of 5.8, and in the present study cheese pH remained unchanged at 5.2. Also, lack of information on the state of calcium and phosphate in most studies limit the possibility of further understanding the effect of citrate on cheese properties.

At the highest level of injection (1.39% citric acid content), increased cheese hardness was accompanied by expansion of the protein matrix. Thus, it is possible to argue in favor of a relationship between the structure and hardness of cheese. Increased citric acid content and the concomitant phosphate solubilization apparently resulted in decreased protein-protein interactions, whereas protein-water interactions seemed to be favored (expanded protein matrix). Increased protein-water interactions could lead to more extensive short-range interactions, such as increased hydration and thickness of protein strands in the protein matrix, which would involve extensive hydrogen bonding and could contribute to increased hardness of cheese. This is in agreement with previous observations in which increased content of sodium chloride resulted in expansion of the protein matrix and increased hardness of cheese (Pastorino et al., 2003b). In addition, phosphate solubilization and decreased protein-protein interactions could lead to a more uniform protein matrix that would allow for a more even distribution of stress between protein aggregates, thus increasing cheese hardness (Marchesseau et al., 1997).

Flow.
Cavalier-Salou and Cheftel (1991) reported increased melting of cheese analogs made from calcium caseinate with increased citrate content (from 0.25 to 3.0%). However, the increase in citrate was confounded with an increase in cheese pH, from 6.1 to 6.8, and increased degree of casein dissociation. Thus, higher pH may have led to increased casein solubilization, which would facilitate cheese melting (Savello et al., 1989). In contrast, in the present experiment, the pH of cheese remained unchanged (5.2), and adding citrate had no effect on either the rate or final extent of cheese flow.

Calcium is a strong promoter of interactions between caseins in the matrix of cheese (Pastorino et al., 2003c), and as a result, calcium content significantly affects cheese melting (Lawrence et al., 1993; Paulson et al 1998a; Pastorino et al., 2003c). Even more, calcium seems to be the major chemical parameter affecting protein interactions in the matrix of cheese, and thus, in determining cheese properties when the pH of cheese is greater than 5.0 (Pastorino et al., 2003a). Therefore, even though increased citric acid content induced phosphate solubilization, the rate and extent of cheese flow were unaffected as the content of bound calcium remained the same.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
ACKNOWLEDGEMENTS
 REFERENCES
 
Increasing the citric acid content of cheese affects the structure and hardness of cheese. Adding citrate promotes phosphate solubilization, possibly by an exchange mechanism through which citrate increasingly participates in the formation of insoluble complexes with calcium while displacing phosphate. This results in decreased protein-protein interactions and increased protein-water interactions. Consequently, the protein matrix partially relaxes and expands, occupying increased area of cheese matrix. A more expanded and possibly homogenous protein matrix increases cheese hardness. However, there is no exchange of calcium by citrate, and the content of bound calcium stays unchanged. As a result, calcium-mediated protein interactions seem to remain the major factor limiting functional properties of cheese, as the rate and extent of cheese flow, and cheese cohesiveness are unaffected by citrate addition.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
REFERENCES
 
This research was funded by Dairy Management Incorporated and the Utah Agricultural Experiment Station. The authors thank Stephen Larsen for cheese making.


   FOOTNOTES
 
1 Contribution number 7545 of the Utah Agricultural Experiment Station. Approved by the director. Back

Received for publication April 26, 2003. Accepted for publication May 27, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 


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