J. Dairy Sci. 88:3798-3809
© American Dairy Science Association, 2005.
Impact of Modifications in Acid Development on the Insoluble Calcium Content and Rheological Properties of Cheddar Cheese
M.-R. Lee1,
M. E. Johnson2 and
J. A. Lucey1
1 Department of Food Science, and
2 Wisconsin Center for Dairy Research, University of Wisconsin-Madison, Madison 53706
Corresponding author: J. A. Lucey; e-mail: jalucey{at}wisc.edu.
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ABSTRACT
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Cheddar cheese was made from milk concentrated by reverse osmosis (RO) to increase the lactose content or from whole milk. Manufacturing parameters (pH at coagulant addition, whey drainage, and milling) were altered to produce cheeses with different total Ca contents and low pH values (i.e., <5.0) during ripening. The concentration of insoluble (INSOL) Ca in cheese was measured by cheese juice method, buffering by acid-base titration, rheological properties by small amplitude oscillatory rheometry, and melting properties by UW-Melt Profiler. The INSOL Ca content as a percentage of total Ca in all cheeses rapidly decreased during the first week of aging but surprisingly did not decrease below approximately 41% even in cheeses with a very low pH (e.g., ~4.7). Insoluble Ca content in cheese was positively correlated (r = 0.79) with cheese pH in both RO and nonRO treatments, reflecting the key role of pH and acid development in altering the extent of solubilization of INSOL Ca. The INSOL Ca content in cheese was positively correlated with the maximum loss tangent value from the rheology test and the degree of flow from the UW-Melt Profiler. When cheeses with pH <5.0 where heated in the rheometer the loss tangent values remained low (<0.5), which coincided with limited meltability of Cheddar cheeses. We believe that this lack of meltability was due to the dominant effects of reduced electrostatic repulsion between casein particles at low pH values (<5.0).
Key Words: calcium • colloidal calcium phosphate • cheese functionality • casein interaction
Abbreviation key: CCP = colloidal calcium phosphate, DOF = degree of flow, G' = storage modulus, G'' = loss modulus, HPHM = high-pH method, INSOL = insoluble, LPHM = low-pH method, LT = loss tangent, LTmax = loss tangent maximum, NONRO = whole milk without reverse osmosis, pH4.6SN = pH 4.6 water-soluble nitrogen, RO = reverse osmosis, SAOR = small amplitude oscillatory rheology
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INTRODUCTION
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It is well recognized that total Ca content, pH, and proteolysis are critical parameters that influence the textural and physical properties of cheeses (Lawrence et al., 1987; Lucey and Fox, 1993; Watkinson et al., 2001; Guinee et al., 2002; Joshi et al., 2003; Lucey et al., 2003; Pastorino et al., 2003a,b; Sheehan and Guinee, 2004). It is difficult to independently study these parameters because rate and extent of acid development, pH, and Ca contents of cheese are interrelated; Ca is lost from casein particles as the pH decreases during cheese manufacture (Lucey and Fox, 1993). The texture of Cheddar cheese at high pH (5.4) is elastic but at low pH (e.g., 4.8), cheese is brittle and crumbly (Lawrence et al., 1987; Pastorino et al., 2003b). Watkinson et al. (2001) observed that both fracture strain and fracture stress increased (i.e., cheese became longer and firmer) when the pH of model Cheddar cheeses increased from 5.2 to 6.2. However, pH itself is only one of the factors that can be responsible for differences in cheese texture (others include composition and age).
Lower total Ca levels generally result in softer cheeses and an increase in melt (Lucey and Fox, 1993). Stretch and flow of cheese when heated increase with a reduction in Ca content (Guinee et al., 2002; Joshi et al., 2003; Sheehan and Guinee, 2004). However, total Ca content alone is not the most useful predictor of the physical properties of cheese (Lawrence et al., 1987). Lucey and Fox (1993) suggested that there was a significant amount of Ca in cheese still associated with casein, which is described as insoluble (INSOL) Ca. The amount of INSOL Ca in cheese plays a key role in controlling cheese texture as it has a direct influence on casein–casein interactions (Lucey et al., 2003). An example in which the state of Ca in cheese could be more important than pH is in direct-acid Mozzarella cheese making, where a much higher stretching pH (~5.6) is used compared with that used in traditional cultured cheese (~5.2) to have similar stretching properties. This is probably due to different levels of total or INSOL Ca in these cheeses (Lucey and Fox, 1993; Lucey et al., 2003).
Recently, Hassan et al. (2004) demonstrated that the INSOL Ca content as a percentage of total Ca of Cheddar cheeses decreased from ~70% after manufacture to ~57% after 3 mo of ripening. It is therefore possible that a reduction in INSOL Ca could be partly responsible for the changes in textural and melting properties of Cheddar cheese during ripening (Lucey et al., 2005). In the study of Hassan et al. (2004), cheese pH did not vary significantly during ripening (~5.2). Pastorino et al. (2003b) demonstrated that decreasing Cheddar cheese pH (postmanufacture) from 5.3 to 5.0 by injection of a 20% glucono-
-lactone solution increased the solubilization of Ca, which contributed to increased flow rate during melt and decreased hardness. When cheese pH was decreased below 5.0 by further injections of glucono--lactone, the flow rate during melt decreased. Thus, it appears that solubilization of INSOL Ca is a critically important parameter affecting cheese texture but this impact may only be observed in a certain pH range.
The objectives of this study were to determine the effects of altering pH values during cheese manufacture and acid levels in cheese on the INSOL Ca content and the physical properties of Cheddar cheese during ripening. Strategies were used to obtain cheese with low pH values (<5.0) during ripening, as studies on cheeses with higher pH values have been previously reported (Hassan et al., 2004; Lucey et al., 2005).
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MATERIALS AND METHODS
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Reverse Osmosis of Milk
Whole milk was concentrated to ~14% total solids content using the reverse osmosis (RO) unit in the pilot plant of the University of Wisconsin-Madison. This RO unit was fitted with 2 spiral-wound elements that were arranged in parallel and composed of thin film composites. Each element had a membrane area of 7.4 m2 and a typical NaCl rejection of 99.5% (PTI Advanced Filtration, Oxford, CA). The unit was operated at 4°C and at ~1655 kPa outlet pressure. Reverse osmosis milk concentrate was pasteurized at 73°C for 15 s, and cooled to 4°C. Pasteurized milk at 74°C for 18 s that was not concentrated (NONRO) was used to make control Cheddar cheeses.
Cheese Manufacturing
Licensed Wisconsin cheese makers manufactured 2 types of full-fat, milled curd Cheddar cheese, designated high pH method (HPHM) and low pH method (LPHM) at the University of Wisconsin-Madison Dairy Plant. The HPHM cheese had a higher rennet, drain, and mill pH, whereas LPHM cheese had lower rennet, drain, and mill pH (Table 1
). It should be noted the HPHM cheese had lower drain and mill pH values compared with many commercial US Cheddar cheeses. Three separate cheese-making trials were performed using RO milk, and 3 separate trials using NONRO milk were conducted over a period of 18 mo. An outline of the cheese manufacturing conditions used is given in Table 1. A mixed-strain starter culture containing Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis was inoculated into the milk at the rate of 1490 g per vat (226 kg) of milk. Double-strength chymosin (Chymostar; Rhodia, Madison, WI) was added at the rate of 17 mL per 226 kg of milk at 32°C. The coagula were cut at similar firmness, as subjectively determined by cheese makers, using 0.63-cm knives, and the curd was given a 5-min healing time, followed by 10 to 15 min of gentle agitation before heating. The temperature of the curd-whey mixture was raised from 32 to 39°C and curd was continuously stirred at 39°C until the curd reached pH ~6.1 and ~5.8 for HPHM and LPHM cheeses, respectively. Curd slabs were cheddared and milled at pH 5.2 to 5.3 and pH 5.0 to 5.1 for HPHM and LPHM cheeses, respectively. Curd was salted at the rate of 0.72 kg per 226 kg of milk. Curd was packed in 9-kg Wilson-style hoops, pressed for 4 h, and then stored overnight at ambient temperature. Cheeses were packaged and stored at 10°C for 1 wk and then 5°C for the rest of ripening. Two 9-kg blocks of cheese were produced from each vat of cheese.
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Table 1. Cheese manufacturing protocol for Cheddar cheese from reverse osmosis (RO) and non-RO (NONRO) treatments (n = 3).
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Compositional Analysis
Milk was analyzed for fat, protein, and casein (Marshall, 1992), total Ca (IDF, 2003) and buffering by the acid-base titration method (Lucey et al., 1993; Hassan et al., 2004). Lactose and lactic acid (both D- and L-lactate) were determined by enzymatic methods (AOAC, 2000; Boehringer Mannheim Biochemicals, Mannheim, Germany). Rennet whey was made from milk on the same cheese-making day and was analyzed for Ca content (IDF, 2003). Compositional analysis on cheese was done after 1 mo for moisture, fat, protein (Marshall, 1992), total Ca (IDF, 2003), and salt by Corning Salt Analyzer (Marshall, 1992). Cheese pH (Marshall, 1992), pH 4.6 soluble nitrogen (pH4.6SN; Kuchroo and Fox, 1982), and the INSOL Ca contents of cheese were determined by the cheese juice method as described by Hassan et al. (2004) after 1 d, 1 wk, 2 wk, 3 wk, 1 mo, and 3 mo. Buffering of cheese was determined by acid-base titration (Hassan et al., 2004) and buffering capacity reported as volume of 0.5 N HCl required to decrease the pH of cheese dispersions (8 g of grated cheese in 40 mL of distilled water) by 1 pH unit (from the initial cheese pH). All INSOL Ca, melt, rheology, and texture measurements were performed at the same intervals.
Melt Profile Analysis
To evaluate melt and flow characteristics of cheese samples, the UW Melt Profiler at Wisconsin Center for Dairy Research was used (Muthukumarappan et al., 1999). At each time point, cheese samples were cut into approximately 200-g blocks and kept in the refrigerator (~5°C) for 2 to 3 h. Cheese samples were then cut into slices (7-mm thick) with a Hobart meat slicer, and cut into 30-mm diameter discs using a stainless steel borer. Cheese samples were stored in a plastic bag in a refrigerator at ~5°C for at least 6 h before testing. Cheese samples were taken from the refrigerator just before testing. The oven temperature was maintained at 72°C during the test. Changes in cheese height and cheese temperature were measured until the cheese temperature reached 62°C. Degree of flow (DOF) was calculated as the change in height when the cheese temperature reached 60°C as compared with the cheese height at the beginning of the test. At least 4 replicates were performed.
Dynamic Small Amplitude Oscillatory Rheometry
The rheological properties of cheese were evaluated using a Paar Physica universal dynamic spectrometer (UDS 200; Physica Messtechnik, Stuttgart, Germany). The dynamic small amplitude oscillatory rheometry (SAOR) technique was used. Cheese samples were cut into ~200-g blocks and kept in a refrigerator for 2 to 3 h. Using a Hobart meat slicer, cheese samples were sliced to approximately 2.2- to 2.4-mm thick, and then cut into 25-mm diameter discs using a cylindrical stainless steel borer. Cheese slices were stored in a plastic bag in a refrigerator at ~5°C for at least 6 h before testing. The rheometer was fitted with a 25-mm diameter serrated parallel plate. All cheese samples were glued onto the bottom heating (peltier) plate of the rheometer with cyanoacrylate glue to prevent slippage of the cheese sample during the test (Nolan et al., 1989). Cheese samples were carefully mounted on the rheometer to minimize deformation by the measuring system. The normal force readings were kept 
1.0 N during sample loading to have good contact with the measuring system without excessive deformation. To minimize dehydration of cheese samples at the exposed area during heating, a layer of vegetable oil was applied. The viscoelastic properties of cheese samples were examined with an applied strain of 0.2% (which was within the linear viscoelastic region of our cheese samples) and a frequency of 0.1 Hz. Cheese samples were heated at a constant rate of 1°C/min from 5 to 80°C. The rheological parameters, such as storage modulus (G') or stiffness, loss modulus (G''), and loss tangent (LT), which is the ratio between the viscous and the elastic properties of the material (LT = G''/G'), were determined from SAOR tests. At least 3 replicates were measured for each cheese sample.
Texture Measurements
Uniaxial compression test was performed using a TA.XT2 Texture Analyzer (Texture Technologies Corporation, Scarsdale, NY). Cheese samples were prepared as recommended by the IDF draft standard for uniaxial compression of cheese (IDF, 2005). Cylindrical cheese samples (height: 19.2 mm, and diameter: 17.5 mm) were prepared using a cork borer. The samples were stored in a plastic bag in the refrigerator for at least 3 h at ~6°C before the start of the test. The diameter of the cross-head was 50 mm. A cross-head speed of 0.83 mm/s was used to compress the sample at each ripening time point and cheese samples were compressed to 80% of the original height. At least 8 replicates were performed.
Statistical Analyses
Data were analyzed using the Statistical Analysis System, version 8.02 (SAS Institute Inc., Cary, NC). Experimental effects of method (RO and NONRO treatment), manufacturing pH treatments and week (aging time) on INSOL Ca content were evaluated using the Proc MIXED procedure for repeated measurement of SAS. Effects included method, manufacturing pH, week, and method x week, week x manufacturing pH, method x week x manufacturing pH interactions. The least squares mean for cheese, nested within method and manufacturing pH, was used as random error term to test method and manufacturing pH. Fisher’s protected least significant difference test was used to compare means, and differences between means were considered significant at P < 0.05. Pearson’s correlation coefficients were estimated between the various responses (i.e., INSOL Ca, pH 4.6SN, rheological parameters, and DOF).
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RESULTS
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Cheddar cheese manufacturing protocols for HPHM and LPHM cheeses are shown in Table 1
. The times required to attain specific pH values varied between trials (due to variable starter activity), but the pH values at key processing points did not vary greatly.
Significantly (P < 0.05) higher concentrations of total and soluble Ca, casein, lactose, and total solids were observed in the RO milk compared with NONRO milk (Table 2). Moisture contents in RO cheeses (~36%) were slightly lower than in NONRO cheeses (~37%). Otherwise, the gross chemical composition of cheeses from both RO and NONRO treatments were similar. The LPHM cheeses had a significantly (P < 0.05) lower total Ca content in cheese for both RO and NONRO treatments (Table 2), as expected. The buffering capacity of HPHM cheeses was slightly higher than that of LPHM cheeses but this difference was only significant for RO cheese (Table 2). The INSOL Ca content of cheese was expressed as milligrams per gram of protein because Ca is an important structural material when it is attached to protein (Lucey and Fox, 1993). The INSOL Ca content (mg/g of protein) decreased during ripening in all cheeses (Table 2). A higher concentration of INSOL Ca was observed in HPHM cheese treatments than in LPHM cheeses in both RO and NONRO treatments (Table 2). There was a decrease in the INSOL Ca content as a function of total Ca during the first 1 to 2 wk of ripening (Figure 1), in agreement with the trend reported by Hassan et al. (2004). After the first 2 wk, there was little further change in the INSOL Ca content as a function of total Ca in all the cheeses during the remainder of the ripening period (Figure 1). The differences in INSOL Ca content of cheeses during ripening were greater between LPHM and HPHM cheeses (~15%) from RO milk than were those from NONRO milk (~10%; Figure 1). The INSOL Ca profiles of LPHM and HPHM cheeses remained different during the entire ripening period; that is, they did not overlap at any point during ripening (Figure 1). The amounts of INSOL Ca in cheeses at 3 mo ranged from 41 to 57% of total Ca in cheese (Figure 1). These INSOL Ca levels were smaller (~58%) than the amounts reported previously for Cheddar cheeses with more typical manufacturing procedures (i.e., acidification profile) (Hassan et al., 2004; Lucey et al., 2005).
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Table 2. Composition of milks and Cheddar cheeses from reverse osmosis (RO) and non-RO (NONRO) treatments (means ± SD).
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Figure 1. Changes in the percentage insoluble Ca content (% of total Ca in cheese) from reverse osmosis (a), and non-reverse osmosis (b) treatment of high-pH method (•) and low-pH method ( ) cheeses. The data represent the means (n = 3), and the error bars represent the standard deviations for each time point.
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Initial (d 1) cheese pH was lower in cheeses made from LPHM in both RO (pH ~4.91; Figure 2b) and NONRO (pH ~4.90; Figure 2d) compared with the initial pH of HPHM RO (pH ~5.06; Figure 2a) and of HPHM NONRO (pH ~5.00; Figure 2b) cheeses. There was a decrease in cheese pH during the first week in all cheeses (Figure 2) due to the fermentation of residual lactose to lactic acid (Table 2). There were no significant differences in the lactic acid levels at 2 wk in the cheeses (Table 2). Cheeses made from RO and NONRO milk decreased in pH during ripening (RO cheese decreased from pH 5.06 to 4.91 and 4.91 to 4.77 for HPHM and LPHM cheeses, respectively, whereas NONRO cheese decreased from pH 5.0 to 4.95 and 4.9 to 4.82 for HPHM and LPHM cheeses, respectively). The pH values of all cheeses did not change greatly after 1 wk of ripening (Figure 2).
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Figure 2. Changes in degree of flow (DOF, ) from UW Melt Profiler test and cheese pH (•) for reverse osmosis, high-pH method (a); reverse osmosis, low-pH method (b); non-reverse osmosis, high-pH method (c); and non-reverse osmosis, low-pH method (d) as a function of ripening time. The data represent the means (n = 3), and the error bars represent the standard deviations for each time point.
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The melting behavior of cheese (i.e., the DOF as measured by UW Melt Profiler) changed during the first week in most cheeses (Figure 2). This change in DOF during the first few weeks occurred concomitantly with the initial decrease in INSOL Ca content of cheese (Figure 1) and a decrease in cheese pH (Figure 2). After the first week, there were only small changes in pH in all cheeses whereas DOF showed no consistent trends for each treatment during the remainder of the ripening period.
The G' values of cheeses determined at 5 and 40°C showed no significant change during ripening (Figure 3a,b). The G' values of cheese determined at 80°C appeared to decrease during the first week in all cheeses and showed no consistent trends during the rest of ripening; some cheeses slightly increased (LPHM cheese from NONRO milk), whereas others slightly decreased (HPHM cheeses from NONRO milk; Figure 3c).
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Figure 3. Changes in storage modulus (G') as a function of ripening time for cheese tested at 5 (a), 40 (b), and 80°C (c) from the small amplitude oscillatory rheology test for non-reverse osmosis, high-pH method ( ), non-reverse osmosis, low-pH method ( ), reverse osmosis, high-pH method (•), and reverse osmosis, low-pH method () cheeses. The data represent the means (n = 3), and the error bars represent the standard deviations for each time point.
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Changes in LT from the SAOR test as a function of temperature for 3-mo-old cheeses are shown in Figure 4. The NONRO HPHM cheese exhibited a large change in LT during heating (from ~0.3 to >1.0), whereas the LT value of other cheeses did not change (i.e., values ranged from ~0.3 to ~0.5).
Changes in the temperature at the maximum in LT (LTmax) during ripening are shown in Figure 5. During heating, the temperature of LTmax was initially around 70°C and gradually decreased to around 58°C during aging, except for HPHM cheeses from NONRO milk, which only decreased to ~62°C during ripening (Figure 5c). A decrease in the temperature of the LTmax during ripening of Cheddar cheese was recently reported by Lucey et al. (2005).
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Figure 5. Changes in the temperature at the maximum loss tangent from reverse osmosis (a), and non-reverse osmosis (b) treatment of high-pH method (•) and low-pH method () cheeses. The data represent the means (n = 3), and the error bars represent the standard deviations for each time point.
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A lower and a more distinct fracture was observed during the compression of the LPHM cheeses from RO milk compared with the other cheeses (Figure 6b). This was also observed during sample preparation—these cheeses were brittle, which made it harder to obtain samples without visual cracks. The LPHM cheeses also exhibited watering-off that started a few weeks after ripening (results not shown), which is consistent with the low pH of these cheeses. Levels of pH4.6SN gradually increased in all cheeses during ripening (Figure 7). The LPHM cheeses had significantly (P < 0.05) higher levels of pH4.6SN compared with those of HPHM cheeses at the 1-d and 4-wk times, but the levels were not significantly different at 12 wk.
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Figure 6. Typical profiles from uniaxial compression tests where 1-mo-old cheese samples were compressed by 80% for reverse osmosis, high-pH method (a), reverse osmosis, low-pH method (b), non-reverse osmosis, high-pH method (c) and non-reverse osmosis, low-pH method (d) treatments.
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Figure 7. Changes in pH 4.6 soluble nitrogen (as % of the total nitrogen) for high-pH method (•) and low-pH method () cheeses from reverse osmosis (a) and non-reverse osmosis (b) treatments. The data represent the means (n = 3), and the error bars represent the standard deviations for each time point.
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In the serum phase of cheese, just after manufacture, the soluble Ca concentration was relatively low but it rapidly increased within the first few weeks (Figure 8). Initially, the majority (between 57 and 74%) of the residual Ca in cheese was in the INSOL form (Figure 1).
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Figure 8. Changes in the serum Ca content of reverse osmosis (a) and non-reverse osmosis (b) treatments for high-pH method (•) and low-pH method () cheeses. The data represent the means (n = 3), and the error bars represent the standard deviations for each time point.
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Changes in cheese pH and INSOL Ca content of cheese during aging were significantly positively correlated (P < 0.0001, r = 0.79; Table 3). Insoluble Ca content was significantly positively correlated with LTmax (r = 0.51) and with DOF (r = 0.40). There were significant positive correlations between INSOL Ca content of cheese and the temperature at LTmax (P < 0.0001, r = 0.59). Maximum LT exhibited a highly positive relationship with DOF (P < 0.0001, r = 0.78). The highly positive correlation between LTmax and meltability of cheese (Table 3) has been previously reported (Ustunol et al., 1994; Mounsey and O’Riordan, 1999; Lucey et al., 2005). The G' values at 5, 40, and 80°C were not significantly correlated with INSOL Ca content.
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Table 3. Pearson’s correlation coefficients between different parameters1 during cheese ripening from reverse osmosis (RO) and non-reverse osmosis (NONRO) treatments.
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Changes in INSOL Ca content of cheese during ripening were significantly affected by cheese manufacturing pH (P < 0.001), but not by milk treatment (Table 4). Moreover, aging time significantly influenced (P < 0.0001) the INSOL Ca content of cheese. There was a significant (P < 0.05) interaction between milk treatment and aging time, suggesting that there were differences in the trends in INSOL Ca content of cheese during ripening between the RO and NONRO treatments (Figure 1), which was probably due to their different pH profiles during ripening.
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Table 4. Mean squares, probabilities, and degrees of freedom for factors that may influence the insoluble (INSOL) Ca content1 of cheese.
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Figure 9 is a scatter plot of the LTmax values of cheeses and their respective pH values. If LTmax values can be used as an indicator of melting, it is clear that the melting behavior of cheese was greatly affected by pH. In the low cheese pH region (4.94), the LTmax values were low indicating poor melting was likely during heating. In this low pH region, neither the reduction in INSOL Ca or proteolysis, both of which occur during ripening, were able to improve the melting properties. In cheeses with pH values >4.94, there was greater variation in the LTmax values, suggesting that the loss of INSOL Ca and proteolysis, which occur during ripening, help in altering meltability.
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Figure 9. Scatter plot for cheese pH vs. maximum loss tangent values from the small amplitude oscillatory rheology test for all cheeses.
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DISCUSSION
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The INSOL Ca content of Cheddar cheese changes during ripening (Hassan et al., 2004). The initial INSOL Ca content of Cheddar cheese and the extent of solubilization during ripening appear to depend on a number of factors. Cheeses with very low pH values (e.g., LPHM with RO treatment, pH ~4.7) still had 
41% of total Ca in the INSOL form even after 3 mo of ripening (Figure 1). The lower Ca of LPHM cheeses was due to greater solubilization of colloidal calcium phosphate (CCP) at the lower pH values used during manufacture of LPHM cheese (Pyne and McGann, 1960; Lawrence and Gilles, 1982; Dalgleish and Law, 1989; Lucey and Fox, 1993). When milk is acidified to pH ~5.0, nearly all the INSOL Ca is dissolved within several hours (Pyne and McGann, 1960; Dalgleish and Law, 1989). Hassan et al. (2004) and Lucey et al. (2005) also reported that the proportion of Ca in the INSOL form in Cheddar cheese remained high (~58%) even after 3 mo of ripening in a cheese that was pH ~5.2. It seems that in cheese, the solubilization of CCP is very different to that in milk (Lucey and Fox, 1993; Lucey et al., 2003; Hassan et al., 2004). It is our hypothesis that the ongoing loss of INSOL Ca could be due to the initial concentrations of INSOL and soluble Ca in cheese not being close to the concentrations that provide a stable equilibrium between the INSOL and soluble phases in the cheese system. Thus, a driving force of the solubilization of INSOL Ca exists in cheese during the initial stage of ripening. A low cheese pH and further acid development are likely to contribute to this initial solubilization reaction, as they would in milk. High residual lactose content in cheese at the end of manufacture results in a large decrease in pH during Cheddar cheese ripening, if it is fermented (Huffman and Kristoffersen, 1984; Shakeel-Ur-Rehman et al., 2004).
There may have been insufficient time during the cheese making process to dissolve the CCP, as most CCP dissolves in milk between 6.0 and ~5.0 (Pyne and McGann, 1960) depending on the acidification conditions (e.g., time). However, this acidification region (pH 6.0) occurs when milk has already been converted into curd particles and mostly after whey draining when the curd particles have undergone considerable syneresis (loss of moisture). It is possible that the solubilization of CCP is slower as the moisture content of the system decreases, although this is conjecture.
There are a few well-known examples of differences in Ca concentrations causing Ca migration in cheese systems. When cheese is placed in a new brine solution that has a low Ca content, Ca is leached from the cheese to the brine until a certain concentration of Ca is attained in the brine, when no further loss of Ca from cheese is observed (Geurts et al., 1972). This loss of cheese Ca can be avoided by the addition of Ca to the freshly prepared brine. It is proposed that a similar phenomenon could be responsible for the loss of INSOL Ca from the casein matrix to the cheese serum during the initial period of ripening. When a certain level of serum Ca content was attained (e.g., ~700 mg/100 g), the solubilization of INSOL slowed (Figure 8). Another example of this Ca migration phenomenon is seen in Camembert cheese, in which the high surface pH causes Ca phosphate to precipitate near the surface (Brooker, 1987). This creates a Ca gradient between the surface and interior of the cheese and results in migration of Ca to the surface (Lucey and Fox, 1993). The decrease in serum Ca observed in some cheese after ripening for 2 to 3 mo (Figure 8) coincided with the appearance of Ca lactate crystals (results not shown). Presumably, the crystals nucleated from the serum phase and thus the effective serum Ca content decreased, although it is possible that crystal formation encouraged further loss of INSOL Ca from casein particles to the cheese serum phase.
The properties of caseins in dairy products are determined by the types of casein interactions present in that system (Horne, 1998). It was recently proposed that cheese texture is controlled by the balance between repulsive and attractive interactions between caseins that form the cheese matrix structure (Lucey et al., 2003). Repulsive interactions include charge repulsion, and attractive interactions include CCP crosslinks, hydrogen bonds, and hydrophobic interactions. It was proposed that a reduction in the attractive interactions, such as solubilization of INSOL Ca, would facilitate greater mobility of caseins and thereby increase cheese melt and flow (Lucey et al., 2003). Conversely, it was proposed that a decrease in repulsive interactions (which causes a concomitant increase in attractive interactions) should reduce melt and flow (Lucey et al., 2003).
The G' values of cheeses determined at 5 and 40°C showed no significant change during ripening, although there was a slight, nonsignificant increase observed in some cheeses during ripening (Figure 3). Lucey et al. (2005) reported that there was an increase in the G' values of high pH (>5.1) Cheddar cheeses determined at 5°C, especially when cheese was aged for a long period (e.g., 9 mo). During ripening, the INSOL Ca content of cheese decreased, which leads to more flexible (due to less crosslinking) casein particles. At low temperatures, hydrophobic interactions are very weak, which could result in increased swelling, an increase in contact area between casein particles, and an increase in attractive interactions (Lucey, 2002). The net result is that, at low temperatures, the Cheddar cheese matrix usually appears to become stiffer during ripening (Lucey et al., 2005). In the present study, because no significant increase was observed in the G' values of cheeses determined at 5°C, we assume that this could be due to the greater loss of cross-linking from INSOL Ca in our very low pH cheeses compared with typical high pH (>5.1) cheese.
The G' values of cheese determined at 80°C appeared to decrease during the first week in all cheeses and showed no consistent trends during the rest of ripening. The initial decrease coincided with the decrease in the INSOL Ca content (Figure 1) and pH (Figure 2) of cheese. The loss of CCP cross-linking between or within casein particles presumably weakens the casein matrix. With further ripening, these G' values remained relatively constant, in contrast to the results of Venugopal and Muthukumarappan (2003) and Lucey et al. (2005), in which the G' values of Cheddar cheese measured at 40°C decreased during ripening. Milk fat is completely liquid by 40°C so the melt and flow of cheese at 40°C is due to action of specific casein interactions (Lucey et al., 2003). During ripening, the pH of most of the LPHM cheeses decreased below 4.9. It is known that low pH cheeses, such as cottage or Feta cheese, do not melt. The crumbly/brittle texture of low pH (5.0) cheeses is in agreement with previous reports (Creamer and Olson, 1982; Lawrence et al., 1987; Luyten et al., 1987; Creamer et al., 1988; Watkinson et al., 2001; Pastorino et al., 2003a). Applying the cheese texture model proposed by Lucey et al. (2003), the very low cheese pH implies a decrease in charge repulsion with the approach of the isoelectric point of casein, an increase in plus/minus (+/– charges) electrostatic interactions, an increase in hydrophobic interaction (due to reduced charge repulsion), and a decrease in the number of CCP cross-links. It appeared that in low pH cheeses, there was a shift toward increased attractive interactions, which helped to maintain high G' values and inhibited the LT values from increasing when cheese was heated to high temperatures (Figure 4). These changes also influenced flow, which was inhibited in low pH cheeses (Figure 2). When Mozzarella cheese pH was decreased from 5.3 to 4.8 by exposing the cheese to volatile acetic acid, cheeses also had greatly reduced meltability (Ge et al., 2002). The flow rate of Cheddar cheeses determined by UW-Melt Profiler significantly decreased when cheese pH was lowered (from 5.3 to 4.7) by the injection of 20% glucono--lactone into cheeses (Pastorino et al., 2003a,b).
Maximum LT and INSOL Ca were significantly positively correlated in both RO and NONRO treatments (Table 3), whereas the opposite results were reported by Lucey et al. (2005). We assume that there were substantial differences in the nature of the protein interactions as many of our cheeses had very low pH values (pH <4.94) compared with the cheeses (pH 5.1) reported by Lucey et al. (2005). In a Cheddar cheese with pH >5.0, the loss of cross-linking material, due to the reduction of INSOL Ca, contributes to the increase in the LTmax value or DOF, i.e., improved melting behavior during ripening (Lucey et al., 2003, 2005). However, in the very low pH cheese (<5.0), there were excessive attractive protein—protein interactions, which may overwhelm any beneficial effects of the solubilization of INSOL Ca on melting (Lucey et al., 2003; Pastorino et al., 2003a).
Proteolysis also contributes to texture changes during cheese ripening. Higher levels of pH4.6SN in LPHM cheeses were probably assisted by the low cheese pH (Fox, 1970; Watkinson et al., 2001; Feeney et al., 2002; Sheehan and Guinee, 2004). At lower pH values, 
s1-casein breakdown is faster because chymosin is more active at lower pH. In addition, the susceptibility of casein to hydrolysis may have increased due to greater loss of CCP from caseins in LPHM cheeses compared with HPHM cheeses (Fox, 1970).
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CONCLUSIONS
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The INSOL Ca content of cheeses was significantly affected by the manufacturing pH values and acid development during ripening. The INSOL Ca content of cheese decreased rapidly in the first few weeks and remained high even in very low pH (e.g., 4.7) cheeses. The G' values of cheese determined at high temperatures hardly changed during ripening and this coincided with the LT values staying relatively constant during heating of cheese. The texture and melting behavior of cheeses was explained by the cheese texture model proposed by Lucey et al. (2003). Although there was a reduction in CCP cross-links, and proteolysis occurred in cheeses during ripening (both of which usually increase melt), the dominant impact of the low pH (especially the increased +/– charge type interactions) overwhelmed these factors. In higher pH cheese, melt was considerably improved compared with the low pH cheese (<4.9). We conclude that cheese with pH values less than 5.0 exhibit markedly different interactions compared with cheese with pH values greater than 5.0.
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ACKNOWLEDGEMENTS
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The authors appreciate the funding of Dairy Management Inc. and the Wisconsin Milk Marketing Board. The authors thank John Jaeggi and Bill Hoesly of the Wisconsin Center for Dairy Research for cheesemaking, and Gene Barmore of the Wisconsin Center for Dairy Research for reverse osmosis processing. The authors also appreciate the useful suggestions from Selvarani Govindasamy-Lucey.
Received for publication April 15, 2005.
Accepted for publication July 15, 2005.
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ABSTRACT
INTRODUCTION
