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Rheological and calcium equilibrium changes during the ripening of Cheddar cheese
ARTICLE IN PRESS
International Dairy Journal 15 (2005) 645–653 www.elsevier.com/locate/idairyj
Rheological and calcium equilibrium changes during the ripening of Cheddar cheese J.A. Luceya,, R. Mishraa, A. Hassana, M.E. Johnsonb a
Department of Food Science, University of Wisconsin-Madison, 1605 Linden Drive, Madison, WI 53706-1565, USA b Wisconsin Center for Dairy Research, 1605 Linden Drive, Madison, WI 53706-1565, USA Received 15 April 2004; accepted 21 August 2004
Abstract The small deformation rheological properties and the calcium (Ca) equilibrium of Cheddar cheese were investigated as a function of ripening time. The proportion of insoluble Ca as a percentage of the total Ca decreased from 72 to 57% between 3 days and 9 months; most changes occurred within the first 4 weeks. During ripening, the storage modulus (G0 ) of the cheese increased at low temperature, but decreased rapidly at high temperature. At temperatures 440 1C, the loss tangent increased to reach a maximum at a temperature of 70 1C in young cheese and there was a steady decline in this temperature during ripening. The maximum loss tangent values increased substantially during the first 4 weeks and then showed little change. Changes in the insoluble Ca content significantly correlated with pH 4.6 soluble nitrogen (pH 4.6 SN). Partial correlation analysis indicated that the insoluble Ca content was more significantly correlated with the rheological properties than was pH 4.6 SN. r 2005 Elsevier Ltd. All rights reserved. Keywords: Rheology; Calcium; Melt; Cheese ripening
1. Introduction Most studies on the ripening of Cheddar cheese have focused on proteolysis and textural properties. It is well known that the calcium (Ca) concentration influences the textural properties of cheese (Lucey & Fox, 1993). The textural properties of Cheddar cheese are widely recognized as critical parameters that determine its sensory attributes and functionality. Proteolysis and flavor development in Cheddar cheese have been intensively studied (Fox, 1989; Fox, O’Connor, McSweeney, Guinee, & O’Brien, 1996; McSweeney & Sousa, 2000). It is well recognized that the texture of Cheddar cheese is influenced by factors such as pH, Ca content, composition (fat, protein, salt, moisture) of the cheese, and proteolysis. Corresponding author. Tel.: +1 608 265 1195; fax: +1 608 262 6872. E-mail address: [email protected] (J.A. Lucey).
0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.08.018
Changes in the texture of Cheddar cheese during ripening are widely considered to be caused by proteolysis (e.g., Lawrence, Gilles, & Creamer, 1999). During maturation, proteolysis causes the network to become shorter in texture (i.e., lower fracture strain) (Lawrence et al., 1999), but variable trends for the firmness/hardness attributes of Cheddar cheese have been reported. The use of different textural terms could be one reason for part of the confusing results. The force at fracture decreases with age for Cheddar cheese (Creamer & Olson, 1982; Lane, Fox, Johnston, & McSweeney, 1997; Halmos, Pollard, Sherkat, & Seuret, 2003), which could be due in part to the reduction in the degree of compression (strain) needed to cause fracture (shorter texture). Cheese rheological properties have also been studied at small deformation (i.e., non-destructively) using small amplitude oscillatory rheology (Taneya, Isutsu, & Sone, 1979; Nolan, Holsinger, & Shieh, 1989; Tunick et al., 1990; Horne, Banks, Leaver, & Law, 1994; Ustunol,
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Kawachi, & Steffe, 1994; Rosenberg, Wang, Chuang, & Shoemaker, 1995; Ustunol, Kawachi, & Steffe, 1995; Guinee, Auty, & Mullins, 1999; Brown, Foegeding, Daubert, Drake, & Gumpertz, 2003; Lucey, Johnson, & Horne, 2003; Venugopal & Muthukumarappan, 2003). Only a few studies on the small deformation rheological properties of Cheddar cheese have investigated the impact of a wide temperature range (e.g., from 5 to 80 1C) on these properties. When Cheddar cheese is heated, the storage modulus (G0 ) decreases considerably and the loss tangent (LT) increases at X40 1C (Horne et al., 1994; Rosenberg et al., 1995; Ustunol et al., 1995; Guinee et al., 1999; Lucey et al., 2003; Venugopal & Muthukumarappan, 2003). In young Cheddar cheese, the LT values at X40 1C are low but increase greatly within the first month (Lucey et al., 2003). Recently, Lucey et al. (2003) proposed that, during cheese maturation, both proteolysis and the slow solubilization of some of the residual insoluble (INSOL) Ca change the curd from the initial ‘‘green’’ rubbery product into the mature cheese texture. Methods to monitor the solubilization of INSOL Ca in cheese have recently been reported (Lucey & Fox, 1993; Hassan, Johnson, & Lucey, 2004). The objectives of this study were to investigate the concomitant changes in proteolysis and solubilization of INSOL Ca during the maturation of Cheddar cheese and to compare these two ripening parameters with a range of textural and functional properties of the cheese.
slabs were cheddared, milled, and salted at cheese pH 5.4 at the rate of 30 g kg1 of the curd weight. The salted curd was packed in hoops, lined with cheesecloth, and pressed overnight at ambient temperature, followed by vacuum packaging and storage at 7 1C until further analysis. Changes in the cheese textural properties were measured at time points of 3 days and 1, 2, 3, and 9 months. 2.2. Compositional analysis
2. Materials and methods
The milk was analysed for total solids, fat, protein, and casein (Marshall, 1992), total Ca (International Dairy Federation, 2003), and INSOL Ca by the acid–base titration method (Lucey, Hauth, Gorry, & Fox, 1993; Hassan et al., 2004). The whey was analysed for total Ca content (International Dairy Federation, 2003). The cheese was analysed at 3 days for moisture, fat, and protein (Marshall, 1992), total Ca (International Dairy Federation, 2003), and salt (Chloride Analyzer 929, Nelson-Jameson, WI). Changes in pH (Marshall, 1992), pH 4.6 soluble nitrogen (pH 4.6 SN) (Kuchroo & Fox, 1982), and the INSOL Ca content in the cheese were measured at time points of 3 days and 1, 2, 3, and 9 months. Acid–base titration of the cheese was performed as described by Lucey, Gorry, and Fox (1993). The INSOL Ca in the cheese was calculated by the acid–base titration method as described by Hassan et al. (2004). The total Ca contents of the cheese (mg 100 g1), milk, and whey were determined using atomic absorption spectroscopy. All analyses were done at least in triplicate.
2.1. Cheese manufacture
2.3. Melt profile analysis
Standard full-fat Cheddar cheese was made at the University of Wisconsin-Madison in 20 L mini-cheese vats that included a computer-controlled cooking program and an automatic variable speed agitator. Five cheese trials (yielding a total of eight cheese vats) were carried out over a period of 1 month. Whole milk was pasteurized at 73 1C for 15 s and was cooled to 4 1C. The milk was heated in the cheese vats to 32.2 1C and inoculated with a mixed starter culture consisting of Lactococcus lactis ssp. cremoris and L. lactis ssp. lactis (DVS 850, Chr. Hansen, Milwaukee, WI) at the rate of 0.2 g kg1 of milk. After a 55-min ripening period, 0.125 mL of calcium chloride kg1 and, 5 min later, 0.072 mL of double strength chymosin kg1 (ChymostarTM, Rhodia, Madison, WI) were added to the milk. The coagulum was cut with 0.63 cm knives, allowed to heal for 5 min, and then gently (manually) agitated for 10 min before cooking. The curd/whey mixture was heated from 32.2 to 38.3 1C over a period of 30 min. The whey was drained after holding for 30 min at the cooking temperature (draining pH 6.2). The curd
Melt profile analysis (MPA) was performed using a UW Melt Profiler according to the procedure described by Muthukumarappan, Wang, and Gunasekaran (1999). Cheese samples were sliced to 7 mm thick and 30 mm diameter disks. Samples were stored in a plastic bag in the refrigerator at 6 1C for at least 3 h before testing. The top and bottom plates were sprayed with a dry film lubricant to prevent the samples from sticking to the plates. A layer of mineral oil was applied to prevent dehydration of the samples during heating. A thermocouple was inserted into the centre of the sample and the cheese disk was placed on the bottom stainless steel plate in an oven maintained at 72 1C (750F Forced Air Oven, Fisher Scientific, Pittsburgh, PA). The top aluminium plate was connected to a linear variable differential transformer and lowered on to the top of the sample. The change in height as a function of time/ sample temperature was measured until the sample temperature reached 63 1C. The cheese height versus time curve has been divided into three regions by Muthukumarappan et al. (1999): an initial slow
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Cheese temperature (°C)
deformation region (softening), a rapid linear deformation region (flow), and a slow linear deformation region (complete melt). The softening temperature corresponds to the point of intersection of tangents drawn at the first two linear regions. The degree of flow (DOF) was calculated as the change in height of the cheese sample at 60 1C as compared with the cheese height at the beginning of the test. The cheese samples were analyzed at 1, 2, 4, and 12 weeks of ripening.
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60 50 40 30 20 10 0 0
200
400
600
800
1000
1200
Heating time (s)
2.4. Dynamic small amplitude oscillatory rheometry A Paar Physica universal dynamic spectrometer (UDS 200 Physica Messtechnik, D-70567, Stuttgart, Germany) was used to study the viscoelastic properties of the cheese. The dynamic small amplitude oscillatory rheometry (SAOR) technique was used. The parameters studied were G0 , loss modulus (G00 ), and LT, which is the ratio between the viscous properties and the elastic properties of the material (LT ¼ G00 /G0 ). Cheese samples were sliced into disks of 2.2 mm thickness and 50 mm diameter. The slices were stored in a plastic bag in the refrigerator at 6 1C for at least 3 h before testing. The rheometer was fitted with a 50-mm diameter serrated parallel plate. Samples were mounted and glued on to the bottom heating (peltier) plate of the rheometer using cyanoacrylate glue. Use of the glue and the serrated plate prevented slippage of the sample (Nolan et al., 1989; Tunick et al., 1990). The exposed surface of the sample was covered with a thin layer of vegetable oil to prevent it drying out. A frequency of 0.1 Hz and a strain of 0.2% (which was within the linear viscoelastic region of our cheese samples) were applied. During loading, the normal force readings were kept at p1.0 N to have good contact between the serrated plate and the cheese sample without excessive deformation of the sample; data acquisition was started only once a relatively constant normal force reading of 0.7 N was obtained (i.e., a low and relatively stable normal force after relaxation of the stress applied during loading). Two types of heating profile were used to study the rheological properties of the cheese samples: a long heating profile (LHP) and a short heating profile (SHP). In the LHP, the cheese samples were heated at a constant rate of 1 1C min1 from 5 to 80 1C. In the SHP, a non-linear heating rate was applied (as shown in Fig. 1) to simulate the heating profile experienced by the cheese samples in the UW Melt Profiler. This was achieved by dividing the temperature profile into eight individual heating regions and applying different heating rates in each, and the rheometer combined all these intervals in a single continuous test. G0 , G00 , and LT were measured as a function of temperature for both the LHP and the SHP tests. The cheese samples were analyzed at time points of 3 days and 1, 2, 3, and 9 months.
Fig. 1. Heating conditions used in the short heating profile of the dynamic small amplitude oscillatory rheology test, which had a similar heating profile to that experienced by the cheese in the UW Melt Profiler test.
2.5. Statistical analysis The data were analyzed using the Statistical Analysis System (SAS, 1999). Pearson’s correlation coefficients between the various responses (i.e., INSOL Ca, pH 4.6 SN, rheological parameters, and DOF) were estimated. The partial correlation coefficient is defined as the correlation between two variables that remains after adjusting for (e.g., partialling out or controlling for) one or more other variables (Steel & Torrie, 1980). It was expected that both INSOL Ca and pH 4.6 SN would change during ripening. Partial correlation coefficients were estimated for the effects of INSOL Ca on the rheological properties when the effects of pH 4.6 SN were adjusted for and vice versa by carrying out multivariate analysis of variance (MANOVA) in SAS. A Pp0:05 was considered to be statistically significant.
3. Results The cheese pH increased slightly during ripening (Table 1). The moisture content was slightly lower than for most typical Cheddar cheese made in the US. The rest of the composition was typical for Cheddar cheese (Table 1). The changes in G0 and the LT with ripening time and heating temperature are shown in Fig. 2. During heating, G0 decreased greatly and the differences between cheeses of different ages increased at temperatures X40 1C (Fig. 2a). At lower temperatures (5–35 1C), the LT values were similar at all ripening times (Fig. 2b). The LT curves increased at 435 1C and the maximum LT (LTmax) tended to occur at lower temperature with increasing ripening time. The G0 value determined at 5 1C (GT ¼ 5 1C) increased during ripening (Fig. 3). In contrast, the G0 values determined at 40 1C (GT ¼ 40 1C) and 80 1C (GT ¼ 80 1C) decreased during ripening and there was a large decrease during the first 60 days
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Average7standard deviation (n ¼ 5)
a
35.470.73 25.071.05 33.270.44 53.070.35 1.770.04 4.770.11 5.1670.07 5.1770.04 5.1570.04 5.2170.06 5.2770.06 769726 72.071.18 62.470.86 59.772.78 58.472.58 57.471.17
Storage modulus (Pa)
8.0e+4 1000
7.0e+4 6.0e+4 5.0e+4
100 0
50
100 150 200 Ripening time (days)
300 250 200 150 100 50 0
250
Fig. 3. Changes in the storage modulus as a function of ripening time for cheese tested at 5 (K), 40 (’), and 80 1C (m), obtained from the long heating profile of the small amplitude oscillatory rheology test. The data represent the means (n ¼ 8) and the error bars represent the standard deviations for each time point.
d ¼ days; m ¼ months. Insoluble Ca as a % of the total Ca in the cheese.
(a)
1e+4
80 75 70 65 60 55 50 0 0
50
100
150
200
250
Ripening time (days) 1e+3
Fig. 4. Changes in the temperature at the maximum loss tangent (K) as a function of the ripening time, obtained from the long heating profile of the small amplitude oscillatory rheology test. The data represent the means (n ¼ 8) and the error bars represent the standard deviations for each time point.
1e+2
1e+1
(b)
2.5
2.0
Loss tangent
9.0e+4
4.0e+4 0.0
b
1e+5
10000
1.0e+5
Temperature at maximum loss tangent (˚C)
Moisture content (%) Protein (%) Fat (%) Moisture in non-fat solids (%) Salt (%) Salt-in-moisture (%) pH at 3 da pH at 1 m pH at 2 m pH at 3 m pH at 9 m Total Ca (mg 100 g1) INSOL Cab at 3 d INSOL Ca at 1 m INSOL Ca at 2 m INSOL Ca at 3 m INSOL Ca at 9 m
Storage modulus at 5ºC (Pa)
1.1e+5
Storage modulus at 80ºC (Pa)
Table 1 Composition of Cheddar cheese
Storage modulus at 40ºC (Pa)
648
1.5
1.0
0.5
0.0 0
10
20
30
40
50
60
70
80
Heating temperature (ºC) Fig. 2. Changes in the storage modulus (a) and the loss tangent (b) as a function of temperature for cheese ripened for 3 days (K), 1 month (J), 2 months (.), 3 months (,), and 9 months (’), obtained from the long heating profile of the small amplitude oscillatory rheology test. The data represent the means (n ¼ 8) and the error bars represent the standard deviations for each time point.
(Fig. 3). During heating, the LTmax occurred at 70 1C in young cheese but decreased during ripening to 62 1C by 9 months (Fig. 4). The LTmax values increased substantially during the first 3 weeks from 1.3 at 3 days to 2.3 by the end of 3 weeks, and then showed little change or a slight increase for the rest of the ripening period (Fig. 5). This agrees with the trend for an increase in the LT during Cheddar cheese ripening reported by Lucey et al. (2003). The value of the LT during heating has been used as an index of the ‘‘meltability’’ of cheese (Ustunol et al., 1994). During maturation, the proportion of soluble Ca in the cheese increased from 25 to 44% between 3 days and 9 months; the majority of the increase occurred during the first 30 days (Fig. 5). This agrees with the trends reported recently by Hassan et al. (2004) for Cheddar cheese. The changes in the LTmax very closely paralleled the changes in the proportion of soluble Ca (Fig. 5). In contrast, the changes in proteolysis (pH 4.6 SN) increased at a slower
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Maximum loss tangent
18 2.5
16 14
2.0
12 1.5
10 8
1.0
6 4
0.5
2 0.0
0 0
50
100
150
200
250
Ripening time (days)
50 45 40 35 30 25 20 15 10 5 0
SOL Ca (% of total Ca in cheese)
3.0
pH 4.6 Soluble nitrogen (% of total nitrogen)
J.A. Lucey et al. / International Dairy Journal 15 (2005) 645–653
Fig. 5. Changes in the soluble Ca content (% of total Ca in the cheese) (K), the maximum loss tangent (J) and pH 4.6 soluble nitrogen (as a % of total nitrogen) (m). The data represent the means (n ¼ 8) and the error bars represent the standard deviations for each time point.
90 85
45 80 40
75 70
35 65 30 0
Degree of flow at 60˚C (%)
Softening temperature (˚C)
50
60 0 0
50
100
150
200
250
Ripening time (days) Fig. 6. Changes in the degree of flow (&) and the softening temperature (1C) (’) of cheese as a function of the ripening time, obtained from the UW Melt Profiler test. The data represent the means (n ¼ 8) and the error bars represent the standard deviations for each time point.
rate during the first 90 days and continued to increase during the whole ripening period (Fig. 5). The melting properties of Cheddar cheese obtained from the UW Melt Profiler changed greatly in the first month, with a rapid increase in the DOF, after which there was little further change (Fig. 6). The temperature at which there is a decrease in the cheese height caused by flow corresponds to the softening temperature (Muthukumarappan et al., 1999). The softening temperature first decreased with ripening time (up to 2 months) and then increased from 2 to 9 months of ripening time (Fig. 6). The softening temperature did not seem to be highly correlated with changes in melting behaviour, as there were no clear trends in the softening point with ripening time. As most of the changes in the DOF occurred during the first 12 weeks of ripening, the melt properties of Cheddar cheese from the UW Melt Profiler at 1, 2, 4,
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and 12 weeks of age were compared with the LT from the SHP of the rheometer (Fig. 7). The cheese height versus cheese temperature curves from the UW Melt Profiler and the LT versus cheese temperature curves (SHP) are shown in Fig. 7. The temperature at which there was an increase in the LT value was similar to the temperature at which there was a decrease in cheese height at all stages of ripening. Both the LT values and the cheese height curves showed similar trends. This suggests that the increase in the LT (i.e., increased viscous contribution or more fluid-like character) coincides with the flow of the cheese during heating. With an increase in the age of the cheese, the shapes of the curves were generally similar, but not identical. At 4 (Fig. 7c) and 12 weeks (Fig. 7d) of ripening, the LT value decreased at around 60 1C. This occurred because the LTmax occurred at a lower temperature in older cheeses (Fig. 4). It should be noted that, in the UW Melt Profiler, the pressure (load) from the top plate caused the cheese to flow when it reached X40 1C whereas, in the SAOR test, a constant gap was maintained for the entire heating step and no flow was observed. Pearson’s correlation coefficients between the rheological data, proteolysis, and INSOL Ca content are given in Table 2. Highly significant positive correlations (Po0:001; rX0:70) were found for INSOL Ca with the temperature at the point of the LTmax, GT ¼ 40 1C, and GT ¼ 80 1C. INSOL Ca was weakly negatively correlated with GT ¼ 5 1C (Po0:05; r ¼ 0:50) and was not correlated with the value of the LT at 8 1C. Highly significant negative correlations (Po0:001; rX 0:92) were found for INSOL Ca with LTmax and DOF. INSOL Ca was significantly negatively correlated (Po0:001; r ¼ 0:79) with pH 4.6 SN. Highly significant negative correlations (Po0:001; rX 0:74) were found for pH 4.6 SN with the temperature at the point of the LTmax, GT ¼ 40 1C, and GT ¼ 80 1C. Highly significant positive correlations (Po0:01; rX0:63) were found for pH 4.6 SN with LTmax, GT ¼ 5 1C, and DOF. The DOF was significantly positively correlated (Po0:001; r40:70) with pH 4.6 SN and LTmax and was negatively correlated with INSOL Ca, the temperature at the point of the LTmax, GT ¼ 40 1C, and GT ¼ 80 1C. Partial correlation analysis (Steel & Torrie, 1980) was performed to separate the effects of the INSOL Ca content from concomitant proteolysis changes during ripening (because they were significantly correlated). When the effect of pH 4.6 SN was excluded, partial correlation analysis indicated that the INSOL Ca content was still very significantly correlated (Po0:001; rX0:80) with the rheological properties, such as LTmax, GT ¼ 40 1C, and GT ¼ 80 1C, and the DOF (Table 3). In contrast, when the effect of INSOL Ca was excluded from the partial correlation analysis, it was found that pH 4.6 SN was only weakly correlated with a single rheological parameter, the temperature at the point
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650
120
0.0
(b)
0.5
100
1.0
80
1.5
60
2.0
40
2.5
20
3.0
0
0.0
120
(c)
0.5
(d)
100
1.0
80
1.5
60
2.0
40
2.5
20
3.0
Cheese height as a % of original height
Loss tangent
(a)
0 0 20 25 30 35 40 45 50 55 60 65 0 20 25 30 35 40 45 50 55 60 65
Cheese temperature (˚C) Fig. 7. Changes in the loss tangent (&) and the cheese height (as a % of the original height) (’), as a function of the cheese temperature during the heating conditions used in the short heating profile of the dynamic small amplitude oscillatory rheology test, which had a similar heating profile to that experienced in the UW Melt Profiler test, for cheese at (a) 1, (b) 2, (c) 4, and (d) 12 weeks of ripening.
Table 2 Pearson’s correlation coefficients between different parameters during cheese ripening
INSOL Ca pH 4.6 SN LTmax Temp. at LTmax G5 1C G40 1C G80 1C
pH 4.6 SN
LTmax
Temp. at LTmax
G5 1C
G40 1C
G80 1C
0.79***
0.92*** 0.76***
0.70*** 0.79*** 0.55**
0.50* 0.63**
0.93*** 0.82*** 0.91*** 0.63** 0.45*
0.92*** 0.74*** 0.93*** 0.65***
0.47*
LT (8 1C)
0.45* 0.55**
0.88***
DOF 0.94*** 0.81*** 0.93*** 0.75*** 0.47* 0.91*** 0.95***
***Po0:001; **Po0:01; *Po0:05:
of the LTmax (Po0:05; r ¼ 0:53) (Table 4). Partial correlation coefficients indicated that the INSOL Ca content was a more important factor than pH 4.6 SN in determining the changes in the rheological properties of Cheddar cheese during maturation.
4. Discussion It is well recognized that, during ageing, Cheddar cheese changes from the ‘‘green’’ initial texture of fresh curd to a smoother and crumblier texture when mature. There have been a number of recent reviews on cheese texture
(Prentice, Langley, & Marshall, 1999; Fox, Guinee, Cogan, & McSweeney, 2000; Gunasekeran & Ak, 2002; Foegeding, Brown, Drake, & Daubert, 2003; Lucey et al., 2003). It is widely believed that proteolysis is responsible for these textural changes in Cheddar cheese (Creamer & Olson, 1982; Fox et al., 2000). For example, the rapid cleavage of as1-casein at the Phe23–Phe24 bond by the coagulant during the first 30 days is considered to be responsible for the initial softening of Cheddar cheese texture (Creamer & Olson, 1982; Fox et al., 2000). In cheeses with a surface rind (e.g., Gouda), moisture loss during maturation results in an increase in firmness (Visser, 1991).
ARTICLE IN PRESS J.A. Lucey et al. / International Dairy Journal 15 (2005) 645–653 Table 3 Partial correlation coefficients between different parameters after controlling for the effect of pH 4.6 soluble nitrogen during cheese ripening
INSOL Ca LTmax G40 1C G80 1C
LTmax
G40 1C
G80 1C
DOF
0.80***
0.80*** 0.83***
0.82*** 0.96*** 0.81***
0.84*** 0.85*** 0.89*** 0.90***
***Po0.001; **Po0.01; *Po0.05.
Table 4 Partial correlation coefficients between different parameters after controlling for the effect of insoluble Ca during cheese ripening
DOF LTmax pH 4.6 SN
LTmax
G40 1C
G80 1C
0.56*
0.71** 0.52*
0.66** 0.89***
Temp. at LTmax
0.53*
***Po0.001; **Po0.01; *Po0.05.
There was an increase in the GT ¼ 5 1C value during ripening (Fig. 3). Rosenberg et al. (1995) reported that the G0 value determined at 25 1C increased with age for Cheddar cheese tested at 19, 240, and 470 days. Venugopal and Muthukumarappan (2003) reported that, in Cheddar cheeses with typical moisture and fat contents, there was little difference in the G0 values of the cheeses determined at 25 1C during ripening. In a cheese with higher moisture content (44.4%), they reported that G0 increased during ripening (Venugopal & Muthukumarappan, 2003). Watkinson et al. (1997) reported that the stiffness or modulus of deformability (MD), which is defined as the slope of the stress–strain curve at the initial, linear low strain region, increased during Cheddar cheese maturation. Watkinson et al. (2001) also reported a similar increase in the MD with the maturation of model cheese (there was no change in moisture, as the cheeses were sealed in polyethylene bags, and very little change in pH). An increase in the initial slope of the force–compression curve with age has also been reported in other studies (Creamer & Olson, 1982). It should be noted that the complex modulus (G*) is the ratio of stress to strain in the linear region (Foegeding et al., 2003) for dynamic oscillatory tests; the differences between G* and MD are the strain, rate of deformation, and frequency used in their determination. G0 is derived from G*, i.e., G0 ¼ G* cos (d), where d is the phase angle (Foegeding et al., 2003). One mechanism that could be involved in proteolysis-related texture changes is the formation of two new ionic groups as each peptide bond is cleaved; these ionic groups compete
651
for available water in the cheese and less water is available for solution of the peptides (Creamer & Olson, 1982). It is presumed that this makes the matrix firmer and that this process occurs gradually during ripening. There was an increase in the G0 of Cheddar cheese with a decrease in the measuring temperature. A similar trend of increasing G0 with a decrease in the measuring temperature has been observed in various types of milk gel (Lucey, 2002). The likely cause of the increase in G0 of casein networks at low measuring temperature is the loosening of casein particles, as hydrophobic interactions are weak and the increased contact area between casein particles leads to increased casein–casein interaction, e.g., hydrogen bonding and electrostatic interactions (Lucey, 2002). At low temperature, milk fat is mostly solid, which could also contribute to the increase in the G0 of the cheese. During the maturation of Cheddar cheese, it is likely that both proteolysis and loss of INSOL Ca increase the flexibility of the casein particles at low temperatures (but only weak correlations between INSOL Ca or pH 4.6 SN and GT ¼ 5 1C were obtained; Table 2). The increase in the contact area between casein particles should then result in an increase in GT ¼ 5 1C, as was observed in our study (Fig. 3). However, the loss of cross-linking material (loss of INSOL Ca and proteolysis) may have the direct effect of weakening the matrix. At higher temperature, the strengthening of the hydrophobic interactions causes the casein particles to shrink and the loss of crosslinking material due to solubilization of some INSOL Ca probably caused more of the reduction in the high temperature G0 values during maturation because it was significantly correlated with LTmax, G40 1C, and GT ¼ 80 1C, and the DOF (Table 3). The loss of intact casein (i.e., structure-forming protein) was less important as it was not significantly correlated with LTmax, GT ¼ 40 1C, and GT ¼ 80 1C, and the DOF when the changes in INSOL Ca were statistically controlled (Table 4). This increase in G0 at low temperatures could be an important factor in explaining some conflicting results of texture tests (which are often conducted at 5 1C and nearly all are tested at p20 1C). Hort and Le Grys (2001) studied an individual Cheddar block during ageing (from 8 to 64 weeks) and observed that, during the maturation process, there was a period during which hardness (derived from both sensory tests and instrumental tests) and stress at fracture increased compared with young (8 weeks) cheese, which had lower hardness and stress at fracture than ‘‘green’’ cheese (1 day after manufacture). A number of other studies (e.g., Piggott & Mowat, 1991; Roberts & Vickers, 1994) have investigated the relationship between Cheddar cheese texture and ageing and most have concluded that the textural attributes are not significantly correlated with age.
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The increase in the LTmax during ripening occurred mainly in the first 4 weeks and then hardly changed (Fig. 5). The LT did not increase until X40 1C, at which point milk fat is completely liquid; therefore, the increase in the LT is due to a modification in protein interactions and not directly to the melting of milk fat. The value of the LT at a high temperature has been correlated with cheese meltability (Ustunol et al., 1994; Mounsey & O’Riordan, 1999). The DOF was significantly correlated (Po0:001; r ¼ 0:85) with the LTmax (Table 3). The temperature at which the LTmax occurred continued to decrease during maturation, but most changes occurred in the first 100 days (Fig. 4). The lower temperature of the LTmax suggested that less thermal energy needed to be given to aged cheese in order for it to reach the state at which it was most fluid-like (or most meltable). Probably, both the loss of intact casein (due to proteolysis) and the loss of cross-linking material (due to the solubilization of some INSOL Ca) are responsible for this reduction in the temperature of the LTmax during maturation, but pH 4.6 SN was more significantly correlated with this change (Table 4). Changes in the value of the LTmax closely followed the trend for the increase in soluble Ca during maturation but not the trend for the production of pH 4.6 SN (Fig. 5). This suggests that solubilization of some INSOL Ca was mostly responsible for the increase in the LTmax. This was also supported by our partial correlation analysis (Tables 3 and 4). It is also possible that some specific proteolytic event, such as the rate of hydrolysis of as1-casein at the Phe23–Phe24 bond by chymosin (which occurs mostly in the first 30 days of Cheddar cheese ripening), could more closely follow the trend for the changes in the LTmax. Changes in the INSOL Ca content during ripening were significantly correlated with pH 4.6 SN (Table 2). It is known that proteolysis in Cheddar cheese releases phosphopeptides (Singh, Fox, & Healy, 1997) and this could have contributed to the decrease in casein-bound Ca. Alternatively, the solubilization of INSOL Ca could have made the caseins more susceptible to proteolysis (Fox, 1970), and thus the loss of INSOL Ca could have contributed to the increase in proteolysis with age.
5. Conclusions During maturation, the proportion of soluble Ca in Cheddar cheese increased, with the majority of the increase occurring during the first 60 days. The changes in the Ca equilibrium were highly correlated with the changes in the rheological properties, especially the increase in the LT and the decrease in G0 at high temperatures. The INSOL Ca content was significantly correlated with the rheological properties of Cheddar cheese measured at high temperatures, similar to those
occurring during melting applications (e.g. DOF). Partial correlation analysis suggested that the changes in the INSOL Ca content during maturation were more significantly correlated with the rheological and melting properties than was the formation of pH 4.6 SN. During maturation, there was a slight increase in G0 at low temperatures, possibly due to greater swelling of the matrix when the hydrophobic interactions were weak. Specific proteolytic events could still be important for cheese textural changes, but these effects need to be separated from the effects of changes in the Ca equilibrium.
Acknowledgments The authors appreciate the funding of Dairy Management Inc. and the Wisconsin Center for Dairy Research. References Brown, J. A., Foegeding, E. A., Daubert, C. R., Drake, M. A., & Gumpertz, M. (2003). Relationships among rheological properties and sensorial properties of young cheeses. Journal of Dairy Science, 86, 3054–3067. Creamer, L. K., & Olson, N. F. (1982). Rheological evaluation of maturing Cheddar cheese. Journal of Food Science, 47, 631–636, 646. Foegeding, E. A., Brown, J., Drake, M., & Daubert, C. R. (2003). Sensory and mechanical aspects of cheese texture. International Dairy Journal, 13, 585–591. Fox, P. F. (1970). Influence of aggregation on the susceptibility of casein to proteolysis. Journal of Dairy Research, 37, 173–180. Fox, P. F. (1989). Proteolysis during cheese manufacture and ripening. Journal of Dairy Science, 72, 1379–1400. Fox, P. F., O’Connor, T. P., McSweeney, P. L. H., Guinee, T. P., & O’Brien, N. M. (1996). Cheese: physical, biochemical, and nutritional aspects. Advances in Food and Nutrition Research, 39, 163–328. Fox, P. F., Guinee, T. P., Cogan, T. M., & McSweeney, P. L. H. (2000). Fundamentals of cheese science. Gaithersburg, MD: Aspen. Guinee, T. P., Auty, M. A. E., & Mullins, C. (1999). Observations on the microstructure and heat-induced changes in the viscoelasticity of commercial cheeses. Australian Journal of Dairy Technology, 54, 84–89. Gunasekeran, S., & Ak, M. M. (2002). Cheese rheology and texture. Boca Raton, FL: CRC Press. Halmos, A. L., Pollard, A., Sherkat, F., & Seuret, M. G. (2003). Natural Cheddar cheese texture variation as a result of milk seasonality. Journal of Texture Studies, 34, 21–40. Hassan, A., Johnson, M. E., & Lucey, J. A. (2004). Changes in the proportion of soluble and insoluble calcium during the ripening of Cheddar cheese. Journal of Dairy Science, 87, 854–862. Horne, D. S., Banks, J. M., Leaver, J., & Law, A. J. R. (1994). Dynamic mechanical spectroscopy of Cheddar cheese. Cheese yield and factors affecting its control (pp. 507–512), Brussels, Belgium: International Dairy Federation. Hort, J., & Le Grys, G. (2001). Developments in the textural and rheological properties of UK Cheddar cheese during ripening. International Dairy Journal, 11, 475–481. International Dairy Federation. (2003). Determination of calcium, sodium, potassium and magnesium contents—atomic absorption
ARTICLE IN PRESS J.A. Lucey et al. / International Dairy Journal 15 (2005) 645–653 spectrometric method. IDF Standard 119. Brussels, Belgium: International Dairy Federation. Kuchroo, C. N., & Fox, P. F. (1982). Soluble nitrogen in Cheddar cheese: comparison of extraction procedures. Milchwissenschaft, 37, 331–335. Lane, C. N., Fox, P. F., Johnston, D. E., & McSweeney, P. L. H. (1997). Contribution of coagulant to proteolysis and textural changes in Cheddar cheese during ripening. International Dairy Journal, 7, 453–464. Lawrence, R. C., Gilles, J., & Creamer, L. K. (1999). Cheddar cheese and related dry-salted cheese varieties. In P. F. Fox (Ed.), Cheese: chemistry, physics and microbiology. 2. Major cheese groups, (2nd ed) (pp. 1–38). Gaithersburg, MD: Aspen. Lucey, J. A. (2002). Formation and physical properties of milk protein gels. Journal of Dairy Science, 85, 281–294. Lucey, J. A., & Fox, P. F. (1993). Importance of calcium and phosphate in cheese manufacture: a review. Journal of Dairy Science, 76, 1714–1724. Lucey, J. A., Hauth, B., Gorry, C., & Fox, P. F. (1993). The acid–base buffering properties of milk. Milchwissenschaft, 48, 268–272. Lucey, J. A., Gorry, C., & Fox, P. F. (1993). Changes in the acid–base buffering curves during the ripening of Emmental cheese. Milchwissenschaft, 48, 183–186. Lucey, J. A., Johnson, M. E., & Horne, D. S. (2003). Perspectives on the basis of the rheology and texture properties of cheese. Journal of Dairy Science, 86, 2725–2743. Marshall, R. T. (1992). Standard methods for the examination of dairy products (16th ed.). Washington, DC: American Public Health Association. McSweeney, P. L. H., & Sousa, M. J. (2000). Biochemical pathways for the production of flavour compounds in cheese during ripening: a review. Le Lait, 80, 293–324. Mounsey, J. S., & O’Riordan, E. D. (1999). Empirical and dynamic rheological data correlation to characterize melt characteristics of imitation cheese. Journal of Food Science, 64, 701–703. Muthukumarappan, K., Wang, Y.-C., & Gunasekaran, S. (1999). Estimating softening point of cheeses. Journal of Dairy Science, 82, 2280–2286. Nolan, E. J., Holsinger, V. H., & Shieh, J. J. (1989). Dynamic rheological properties of natural and imitation Mozzarella cheeses. Journal of Texture Studies, 20, 179–183. Piggott, J. R., & Mowat, R. G. (1991). Sensory aspects of maturation of Cheddar cheese by descriptive analysis. Journal of Sensory Studies, 6, 49–62.
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Prentice, J. H., Langley, K. R., & Marshall, R. J. (1999). Cheese rheology. In P. F. Fox (Ed.), Cheese: chemistry, physics and microbiology. 1. General aspects, (2nd ed) (pp. 303–340). Gaithersburg, MD: Aspen. Roberts, A. K., & Vickers, Z. M. (1994). Cheddar cheese aging: changes in sensory attributes and consumer acceptance. Journal of Food Science, 59, 328–334. Rosenberg, M., Wang, Z., Chuang, S. L., & Shoemaker, C. F. (1995). Viscoelastic property changes in Cheddar cheese during ripening. Journal of Food Science, 60, 640–644. SAS (1999). SAS user’s guide. Statistics, version 8.0 Ed. Cary, NC: SAS Institute, Inc. Singh, T. K., Fox, P. F., & Healy, A. (1997). Isolation and identification of further peptides in the diafiltration retentate of the water-soluble fraction of Cheddar cheese. Journal of Dairy Research, 64, 433–443. Steel, R. G. D., & Torrie, J. H. (1980). Multiple and partial regression and correlation. Principles and procedures of statistics: a biometrical approach (2nd ed.) (pp. 311–335). New York: McGraw-Hill. Taneya, S., Isutsu, T., & Sone, T. (1979). Dynamic viscoelasticity of natural and process cheese. In P. Sherman (Ed.), Food texture and rheology (pp. 369–383). London: Academic Press. Tunick, M. H., Nolan, E. J., Shieh, J. J., Thompson, M. P., Maleeff, B. E., & Holsinger, V. H. (1990). Cheddar and Cheshire cheese rheology. Journal of Dairy Science, 73, 1671–1675. Ustunol, Z., Kawachi, K., & Steffe, J. (1994). Arnott test correlates with dynamic rheological properties for determining Cheddar cheese meltability. Journal of Food Science, 59, 970–971. Ustunol, Z., Kawachi, K., & Steffe, J. (1995). Rheological properties of Cheddar cheese as influenced by fat reduction and ripening time. Journal of Food Science, 60, 1208–1210. Venugopal, V., & Muthukumarappan, K. (2003). Rheological properties of Cheddar cheese during heating and cooling. International Journal of Food Properties, 6, 99–114. Visser, J. (1991). Factors affecting the rheological and fracture properties of hard and semi-hard cheese. Bulletin of the IDF, 268, 49–61. Watkinson, P., Boston, G., Campanella, O., Coker, C., Johnston, K., Luckman, M., & White, N. (1997). Rheological properties and maturation of New Zealand Cheddar cheese. Le Lait, 77, 109–120. Watkinson, P., Coker, C., Crawford, R., Dodds, C., Johnston, K., McKenna, A., & White, N. (2001). Effect of cheese pH and ripening time on model cheese textural properties and proteolysis. International Dairy Journal, 11, 455–464.
International Dairy Journal 15 (2005) 645–653 www.elsevier.com/locate/idairyj
Rheological and calcium equilibrium changes during the ripening of Cheddar cheese J.A. Luceya,, R. Mishraa, A. Hassana, M.E. Johnsonb a
Department of Food Science, University of Wisconsin-Madison, 1605 Linden Drive, Madison, WI 53706-1565, USA b Wisconsin Center for Dairy Research, 1605 Linden Drive, Madison, WI 53706-1565, USA Received 15 April 2004; accepted 21 August 2004
Abstract The small deformation rheological properties and the calcium (Ca) equilibrium of Cheddar cheese were investigated as a function of ripening time. The proportion of insoluble Ca as a percentage of the total Ca decreased from 72 to 57% between 3 days and 9 months; most changes occurred within the first 4 weeks. During ripening, the storage modulus (G0 ) of the cheese increased at low temperature, but decreased rapidly at high temperature. At temperatures 440 1C, the loss tangent increased to reach a maximum at a temperature of 70 1C in young cheese and there was a steady decline in this temperature during ripening. The maximum loss tangent values increased substantially during the first 4 weeks and then showed little change. Changes in the insoluble Ca content significantly correlated with pH 4.6 soluble nitrogen (pH 4.6 SN). Partial correlation analysis indicated that the insoluble Ca content was more significantly correlated with the rheological properties than was pH 4.6 SN. r 2005 Elsevier Ltd. All rights reserved. Keywords: Rheology; Calcium; Melt; Cheese ripening
1. Introduction Most studies on the ripening of Cheddar cheese have focused on proteolysis and textural properties. It is well known that the calcium (Ca) concentration influences the textural properties of cheese (Lucey & Fox, 1993). The textural properties of Cheddar cheese are widely recognized as critical parameters that determine its sensory attributes and functionality. Proteolysis and flavor development in Cheddar cheese have been intensively studied (Fox, 1989; Fox, O’Connor, McSweeney, Guinee, & O’Brien, 1996; McSweeney & Sousa, 2000). It is well recognized that the texture of Cheddar cheese is influenced by factors such as pH, Ca content, composition (fat, protein, salt, moisture) of the cheese, and proteolysis. Corresponding author. Tel.: +1 608 265 1195; fax: +1 608 262 6872. E-mail address: [email protected] (J.A. Lucey).
0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.08.018
Changes in the texture of Cheddar cheese during ripening are widely considered to be caused by proteolysis (e.g., Lawrence, Gilles, & Creamer, 1999). During maturation, proteolysis causes the network to become shorter in texture (i.e., lower fracture strain) (Lawrence et al., 1999), but variable trends for the firmness/hardness attributes of Cheddar cheese have been reported. The use of different textural terms could be one reason for part of the confusing results. The force at fracture decreases with age for Cheddar cheese (Creamer & Olson, 1982; Lane, Fox, Johnston, & McSweeney, 1997; Halmos, Pollard, Sherkat, & Seuret, 2003), which could be due in part to the reduction in the degree of compression (strain) needed to cause fracture (shorter texture). Cheese rheological properties have also been studied at small deformation (i.e., non-destructively) using small amplitude oscillatory rheology (Taneya, Isutsu, & Sone, 1979; Nolan, Holsinger, & Shieh, 1989; Tunick et al., 1990; Horne, Banks, Leaver, & Law, 1994; Ustunol,
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Kawachi, & Steffe, 1994; Rosenberg, Wang, Chuang, & Shoemaker, 1995; Ustunol, Kawachi, & Steffe, 1995; Guinee, Auty, & Mullins, 1999; Brown, Foegeding, Daubert, Drake, & Gumpertz, 2003; Lucey, Johnson, & Horne, 2003; Venugopal & Muthukumarappan, 2003). Only a few studies on the small deformation rheological properties of Cheddar cheese have investigated the impact of a wide temperature range (e.g., from 5 to 80 1C) on these properties. When Cheddar cheese is heated, the storage modulus (G0 ) decreases considerably and the loss tangent (LT) increases at X40 1C (Horne et al., 1994; Rosenberg et al., 1995; Ustunol et al., 1995; Guinee et al., 1999; Lucey et al., 2003; Venugopal & Muthukumarappan, 2003). In young Cheddar cheese, the LT values at X40 1C are low but increase greatly within the first month (Lucey et al., 2003). Recently, Lucey et al. (2003) proposed that, during cheese maturation, both proteolysis and the slow solubilization of some of the residual insoluble (INSOL) Ca change the curd from the initial ‘‘green’’ rubbery product into the mature cheese texture. Methods to monitor the solubilization of INSOL Ca in cheese have recently been reported (Lucey & Fox, 1993; Hassan, Johnson, & Lucey, 2004). The objectives of this study were to investigate the concomitant changes in proteolysis and solubilization of INSOL Ca during the maturation of Cheddar cheese and to compare these two ripening parameters with a range of textural and functional properties of the cheese.
slabs were cheddared, milled, and salted at cheese pH 5.4 at the rate of 30 g kg1 of the curd weight. The salted curd was packed in hoops, lined with cheesecloth, and pressed overnight at ambient temperature, followed by vacuum packaging and storage at 7 1C until further analysis. Changes in the cheese textural properties were measured at time points of 3 days and 1, 2, 3, and 9 months. 2.2. Compositional analysis
2. Materials and methods
The milk was analysed for total solids, fat, protein, and casein (Marshall, 1992), total Ca (International Dairy Federation, 2003), and INSOL Ca by the acid–base titration method (Lucey, Hauth, Gorry, & Fox, 1993; Hassan et al., 2004). The whey was analysed for total Ca content (International Dairy Federation, 2003). The cheese was analysed at 3 days for moisture, fat, and protein (Marshall, 1992), total Ca (International Dairy Federation, 2003), and salt (Chloride Analyzer 929, Nelson-Jameson, WI). Changes in pH (Marshall, 1992), pH 4.6 soluble nitrogen (pH 4.6 SN) (Kuchroo & Fox, 1982), and the INSOL Ca content in the cheese were measured at time points of 3 days and 1, 2, 3, and 9 months. Acid–base titration of the cheese was performed as described by Lucey, Gorry, and Fox (1993). The INSOL Ca in the cheese was calculated by the acid–base titration method as described by Hassan et al. (2004). The total Ca contents of the cheese (mg 100 g1), milk, and whey were determined using atomic absorption spectroscopy. All analyses were done at least in triplicate.
2.1. Cheese manufacture
2.3. Melt profile analysis
Standard full-fat Cheddar cheese was made at the University of Wisconsin-Madison in 20 L mini-cheese vats that included a computer-controlled cooking program and an automatic variable speed agitator. Five cheese trials (yielding a total of eight cheese vats) were carried out over a period of 1 month. Whole milk was pasteurized at 73 1C for 15 s and was cooled to 4 1C. The milk was heated in the cheese vats to 32.2 1C and inoculated with a mixed starter culture consisting of Lactococcus lactis ssp. cremoris and L. lactis ssp. lactis (DVS 850, Chr. Hansen, Milwaukee, WI) at the rate of 0.2 g kg1 of milk. After a 55-min ripening period, 0.125 mL of calcium chloride kg1 and, 5 min later, 0.072 mL of double strength chymosin kg1 (ChymostarTM, Rhodia, Madison, WI) were added to the milk. The coagulum was cut with 0.63 cm knives, allowed to heal for 5 min, and then gently (manually) agitated for 10 min before cooking. The curd/whey mixture was heated from 32.2 to 38.3 1C over a period of 30 min. The whey was drained after holding for 30 min at the cooking temperature (draining pH 6.2). The curd
Melt profile analysis (MPA) was performed using a UW Melt Profiler according to the procedure described by Muthukumarappan, Wang, and Gunasekaran (1999). Cheese samples were sliced to 7 mm thick and 30 mm diameter disks. Samples were stored in a plastic bag in the refrigerator at 6 1C for at least 3 h before testing. The top and bottom plates were sprayed with a dry film lubricant to prevent the samples from sticking to the plates. A layer of mineral oil was applied to prevent dehydration of the samples during heating. A thermocouple was inserted into the centre of the sample and the cheese disk was placed on the bottom stainless steel plate in an oven maintained at 72 1C (750F Forced Air Oven, Fisher Scientific, Pittsburgh, PA). The top aluminium plate was connected to a linear variable differential transformer and lowered on to the top of the sample. The change in height as a function of time/ sample temperature was measured until the sample temperature reached 63 1C. The cheese height versus time curve has been divided into three regions by Muthukumarappan et al. (1999): an initial slow
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Cheese temperature (°C)
deformation region (softening), a rapid linear deformation region (flow), and a slow linear deformation region (complete melt). The softening temperature corresponds to the point of intersection of tangents drawn at the first two linear regions. The degree of flow (DOF) was calculated as the change in height of the cheese sample at 60 1C as compared with the cheese height at the beginning of the test. The cheese samples were analyzed at 1, 2, 4, and 12 weeks of ripening.
647
60 50 40 30 20 10 0 0
200
400
600
800
1000
1200
Heating time (s)
2.4. Dynamic small amplitude oscillatory rheometry A Paar Physica universal dynamic spectrometer (UDS 200 Physica Messtechnik, D-70567, Stuttgart, Germany) was used to study the viscoelastic properties of the cheese. The dynamic small amplitude oscillatory rheometry (SAOR) technique was used. The parameters studied were G0 , loss modulus (G00 ), and LT, which is the ratio between the viscous properties and the elastic properties of the material (LT ¼ G00 /G0 ). Cheese samples were sliced into disks of 2.2 mm thickness and 50 mm diameter. The slices were stored in a plastic bag in the refrigerator at 6 1C for at least 3 h before testing. The rheometer was fitted with a 50-mm diameter serrated parallel plate. Samples were mounted and glued on to the bottom heating (peltier) plate of the rheometer using cyanoacrylate glue. Use of the glue and the serrated plate prevented slippage of the sample (Nolan et al., 1989; Tunick et al., 1990). The exposed surface of the sample was covered with a thin layer of vegetable oil to prevent it drying out. A frequency of 0.1 Hz and a strain of 0.2% (which was within the linear viscoelastic region of our cheese samples) were applied. During loading, the normal force readings were kept at p1.0 N to have good contact between the serrated plate and the cheese sample without excessive deformation of the sample; data acquisition was started only once a relatively constant normal force reading of 0.7 N was obtained (i.e., a low and relatively stable normal force after relaxation of the stress applied during loading). Two types of heating profile were used to study the rheological properties of the cheese samples: a long heating profile (LHP) and a short heating profile (SHP). In the LHP, the cheese samples were heated at a constant rate of 1 1C min1 from 5 to 80 1C. In the SHP, a non-linear heating rate was applied (as shown in Fig. 1) to simulate the heating profile experienced by the cheese samples in the UW Melt Profiler. This was achieved by dividing the temperature profile into eight individual heating regions and applying different heating rates in each, and the rheometer combined all these intervals in a single continuous test. G0 , G00 , and LT were measured as a function of temperature for both the LHP and the SHP tests. The cheese samples were analyzed at time points of 3 days and 1, 2, 3, and 9 months.
Fig. 1. Heating conditions used in the short heating profile of the dynamic small amplitude oscillatory rheology test, which had a similar heating profile to that experienced by the cheese in the UW Melt Profiler test.
2.5. Statistical analysis The data were analyzed using the Statistical Analysis System (SAS, 1999). Pearson’s correlation coefficients between the various responses (i.e., INSOL Ca, pH 4.6 SN, rheological parameters, and DOF) were estimated. The partial correlation coefficient is defined as the correlation between two variables that remains after adjusting for (e.g., partialling out or controlling for) one or more other variables (Steel & Torrie, 1980). It was expected that both INSOL Ca and pH 4.6 SN would change during ripening. Partial correlation coefficients were estimated for the effects of INSOL Ca on the rheological properties when the effects of pH 4.6 SN were adjusted for and vice versa by carrying out multivariate analysis of variance (MANOVA) in SAS. A Pp0:05 was considered to be statistically significant.
3. Results The cheese pH increased slightly during ripening (Table 1). The moisture content was slightly lower than for most typical Cheddar cheese made in the US. The rest of the composition was typical for Cheddar cheese (Table 1). The changes in G0 and the LT with ripening time and heating temperature are shown in Fig. 2. During heating, G0 decreased greatly and the differences between cheeses of different ages increased at temperatures X40 1C (Fig. 2a). At lower temperatures (5–35 1C), the LT values were similar at all ripening times (Fig. 2b). The LT curves increased at 435 1C and the maximum LT (LTmax) tended to occur at lower temperature with increasing ripening time. The G0 value determined at 5 1C (GT ¼ 5 1C) increased during ripening (Fig. 3). In contrast, the G0 values determined at 40 1C (GT ¼ 40 1C) and 80 1C (GT ¼ 80 1C) decreased during ripening and there was a large decrease during the first 60 days
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Average7standard deviation (n ¼ 5)
a
35.470.73 25.071.05 33.270.44 53.070.35 1.770.04 4.770.11 5.1670.07 5.1770.04 5.1570.04 5.2170.06 5.2770.06 769726 72.071.18 62.470.86 59.772.78 58.472.58 57.471.17
Storage modulus (Pa)
8.0e+4 1000
7.0e+4 6.0e+4 5.0e+4
100 0
50
100 150 200 Ripening time (days)
300 250 200 150 100 50 0
250
Fig. 3. Changes in the storage modulus as a function of ripening time for cheese tested at 5 (K), 40 (’), and 80 1C (m), obtained from the long heating profile of the small amplitude oscillatory rheology test. The data represent the means (n ¼ 8) and the error bars represent the standard deviations for each time point.
d ¼ days; m ¼ months. Insoluble Ca as a % of the total Ca in the cheese.
(a)
1e+4
80 75 70 65 60 55 50 0 0
50
100
150
200
250
Ripening time (days) 1e+3
Fig. 4. Changes in the temperature at the maximum loss tangent (K) as a function of the ripening time, obtained from the long heating profile of the small amplitude oscillatory rheology test. The data represent the means (n ¼ 8) and the error bars represent the standard deviations for each time point.
1e+2
1e+1
(b)
2.5
2.0
Loss tangent
9.0e+4
4.0e+4 0.0
b
1e+5
10000
1.0e+5
Temperature at maximum loss tangent (˚C)
Moisture content (%) Protein (%) Fat (%) Moisture in non-fat solids (%) Salt (%) Salt-in-moisture (%) pH at 3 da pH at 1 m pH at 2 m pH at 3 m pH at 9 m Total Ca (mg 100 g1) INSOL Cab at 3 d INSOL Ca at 1 m INSOL Ca at 2 m INSOL Ca at 3 m INSOL Ca at 9 m
Storage modulus at 5ºC (Pa)
1.1e+5
Storage modulus at 80ºC (Pa)
Table 1 Composition of Cheddar cheese
Storage modulus at 40ºC (Pa)
648
1.5
1.0
0.5
0.0 0
10
20
30
40
50
60
70
80
Heating temperature (ºC) Fig. 2. Changes in the storage modulus (a) and the loss tangent (b) as a function of temperature for cheese ripened for 3 days (K), 1 month (J), 2 months (.), 3 months (,), and 9 months (’), obtained from the long heating profile of the small amplitude oscillatory rheology test. The data represent the means (n ¼ 8) and the error bars represent the standard deviations for each time point.
(Fig. 3). During heating, the LTmax occurred at 70 1C in young cheese but decreased during ripening to 62 1C by 9 months (Fig. 4). The LTmax values increased substantially during the first 3 weeks from 1.3 at 3 days to 2.3 by the end of 3 weeks, and then showed little change or a slight increase for the rest of the ripening period (Fig. 5). This agrees with the trend for an increase in the LT during Cheddar cheese ripening reported by Lucey et al. (2003). The value of the LT during heating has been used as an index of the ‘‘meltability’’ of cheese (Ustunol et al., 1994). During maturation, the proportion of soluble Ca in the cheese increased from 25 to 44% between 3 days and 9 months; the majority of the increase occurred during the first 30 days (Fig. 5). This agrees with the trends reported recently by Hassan et al. (2004) for Cheddar cheese. The changes in the LTmax very closely paralleled the changes in the proportion of soluble Ca (Fig. 5). In contrast, the changes in proteolysis (pH 4.6 SN) increased at a slower
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Maximum loss tangent
18 2.5
16 14
2.0
12 1.5
10 8
1.0
6 4
0.5
2 0.0
0 0
50
100
150
200
250
Ripening time (days)
50 45 40 35 30 25 20 15 10 5 0
SOL Ca (% of total Ca in cheese)
3.0
pH 4.6 Soluble nitrogen (% of total nitrogen)
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Fig. 5. Changes in the soluble Ca content (% of total Ca in the cheese) (K), the maximum loss tangent (J) and pH 4.6 soluble nitrogen (as a % of total nitrogen) (m). The data represent the means (n ¼ 8) and the error bars represent the standard deviations for each time point.
90 85
45 80 40
75 70
35 65 30 0
Degree of flow at 60˚C (%)
Softening temperature (˚C)
50
60 0 0
50
100
150
200
250
Ripening time (days) Fig. 6. Changes in the degree of flow (&) and the softening temperature (1C) (’) of cheese as a function of the ripening time, obtained from the UW Melt Profiler test. The data represent the means (n ¼ 8) and the error bars represent the standard deviations for each time point.
rate during the first 90 days and continued to increase during the whole ripening period (Fig. 5). The melting properties of Cheddar cheese obtained from the UW Melt Profiler changed greatly in the first month, with a rapid increase in the DOF, after which there was little further change (Fig. 6). The temperature at which there is a decrease in the cheese height caused by flow corresponds to the softening temperature (Muthukumarappan et al., 1999). The softening temperature first decreased with ripening time (up to 2 months) and then increased from 2 to 9 months of ripening time (Fig. 6). The softening temperature did not seem to be highly correlated with changes in melting behaviour, as there were no clear trends in the softening point with ripening time. As most of the changes in the DOF occurred during the first 12 weeks of ripening, the melt properties of Cheddar cheese from the UW Melt Profiler at 1, 2, 4,
649
and 12 weeks of age were compared with the LT from the SHP of the rheometer (Fig. 7). The cheese height versus cheese temperature curves from the UW Melt Profiler and the LT versus cheese temperature curves (SHP) are shown in Fig. 7. The temperature at which there was an increase in the LT value was similar to the temperature at which there was a decrease in cheese height at all stages of ripening. Both the LT values and the cheese height curves showed similar trends. This suggests that the increase in the LT (i.e., increased viscous contribution or more fluid-like character) coincides with the flow of the cheese during heating. With an increase in the age of the cheese, the shapes of the curves were generally similar, but not identical. At 4 (Fig. 7c) and 12 weeks (Fig. 7d) of ripening, the LT value decreased at around 60 1C. This occurred because the LTmax occurred at a lower temperature in older cheeses (Fig. 4). It should be noted that, in the UW Melt Profiler, the pressure (load) from the top plate caused the cheese to flow when it reached X40 1C whereas, in the SAOR test, a constant gap was maintained for the entire heating step and no flow was observed. Pearson’s correlation coefficients between the rheological data, proteolysis, and INSOL Ca content are given in Table 2. Highly significant positive correlations (Po0:001; rX0:70) were found for INSOL Ca with the temperature at the point of the LTmax, GT ¼ 40 1C, and GT ¼ 80 1C. INSOL Ca was weakly negatively correlated with GT ¼ 5 1C (Po0:05; r ¼ 0:50) and was not correlated with the value of the LT at 8 1C. Highly significant negative correlations (Po0:001; rX 0:92) were found for INSOL Ca with LTmax and DOF. INSOL Ca was significantly negatively correlated (Po0:001; r ¼ 0:79) with pH 4.6 SN. Highly significant negative correlations (Po0:001; rX 0:74) were found for pH 4.6 SN with the temperature at the point of the LTmax, GT ¼ 40 1C, and GT ¼ 80 1C. Highly significant positive correlations (Po0:01; rX0:63) were found for pH 4.6 SN with LTmax, GT ¼ 5 1C, and DOF. The DOF was significantly positively correlated (Po0:001; r40:70) with pH 4.6 SN and LTmax and was negatively correlated with INSOL Ca, the temperature at the point of the LTmax, GT ¼ 40 1C, and GT ¼ 80 1C. Partial correlation analysis (Steel & Torrie, 1980) was performed to separate the effects of the INSOL Ca content from concomitant proteolysis changes during ripening (because they were significantly correlated). When the effect of pH 4.6 SN was excluded, partial correlation analysis indicated that the INSOL Ca content was still very significantly correlated (Po0:001; rX0:80) with the rheological properties, such as LTmax, GT ¼ 40 1C, and GT ¼ 80 1C, and the DOF (Table 3). In contrast, when the effect of INSOL Ca was excluded from the partial correlation analysis, it was found that pH 4.6 SN was only weakly correlated with a single rheological parameter, the temperature at the point
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650
120
0.0
(b)
0.5
100
1.0
80
1.5
60
2.0
40
2.5
20
3.0
0
0.0
120
(c)
0.5
(d)
100
1.0
80
1.5
60
2.0
40
2.5
20
3.0
Cheese height as a % of original height
Loss tangent
(a)
0 0 20 25 30 35 40 45 50 55 60 65 0 20 25 30 35 40 45 50 55 60 65
Cheese temperature (˚C) Fig. 7. Changes in the loss tangent (&) and the cheese height (as a % of the original height) (’), as a function of the cheese temperature during the heating conditions used in the short heating profile of the dynamic small amplitude oscillatory rheology test, which had a similar heating profile to that experienced in the UW Melt Profiler test, for cheese at (a) 1, (b) 2, (c) 4, and (d) 12 weeks of ripening.
Table 2 Pearson’s correlation coefficients between different parameters during cheese ripening
INSOL Ca pH 4.6 SN LTmax Temp. at LTmax G5 1C G40 1C G80 1C
pH 4.6 SN
LTmax
Temp. at LTmax
G5 1C
G40 1C
G80 1C
0.79***
0.92*** 0.76***
0.70*** 0.79*** 0.55**
0.50* 0.63**
0.93*** 0.82*** 0.91*** 0.63** 0.45*
0.92*** 0.74*** 0.93*** 0.65***
0.47*
LT (8 1C)
0.45* 0.55**
0.88***
DOF 0.94*** 0.81*** 0.93*** 0.75*** 0.47* 0.91*** 0.95***
***Po0:001; **Po0:01; *Po0:05:
of the LTmax (Po0:05; r ¼ 0:53) (Table 4). Partial correlation coefficients indicated that the INSOL Ca content was a more important factor than pH 4.6 SN in determining the changes in the rheological properties of Cheddar cheese during maturation.
4. Discussion It is well recognized that, during ageing, Cheddar cheese changes from the ‘‘green’’ initial texture of fresh curd to a smoother and crumblier texture when mature. There have been a number of recent reviews on cheese texture
(Prentice, Langley, & Marshall, 1999; Fox, Guinee, Cogan, & McSweeney, 2000; Gunasekeran & Ak, 2002; Foegeding, Brown, Drake, & Daubert, 2003; Lucey et al., 2003). It is widely believed that proteolysis is responsible for these textural changes in Cheddar cheese (Creamer & Olson, 1982; Fox et al., 2000). For example, the rapid cleavage of as1-casein at the Phe23–Phe24 bond by the coagulant during the first 30 days is considered to be responsible for the initial softening of Cheddar cheese texture (Creamer & Olson, 1982; Fox et al., 2000). In cheeses with a surface rind (e.g., Gouda), moisture loss during maturation results in an increase in firmness (Visser, 1991).
ARTICLE IN PRESS J.A. Lucey et al. / International Dairy Journal 15 (2005) 645–653 Table 3 Partial correlation coefficients between different parameters after controlling for the effect of pH 4.6 soluble nitrogen during cheese ripening
INSOL Ca LTmax G40 1C G80 1C
LTmax
G40 1C
G80 1C
DOF
0.80***
0.80*** 0.83***
0.82*** 0.96*** 0.81***
0.84*** 0.85*** 0.89*** 0.90***
***Po0.001; **Po0.01; *Po0.05.
Table 4 Partial correlation coefficients between different parameters after controlling for the effect of insoluble Ca during cheese ripening
DOF LTmax pH 4.6 SN
LTmax
G40 1C
G80 1C
0.56*
0.71** 0.52*
0.66** 0.89***
Temp. at LTmax
0.53*
***Po0.001; **Po0.01; *Po0.05.
There was an increase in the GT ¼ 5 1C value during ripening (Fig. 3). Rosenberg et al. (1995) reported that the G0 value determined at 25 1C increased with age for Cheddar cheese tested at 19, 240, and 470 days. Venugopal and Muthukumarappan (2003) reported that, in Cheddar cheeses with typical moisture and fat contents, there was little difference in the G0 values of the cheeses determined at 25 1C during ripening. In a cheese with higher moisture content (44.4%), they reported that G0 increased during ripening (Venugopal & Muthukumarappan, 2003). Watkinson et al. (1997) reported that the stiffness or modulus of deformability (MD), which is defined as the slope of the stress–strain curve at the initial, linear low strain region, increased during Cheddar cheese maturation. Watkinson et al. (2001) also reported a similar increase in the MD with the maturation of model cheese (there was no change in moisture, as the cheeses were sealed in polyethylene bags, and very little change in pH). An increase in the initial slope of the force–compression curve with age has also been reported in other studies (Creamer & Olson, 1982). It should be noted that the complex modulus (G*) is the ratio of stress to strain in the linear region (Foegeding et al., 2003) for dynamic oscillatory tests; the differences between G* and MD are the strain, rate of deformation, and frequency used in their determination. G0 is derived from G*, i.e., G0 ¼ G* cos (d), where d is the phase angle (Foegeding et al., 2003). One mechanism that could be involved in proteolysis-related texture changes is the formation of two new ionic groups as each peptide bond is cleaved; these ionic groups compete
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for available water in the cheese and less water is available for solution of the peptides (Creamer & Olson, 1982). It is presumed that this makes the matrix firmer and that this process occurs gradually during ripening. There was an increase in the G0 of Cheddar cheese with a decrease in the measuring temperature. A similar trend of increasing G0 with a decrease in the measuring temperature has been observed in various types of milk gel (Lucey, 2002). The likely cause of the increase in G0 of casein networks at low measuring temperature is the loosening of casein particles, as hydrophobic interactions are weak and the increased contact area between casein particles leads to increased casein–casein interaction, e.g., hydrogen bonding and electrostatic interactions (Lucey, 2002). At low temperature, milk fat is mostly solid, which could also contribute to the increase in the G0 of the cheese. During the maturation of Cheddar cheese, it is likely that both proteolysis and loss of INSOL Ca increase the flexibility of the casein particles at low temperatures (but only weak correlations between INSOL Ca or pH 4.6 SN and GT ¼ 5 1C were obtained; Table 2). The increase in the contact area between casein particles should then result in an increase in GT ¼ 5 1C, as was observed in our study (Fig. 3). However, the loss of cross-linking material (loss of INSOL Ca and proteolysis) may have the direct effect of weakening the matrix. At higher temperature, the strengthening of the hydrophobic interactions causes the casein particles to shrink and the loss of crosslinking material due to solubilization of some INSOL Ca probably caused more of the reduction in the high temperature G0 values during maturation because it was significantly correlated with LTmax, G40 1C, and GT ¼ 80 1C, and the DOF (Table 3). The loss of intact casein (i.e., structure-forming protein) was less important as it was not significantly correlated with LTmax, GT ¼ 40 1C, and GT ¼ 80 1C, and the DOF when the changes in INSOL Ca were statistically controlled (Table 4). This increase in G0 at low temperatures could be an important factor in explaining some conflicting results of texture tests (which are often conducted at 5 1C and nearly all are tested at p20 1C). Hort and Le Grys (2001) studied an individual Cheddar block during ageing (from 8 to 64 weeks) and observed that, during the maturation process, there was a period during which hardness (derived from both sensory tests and instrumental tests) and stress at fracture increased compared with young (8 weeks) cheese, which had lower hardness and stress at fracture than ‘‘green’’ cheese (1 day after manufacture). A number of other studies (e.g., Piggott & Mowat, 1991; Roberts & Vickers, 1994) have investigated the relationship between Cheddar cheese texture and ageing and most have concluded that the textural attributes are not significantly correlated with age.
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The increase in the LTmax during ripening occurred mainly in the first 4 weeks and then hardly changed (Fig. 5). The LT did not increase until X40 1C, at which point milk fat is completely liquid; therefore, the increase in the LT is due to a modification in protein interactions and not directly to the melting of milk fat. The value of the LT at a high temperature has been correlated with cheese meltability (Ustunol et al., 1994; Mounsey & O’Riordan, 1999). The DOF was significantly correlated (Po0:001; r ¼ 0:85) with the LTmax (Table 3). The temperature at which the LTmax occurred continued to decrease during maturation, but most changes occurred in the first 100 days (Fig. 4). The lower temperature of the LTmax suggested that less thermal energy needed to be given to aged cheese in order for it to reach the state at which it was most fluid-like (or most meltable). Probably, both the loss of intact casein (due to proteolysis) and the loss of cross-linking material (due to the solubilization of some INSOL Ca) are responsible for this reduction in the temperature of the LTmax during maturation, but pH 4.6 SN was more significantly correlated with this change (Table 4). Changes in the value of the LTmax closely followed the trend for the increase in soluble Ca during maturation but not the trend for the production of pH 4.6 SN (Fig. 5). This suggests that solubilization of some INSOL Ca was mostly responsible for the increase in the LTmax. This was also supported by our partial correlation analysis (Tables 3 and 4). It is also possible that some specific proteolytic event, such as the rate of hydrolysis of as1-casein at the Phe23–Phe24 bond by chymosin (which occurs mostly in the first 30 days of Cheddar cheese ripening), could more closely follow the trend for the changes in the LTmax. Changes in the INSOL Ca content during ripening were significantly correlated with pH 4.6 SN (Table 2). It is known that proteolysis in Cheddar cheese releases phosphopeptides (Singh, Fox, & Healy, 1997) and this could have contributed to the decrease in casein-bound Ca. Alternatively, the solubilization of INSOL Ca could have made the caseins more susceptible to proteolysis (Fox, 1970), and thus the loss of INSOL Ca could have contributed to the increase in proteolysis with age.
5. Conclusions During maturation, the proportion of soluble Ca in Cheddar cheese increased, with the majority of the increase occurring during the first 60 days. The changes in the Ca equilibrium were highly correlated with the changes in the rheological properties, especially the increase in the LT and the decrease in G0 at high temperatures. The INSOL Ca content was significantly correlated with the rheological properties of Cheddar cheese measured at high temperatures, similar to those
occurring during melting applications (e.g. DOF). Partial correlation analysis suggested that the changes in the INSOL Ca content during maturation were more significantly correlated with the rheological and melting properties than was the formation of pH 4.6 SN. During maturation, there was a slight increase in G0 at low temperatures, possibly due to greater swelling of the matrix when the hydrophobic interactions were weak. Specific proteolytic events could still be important for cheese textural changes, but these effects need to be separated from the effects of changes in the Ca equilibrium.
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