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The Effect of Milk Fat, the Ratio of Casein to Water, and Temperature on the Viscoelastic Properties of Rennet Casein Gels
DAIRY FOODS The Effect of Milk Fat, the Ratio of Casein to Water, and Temperature on the Viscoelastic Properties of Rennet Casein Gels N. ZHOU1 and S. J. MULVANEY Department of Food Science, Cornell University, Ithaca, NY 14853
ABSTRACT The objective of this study was to evaluate the effects of milk fat, the ratio of casein to water, and temperature on the viscoelastic properties of a model system for cheese based on dried rennet casein power. Casein gels were formed by mixing rennet casein, butter (as a source of milk fat), water, and some minor ingredients at 0.89, 0.58, and 0.38 ratios of casein to water; milk fat was added at 0, 12, and 24% at each ratio. Strain sweep, frequency sweep, melting behavior, and stress relaxation were used to characterize the viscoelastic properties of casein gels. The addition of milk fat reduced the linear viscoelastic region of casein gels. The complex modulus increased as milk fat content increased at temperatures ≤20°C, decreased at temperatures ≥30°C, and increased at all temperatures as the ratio of casein to water increased. Any particular value for the complex modulus could be obtained by a number of different combinations of the milk fat content, the ratio of casein to water, and temperature. The casein gels showed three distinctive zones of viscoelastic behavior over a temperature range of 5 to 70°C: rubbery solid, transition, and melt. The melting temperature and relaxation time of the casein gels increased as the ratio of casein to water increased; milk fat content had less effect on these gel properties. The casein matrix alone (0% added milk fat) showed a gradual softening with temperature starting at about 10°C. Thus, these composite gels represent a rather unique system by which the dispersed filler phase (milk fat) melts over the same temperature range as the underlying polymer matrix dissociates. ( Key words: casein, rheology, viscoelastic properties, cheese) Abbreviation key: C:W = ratio of casein to water, G* = complex modulus (subscript is = power law
Received September 22, 1997. Accepted June 3, 1998. 1Nabisco Food Group, East Hanover, NJ 07936-1944. 1998 J Dairy Sci 81:2561–2571
value at 1 Hz), G′ = elastic modulus, G′′ = loss modulus, MF = milk fat, n* = power law exponent, t50 = time required for initial stress in stress relaxation to relax 50%. INTRODUCTION The development of high quality products with reduced fat is a high priority for the food industry because of consumer demand. Both natural and process cheeses are popular dairy foods that contain enough milk fat ( MF) to make them a significant source of dietary fat. Therefore, the ability to reduce the fat content of cheeses is important to the dairy industry, but the same quality has been difficult to maintain in low fat or fat-free versions of many cheeses. The production of acceptable reduced fat or fat-free cheeses is a challenge because the lipid fraction affects rheological properties, and, hence, textural and functional properties as well. Understanding the effect of lipid on the rheological properties of cheese should be a first step in formulating low fat cheeses that have rheological and physical properties that are similar to their full fat counterparts. However, the exact effect of MF content on the rheological properties of cheeses is difficult to determine from published studies because of differences in composition, manufacturing, aging, temperature of measurement (which would affect both the casein matrix and the solid fat content of the lipid component), and the use of procedures for both small-strain and large-strain rheological testing. Also, the moisture content and the ratio of protein to moisture of cheese curd apparently changes with a reduction in fat content of the cheese milk, making it difficult to separate the effects of lipid and from the effects of moisture content on the rheological properties of cheese obtained from curds (5, 15). Thus, there are certain advantages to using model systems to isolate the effect of MF only on the rheological properties of casein-based products without the confounding secondary effects caused by other factors, such as the ratio of protein to moisture, thermomechanical treat-
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ment, and time and temperature history. Thus, much of the published literature regarding the rheological properties of cheese or models thereof can be roughly categorized according to preparation method, either separation of curds and whey (with or without the use of starter culture) or reconstitution of dry milk ingredients, or testing regimen, either small-strain or large-strain methodologies. However, characterization of interactions in a MFcasein system requires the use of small-strain rheological measurements made within the linear viscoelastic region. Otherwise, these short-range interactions are broken, and the rheological measurements reflect the long-range casein polymer matrix only. For example, Pagliarini and Beatrice ( 1 0 ) and Tunick and Shieh ( 1 6 ) using large-strain testing, reported that a reduction in fat content resulted in a firmer (or harder) cheese, but Ma et al. ( 6 ) , using small-strain oscillatory testing, reported that both the elastic modulus ( G′) and the loss modulus ( G′′) were lower in reduced fat Cheddar cheese at 20°C. As pointed out by Tunick and Shieh (16), large-strain texture results would be influenced primarily by the increased concentration of casein per unit volume in reduced fat cheeses. In addition, linear viscoelastic testing gives material properties that are characteristic of the material only (not its size or shape) and are directly related to underlying molecular structures. Model systems based on milk or casein powders have been used in place of cheeses to determine the effect of lipid (10); lipid content and moisture in solids-not-fat ( 7 ) ; and calcium, fat, and total solids ( 1 4 ) on the rheological properties of systems based on casein. Model systems allow for more control of the initial composition of the milk (if curd is to be formed), overall final composition, and hydrolysis of casein after cheese making because the use of starter culture can be avoided. The disadvantages of using fluid milk as the basis for a cheese model is that the curd-forming process makes it difficult to control independently the lipid content, total solids content, and the ratio of protein to water in the final product. Also, the use of skim milk powders may introduce other interference factors; for example, lactose and whey protein both reduce the rate of gelation and the gel strength of rennet casein micelle systems (11). Therefore, the objectives of this study were to determine the effect of MF content, the ratio of casein to water ( C:W) , and temperature on the linear viscoelastic properties of casein gels that were obtained by reconstitution of dried rennet casein powder with butter (as a source of MF) and water. This type of model system was chosen so that both the MF content and Journal of Dairy Science Vol. 81, No. 10, 1998
TABLE 1. Composition of casein gels expressed as the ratio of casein to water (C:W) and the actual ratio of C:W:milk fat ( M F ) in each gel. C:W:MF1
C:W (g/g) 0.89 0.58 0.38
45.2:50.8:0 35.2:60.8:0 26.4:69.6:0
(g/100 g ) 39.6:44.4:12 30.8:53.2:12 23.0:61.0:12
33.9:38.1:24 26.4:45.6:24 19.8:52.2:24
1The other 4 g/100 g of each gel consisted of sodium chloride, sodium citrate, citric acid, and disodium phosphate.
C:W could be independent experimental variables. Although the formulation and method of preparation of these model gels do not result in any one particular type of cheese, they should provide C:W of MF systems that represent the linear viscoelastic properties of cheese in general. Relatively few studies have determined the effect of higher temperatures on the rheological properties of cheese, even though the meltdown and functional properties of melted cheese are important. MATERIALS AND METHODS Rennet casein powder (Alaren 771, 30 mesh) was obtained from New Zealand Milk Products, Inc. (Santa Rosa, CA). Unsalted butter was purchased from a local market and used as a source of MF for this study. Nine casein gels with different MF contents (0, 12, and 24%) and C:W ratios (0.89, 0.58, and 0.38) were made by mixing rennet casein, MF, water, and minor ingredients [sodium chloride (1.80%), anhydrous citric acid (0.90%), anhydrous disodium phosphate (0.75%), and sodium citrate (0.40%)]. All ingredients were weighed on a dry matter basis, and additional water was added to obtain the desired composition. The concentrations of the salts and acid used were the same for all gels. The actual C:W:fat of the model systems are listed in Table 1. A procedure for making a Mozzarella cheese analog using this rennet casein product (supplied by New Zealand Milk Products) was followed to make the casein gels, except that MF replaced vegetable oil to simulate the textural and meltdown properties of natural and process cheeses containing MF. The recommended C:W was 0.58, and the recommended fat content was 24% for the cheese analog. In practice, C:W of 0.38 and 0.89 were found to be the lowest and highest values, respectively, that could be used to obtain gels at all three MF levels, and these C:W values were used to bracket the recommended value. Comparison with nutritional labeling of several regu-
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lar and reduced fat Mozzarella and process cheeses indicated that this range of C:W and MF contents was representative of those found in commercial products. Rennet casein and MF were heated to 95 to 100°C separately in a water bath. The total amount of water added was divided into two parts. Salts (disodium phosphate, sodium chloride, and sodium citrate) were dissolved in 85% of the water and preheated to 100°C. Citric acid was dissolved in the other 15% of the water and kept at room temperature ( ≈25°C). Rennet casein and salt solution were mixed with a spatula for 5 min in a water bath to hydrate the casein. Preheated butter was added during mixing, followed by citric acid solution; the mixture was stirred with a spatula for 15 to 20 min. Casein gels were made in 300-g batches and were stored at 4°C for 10 d before the determination of rheological properties. The gel surface was sealed with two layers of plastic film (no space between gel and films), and the beakers were covered with aluminum foil during storage. All gels were made and tested in duplicate. Rheological Properties Rheological properties were determined using a Bohlin VOR rheometer (Bohlin Instruments Inc., Cranbury, NJ) in parallel plate mode (15 mm in diameter and 2.5-mm gap). Casein gels were carefully cut into 25.4-mm diameter cylinders with a cylindrical cutter and then sliced into 2.5-mm thick discs with a specially designed slicer. The slicer consisted of two hollow cylinders (i.d. of 25.4 mm) lined up on a board next to each other; a slit about 1 mm wide was in between. The cylindrically shaped casein gels were slid into this slicer and sliced by passing a wire through the slit. A plastic cylinder (diameter of 25.4 mm) was inserted into the slicer to adjust the distance between the slit and the end of the inserted plastic cylinder. For this study, this distance was fixed at 2.5 mm to ensure that all of the discs had the same thickness. The cheese disc was glued to the surface of the lower plate. The upper serrated plate was lowered until it reached a 2.5-mm gap distance, and the sample was trimmed so that it has the same diameter as the upper plate. The exposed edge of the sample was coated with a thin layer of mineral oil to minimize moisture loss during the measurement. All samples were rested for 10 min before measurement to relax stresses introduced during the sample mounting. Strain sweeps at temperatures of 5 and 20°C were used to determine the linear viscoelastic range of the gels at these temperatures. Temperature sweeps from
Figure 1. Effect of the ratio of casein to water (open symbols = 0.89, gray symbols = 0.58, and black symbols = 0.38) and milk fat content (circles = 24%, diamonds = 12%, and triangle = 0%) on the complex modulus ( G * ) of casein gels at 5°C.
5 to 70°C at five frequencies (0.628, 3.14, 6.28, 31.4, and 62.8 rad/s) at a strain of 0.02% were used to characterize the dependence of the viscoelastic properties of casein gels on temperature and frequency. The complex modulus ( G*) , G′, and G′′, where the magnitude of G* = ( G′2 + G′′2) 0.5, were obtained during heat treatment at a rate of 2°C/min. Stress relaxation experiments at 5 and 20°C at 0.1% strain (strain rise time of 0.5 s ) were also used to characterize the longer time (low frequency) flow behavior of the samples. Microsoft Excel 6.0 (Microsoft Corp., Redmond, WA) was used for power law fitting and ANOVA. SigmaPlot® 3.01 (Jandel Corp., San Raphael, CA) was used to put the data into contour plots. RESULTS Strain Sweeps Figure 1 shows G* as a function of strain for casein gels with different C:W and MF contents at 5°C. Casein gels with no added MF did not show a decrease of G* values, regardless of C:W up to 2% strain; no disruption of the elastic network structure was indicated up to this level of strain. The decrease in linear viscoelastic region for gels containing added MF depended on the C:W, decreasing to about 0.9, 0.5, and 0.1% for C:W of 0.89, 0.58, and 0.38 respectively. The 0.5% linear viscoelastic range for casein gels with added MF at a C:W ratio of 0.58 (recommended formula for Mozzarella cheese analog) is conJournal of Dairy Science Vol. 81, No. 10, 1998
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Figure 2. Effect of the ratio of casein to water (open symbols = 0.89, gray symbols = 0.58, and black symbols = 0.38) and milk fat content (circles = 24%, diamonds = 12%, and triangle = 0%) on the complex modulus ( G * ) of casein gels at 20°C.
sistent with the data obtained for commercial Mozzarella cheeses (1, 9). At strains above these apparent elastic limits, G* decreased and appeared to approach the G* value of the underlying unfilled (no added MF) casein polymer system. This behavior was quite similar to that of other filled polymer systems, such as gluten and starch or natural rubber and carbon black, for which contacts between polymer and filler are probably involved ( 3 ) . For the unfilled gels, G* was increased from about 70 to 500 kPa as the C:W was increased from 0.38 to 0.89. Also, for each C:W, added MF (12%) increased G* in the linear viscoelastic region. Increased MF content (24%) resulted in increased G* for C:W of 0.38 and 0.89, but not for the gel with a C:W of 0.58. It is also interesting to compare results for the two samples: C:W = 0.38 and MF = 24% or C:W = 0.58 and MF = 0%. For these two gels, G* is essentially equivalent at low strains ( ≤ 0.1%), but the elastic strain limit and, hence, the apparent yield stress [critical stain × G*] are quite different. Thus, these casein gels cannot be considered equivalent based on G* values for the linear viscoelastic region only, as critical strain is also an important material property. Differences in the underlying long-range structures of the casein network only become apparent at larger strains or longer times, as is discussed in the section on stress relaxation. The strain dependence of G* at 20°C and at 5°C were similar in the linear viscoelastic range (Figure 2). Unfilled gels did not show a decrease of G* values up to 2% strain for all C:W ratios. The addition of MF Journal of Dairy Science Vol. 81, No. 10, 1998
to casein gels decreased their linear viscoelastic region to about 0.7, 0.3, and 0.1% for gels with C:W of 0.89, 0.58, and 0.38, respectively. Compared with the results for 5°C, the linear viscoelastic region was decreased more at 20°C with added MF for each C:W, which suggests that MF in liquid form is a less effective reinforcer of the casein network. The MF is about 75% solid at 5°C and about 20% solid at 20°C (13). The G* value of unfilled gels increased from 50 to 400 kPa as C:W increased at 20°C. However, added MF decreased G* at each C:W, which was opposite to the effect of MF on G* at 5°C. These opposite effects could again be explained by the phase change of MF from mostly solid to mostly liquid in this temperature range. Chronakis and Kasapis ( 2 ) also reported a 90% decrease in G′ of spreadable butter from 5 to 20°C, presumably from melting. Studies of cheese microstructure have shown that MF may exist as fat globules in cheese (7, 15, 16). Thus, solid fat globules appear to reinforce the casein gel structure via shortrange interactions with the casein network. Dynamic Mechanical Thermal Analysis Dynamic mechanical thermal analysis was used to obtain information on the effects of temperature and frequency on the viscoelastic properties of the casein gels. Figure 3 shows a representative result for the casein gel with a C:W of 0.58 and MF content of 24%.
Figure 3. Effect of frequency (Hertz) and temperature (O: 5°C, »: 10°C, ◊: 20°C, ♦: 30°C, ∫: 40°C, and o: 50°C ) on the complex modulus ( G * ) for a casein gel with 24% milk fat and a ratio of casein to water of 0.58.
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VISCOELASTIC PROPERTIES OF CASEIN GELS TABLE 2. Power law characterization1 obtained from frequency sweeps for each casein gel. Ratio of casein to water (g/g) 0.89 Milk fat content
Temperature ( °C )
0%
5d 10c 20† 30† 40b 50a
12%
5d 10c 20x 30x 40b 50a 5d 10c 20x 30x 40b 50a
24%
A,B,CThe
*A
0.58 *B
0.38 *C
G0
n*
G0
n*
G0
n*
384.34 375.85 305.65 234.16 165.18 72.33 495.04 481.38 327.12 183.60 112.78 42.01 605.28 589.78 344.89 146.25 84.51 28.97
0.185 0.186 0.198 0.206 0.231 0.302 0.180 0.183 0.203 0.230 0.256 0.339 0.176 0.177 0.199 0.237 0.261 0.362
114.09 106.69 90.70 63.67 41.10 17.83 180.13 169.83 121.11 59.90 34.65 11.65 212.10 198.61 129.08 50.88 24.92 6.68
0.174 0.192 0.206 0.232 0.272 0.362 0.179 0.189 0.199 0.241 0.289 0.389 0.172 0.184 0.196 0.236 0.278 0.406
46.03 43.09 34.93 22.08 13.08 4.84 65.00 61.42 38.18 17.55 9.78 3.31 110.01 104.07 49.92 15.51 8.43 3.10
0.189 0.203 0.219 0.268 0.325 0.416 0.187 0.196 0.219 0.274 0.299 0.366 0.168 0.176 0.198 0.277 0.294 0.269
G*0 at each ratio of casein to water with the same temperature differ ( P < 0.01).
each milk fat content, G*0 at the same temperature with no common superscript letter differ ( P < 0.01). a,b,c,dAt
xThe 1G*
0
G*0 of gels at these temperatures with different milk fat content do not differ ( P > 0.05).
= Complex modulus (kilopascals) at a frequency of 1 Hz; n* = power low exponent (slope).
All gels showed similar trends. Given the nearly linear appearance of the G* versus the frequency data on logarithmic coordinates, a power law model was used to characterize the frequency dependence of G* over this limited frequency range for the gels as follows: G* = G*0 fn* where G*0 = model value of G* at a frequency of 1 Hz, f = frequency in Hertz, and n* = the power law exponent. The G*0 is indicative of the overall stiffness, or resistance to deformation, within the linear viscoelastic region of the gels at a frequency of 1 Hz. This value may be considered as the firmness of the gels when they were subjected to a fairly rapid deformation, such as depressing the gel quickly with one’s thumb and releasing. The n* may be taken as a convenient measure of the relative degree of viscoelasticity of the gels. It would be expected that n* would approach zero for ideal elastic behavior (i.e., no frequency dependence) and would increase as the degree of viscoelasticity increased.
Values of the power law parameters, G*0 and n*, at different temperatures for each casein gel are shown in Table 2. The G*0 values were significantly increased by added MF at temperatures of 5 and 10°C and decreased ( P < 0.01) by MF content at temperatures of 40 and 50°C. At temperatures of 20 and 30°C, however, MF had no ( P > 0.05) effect on G*0 values. A decrease in the C:W ratio of the gel decreased ( P < 0.01) the G*0 values at all temperatures. The ANOVA also showed that the effect of C:W ratio and MF on G*0 did not interact at temperatures of 5, 10, 20, and 30°C but did interact ( P < 0.05) at temperatures of 40 and 50°C. This interaction means that the effect of MF on G*0 is dependent on C:W and vice versa, but the effect of MF and C:W on G*0 is additive in a casein network up to 30°C. The MF probably acts as a plasticizer (softener) of the casein melt at temperatures above 40°C, which is consistent with the results of Marshall ( 7 ) for stiffness of process cheese analogs at 21°C. Contour plots of the change of G*0 with C:W ratio and MF content (Figure 4 ) showed that the effect of Journal of Dairy Science Vol. 81, No. 10, 1998
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MF content on G*0 at constant C:W changed direction
ature by adjusting the composition. However, G*0
at a temperature between 20 and 30°C, increasing G*0
represents only the short-range linear viscoelastic properties of the gels and not necessarily their longrange network properties, as is discussed further in the section on stress relaxation. The n* increased in a similar manner at temperature above 10°C for each C:W, regardless of MF content (Table 2), and probably reflects the loss of physical crosslinks in the casein matrix as the temperature is increased. In general, n* appeared to be more affected by temperature at constant C:W than by C:W at constant temperature. This result is probably due to the overall high concentration of casein that is present in all of the gels (i.e., sufficiently high to
at temperatures ≤20°C and decreasing G*0 at temperatures ≥30°C. One way to interpret these results is that, for each temperature, a corresponding range of G*0 values could be achieved by varying C:W and MF contents in the experimental range. In addition, within this range, any particular value of G*0 could be obtained by a number of different gel compositions. Viewed another way, a particular G*0 value associated with a certain gel composition at one temperature can be reproduced at a somewhat higher or lower temper-
Figure 4. Complex modulus ( G*o ; kilopascals) ) at 1 Hz of casein gels as a function of the ratio of casein to water (C:W) and milk fat ( M F ) content at different temperatures. Journal of Dairy Science Vol. 81, No. 10, 1998
VISCOELASTIC PROPERTIES OF CASEIN GELS
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obtain a gel in the first place). The higher G*0 values and lower n* values at the highest C:W and lowest temperatures are logically due to an increase in the number of physical crosslinks in the gel and the presence of solid fat. The three unfilled gels of different C:W all show a progressive softening (decrease in G*0) as the temperature is increased above 5°C, particularly above 10°C. Thus, the softening and meltdown properties of these gels involve the liquefaction of MF superimposed over the dissociation of the underlying secondary interactions (noncovalent) of the casein network. Melting Behavior of Gels Figure 5 shows the relative contributions of elastic ( G ′) and viscous effects ( G′′) to the overall resistance to deformation of the gels as a function of temperature, C:W, and MF content. The G′ is much greater than the G′′ for all gels at lower temperatures and showed a more dramatic change than did G′′ with temperature up to 30°C, at which temperature most of the MF is liquefied. A comparison of gels with different MF contents indicated that G′ was increased at low temperatures by added MF. The values for G′ and G′′ crossed over each other ( G′′ = G′) in the temperature range of 45°C to 65°C, depending upon the C:W and MF content. The crossover temperature at which tan d ( G ′′/G′) = 1 is a convenient estimate of the melting point of the gels. Table 3 shows that the crossover temperatures for the casein gels ranged from 46 to 64°C, depending on C:W and MF content. A decrease in the C:W from 0.89 to 0.38 reduced the melting point from 64 to 50, 62 to 49, and 64 to 46°C for MF contents of 0, 12, and 24%, respectively. This decrease was much greater than that occurring as the MF content increased at constant C:W; results ranged from essentially no change for a C:W of 0.89 to 6°C for the other
TABLE 3. The effect of the ratio of casein to water (C:W) and milk fat content on the temperature ( T ) at which the loss tangent ( G′′/ G′) = 1 and the corresponding value of the elastic modulus ( G′) = the loss modulus ( G′′) at this temperature. Milk fat 0%
12%
24%
C:W
T
G′
T
G′
T
G′
0.89 0.58 0.38
( °C ) 64 62 50
(kPa) 32 9.5 6.5
( °C ) 62 59 49
(kPa) 20 8.5 4.7
( °C ) 64 56 46
(kPa) 13 6 3.8
Figure 5. Effect of temperature and the ratio of casein to water (C:W) on the elastic ( G′) and loss ( G′′) moduli of casein gels with 0, 12, and 24% added milk fat.
two C:W. These results indicate that the C:W is a critical factor in the meltdown and flow properties of these gels, regardless of the MF content. However, G′ at the crossover temperature (Table 3 ) decreased with either an increase in MF content at constant C:W ratio or a decrease in C:W at constant MF content. Because the energy stored elastically in a cyclic deformation is directly proportional to G′ (12), one might expect a melt with higher G′ to be perceived as more rubbery or tougher. In other words, more work would be needed to achieve the same deformation as G′ increases. Thus, just as the firmness at low temperature ( G * ) is affected by both C:W and MF content, so also are the material properties of the corresponding melt. For any given C:W, the change in overall firmness as given by G* with increased temperature consisted of three distinct zones of viscoelastic behavior, as is illustrated in Figure 6 for C:W of 0.58. The plateau region for G* at temperatures below 10°C (zone 1 in Figure 6 ) represents a firm and highly elastic gel where G′ >> G′′ (Figure 5). As the temperature is increased above about 10°C (zone 2 in Figure 6), the gels soften, primarily because of a decrease in G′, which means that the softening is also accompanied Journal of Dairy Science Vol. 81, No. 10, 1998
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Figure 6. The effect of temperature on the viscoelastic properties of casein gels as three distinct zones, corresponding to rubbery solid (zone 1), transition (zone 2), or melt (zone 3).
by a loss of elasticity. The liquid portion of MF in the casein network increases rapidly between 10 and 25°C (inset in Figure 6), resulting in a faster G* decrease for gels at higher MF content. When the majority of the MF is liquefied (around 20 to 30°C), G* of the filled system actually becomes lower than that of the unfilled system. At the crossover temperature (zone 3 in Figure 6), G* reached another plateau region, which represents a viscoelastic melt. It is important to recognize these distinct zones of viscoelastic behavior for a given composition as they are related to both the textural attributes of the gels and their flowability in the melt state. For example, the temperature at which tan d = 1 was found to be 56°C for the case in which C:W is 0.58 and MF is 24% (Mozzarella cheese analog). This temperature is remarkably close to the typical stretching temperature for Mozzarella cheese of 57°C.
initial stress was applied. However, at longer times, casein gels with the same C:W tended to superimpose without shifting of the time axis, indicating that added MF did not affect the relaxation times of the casein network. The one exception was the gel with a C:W of 0.38 and a MF content of 24%, which also showed strain sweep behavior similar to the gel with a C:W of 0.58 (Figure 1 ) in the linear viscoelastic region. It is possible that, at 5°C, solid fat provides additional crosslinking points for the dilute casein network. A gradual but substantial relaxation for all gels was observed at longer times. Masi and Addeo ( 8 ) have suggested that relaxation in Mozzarella cheese is due to the breakage and reformation of secondary bonds (i.e., a temporary network structure). The presence of a plateau region here at short times (high frequencies), coupled with the extensive stress relaxation, suggests that a similar mechanism applies to these rennet casein gels. The rate of stress relaxation is clearly slower at higher C:W and is not affected by MF content, except for the one aberrant gel just discussed. This evidence indicates that the important functional properties of casein-based products related to viscous or rubbery flow, such as melt and stretch of cheese, may depend more on the C:W than on the fat content. Stress relaxation at 20°C (Figure 8 ) showed trends that were qualitatively similar to those at 5°C, except that all gels with a C:W of 0.38 lacked a well-
Stress Relaxation Stress relaxation was used to determine the viscoelastic properties of the gels at longer times up to 1000 s. Figure 7 shows the stress relaxation curves for casein gels obtained at 5°C, plotted as G/G0 versus time, for which G is the modulus at any time and G0 is the initial modulus. All samples showed very similar relaxation behavior up to about 1 s after the Journal of Dairy Science Vol. 81, No. 10, 1998
Figure 7. Effect of the ratio of casein to water (C:W) (open symbols = 0.89, gray symbols = 0.58, black symbols = 0.38) and milk fat ( M F ) content (circles = 24%, diamonds = 12%, and triangle = 0%) on the stress relaxation modulus ( G ) normalized by the initial modulus ( G 0) at 5°C.
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DISCUSSION
Figure 8. Effect of the ratio of casein to water (C:W) (open symbols = 0.89, gray symbols = 0.58, and black symbols = 0.38) and milk fat ( M F ) content (circles = 24%, diamonds = 12%, and triangle = 0%) on the stress relaxation modulus ( G ) normalized by the initial modulus ( G 0) at 20°C.
defined, short-time plateau. Apparently the crosslink density is not sufficient to provide a resilient, solid gel at 20°C and a C:W of 0.38. This result is consistent with the lower G*0 at 20°C (Table 2 ) for these gels. Table 4 shows the time needed for the shear modulus to relax 50% of its original value ( t50) for casein gels at 5 and 20°C. All samples showed a t50 less than 5 s. A decrease of C:W at either experimental temperature increased the relaxation rate of casein gels. The ANOVA of t50 values showed that a decreased C:W decreased ( P < 0.01) the t50 value. However, MF did not exert a significant effect on the relaxation time. These results showed that casein gels with C:W of 0.38 relaxed much faster than did the other gels. At room temperature, these gels flowed within an hour because of the stress of their own weight (i.e., these gels did not contain the network structure required to hold their shape for a long time under the force of gravity).
Isolation of the main factors that are important to the MF-casein system, namely, the C:W, temperature, and volume (or mass) fraction of filler, made some of the unique aspects of this system apparent. The combination of the low molecular mass (for a polymer) of casein, temperature-dependent crosslinks, and the correspondingly high concentration of casein needed to obtain the sufficient number of crosslinks for solidlike behavior at low temperatures also leads to a viscoelastic melt at relatively low temperatures of around 60°C. Also, MF functions as a uniquely compatible filler of the casein-water matrix and serves a dual role. The MF is solid at low temperatures (e.g., <10°C), which corresponds to extensive crosslinking of the casein matrix, allowing it to act as a rigid filler, but liquefies over essentially the same temperature range that the casein network dissociates, thus diluting or plasticizing the viscoelastic melt as well. Our results for the effect of MF on G* and critical strain for strain sweeps and stress relaxation also support the idea that fat globules may act either as actual crosslinking points for a dilute casein network or as reinforcing filler particles in denser casein networks, as depicted and discussed by Xiong and Kinsella (17). However, in either case, the casein interactions of the MF globule were weaker than the casein network as shown by the decrease in critical strain. Because the short-time plateau region in stress relaxation was not extended at all by the presence of added MF at either 5 or 20°C (exception noted earlier) and because G*0 decreased with added MF at temperatures >30°C, added MF was not shown to form its own network at the levels used here or to affect the existing casein-water matrix significantly. This result implies that maintenance of a constant C: W as the fat content is reduced may help to retain the functional properties of full fat cheeses in lower fat versions. As MF is added to a gel of constant C:W, the volume fraction of the casein matrix is reduced, but the essential character of the remaining casein
TABLE 4. Effect of the ratio of casein to water (C:W) and milk fat ( M F ) content on the time for stress relaxation to 50% of its initial value at 5 and 20°C. 5°C
20°C
C:W
0% MF
12% MF
24% MF
0.89 0.58 0.38
4.85 3.37 2.44
4.42 3.69 2.46
4.51 3.67 3.25
0% MF
12% MF
24% MF
3.17 1.91 0.89
2.40 1.90 0.75
2.74 2.16 0.78
(s)
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matrix is retained. As discussed in the introduction, this relationship is not easily deduced from most reported results on the rheological properties of cheese (or models thereof) because the fat content and ratios of protein to water generally change as the fat level is varied. One might think of the C:W value as indicative of the crosslink density within the casein matrix at a particular temperature (all other factors equal) up to its melting point and as indicative also of the concentration of casein molecules in the casein melt above the gel melting point. The general principles of polymer science would indicate that the C:W is a critical variable for both firmness of the gels and viscosity of the corresponding melt obtained upon treatment and that it may be difficult to decouple the low temperature characteristics of the gels from their functional properties in the melt state. In addition, the effect of liquid MF on the rheological properties of the melt is further complicated by the possible partition of added MF into a fraction that is emulsified into the casein network and into a fraction of free oil, as has been discussed by Kindstedt and Rippe ( 4 ) for Mozzarella cheeses. According to those researchers, the efficiency of emulsification of fat into the casein network could depend upon the ratio of total fat to casein, which could provide an explanation as to why we observed a significant interaction between the C:W and MF level on G*0 only at 40 and 50°C. However, because we did not determine the free oil content of the casein gels obtained here, this result must be considered speculative. Overall, the results shown here for critical strain, values of the viscoelastic moduli, and stress relaxation were quite similar to those previously reported for Mozzarella cheese, even though emulsifiers were used here, and the gels were not stretched as Mozzarella would be. This similarity is probably because results here were limited to viscoelastic phenomena within the linear viscoelastic region, in which effects are dominated by the character of the casein proteins themselves and their interactions with MF. These characteristics would be expected to be similar in Mozzarella cheese, in which casein proteins predominate. However, these intermolecular interactions give rise to the macroscopic functional properties of polymeric systems. Therefore, these casein gel systems might be used to investigate the effect of other important factors, such as pH and calcium content, on the material properties of products based on casein. CONCLUSIONS This work has shown that added MF mainly affects the short-range elastic properties of the casein sysJournal of Dairy Science Vol. 81, No. 10, 1998
tem, G*0 and critical strain; the long-range network properties, as determined by stress relaxation, were primarily a function of the concentration of casein in the hydrated casein network. Thus, the casein:MF system behaves much like a typical filled polymer system, except that the material properties of the filler itself are also temperature dependent. The MF acts as a reinforcing filler at low temperatures and gradually becomes a diluting factor or plasticizer of the casein melt. The latter probably occurs above about 40°C, at which temperature the casein network is substantially dissociated with and the MF is completely liquid. Dynamic mechanical thermal analysis was found to offer a convenient means of characterizing the overall stiffness, relative elasticity, and transition from gel to melt for these rennet casein gels. Determination of linear viscoelastic properties could be used to guide the development of formulations or processes to achieve certain physical characteristics in products such as cheese that are based on caseins. REFERENCES 1 Ak, M. M., and S. Gunasekaran. 1996. Dynamic rheological properties of Mozzarella cheese during refrigerated storage. J. Food Sci. 61:566–568; 584. 2 Chronakis, I. S., and S. Kasapis. 1995. A rheological study on the application of carbohydrate-protein incompatibility to the development of low fat commercial spreads. Carbohydr. Polym. 28:367–373. 3 Ferry, J. D. 1980. Molecular theory for undiluted amorphous polymers and concentrated solutions: networks and entanglements. Pages 224–263 in Viscoelastic Properties of Polymers. 3rd ed. John Wiley & Sons, Inc., New York, NY. 4 Kindstedt, P. S., and J. K. Rippe. 1990. Rapid quantitative test for free oil (oiling off) in melted Mozzarella cheese. J. Dairy Sci. 73:867–873. 5 Lawrence, R. C., H. A. Heap, and J. Gilles. 1984. A controlled approach to cheese technology. J. Dairy Sci. 67:1632–1645. 6 Ma, L., M. A. Drake, G. V. Barbosa-Canovas, and B. G. Swanson. 1996.Viscoelastic properties of reduced-fat and full-fat Cheddar cheeses. J. Food Sci. 61:821–823. 7 Marshall, R. J. 1990. Composition, structure, rheological properties, and sensory texture of processed cheese analogues. J. Sci. Food Agric. 50:237–252. 8 Masi, P., and F. Addeo. 1986. An examination of some mechanical properties of a group of Italian cheeses and their relation to structure and conditions of manufacture. J. Food Eng. 5: 217–229. 9 Nolan, E. J., V. H. Holsinger, and J. J. Shieh. 1989. Dynamic rheological properties of natural and imitation Mozzarella cheese. J. Texture Stud. 20:179–189. 10 Pagliarini, E., and N. Beatrice. 1994. Sensory and rheological properties of low-fat filled ‘pasta filata’ cheese. J. Dairy Res. 61: 299–304. 11 Park, S.-Y., R. Niki, and K. Nakamura. 1996. Rheological behavior of casein micelles and reconstituted skim milk gels: the effects of temperature on gelation induced by rennet. Int. Food Sci. Technol. 2:103–107. 12 Rosen, 1993. Chapter XVIII. Linear Viscoelasticity. Pages 298–349 in Fundamental Principles of Polymeric Materials. 2nd ed. John Wiley & Sons, Inc., New York, NY. 13 Shukla, A., A. R. Bhaskar, S.S.H. Rizvi, and S. J. Mulvaney. 1994. Physiochemical and rheological properties of milk fat
VISCOELASTIC PROPERTIES OF CASEIN GELS made from supercritically fractionated milk fat. J. Dairy Sci. 77: 45–54. 14 Solorza, F. J., and A. E. Bell. 1995. Effect of calcium, fat and total solids on the rheology of a model soft cheese system. J. Soc. Dairy Technol. 48(4):133–139. 15 Tunick, M. H., K. L. Mackey, and J. H. Shieh, P. W. Smith, P. Cooke, and E. L. Malin. 1993. Rheology and microstructure of low-fat Mozzarella cheese. Int. Dairy J. 3:649–662.
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16 Tunick, M. H., and J. J. Shieh. 1995. Rehology of reduced-fat Mozzarella cheese. Page 7–19 in Chemistry of StructureFunction Relationships in Cheese. E. L. Malin and M. H. Tunick, ed. Plenum Press, New York, NY. 17 Xiong, Y. L., and J. E. Kinsella. 1991. Influence of fat globule membrane composition and fat type on the rheological properties of milk based composite gels. II. Results. Milchwissenschaft 46:207–212.
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ABSTRACT The objective of this study was to evaluate the effects of milk fat, the ratio of casein to water, and temperature on the viscoelastic properties of a model system for cheese based on dried rennet casein power. Casein gels were formed by mixing rennet casein, butter (as a source of milk fat), water, and some minor ingredients at 0.89, 0.58, and 0.38 ratios of casein to water; milk fat was added at 0, 12, and 24% at each ratio. Strain sweep, frequency sweep, melting behavior, and stress relaxation were used to characterize the viscoelastic properties of casein gels. The addition of milk fat reduced the linear viscoelastic region of casein gels. The complex modulus increased as milk fat content increased at temperatures ≤20°C, decreased at temperatures ≥30°C, and increased at all temperatures as the ratio of casein to water increased. Any particular value for the complex modulus could be obtained by a number of different combinations of the milk fat content, the ratio of casein to water, and temperature. The casein gels showed three distinctive zones of viscoelastic behavior over a temperature range of 5 to 70°C: rubbery solid, transition, and melt. The melting temperature and relaxation time of the casein gels increased as the ratio of casein to water increased; milk fat content had less effect on these gel properties. The casein matrix alone (0% added milk fat) showed a gradual softening with temperature starting at about 10°C. Thus, these composite gels represent a rather unique system by which the dispersed filler phase (milk fat) melts over the same temperature range as the underlying polymer matrix dissociates. ( Key words: casein, rheology, viscoelastic properties, cheese) Abbreviation key: C:W = ratio of casein to water, G* = complex modulus (subscript is = power law
Received September 22, 1997. Accepted June 3, 1998. 1Nabisco Food Group, East Hanover, NJ 07936-1944. 1998 J Dairy Sci 81:2561–2571
value at 1 Hz), G′ = elastic modulus, G′′ = loss modulus, MF = milk fat, n* = power law exponent, t50 = time required for initial stress in stress relaxation to relax 50%. INTRODUCTION The development of high quality products with reduced fat is a high priority for the food industry because of consumer demand. Both natural and process cheeses are popular dairy foods that contain enough milk fat ( MF) to make them a significant source of dietary fat. Therefore, the ability to reduce the fat content of cheeses is important to the dairy industry, but the same quality has been difficult to maintain in low fat or fat-free versions of many cheeses. The production of acceptable reduced fat or fat-free cheeses is a challenge because the lipid fraction affects rheological properties, and, hence, textural and functional properties as well. Understanding the effect of lipid on the rheological properties of cheese should be a first step in formulating low fat cheeses that have rheological and physical properties that are similar to their full fat counterparts. However, the exact effect of MF content on the rheological properties of cheeses is difficult to determine from published studies because of differences in composition, manufacturing, aging, temperature of measurement (which would affect both the casein matrix and the solid fat content of the lipid component), and the use of procedures for both small-strain and large-strain rheological testing. Also, the moisture content and the ratio of protein to moisture of cheese curd apparently changes with a reduction in fat content of the cheese milk, making it difficult to separate the effects of lipid and from the effects of moisture content on the rheological properties of cheese obtained from curds (5, 15). Thus, there are certain advantages to using model systems to isolate the effect of MF only on the rheological properties of casein-based products without the confounding secondary effects caused by other factors, such as the ratio of protein to moisture, thermomechanical treat-
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ment, and time and temperature history. Thus, much of the published literature regarding the rheological properties of cheese or models thereof can be roughly categorized according to preparation method, either separation of curds and whey (with or without the use of starter culture) or reconstitution of dry milk ingredients, or testing regimen, either small-strain or large-strain methodologies. However, characterization of interactions in a MFcasein system requires the use of small-strain rheological measurements made within the linear viscoelastic region. Otherwise, these short-range interactions are broken, and the rheological measurements reflect the long-range casein polymer matrix only. For example, Pagliarini and Beatrice ( 1 0 ) and Tunick and Shieh ( 1 6 ) using large-strain testing, reported that a reduction in fat content resulted in a firmer (or harder) cheese, but Ma et al. ( 6 ) , using small-strain oscillatory testing, reported that both the elastic modulus ( G′) and the loss modulus ( G′′) were lower in reduced fat Cheddar cheese at 20°C. As pointed out by Tunick and Shieh (16), large-strain texture results would be influenced primarily by the increased concentration of casein per unit volume in reduced fat cheeses. In addition, linear viscoelastic testing gives material properties that are characteristic of the material only (not its size or shape) and are directly related to underlying molecular structures. Model systems based on milk or casein powders have been used in place of cheeses to determine the effect of lipid (10); lipid content and moisture in solids-not-fat ( 7 ) ; and calcium, fat, and total solids ( 1 4 ) on the rheological properties of systems based on casein. Model systems allow for more control of the initial composition of the milk (if curd is to be formed), overall final composition, and hydrolysis of casein after cheese making because the use of starter culture can be avoided. The disadvantages of using fluid milk as the basis for a cheese model is that the curd-forming process makes it difficult to control independently the lipid content, total solids content, and the ratio of protein to water in the final product. Also, the use of skim milk powders may introduce other interference factors; for example, lactose and whey protein both reduce the rate of gelation and the gel strength of rennet casein micelle systems (11). Therefore, the objectives of this study were to determine the effect of MF content, the ratio of casein to water ( C:W) , and temperature on the linear viscoelastic properties of casein gels that were obtained by reconstitution of dried rennet casein powder with butter (as a source of MF) and water. This type of model system was chosen so that both the MF content and Journal of Dairy Science Vol. 81, No. 10, 1998
TABLE 1. Composition of casein gels expressed as the ratio of casein to water (C:W) and the actual ratio of C:W:milk fat ( M F ) in each gel. C:W:MF1
C:W (g/g) 0.89 0.58 0.38
45.2:50.8:0 35.2:60.8:0 26.4:69.6:0
(g/100 g ) 39.6:44.4:12 30.8:53.2:12 23.0:61.0:12
33.9:38.1:24 26.4:45.6:24 19.8:52.2:24
1The other 4 g/100 g of each gel consisted of sodium chloride, sodium citrate, citric acid, and disodium phosphate.
C:W could be independent experimental variables. Although the formulation and method of preparation of these model gels do not result in any one particular type of cheese, they should provide C:W of MF systems that represent the linear viscoelastic properties of cheese in general. Relatively few studies have determined the effect of higher temperatures on the rheological properties of cheese, even though the meltdown and functional properties of melted cheese are important. MATERIALS AND METHODS Rennet casein powder (Alaren 771, 30 mesh) was obtained from New Zealand Milk Products, Inc. (Santa Rosa, CA). Unsalted butter was purchased from a local market and used as a source of MF for this study. Nine casein gels with different MF contents (0, 12, and 24%) and C:W ratios (0.89, 0.58, and 0.38) were made by mixing rennet casein, MF, water, and minor ingredients [sodium chloride (1.80%), anhydrous citric acid (0.90%), anhydrous disodium phosphate (0.75%), and sodium citrate (0.40%)]. All ingredients were weighed on a dry matter basis, and additional water was added to obtain the desired composition. The concentrations of the salts and acid used were the same for all gels. The actual C:W:fat of the model systems are listed in Table 1. A procedure for making a Mozzarella cheese analog using this rennet casein product (supplied by New Zealand Milk Products) was followed to make the casein gels, except that MF replaced vegetable oil to simulate the textural and meltdown properties of natural and process cheeses containing MF. The recommended C:W was 0.58, and the recommended fat content was 24% for the cheese analog. In practice, C:W of 0.38 and 0.89 were found to be the lowest and highest values, respectively, that could be used to obtain gels at all three MF levels, and these C:W values were used to bracket the recommended value. Comparison with nutritional labeling of several regu-
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lar and reduced fat Mozzarella and process cheeses indicated that this range of C:W and MF contents was representative of those found in commercial products. Rennet casein and MF were heated to 95 to 100°C separately in a water bath. The total amount of water added was divided into two parts. Salts (disodium phosphate, sodium chloride, and sodium citrate) were dissolved in 85% of the water and preheated to 100°C. Citric acid was dissolved in the other 15% of the water and kept at room temperature ( ≈25°C). Rennet casein and salt solution were mixed with a spatula for 5 min in a water bath to hydrate the casein. Preheated butter was added during mixing, followed by citric acid solution; the mixture was stirred with a spatula for 15 to 20 min. Casein gels were made in 300-g batches and were stored at 4°C for 10 d before the determination of rheological properties. The gel surface was sealed with two layers of plastic film (no space between gel and films), and the beakers were covered with aluminum foil during storage. All gels were made and tested in duplicate. Rheological Properties Rheological properties were determined using a Bohlin VOR rheometer (Bohlin Instruments Inc., Cranbury, NJ) in parallel plate mode (15 mm in diameter and 2.5-mm gap). Casein gels were carefully cut into 25.4-mm diameter cylinders with a cylindrical cutter and then sliced into 2.5-mm thick discs with a specially designed slicer. The slicer consisted of two hollow cylinders (i.d. of 25.4 mm) lined up on a board next to each other; a slit about 1 mm wide was in between. The cylindrically shaped casein gels were slid into this slicer and sliced by passing a wire through the slit. A plastic cylinder (diameter of 25.4 mm) was inserted into the slicer to adjust the distance between the slit and the end of the inserted plastic cylinder. For this study, this distance was fixed at 2.5 mm to ensure that all of the discs had the same thickness. The cheese disc was glued to the surface of the lower plate. The upper serrated plate was lowered until it reached a 2.5-mm gap distance, and the sample was trimmed so that it has the same diameter as the upper plate. The exposed edge of the sample was coated with a thin layer of mineral oil to minimize moisture loss during the measurement. All samples were rested for 10 min before measurement to relax stresses introduced during the sample mounting. Strain sweeps at temperatures of 5 and 20°C were used to determine the linear viscoelastic range of the gels at these temperatures. Temperature sweeps from
Figure 1. Effect of the ratio of casein to water (open symbols = 0.89, gray symbols = 0.58, and black symbols = 0.38) and milk fat content (circles = 24%, diamonds = 12%, and triangle = 0%) on the complex modulus ( G * ) of casein gels at 5°C.
5 to 70°C at five frequencies (0.628, 3.14, 6.28, 31.4, and 62.8 rad/s) at a strain of 0.02% were used to characterize the dependence of the viscoelastic properties of casein gels on temperature and frequency. The complex modulus ( G*) , G′, and G′′, where the magnitude of G* = ( G′2 + G′′2) 0.5, were obtained during heat treatment at a rate of 2°C/min. Stress relaxation experiments at 5 and 20°C at 0.1% strain (strain rise time of 0.5 s ) were also used to characterize the longer time (low frequency) flow behavior of the samples. Microsoft Excel 6.0 (Microsoft Corp., Redmond, WA) was used for power law fitting and ANOVA. SigmaPlot® 3.01 (Jandel Corp., San Raphael, CA) was used to put the data into contour plots. RESULTS Strain Sweeps Figure 1 shows G* as a function of strain for casein gels with different C:W and MF contents at 5°C. Casein gels with no added MF did not show a decrease of G* values, regardless of C:W up to 2% strain; no disruption of the elastic network structure was indicated up to this level of strain. The decrease in linear viscoelastic region for gels containing added MF depended on the C:W, decreasing to about 0.9, 0.5, and 0.1% for C:W of 0.89, 0.58, and 0.38 respectively. The 0.5% linear viscoelastic range for casein gels with added MF at a C:W ratio of 0.58 (recommended formula for Mozzarella cheese analog) is conJournal of Dairy Science Vol. 81, No. 10, 1998
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Figure 2. Effect of the ratio of casein to water (open symbols = 0.89, gray symbols = 0.58, and black symbols = 0.38) and milk fat content (circles = 24%, diamonds = 12%, and triangle = 0%) on the complex modulus ( G * ) of casein gels at 20°C.
sistent with the data obtained for commercial Mozzarella cheeses (1, 9). At strains above these apparent elastic limits, G* decreased and appeared to approach the G* value of the underlying unfilled (no added MF) casein polymer system. This behavior was quite similar to that of other filled polymer systems, such as gluten and starch or natural rubber and carbon black, for which contacts between polymer and filler are probably involved ( 3 ) . For the unfilled gels, G* was increased from about 70 to 500 kPa as the C:W was increased from 0.38 to 0.89. Also, for each C:W, added MF (12%) increased G* in the linear viscoelastic region. Increased MF content (24%) resulted in increased G* for C:W of 0.38 and 0.89, but not for the gel with a C:W of 0.58. It is also interesting to compare results for the two samples: C:W = 0.38 and MF = 24% or C:W = 0.58 and MF = 0%. For these two gels, G* is essentially equivalent at low strains ( ≤ 0.1%), but the elastic strain limit and, hence, the apparent yield stress [critical stain × G*] are quite different. Thus, these casein gels cannot be considered equivalent based on G* values for the linear viscoelastic region only, as critical strain is also an important material property. Differences in the underlying long-range structures of the casein network only become apparent at larger strains or longer times, as is discussed in the section on stress relaxation. The strain dependence of G* at 20°C and at 5°C were similar in the linear viscoelastic range (Figure 2). Unfilled gels did not show a decrease of G* values up to 2% strain for all C:W ratios. The addition of MF Journal of Dairy Science Vol. 81, No. 10, 1998
to casein gels decreased their linear viscoelastic region to about 0.7, 0.3, and 0.1% for gels with C:W of 0.89, 0.58, and 0.38, respectively. Compared with the results for 5°C, the linear viscoelastic region was decreased more at 20°C with added MF for each C:W, which suggests that MF in liquid form is a less effective reinforcer of the casein network. The MF is about 75% solid at 5°C and about 20% solid at 20°C (13). The G* value of unfilled gels increased from 50 to 400 kPa as C:W increased at 20°C. However, added MF decreased G* at each C:W, which was opposite to the effect of MF on G* at 5°C. These opposite effects could again be explained by the phase change of MF from mostly solid to mostly liquid in this temperature range. Chronakis and Kasapis ( 2 ) also reported a 90% decrease in G′ of spreadable butter from 5 to 20°C, presumably from melting. Studies of cheese microstructure have shown that MF may exist as fat globules in cheese (7, 15, 16). Thus, solid fat globules appear to reinforce the casein gel structure via shortrange interactions with the casein network. Dynamic Mechanical Thermal Analysis Dynamic mechanical thermal analysis was used to obtain information on the effects of temperature and frequency on the viscoelastic properties of the casein gels. Figure 3 shows a representative result for the casein gel with a C:W of 0.58 and MF content of 24%.
Figure 3. Effect of frequency (Hertz) and temperature (O: 5°C, »: 10°C, ◊: 20°C, ♦: 30°C, ∫: 40°C, and o: 50°C ) on the complex modulus ( G * ) for a casein gel with 24% milk fat and a ratio of casein to water of 0.58.
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VISCOELASTIC PROPERTIES OF CASEIN GELS TABLE 2. Power law characterization1 obtained from frequency sweeps for each casein gel. Ratio of casein to water (g/g) 0.89 Milk fat content
Temperature ( °C )
0%
5d 10c 20† 30† 40b 50a
12%
5d 10c 20x 30x 40b 50a 5d 10c 20x 30x 40b 50a
24%
A,B,CThe
*A
0.58 *B
0.38 *C
G0
n*
G0
n*
G0
n*
384.34 375.85 305.65 234.16 165.18 72.33 495.04 481.38 327.12 183.60 112.78 42.01 605.28 589.78 344.89 146.25 84.51 28.97
0.185 0.186 0.198 0.206 0.231 0.302 0.180 0.183 0.203 0.230 0.256 0.339 0.176 0.177 0.199 0.237 0.261 0.362
114.09 106.69 90.70 63.67 41.10 17.83 180.13 169.83 121.11 59.90 34.65 11.65 212.10 198.61 129.08 50.88 24.92 6.68
0.174 0.192 0.206 0.232 0.272 0.362 0.179 0.189 0.199 0.241 0.289 0.389 0.172 0.184 0.196 0.236 0.278 0.406
46.03 43.09 34.93 22.08 13.08 4.84 65.00 61.42 38.18 17.55 9.78 3.31 110.01 104.07 49.92 15.51 8.43 3.10
0.189 0.203 0.219 0.268 0.325 0.416 0.187 0.196 0.219 0.274 0.299 0.366 0.168 0.176 0.198 0.277 0.294 0.269
G*0 at each ratio of casein to water with the same temperature differ ( P < 0.01).
each milk fat content, G*0 at the same temperature with no common superscript letter differ ( P < 0.01). a,b,c,dAt
xThe 1G*
0
G*0 of gels at these temperatures with different milk fat content do not differ ( P > 0.05).
= Complex modulus (kilopascals) at a frequency of 1 Hz; n* = power low exponent (slope).
All gels showed similar trends. Given the nearly linear appearance of the G* versus the frequency data on logarithmic coordinates, a power law model was used to characterize the frequency dependence of G* over this limited frequency range for the gels as follows: G* = G*0 fn* where G*0 = model value of G* at a frequency of 1 Hz, f = frequency in Hertz, and n* = the power law exponent. The G*0 is indicative of the overall stiffness, or resistance to deformation, within the linear viscoelastic region of the gels at a frequency of 1 Hz. This value may be considered as the firmness of the gels when they were subjected to a fairly rapid deformation, such as depressing the gel quickly with one’s thumb and releasing. The n* may be taken as a convenient measure of the relative degree of viscoelasticity of the gels. It would be expected that n* would approach zero for ideal elastic behavior (i.e., no frequency dependence) and would increase as the degree of viscoelasticity increased.
Values of the power law parameters, G*0 and n*, at different temperatures for each casein gel are shown in Table 2. The G*0 values were significantly increased by added MF at temperatures of 5 and 10°C and decreased ( P < 0.01) by MF content at temperatures of 40 and 50°C. At temperatures of 20 and 30°C, however, MF had no ( P > 0.05) effect on G*0 values. A decrease in the C:W ratio of the gel decreased ( P < 0.01) the G*0 values at all temperatures. The ANOVA also showed that the effect of C:W ratio and MF on G*0 did not interact at temperatures of 5, 10, 20, and 30°C but did interact ( P < 0.05) at temperatures of 40 and 50°C. This interaction means that the effect of MF on G*0 is dependent on C:W and vice versa, but the effect of MF and C:W on G*0 is additive in a casein network up to 30°C. The MF probably acts as a plasticizer (softener) of the casein melt at temperatures above 40°C, which is consistent with the results of Marshall ( 7 ) for stiffness of process cheese analogs at 21°C. Contour plots of the change of G*0 with C:W ratio and MF content (Figure 4 ) showed that the effect of Journal of Dairy Science Vol. 81, No. 10, 1998
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MF content on G*0 at constant C:W changed direction
ature by adjusting the composition. However, G*0
at a temperature between 20 and 30°C, increasing G*0
represents only the short-range linear viscoelastic properties of the gels and not necessarily their longrange network properties, as is discussed further in the section on stress relaxation. The n* increased in a similar manner at temperature above 10°C for each C:W, regardless of MF content (Table 2), and probably reflects the loss of physical crosslinks in the casein matrix as the temperature is increased. In general, n* appeared to be more affected by temperature at constant C:W than by C:W at constant temperature. This result is probably due to the overall high concentration of casein that is present in all of the gels (i.e., sufficiently high to
at temperatures ≤20°C and decreasing G*0 at temperatures ≥30°C. One way to interpret these results is that, for each temperature, a corresponding range of G*0 values could be achieved by varying C:W and MF contents in the experimental range. In addition, within this range, any particular value of G*0 could be obtained by a number of different gel compositions. Viewed another way, a particular G*0 value associated with a certain gel composition at one temperature can be reproduced at a somewhat higher or lower temper-
Figure 4. Complex modulus ( G*o ; kilopascals) ) at 1 Hz of casein gels as a function of the ratio of casein to water (C:W) and milk fat ( M F ) content at different temperatures. Journal of Dairy Science Vol. 81, No. 10, 1998
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obtain a gel in the first place). The higher G*0 values and lower n* values at the highest C:W and lowest temperatures are logically due to an increase in the number of physical crosslinks in the gel and the presence of solid fat. The three unfilled gels of different C:W all show a progressive softening (decrease in G*0) as the temperature is increased above 5°C, particularly above 10°C. Thus, the softening and meltdown properties of these gels involve the liquefaction of MF superimposed over the dissociation of the underlying secondary interactions (noncovalent) of the casein network. Melting Behavior of Gels Figure 5 shows the relative contributions of elastic ( G ′) and viscous effects ( G′′) to the overall resistance to deformation of the gels as a function of temperature, C:W, and MF content. The G′ is much greater than the G′′ for all gels at lower temperatures and showed a more dramatic change than did G′′ with temperature up to 30°C, at which temperature most of the MF is liquefied. A comparison of gels with different MF contents indicated that G′ was increased at low temperatures by added MF. The values for G′ and G′′ crossed over each other ( G′′ = G′) in the temperature range of 45°C to 65°C, depending upon the C:W and MF content. The crossover temperature at which tan d ( G ′′/G′) = 1 is a convenient estimate of the melting point of the gels. Table 3 shows that the crossover temperatures for the casein gels ranged from 46 to 64°C, depending on C:W and MF content. A decrease in the C:W from 0.89 to 0.38 reduced the melting point from 64 to 50, 62 to 49, and 64 to 46°C for MF contents of 0, 12, and 24%, respectively. This decrease was much greater than that occurring as the MF content increased at constant C:W; results ranged from essentially no change for a C:W of 0.89 to 6°C for the other
TABLE 3. The effect of the ratio of casein to water (C:W) and milk fat content on the temperature ( T ) at which the loss tangent ( G′′/ G′) = 1 and the corresponding value of the elastic modulus ( G′) = the loss modulus ( G′′) at this temperature. Milk fat 0%
12%
24%
C:W
T
G′
T
G′
T
G′
0.89 0.58 0.38
( °C ) 64 62 50
(kPa) 32 9.5 6.5
( °C ) 62 59 49
(kPa) 20 8.5 4.7
( °C ) 64 56 46
(kPa) 13 6 3.8
Figure 5. Effect of temperature and the ratio of casein to water (C:W) on the elastic ( G′) and loss ( G′′) moduli of casein gels with 0, 12, and 24% added milk fat.
two C:W. These results indicate that the C:W is a critical factor in the meltdown and flow properties of these gels, regardless of the MF content. However, G′ at the crossover temperature (Table 3 ) decreased with either an increase in MF content at constant C:W ratio or a decrease in C:W at constant MF content. Because the energy stored elastically in a cyclic deformation is directly proportional to G′ (12), one might expect a melt with higher G′ to be perceived as more rubbery or tougher. In other words, more work would be needed to achieve the same deformation as G′ increases. Thus, just as the firmness at low temperature ( G * ) is affected by both C:W and MF content, so also are the material properties of the corresponding melt. For any given C:W, the change in overall firmness as given by G* with increased temperature consisted of three distinct zones of viscoelastic behavior, as is illustrated in Figure 6 for C:W of 0.58. The plateau region for G* at temperatures below 10°C (zone 1 in Figure 6 ) represents a firm and highly elastic gel where G′ >> G′′ (Figure 5). As the temperature is increased above about 10°C (zone 2 in Figure 6), the gels soften, primarily because of a decrease in G′, which means that the softening is also accompanied Journal of Dairy Science Vol. 81, No. 10, 1998
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Figure 6. The effect of temperature on the viscoelastic properties of casein gels as three distinct zones, corresponding to rubbery solid (zone 1), transition (zone 2), or melt (zone 3).
by a loss of elasticity. The liquid portion of MF in the casein network increases rapidly between 10 and 25°C (inset in Figure 6), resulting in a faster G* decrease for gels at higher MF content. When the majority of the MF is liquefied (around 20 to 30°C), G* of the filled system actually becomes lower than that of the unfilled system. At the crossover temperature (zone 3 in Figure 6), G* reached another plateau region, which represents a viscoelastic melt. It is important to recognize these distinct zones of viscoelastic behavior for a given composition as they are related to both the textural attributes of the gels and their flowability in the melt state. For example, the temperature at which tan d = 1 was found to be 56°C for the case in which C:W is 0.58 and MF is 24% (Mozzarella cheese analog). This temperature is remarkably close to the typical stretching temperature for Mozzarella cheese of 57°C.
initial stress was applied. However, at longer times, casein gels with the same C:W tended to superimpose without shifting of the time axis, indicating that added MF did not affect the relaxation times of the casein network. The one exception was the gel with a C:W of 0.38 and a MF content of 24%, which also showed strain sweep behavior similar to the gel with a C:W of 0.58 (Figure 1 ) in the linear viscoelastic region. It is possible that, at 5°C, solid fat provides additional crosslinking points for the dilute casein network. A gradual but substantial relaxation for all gels was observed at longer times. Masi and Addeo ( 8 ) have suggested that relaxation in Mozzarella cheese is due to the breakage and reformation of secondary bonds (i.e., a temporary network structure). The presence of a plateau region here at short times (high frequencies), coupled with the extensive stress relaxation, suggests that a similar mechanism applies to these rennet casein gels. The rate of stress relaxation is clearly slower at higher C:W and is not affected by MF content, except for the one aberrant gel just discussed. This evidence indicates that the important functional properties of casein-based products related to viscous or rubbery flow, such as melt and stretch of cheese, may depend more on the C:W than on the fat content. Stress relaxation at 20°C (Figure 8 ) showed trends that were qualitatively similar to those at 5°C, except that all gels with a C:W of 0.38 lacked a well-
Stress Relaxation Stress relaxation was used to determine the viscoelastic properties of the gels at longer times up to 1000 s. Figure 7 shows the stress relaxation curves for casein gels obtained at 5°C, plotted as G/G0 versus time, for which G is the modulus at any time and G0 is the initial modulus. All samples showed very similar relaxation behavior up to about 1 s after the Journal of Dairy Science Vol. 81, No. 10, 1998
Figure 7. Effect of the ratio of casein to water (C:W) (open symbols = 0.89, gray symbols = 0.58, black symbols = 0.38) and milk fat ( M F ) content (circles = 24%, diamonds = 12%, and triangle = 0%) on the stress relaxation modulus ( G ) normalized by the initial modulus ( G 0) at 5°C.
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DISCUSSION
Figure 8. Effect of the ratio of casein to water (C:W) (open symbols = 0.89, gray symbols = 0.58, and black symbols = 0.38) and milk fat ( M F ) content (circles = 24%, diamonds = 12%, and triangle = 0%) on the stress relaxation modulus ( G ) normalized by the initial modulus ( G 0) at 20°C.
defined, short-time plateau. Apparently the crosslink density is not sufficient to provide a resilient, solid gel at 20°C and a C:W of 0.38. This result is consistent with the lower G*0 at 20°C (Table 2 ) for these gels. Table 4 shows the time needed for the shear modulus to relax 50% of its original value ( t50) for casein gels at 5 and 20°C. All samples showed a t50 less than 5 s. A decrease of C:W at either experimental temperature increased the relaxation rate of casein gels. The ANOVA of t50 values showed that a decreased C:W decreased ( P < 0.01) the t50 value. However, MF did not exert a significant effect on the relaxation time. These results showed that casein gels with C:W of 0.38 relaxed much faster than did the other gels. At room temperature, these gels flowed within an hour because of the stress of their own weight (i.e., these gels did not contain the network structure required to hold their shape for a long time under the force of gravity).
Isolation of the main factors that are important to the MF-casein system, namely, the C:W, temperature, and volume (or mass) fraction of filler, made some of the unique aspects of this system apparent. The combination of the low molecular mass (for a polymer) of casein, temperature-dependent crosslinks, and the correspondingly high concentration of casein needed to obtain the sufficient number of crosslinks for solidlike behavior at low temperatures also leads to a viscoelastic melt at relatively low temperatures of around 60°C. Also, MF functions as a uniquely compatible filler of the casein-water matrix and serves a dual role. The MF is solid at low temperatures (e.g., <10°C), which corresponds to extensive crosslinking of the casein matrix, allowing it to act as a rigid filler, but liquefies over essentially the same temperature range that the casein network dissociates, thus diluting or plasticizing the viscoelastic melt as well. Our results for the effect of MF on G* and critical strain for strain sweeps and stress relaxation also support the idea that fat globules may act either as actual crosslinking points for a dilute casein network or as reinforcing filler particles in denser casein networks, as depicted and discussed by Xiong and Kinsella (17). However, in either case, the casein interactions of the MF globule were weaker than the casein network as shown by the decrease in critical strain. Because the short-time plateau region in stress relaxation was not extended at all by the presence of added MF at either 5 or 20°C (exception noted earlier) and because G*0 decreased with added MF at temperatures >30°C, added MF was not shown to form its own network at the levels used here or to affect the existing casein-water matrix significantly. This result implies that maintenance of a constant C: W as the fat content is reduced may help to retain the functional properties of full fat cheeses in lower fat versions. As MF is added to a gel of constant C:W, the volume fraction of the casein matrix is reduced, but the essential character of the remaining casein
TABLE 4. Effect of the ratio of casein to water (C:W) and milk fat ( M F ) content on the time for stress relaxation to 50% of its initial value at 5 and 20°C. 5°C
20°C
C:W
0% MF
12% MF
24% MF
0.89 0.58 0.38
4.85 3.37 2.44
4.42 3.69 2.46
4.51 3.67 3.25
0% MF
12% MF
24% MF
3.17 1.91 0.89
2.40 1.90 0.75
2.74 2.16 0.78
(s)
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matrix is retained. As discussed in the introduction, this relationship is not easily deduced from most reported results on the rheological properties of cheese (or models thereof) because the fat content and ratios of protein to water generally change as the fat level is varied. One might think of the C:W value as indicative of the crosslink density within the casein matrix at a particular temperature (all other factors equal) up to its melting point and as indicative also of the concentration of casein molecules in the casein melt above the gel melting point. The general principles of polymer science would indicate that the C:W is a critical variable for both firmness of the gels and viscosity of the corresponding melt obtained upon treatment and that it may be difficult to decouple the low temperature characteristics of the gels from their functional properties in the melt state. In addition, the effect of liquid MF on the rheological properties of the melt is further complicated by the possible partition of added MF into a fraction that is emulsified into the casein network and into a fraction of free oil, as has been discussed by Kindstedt and Rippe ( 4 ) for Mozzarella cheeses. According to those researchers, the efficiency of emulsification of fat into the casein network could depend upon the ratio of total fat to casein, which could provide an explanation as to why we observed a significant interaction between the C:W and MF level on G*0 only at 40 and 50°C. However, because we did not determine the free oil content of the casein gels obtained here, this result must be considered speculative. Overall, the results shown here for critical strain, values of the viscoelastic moduli, and stress relaxation were quite similar to those previously reported for Mozzarella cheese, even though emulsifiers were used here, and the gels were not stretched as Mozzarella would be. This similarity is probably because results here were limited to viscoelastic phenomena within the linear viscoelastic region, in which effects are dominated by the character of the casein proteins themselves and their interactions with MF. These characteristics would be expected to be similar in Mozzarella cheese, in which casein proteins predominate. However, these intermolecular interactions give rise to the macroscopic functional properties of polymeric systems. Therefore, these casein gel systems might be used to investigate the effect of other important factors, such as pH and calcium content, on the material properties of products based on casein. CONCLUSIONS This work has shown that added MF mainly affects the short-range elastic properties of the casein sysJournal of Dairy Science Vol. 81, No. 10, 1998
tem, G*0 and critical strain; the long-range network properties, as determined by stress relaxation, were primarily a function of the concentration of casein in the hydrated casein network. Thus, the casein:MF system behaves much like a typical filled polymer system, except that the material properties of the filler itself are also temperature dependent. The MF acts as a reinforcing filler at low temperatures and gradually becomes a diluting factor or plasticizer of the casein melt. The latter probably occurs above about 40°C, at which temperature the casein network is substantially dissociated with and the MF is completely liquid. Dynamic mechanical thermal analysis was found to offer a convenient means of characterizing the overall stiffness, relative elasticity, and transition from gel to melt for these rennet casein gels. Determination of linear viscoelastic properties could be used to guide the development of formulations or processes to achieve certain physical characteristics in products such as cheese that are based on caseins. REFERENCES 1 Ak, M. M., and S. Gunasekaran. 1996. Dynamic rheological properties of Mozzarella cheese during refrigerated storage. J. Food Sci. 61:566–568; 584. 2 Chronakis, I. S., and S. Kasapis. 1995. A rheological study on the application of carbohydrate-protein incompatibility to the development of low fat commercial spreads. Carbohydr. Polym. 28:367–373. 3 Ferry, J. D. 1980. Molecular theory for undiluted amorphous polymers and concentrated solutions: networks and entanglements. Pages 224–263 in Viscoelastic Properties of Polymers. 3rd ed. John Wiley & Sons, Inc., New York, NY. 4 Kindstedt, P. S., and J. K. Rippe. 1990. Rapid quantitative test for free oil (oiling off) in melted Mozzarella cheese. J. Dairy Sci. 73:867–873. 5 Lawrence, R. C., H. A. Heap, and J. Gilles. 1984. A controlled approach to cheese technology. J. Dairy Sci. 67:1632–1645. 6 Ma, L., M. A. Drake, G. V. Barbosa-Canovas, and B. G. Swanson. 1996.Viscoelastic properties of reduced-fat and full-fat Cheddar cheeses. J. Food Sci. 61:821–823. 7 Marshall, R. J. 1990. Composition, structure, rheological properties, and sensory texture of processed cheese analogues. J. Sci. Food Agric. 50:237–252. 8 Masi, P., and F. Addeo. 1986. An examination of some mechanical properties of a group of Italian cheeses and their relation to structure and conditions of manufacture. J. Food Eng. 5: 217–229. 9 Nolan, E. J., V. H. Holsinger, and J. J. Shieh. 1989. Dynamic rheological properties of natural and imitation Mozzarella cheese. J. Texture Stud. 20:179–189. 10 Pagliarini, E., and N. Beatrice. 1994. Sensory and rheological properties of low-fat filled ‘pasta filata’ cheese. J. Dairy Res. 61: 299–304. 11 Park, S.-Y., R. Niki, and K. Nakamura. 1996. Rheological behavior of casein micelles and reconstituted skim milk gels: the effects of temperature on gelation induced by rennet. Int. Food Sci. Technol. 2:103–107. 12 Rosen, 1993. Chapter XVIII. Linear Viscoelasticity. Pages 298–349 in Fundamental Principles of Polymeric Materials. 2nd ed. John Wiley & Sons, Inc., New York, NY. 13 Shukla, A., A. R. Bhaskar, S.S.H. Rizvi, and S. J. Mulvaney. 1994. Physiochemical and rheological properties of milk fat
VISCOELASTIC PROPERTIES OF CASEIN GELS made from supercritically fractionated milk fat. J. Dairy Sci. 77: 45–54. 14 Solorza, F. J., and A. E. Bell. 1995. Effect of calcium, fat and total solids on the rheology of a model soft cheese system. J. Soc. Dairy Technol. 48(4):133–139. 15 Tunick, M. H., K. L. Mackey, and J. H. Shieh, P. W. Smith, P. Cooke, and E. L. Malin. 1993. Rheology and microstructure of low-fat Mozzarella cheese. Int. Dairy J. 3:649–662.
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16 Tunick, M. H., and J. J. Shieh. 1995. Rehology of reduced-fat Mozzarella cheese. Page 7–19 in Chemistry of StructureFunction Relationships in Cheese. E. L. Malin and M. H. Tunick, ed. Plenum Press, New York, NY. 17 Xiong, Y. L., and J. E. Kinsella. 1991. Influence of fat globule membrane composition and fat type on the rheological properties of milk based composite gels. II. Results. Milchwissenschaft 46:207–212.
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