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* Western Dairy Center, Department of Nutrition and Food Sciences Utah State University, Logan 84322;
Dairy Science Department South Dakota State University, Brookings 57007; and
Department of Microbiology Weber State University, Ogden, UT 84408
Corresponding author:
D. J. McMahon; e-mail:
djm{at}cc.usu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Abbreviation key: SEM = scanning electron microscopy, TEM = transmission electron microscopy
Key Words: hydrophobic interactions • protein matrix • color • whiteness
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Electron Microscopy
Scanning electron microscopy (SEM) was used to prepare low magnification (3000x) micrographs for image analysis of bulk arrangements of the cheese matrix, whereas transmission electron microscopy (TEM) was used to prepare both low (3000x) and high (85,000x) magnification micrographs. The high magnification images were then used to characterize protein-to-protein arrangements within the protein matrix.
SEM.
Cheese samples, (approximately 1 mm x 1 mm x 10 mm), were taken at 10°C and 50°C on d 1, and fixed in fresh 3% glutaraldehyde for 3 h (also at 10°C and 50°C, respectively). The glutaraldehyde solution was changed every 30 min. Once fixed, the samples were stored in new glutaraldehyde solution at 4°C until further processed according to McManus et al. (1993). Samples were frozen in liquefied Freon 22 (–159°C; Mallinckrodt Inc., Paris, KY), transferred to liquid nitrogen, cryofractured perpendicular to their long axis, and thawed in 2% glutaraldehyde. They were then dehydrated in a graded ethanol series followed by fat extraction with Freon 113 (Mallinckrodt Inc., Paris, KY). After overnight storage in Freon 113 at 4°C, the samples were rehydrated by reversing the graded ethanol series, and washed with a 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences, Fort Washington, PA), pH 7.2. The samples were then postfixed for 2 h with a solution containing 1% OsO4 (Electron Microscopy Sciences, Fort Washington, PA) and 1.5% K4Fe(CN)6·3H2O (Fisher Scientific Co., Fair Lawn, NJ). This solution was replaced by a 2% tannic acid (Mallinckrodt Inc., Paris, KY) solution in cacodylate buffer, and the samples were left for 3 h at 20°C. The tannic solution was then replaced with osmium tetroxide-potassium ferrocyanate solution, and samples left for 4 h. This solution was later replaced with an aqueous solution of 1% hydroquinone (Mallinckrodt Inc., Paris, KY), and samples left overnight. After postfixing, the samples were washed with distilled water, dehydrated in a graded ethanol series, and critical-point dried in a critical-point drier (Model 1200; Polaron, Waterford, England) with CO2. Samples were viewed in a field emission scanning electron microscope (Model S-4000T FESEM; Hitachi Scientific Instruments, Mountain View, CA) operated at 3 kV. Images from each sample, at 3000x magnification, from two randomly selected fields were recorded on Kodak TMX 120 film.
TEM.
Cheese samples, approximately 1 mm3, were obtained from previously taken and fixed samples for SEM. The samples were stored in new glutaraldehyde solution at 4°C until further processed according to Paulson et al. (1998). Samples were then placed into 1% OsO4 (Electron Microscopy Sciences, Fort Washington, PA) in 0.2 M cacodylate buffer for 1 h (Electron Microscopy Sciences, Fort Washington, PA). After dehydration in a graded ethanol series, the samples were infiltrated with Spurr’s epoxy overnight, transferred to Beem capsules filled with Spurr’s epoxy, and heated to 70°C for 24 h. Sections, 70 to 80 nm thick, were cut on an Ultracut ultramicrotome (Leica Inc., Deerfield, IL), transferred to 300-hex mesh grids, and counterstained with uranyl acetate and lead citrate. They were then viewed on an electron microscope (Model 902; Carl Zeiss, Inc. Thornwood, NY) at an accelerating voltage of 80 kV. Transmission electron micrographs, at 3000x and 85,000x magnifications, from two randomly selected fields were then recorded on Kodak SO 163 film.
Image Analysis
SEM micrographs.
Negatives of scanning electron micrographs were scanned and their digital images recorded. The images were then uploaded into Adobe Photoshop 4.0 (Adobe Systems Inc., San Jose, CA), and their grayscale values analyzed. Dark areas (corresponding to areas previously occupied by serum) were differentiated from light areas (corresponding to protein matrix) by applying the threshold function. A threshold level of either 60 or 75 was selected for each micrograph, depending on the individual image characteristics, so that a precise differentiation between dark and light areas was obtained. The proportions of dark and light pixels were then determined by applying the histogram function.
TEM micrographs.
Negatives of transmission electron micrographs taken at low and high magnification were scanned and their digital images recorded. High-magnification images were then uploaded into Adobe Photoshop 4.0, and a representative 512 x 512-pixel area was selected by applying the crop function. The cropped images were then uploaded into NIH Image 1.60 (National Institutes of Health, Washington, DC), and the threshold function, at a level equal to the mean density value of each image, was applied to allow for differentiation between high- (dark) and low- (light) density regions. Regions of the micrograph with a high-density of electrons were considered to represent protein aggregates within the protein matrix. The percentage of area of the micrographs representing protein aggregates and their mean density value were determined by applying the measure function of the software, which computes the area and mean gray value of the current selection. Density was measured in the grayscale from 0 to 255, with 0 corresponding to white and 255 to black. Aggregate size (area) was determined in a 250 x 250-pixel image area, cropped from the 512 x 512-pixel image, by applying the measure function. Then, by assuming the aggregates to be spheres, their mean volume was determined. The number of aggregates was determined in a 170 x 170-pixel image by applying the measure function. Considering the width of the cheese section to be 75 nm in average, and assuming no superposition of aggregates within the three-dimensional section, the concentration, or number of aggregates per unit volume of protein matrix could be calculated. Then, by considering the aggregates to be evenly distributed within the section, the distance between adjacent aggregates was determined. Also, the volume of the section occupied by them was determined.
Experimental Design and Statistical Analysis
The experiment was conducted in quadruplet with treatments, cheese temperatures of 10°C and 50°C, randomly assigned. Two replications were considered for SEM and TEM image analysis. For TEM, two samples from each replicate were analyzed to determine the number, size, and area occupied by high-density protein aggregates. When determining size, up to 200 aggregates per sample were measured, and values greater than four standard deviations were disregarded. The mean aggregate size from each sample was then considered for analysis. Statistical analysis, ANOVA, was performed using SAS (1991).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Cheese Microstructure
Differences between cheeses at 10°C and 50°C could be visually observed in images obtained by SEM (Figure 1
) and low-magnification TEM (Figure 2). In the TEM images, cheese at 10°C (Figure 2a) had a less dense protein matrix with fewer and smaller serum pockets present in the cheese matrix, as compared with cheese at 50°C (Figure 2b). This was also observed in scanning electron micrographs. In cheese at 10°C (Figure 1a), spherical serum pockets, ranging in size between 0.6 and 1.0 µm in diameter, were observed in the cheese matrix occupying 1% of the matrix area (Figure 1c). In contrast, at 50°C (Figure 1b), the cheese matrix had bigger pockets, ranging in size between 0.6 and 7.0 µm in diameter and occupying 31% of the cheese matrix area (Figure 1d). There was, however, no difference in number of pockets observed. In terms of protein matrix, there was a corresponding reduction in area, with cheese at 50°C having a reduced area occupied by protein matrix, as compared with cheese at 10°C, 69% versus 99% (P = 0.022).
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). At 10°C (Figure 3a), the protein matrix appeared less dense and was more homogenous than at 50°C (Figure 3b). The lower temperature was also associated with the presence of a higher number of aggregates and smaller mean aggregate size (Table 1). Even though the largest aggregates were observed in cheese at 10°C, a higher frequency of small aggregates led to their lower mean size. These aggregates were located 15 nm apart and occupied 48% of the area of the protein matrix (Table 1). In contrast, at 50°C, a bigger size but lower number of aggregates in the protein matrix was observed (Table 1). This resulted in an increase of area (+15%) and volume (+40%) of protein matrix occupied by aggregates in cheese at 50°C. On average, the aggregates were located 24 nm apart. Also, the mean density value was higher for cheese at 50°C than at 10°C (Table 1). Assuming the aggregates to be spheres and uniformly distributed within the protein matrix, a graphical two-dimensional representation of their arrangement in a 100 x 100 x 75-nm section of matrix was developed (Figure 4). At 10°C the aggregates were smaller and located closer together (Figure 4a) as compared with aggregates at 50°C (Figure 4b). Figure 4c shows that if the aggregates at 10°C were drawn to the same size as observed at 50°C there would be insufficient protein material to account for the increased size of protein aggregates observed at 50°C. Thus, at 50°C there was more protein material per unit volume of protein matrix than at 10°C.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Although we did not determine aggregate composition, it can be assumed that the composition of the aggregates was probably different, with aggregates at 50°C containing more ß-casein. Among the caseins, ß-casein has the greatest average hydrophobicity and its solubility is greatly affected by temperature (Bingham, 1971). The decreased solubility of ß-casein as it is heated is thought to be the major parameter affecting heat-induced opacity of cheese (Metzger et al., 2000; Dave et al., 2001). Dissociation of ß-casein from the micelle occurs at low temperature as a consequence of weakening of hydrophobic interactions and removal of colloidal calcium phosphate (Pierre and Brule, 1981). This dissociation is reversible and dependent on temperature. Dissociation of ß-casein from the protein matrix at 10°C possibly contributed to the occurrence of a higher number of smaller aggregates observed at that temperature.
In analyzing electron micrographs, dark areas have high electron density and light areas have low electron density. This contrast is based on binding of heavy metal atoms (Os, Ur, and Pb) by proteins. Thus, heavy metals are used for "staining" the protein fraction of the cheese matrix. The higher the retention of heavy metals, the more electrons that are scattered, and consequently, the darker the micrograph image. Therefore, the higher electron density of cheese at 50°C (Table 1) was indicative of more protein present per unit volume of protein matrix. To obtain this increased concentration of protein requires a contraction of the protein matrix.
As hydrophobic interactions become more important with temperature increase, there would be a reassociation of ß-casein (and possibly calcium) with the other proteins. Calcium promotes protein-to-protein interactions (Bloomfield, 1979), and its reassociation at the higher temperature may have also contributed to the occurrence of bigger aggregates by affecting interactions involving 
s1- and s2-casein as well as ß-casein. This promotion of aggregation at the molecular level is then manifested as changes in cheese structure. Contraction of the protein matrix increases the overall density of the matrix, and serum pockets throughout the cheese matrix become larger (Figure 1b compared to 1a). Thus, changes in cheese structure, as the temperature of the cheese increases, are ultimately brought about by changes in the extent and nature of protein interactions.
Cheese Opacity
A nonfat cheese can be considered to be composed of two interacting phases, the serum and the protein matrix. The effect of changes in the serum phase of cheese during heating and cooling on cheese whiteness was suggested by Metzger et al. (2000). Based on in vitro formation of a white ß-casein gel from cheese serum, they suggested that formation of such a gel during heating of cheese would increase the whiteness of the cheese. However, it has not been demonstrated that such a gel would be formed in vivo within the cheese matrix. When cheese is heated, ß-casein would be surrounded by other proteins, and formation of a ß-casein homopolymeric gel would have to compete with reassociation of ß-casein with other proteins in the cheese matrix. Such association of ß-casein with s1-, s2-, and para-
-casein would deplete the serum of protein, and a serum gel would not form.
In the present study, no evidence of such a separate serum gel was found either in scanning or transmission electron micrographs of the cheese. If a protein gel were formed, it should have been at least partially retained by means of fixation as a consequence of sample preparation for electron microscopy. Also, when the ultrastructure of the cheese was examined, no apparent discontinuity in the protein matrix was observed, as would be the case when two different gel matrices were present. Therefore, the contribution of such a heat-induced serum gel to the observed increased opacity of cheese does not appear feasible.
The structure of the protein matrix at 50°C can be considered similar to the particulate type of gel that can be formed by aggregation of whey proteins. In Langton and Hermansson’s study (1992) of the effect of pH on the formation of ß-lactoglobulin and whey protein gels, the occurrence of such a gel network was associated with the observation of a white rather than a translucent gel. A translucent gel was observed when a fine-stranded network was formed. At pH 4.0, a white ß-lactoglobulin gel was formed that had an open and irregular network, with increased density fluctuations as compared with translucent gel, which was formed at pH 3.5. Accordingly, the protein matrix of cheese at 50°C appeared less uniform, with more density fluctuations and bigger openings, as was observed by Langton and Hermansson (1992), when a white ß-lactoglobulin gel was formed.
In their study, bigger aggregates were present in the white gel than in the more translucent gel. Similarly, larger aggregates were observed in the protein matrix of cheese at 50°C. Therefore, in agreement with previous observations, the occurrence of larger aggregates and more density fluctuations in the protein matrix was associated with increased light reflection and opacity of the cheese. Whether such changes in protein aggregation contribute to differences in reflection of light should be considered according to light scattering principles.
The scattering of light by a small particle (longest dimension being less than 
/20; i.e., spherical particles with diameter less than 25 nm, for an average wavelength of light of 500 nm) is determined by Rayleigh’s equation:
 | (Eq. 1) |
where Io,u is the initial intensity of unpolarized light of wavelength in the surrounding medium; I
is the intensity of scattered light; is the angle measured in the plane of the initial and scattered beam; r is the distance from the particle at which the scattered light is measured; n0 is the refractive index of the surrounding medium; n1 is the refractive index of the particle; v is the particle volume; and Np is the number of particles per unit volume (Hunter, 1989).
Protein matrix.
In the case of a solid, a large number of small scattering centers will scatter light, and the waves will interfere either constructively or destructively, depending on the phase difference (Dickinson and Stainsby, 1982). However, in practice, an interparticle distance about three times the radius of the particle may be considered as a sufficient condition for independence (Zemb, 1990). This means that if the particles are sufficiently apart from one another their scattering properties can be considered without reference to the surrounding scatterers. Therefore, based upon the small size of the aggregates (< /20), and the distance between them (>3 radii), they can be considered to behave independently as Rayleigh scattering elements. According to Eq. 1, and considering that , , and r would remain constant when cheese is heated, the intensity of scattered light becomes a function of the difference in refractive index of the particles and surrounding medium (n1 and n0), the volume of the particles (v), and the number of particles per unit volume (Np). Consequently, the bigger the size of the particle and the higher its concentration, the more light will be scattered.
When cheese temperature increased from 10°C to 50°C, the size of the aggregates increased from 1.6 nm to 3.0 nm in diameter. This was accompanied by a decrease in aggregate concentration from 181 to 37 aggregates per 7.5 x 105 nm3-protein matrix section. However, when the change in aggregate volume and concentration were considered in Eq. 1, the product v2Np was bigger for cheese at 50°C than at 10°C (9.1 x 10–3 versus 9.5 x 10–4, respectively). Thus, the increase in average size of the aggregates more than compensates for the decrease in particle concentration and would contribute to increased scattering of light. And since the particles are far enough apart to be considered independent light scatterers, they increase the opacity of the cheese when heated to 50°C.
In addition to the changes in aggregate size that promote light scattering of cheese at 50°C, changes in refractive index should also be considered. The refractive index of the continuous and dispersed phases of the protein matrix (serum and protein aggregates, respectively) was not determined, but any changes would have also contributed to increased light scattering. If ß-casein moves from very soluble to insoluble as cheese is heated, its concentration in the serum would decrease, thus decreasing the refractive index of the serum. At 50°C, the protein aggregates were more compact (i.e., had higher electron density), so if anything, their refractive index would increase. Therefore, as aggregation was promoted, differences in chemical composition and physical properties between the serum and protein aggregates probably increased. This would have resulted in an increased difference between the refractive index of the continuous and dispersed phases, thus further promoting light scattering in accordance with Eq. 1.
Serum pockets.
Another consequence of increasing protein-to-protein interactions as cheese was heated was the increase in size of serum pockets in the cheese matrix (Figures 1 and 2). This can be considered a manifestation of microphase separation. Such phase separation has been recognized to occur in the formation of turbid or opaque gels from globular proteins (Clark, 1998). As the proteins approach their isoelectric point, or are subject to high ionic strength, a gel is formed which normally releases water and is characterized by presenting a heterogeneous microstructure. Also, when heating cycles are applied to globular protein gels, the system may become progressively demixed and phases may separate.
In the present experiment, as the continuous phase (serum) became increasingly different in composition and physical properties with respect to the dispersed phase (protein aggregates), phase separation occurred. Thus, serum pockets grew in size from a maximum size of 1.0 µm in diameter at 10°C to 7.0 µm at 50°C (Figure 1). This indicates that increased protein-to-protein interactions, promoted by an increase in cheese temperature, results in a decreased capacity of proteins to interact with water. As a consequence, protein hydration decreases and serum separates from the protein matrix, increasing the size of the serum pockets. Such an increase in size of serum pockets increases the heterogeneity of the system at the microstructural level. This increases the difference between the refractive index of the continuous (protein matrix) and dispersed (serum pockets) phases as they separate from one another. Consequently, and according to Eq. 1, this would also promote light scattering.
Serum pockets themselves may also be considered scattering elements. According to the Mie scattering theory, as particles grow to larger than the wavelength of light, all wavelengths of light tend to be equally scattered, which usually results in a white sample appearance (Waldman, 1983). Therefore, as serum pockets grow in size they could promote scattering of light, scattering all wavelengths equally. As we did not collect data regarding either the composition or refractive index of the serum, the actual independent contribution of the serum pockets to light scattering remains uncertain. Still, in general, the increase in heterogeneity of the system brought about by the increase in size of the serum pockets would have contributed to promoting light scattering and opacity of the cheese.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Received for publication May 24, 2001. Accepted for publication January 23, 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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s1-, ß-, and -casein. J. Dairy Sci. 54:1077–1080.This article has been cited by other articles:
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