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Reorganization of casein submicelles in Mozzarella cheese during storage
0 PII:
ELSEVIER
IHI. Duirj~ Journtrl7 ( 1997) I49- I55 1997 Published by Elsevier Science Ltd Printed in Great 0958-6946:97/$17.00
SO9S8-6946(96)00057-X
Britain + 0.00
Reorganization of Casein Submicelles in Mozzarella Cheese During Storage Michael H. Tunick, Peter H. Cooke, Edyth L. Malin, Philip W. Smith and V. H. Holsinger United Stutes Department
of’ Agriculture, Agricultural
(Received
Research Serviw, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmnor. PA 19038, USA
20 October
1995; accepted
16 October
1996)
ABSTRACT To gain a better understanding of the factors involved in textural changes in Mozzarella cheese during storage, electron microscopic imaging was used to compare the ultrastructure of the protein matrix on the day of stretching (0 weeks) and after 6 weeks of storage at 4°C. Low-fat and full-fat cheeses were prepared from nonhomogenized milk and from milk homogenized at 10.3 and 17.2 MPa. Analysis of the stained electron density distribution in image transforms of the protein matrix revealed that spacing patterns of regular structures with the dimensions of casein submicelles changed during storage, in most cases forming larger aggregates at longer average spacings, but there were no significant changes in total electron density between fresh and stored cheeses. These effects were related to proteolysis. Submicelles in low-fat cheeses homogenized at 17.2 MPa reorganized differently, a likely result of decreased proteolysis. Submicelles formed part of a new membrane around fat globules in cheeses made from homogenized milk, as previously observed. Changes in submicelle size and distribution could explain some of the differences in products made from homogenized and texture properties between fresh and stored Mozzarella cheeses, and between nonhomogenized milk. 0 1997 Published by Elsevier Science Ltd
INTRODUCTION
indicated that casein micelles dissociate upon removal of calcium to form noncolloidal protein complexes called submicelles (Schmidt, 1982; Farrell, 1988; Holt, 1992). This phenomenon has been observed in cheese and has been attributed to conversion of dicalcium paracaseinate to monocalcium phosphate in the curd (Kimura et al., 1992). Theoretical structures of casein submicelles have been proposed (Kumosinski et al., 1994, 1996). Reports on studies of submicelles in cheese are scarce. Age-related changes of the submicelle structure of cheeses such as Domiati (Abd El-Salam and El-Shibiny, 1973), Camembert, Harzer, and Tilsit (Knoop and Buchheim, 1980) have been investigated by transmission electron microscopy (TEM). TEM was also used to examine the casein particles in fresh string cheese that was prepared in a manner similar to that of Mozzarella (Kimura et al., 1992; Taneya et al., 1992). The patterns in the spacing between casein submicelles lend themselves to analysis using Fourier transform techniques, which has many applications such as characterizing textile patterns (Wood, 1990), but apparently has not been used to examine cheese structure. An earlier study from this laboratory dealt with microstructure and ultrastructure of low-fat Mozzarella made from nonhomogenized milk (Cooke et al., 1995). The purpose of this investigation was to examine changes during storage in the submicelle structure of low-fat and full-fat Mozzarella made from nonhomogenized and homogenized milk and relate them to factors affecting the characteristics of the cheese.
Mozzarella cheese is being consumed in the US at the rate of 3.4kg per capita, more than any cheese except Cheddar (Putnam and Allshouse, 1994). An understanding of the ultrastructure of Mozzarella and the factors that affect it will help improve the quality of this popular product. Although Mozzarella is considered to be an unripened cheese, it does undergo changes during storage (Kindstedt, 1993). The action of residual coagulant hydrolyzes asIcasein, and the resulting ccsl-casein peptide can be observed by urea-polyacrylamide gel electrophoresis (urea-PAGE) (Kindstedt, 1993) and by SDS-PAGE (Kiely et al., 1993; Tunick rt al., 1993a, b, 1995; Malin et al., 1995). Proteolysis of casein allows fat globules, which are initially dispersed in the protein matrix, to coalesce when the cheese is heated, thus increasing meltability and free oil (Kiely et al., 1993; Tunick, 1994). Proteolysis has been correlated also with decreases in the values of texture parameters such as hardness and springiness (Tunick et al., 1993a,b, 1995). The protein matrix of Mozzarella can be visualized by scanning electron microscopy (SEM), as shown in several reports on the microstructure of Mozzarella (Kalab, 1977; Taranto et al., 1979; Masi and Addeo, 1986; Paquet and Kalab, 1988; Kiely et ul., 1992 1993; Tunick et al., 1993a). Only two of these studies (Kiely et al., 1993; Tunick et al., 1993a) described changes during storage. Alterations in the casein matrix also may be present at the micelle level. Physical chemistry and electron microscopy have 149
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M. H. Tunick et al.
MATERIALS AND METHODS Cheese preparation Two batches of cheese were prepared each week on different days over a period of several months. Milk (22.7 kg) was standardized with cream (35% fat) or skim milk (0.14% fat) to 3.5% milk fat for full-fat Mozzarella or 1.0% milk fat for low-fat Mozzarella. The milk was then pasteurized at 63°C for 30min and, if desired for homogenized-milk cheese, homogenized in a two-stage Manton-Gaulin’ (Everett, MA, USA) homogenizer at 63°C. The second stage pressure was 3430 kPa, and the first stage pressure was 6870 kPa for the 10.3 MPa cheeses and 13740 kPa for the 17.2 MPa cheeses. Mozzarella cheese was prepared according to procedures previously described (Tunick et al., 1995). Cheese milk was held at 32.4”C and inoculated by direct addition of 125mL of CR7 starter culture (MarschallRh6ne Poulenc, Madison, WI, USA), described by the manufacturer as 50% Streptococcus salivarius, subsp. thermophilus and 50% Lactobacillus delbrueckii, subsp. bulgaricus. After the pH decreased 0.1 unit, 4.4g of #01034 single strength calf rennet (Chr. Hansen’s Laboratory, Milwaukee, WI, USA) were added. The curd was cut after 35 min and held for another 15 min. Low-fat cheeses were stirred at 32.4”C for 10min and held for 90min. The whey (pH 6.36.4) was then drained and the curd rinsed, covered with 32.4”C water for 30min, then cut into slabs. Full-fat cheeses were treated similarly except that the curd was heated to 459°C for 45 min and held for 50 min before draining and cutting. The rinsing step was omitted. When the pH of both cheeses decreased to 5.2-5.3, the slabs were covered with cheesecloth and stored at -20°C overnight. The next day, the curd was divided into eight parts and stretched and kneaded multidirectionally by hand for 7min in 70-80°C water. The samples were pressed cups 224 mL polyethylene measuring into approximately 80mm in diameter and 55 mm high, cooled, removed from the cups, brined for 2h in 23% salt solution, blotted dry with paper towels, and stored in vacuum-sealed pouches at 4°C for 6 weeks. Two cheeses were prepared each week over a period of several months according to a 3x2 factorial arrangement in a completely random design. The design parameters included two fat levels (low-fat and full-fat) and three homogenization pressures (0, 10.3, and 17.2 MPa). There were at least two replicates of each type of cheese. Samples for ultrastructural analysis were taken on the day of stretching and after 6 weeks of storage. The full-fat cheeses contained 47.049.5% moisture and 47.2-51.3% fat in dry matter (FDM), and the low-fat cheeses contained 54.1-56.8% moisture and 21.3-24.6% FDM (Tunick et al., 1993b). All of the cheeses had a final pH value of 5.2-5.3.
in a solution of 1% glutaraldehyde in 0.1 M sodium cacodylate at pH 7.2. The samples were washed in 0.1 M sodium cacodylate buffer, soaked in 2.0 mL of 2% osmium tetroxide-0.1 M sodium cacodylate buffer for 2 h, and rinsed with distilled water. The samples were then dehydrated in a graded series of ethanol solutions (50, 80, 90, 100 and loo%), transferred to propylene oxide, infiltrated overnight with 50% propylene oxide-50% epoxy resin embedding medium (Electron Microscopy Sciences, Fort Washington, PA, USA), and embedded in 100% epoxy resin. Sections 70-90nm thick were cut with a microtome, stained with lead citrate and uranyl acetate, and imaged with a Philips model CM 12 transmission electron microscope (Philips Electronics, Mahwah, NJ, USA) in the bright field image mode. Images were recorded on 8.26x 10.16 cm Kodak type 4489 photographic film (Eastman Kodak Co., Rochester, NY, USA) at an instrumental magnification of 60,000 x . Selected areas of trans-illuminated photographic negatives, equivalent to 0.85pm square, were digitized at a resolution of 1.6nm/pixel and frame averaged using a series 68 television camera (DAGE-MTI, Michigan City, IN, USA), synchronized with a DT2853 frame grabber (Data Translation, Marlboro, MA, USA), and controlled with Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA) (Kumosinski et al., 1996). Ten-frame averages were processed to flatten the background, and brightness and contrast were adjusted to span 256 gray levels. Regular (reciprocal) spacings of electron dense regions were measured from peaks of intensity in radial distribution plots of Fast Fourier Transforms, computed from averaged images. Total areas of electron density were determined by integration of binary images, derived from gray level contours (Cooke et al., 1995). Average spacings of near-neighbor distances in electron density related to subinicelles were estimated from peaks of intensity in radial distribution profiles of the transforms.
RESULTS AND DISCUSSION The effect of the sequence of digital image processing steps on image features is illustrated in Fig. 1 (a low-fat nonhomogenized Mozzarella at 0 weeks) and Fig. 2 (the same sample at 6 weeks). The size and organization of discrete stained, electron dense phases in cheese samples remain proceeding essentially the same from (A) photographic prints of the electron microscope negatives to (B) digitized images to (C) flattened, brightness-enhanced and contrast-enhanced images to (D) binary images. Cheeses from nonhomogenized milk
TEM Cubes of approximately 5 mm sides were removed from the interior of the cheese with a razor and fixed ‘Mention of brand or firm name does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.
Electron dense regions measuring 8-20nm in diameter were observed in transmission electron micrographic images of thin sections of the matrix of all cheese samples obtained on the day of stretching (Figs 1, 3A and 4A). The size range of these regions indicates they are casein submicelles (Schmidt and Buchheim, 1970, 1976; Buchheim and Welsch, 1973),
Casein submicelles in Mozzarella cheese storage
Fig. 1. Sequential processing steps on images of low-fat nonhomogenized Mozzarella on day of stretching. (B) Digitized image of photographic negative. (C) Flattened, brightness-enhanced. and contrast-enhanced. gray level segmentation. Scale bar corresponds to IOOnm.
which have been observed previously in string cheeses at pH 4.9-5.5 (Kimura et al., 1992; Taneya et crl., 1992). A full-fat nonhomogenized Mozzarella at 0 weeks, shown in Fig. 3A, clearly revealed these electron dense regions. After 6 weeks of storage, numerous electron dense regions, measuring 3040nm in diameter, were found (Fig. 3B). The same regions were observed in low-fat nonhomogenized samples (Figs 1 and 2). These large, dense regions closely resembled objects observed in other cheeses coagulated by calf rennet (Knoop and Buchheim, 1980). In each case, the 0- and 6-week samples contain a different distribution of electron density, relating to a change in organization of stained material at dimensions corresponding to submicelles. The average spacing for regions related to submicelles was around 17 nm at 0 weeks and increased to around 40nm by 6 weeks (Fig. 5A). Similar changes in average size and spacing of submicelles at 0 and 6 weeks were observed in both low-fat and full-fat samples. The O-week sample exhibits a shoulder between I5 and 30nm-‘, with the top of the shoulder corresponding to gray level around 90. The spacing is approximately 20 nrn-’ After 6 weeks, the shoulder of intensity has shifted to 30_50nm-‘. Despite the apparent differences in size and spacing of submicellar structures between cheeses at 0 and 6 weeks, the total area of electron density was comparable (Table l), suggesting that submicelles were reorganized during storage.
151
(A) Photographic print. (D) Binary image from
Proteolysis of x,~-casein in Mozzarella cheese results from the action of the coagulant, which can resist thermal inactivation if cooking and stretching conditions are extreme 1989). As not (Fox, previously reported (Tunick et al., 1993b), much of the a,,-casein in the low-fat and full-fat cheeses was degraded into the cc,,-I-casein peptide during the storage period. Although a,,-casein is primarily responsible for the structure of the protein matrix in cheese (Fox, 1989), over half the casein in Mozzarella includes CQ- and p-caseins, which are not as readily attacked by calf rennet in cheese (Fox et al., 1995). Our earlier studies showed that the percentages of c(,~- and /I-casein in low-fat and fullfat Mozzarella coagulated by calf rennet do not decrease significantly during storage (Tunick et al., 1993a,b, 1995). Therefore, the images suggest that the loss of significant amounts of x,,-casein and concurrent formation of peptides may facilitate rearrangement of the remaining submicelle particles into a pattern of clusters open spaces, possibly and through hydrophobic or electrostatic interactions. Molecular modeling of a,,-I-casein peptides ranging from 9 to 69 residues in length shows that they have a strong tendency to become more compact (Malin and Brown, 1995) which could also facilitate clustering. In addition, these compact peptides may replace fat globules in maintaining a looser density of the protein matrix.
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Fig. 2. Sequential processing steps on images of low-fat nonhomogenized Mozzarella after 6 weeks of refrigerated storage. (A) Photographic print. (B) Digitized image of photographic negative. (C) Flattened, brightness-enhanced, and contrast-enhanced. (D) Binary image from gray level segmentation. Scale bar corresponds to 100 nm.
Cheese made from homogenized milk The number and size of the fat droplets in the cheeses made from homogenized milk were related to homogenization pressure. The fresh cheeses made from milk homogenized at 10.3 MPa contained submicelles of the same diameter as observed with the nonhomogenized cheeses, along with fat droplets measuring 60400nm in diameter. The droplets were not spherical due to stretching of the curd. A low-fat 10.3 MPa Mozzarella at 0 weeks, containing a uniform distribution of regular submicellar regions, is shown in Fig. 3C. At 6 weeks, the average size of the regions increased (Fig. 3D), and the average spacing increased from 15 nm at 0 weeks to 35-40 nm at 6 weeks (Fig. 5B). The integrated areas occupied by submicelles in these projected images again remained constant during the storage period (Table 1). The membrane border was visible in many of the droplets. The diameter of fat globules increases to < 1 pm when milk is homogenized (Walstra, 197.5), and the surface area of the fat increases 4- to 6-fold as a result (McPherson et al., 1984). TEM micrographs demonstrate the partial layer of casein particles first reported by Henstra and Schmidt (1970) along with some of the original fat globule membrane first observed by Keenan et al. (1988). The clustering of particles at 6 weeks (Fig. 3D) and increase in average spacing (Fig. 4B) were again
evident and were apparently unaffected by homogenization. The full-fat cheeses made from milk homogenized at 17.2 MPa contained small regions of electron density that were of l&12nm diameter at 0 weeks (Fig. 4A) and 30-40nm at 6 weeks (Fig. 4B). The average spacing increased from around 18 nm at 0 weeks to approximately 40nm at 6 weeks (Fig. 6A). The total integrated areas identified with submicelles before and after storage were comparable (Table 1). The ultrastructure of low-fat cheeses homogenized at 17.2MPa resembled the other cheeses at 0 weeks with regular small electron dense regions around lo-20 nm diameter (Fig. 4C), but at 6 weeks the submicelles were not uniformly reorganized into large regions (Fig. 4D), and their distribution contained a broad range of spacings (Fig. 6B). These effects may be related to retardation of proteolysis in both low-fat and full-fat cheeses at higher homogenization pressures (Malin et effect of al., 1993, although the precise homogenization on cheese proteins is not yet understood. The fat droplets had diameters ranging from 60_280nm, and appeared to be coated by membranes.
Effects on physical properties Hardness and meltability of Mozzarella are strongly correlated with homogenization pressure (Tunick et al., 1993b), an indication that fat-casein interactions
Casein submicelles in Mozzarella cheese storage
Fig. 3. Digitized image areas of electron microscope negatives. Dense (black) areas represent electron dense clusters separated electron-lucent areas; larger lucent (white) circular areas represent fat droplets. (A) Full-fat nonhomogenized cheese on day stretching. (B) Same, after 6 weeks of refrigerated storage. (C) Low-fat Mozzarella cheese made from milk homogenized 10.3 MPa, on day of stretching. (D) Same, after 6 weeks of refrigerated storage. Ruled grating corresponds to a scaled spacing 16.7 nm.
Table 1. Total Integrated Areas of Electron Dense Regions in Mozzarella Cheeses During Refrigerated Storage. Average of Six Determinations Homogenization Pressure (MPa) 0
10.3 17.2
(% of total area) 0 Weeks
6 Weeks
44 48 47
48 52 48
take place in cheese made from homogenized milk. Homogenization breaks up fat globules into smaller entities and ruptures the fat globule membrane. The casein submicelles that become incorporated into a new membrane material surrounding fat globules in Mozzarella prepared from homogenized milk interact with the casein matrix, thereby allowing the fat to become part of the matrix (Lelievre et al., 1990). The insulation of the smaller fat droplets in the matrix prevents the fat from melting easily (Tunick, 1994), decreasing meltability. The protein-protein interactions between the new membrane and the matrix reduce flexibility of the casein, making the cheese harder. The ultrastructure of the casein submicelles in lowfat and full-fat Mozzarella cheeses made from
153
by of at of
nonhomogenized or homogenized milk was more porous after 6 weeks of storage than at the outset, which should lead to fewer physical interactions between casein micelles and result in a weaker casein matrix. This effect has been observed in other cheeses such as Domiati, in which TEM micrographs have shown that casein aggregates disintegrate with proteolysis into a loose structure, resulting in a cheese with a weak body (Abd El-Salam and ElShibiny, 1973). As mentioned previously, a significant decrease in the strength of the matrix would also allow the fat globules to coalesce, a phenomenon observed by SEM (Kiely et al., 1993; Tunick et al., 1993a). Such changes would make the cheese more compressible and meltable and less elastic. Texture profile studies have shown that hardness and springiness values in Mozzarella decrease during storage; other tests revealed that elastic and viscous moduli decrease and Schreiber meltability values increase. All these effects have been correlated with proteolysis of cr,t-casein (Tunick et al., 1993a,b, 1995). It therefore appears that the proteolytic breakdown of cr,,-casein in Mozzarella cheese leads to a reorganization and weakening of the protein matrix, resulting in a softer, less elastic, more meltable cheese. The protein matrix in cheese is probably a more fluid environment than is generally believed.
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250 1
B
40 20
10 nm
-
0 weeks
5
-l ------ 6 weeks
Fig. 5. Radial distribution profiles of reciprocal spacings in electron density in Fast Fourier Transforms of protein matrix images of Mozzarella cheeses made from nonhomogenized and homogenized milk after 0 and 6 weeks of storage. Relative intentsity is measured in gray levels. Spacings are measured in nm . (A) Nonhomogenized full-fat cheese. (B) Low-fat cheese homogenized at 10.3 MPa.
40 23
10 nm
Fig. 4. Digitized image areas of electron microscope tives. (A) Full-fat Mozzarella cheese made from milk genized at 17.2 MPa, on day of stretching. (B) Same, weeks of refrigerated storage. (C) Low-fat Mozzarella made from milk homogenized at 17.2 MPa, on day of ing. (D) Same, after 6 weeks of refrigerated storage. grating corresponds to a scaled spacing of 16.7 nm.
negahomoafter 6 cheese stretchRuled
-
Oweeks
5
-l ------ 6 weeks
Fig. 6. Radial distribution profiles of reciprocal spacings in electron density in Fast Fourier Transforms of protein matrix images of Mozzarella cheeses made from milk homogenized at 17.2MPa. (A) Full-fat cheese at 0 and 6 weeks. (B) Low-fat cheese at 0 and 6 weeks.
Cusein .suhmicelle.s in Mozxrella
ACKNOWLEDGEMENTS The authors thank Dr Harold M. Farrell, Jr. for helpful discussions, Joe Uknalis for TEM sample preparation, Melinda Yin for image analyses, and Peggy Greb for photographic services.
REFERENCES Abd El-Salam, M.H. and El-Shibiny, S. (1973) An electronmicroscope study of the structure of Domiati cheese. J. Dairv Res., 40, 113. I 15. Buchheim, W. and Welsch, U. (1973) Evidence for the submicellar composition of casein micelles on the basis of electron microscopical studies. Neth. Milk Da+), J., 27, 163-180. Cooke, P.H., Tunick, M.H., Malin, E.L., Smith, P.W. and Holsinger, V.H. (I 995). Electron density patterns in low-fat Mozzarella cheeses during refrigerated storage. In Chemistry ~f.Strll(.ture/Function Relationships in Cheese, eds E.L. Malin and M.H. Tunick. Plenum Press, New York, pp. 31 I-320. Farrell, H.M., Jr. (1988). Physical equilibria: proteins. In Fundumenta1.v oj’ Dairy Chemistry, ed. N.P. Wong. Van Nostrand Reinhold Co., New York, pp. 461-510. Fox, P.F. (1989) Proteolysis during cheese manufacture and ripening. J. Dairy Sri., 72, 1379-1400. Fox, P.F., Singh, T.K. and McSweeney, P.L.H. (1995). Biogenesis of flavour compounds in cheese. In Chemistry ?f‘ Struc.turelFut?~tion Relationships in Cheese, eds E. L. Malin & M.H. Tunick. Plenum Press. New York, pp. 5998. Henstra, S. and Schmidt, D.G. (1970) On the structure of the fat-protein complex in homogenized cow’s milk. Neth. Milk Dairy J., 24, 45-5 I. Holt. C. (1992) Structure and stability of bovine casein micelles. Adv. Prot. Chem., 43, 63-I 13. Kalab, M. (1977) Milk gel structure. VI. Cheese texture and microstructure. Milchwissenschqft, 32, 449458. Keenan, T.W.. Mather, I.H. and Dylewski, D.P. (1988). Physical equilibria: lipid phase. In Fundamentals of Da&y Chemistr,,, ed. N.P. Wong. Van Nostrand Reinhold Co., New York, pp. 51 l-582. Kiely. L.J., Kindstedt, P.S., Hendricks, G.M., Levis, J.E., Yun. J.J. and Barbano, D.M. (1992) Effect of draw pH on the development of curd structure during the manufacture of Mozzarella cheese. Food Struct., 11, 217-224. Kiely, L.J., Kindstedt, P.S., Hendricks, G.M., Levis, J.E.. Yun, J.J. and Barbano, D.M. (1993) Age related changes in the microstructure of Mozzarella cheese. Food Struct., 12, 13-20. Kimura, T., Sagara. Y., Fukushima, M. and Taneya, S. (1992) Effect of pH on submicroscopic structure of String cheese. Milch,cissenschqft. 47, 547-552. Kindstedt, P.S. (1993) Effect of manufacturing factors, composition, and proteolysis on the functional characteristics of Mozzarella cheese. Crit. Revs. Food Sci. Nutr., 33, 167-187. Knoop, A.-M. and Buchheim, W. (1980) The different development of the structure in Harzer-. Tilsit- and Camembert-cheese during ripening. Milch~~issenschqft, 35, 482-488. Kumosinski, T.F., King, G. and Farrell, H.M. Jr. (1994) An energy-minimized casein submicelle working model. J. Protein Chem., 13, 681-700. Kumosinski, T.F., Uknalis, J.J., Cooke, P.H. and Farrell,
cheese storage
155
H.M. Jr. ( 1996) Correlation of refined models for casein submicelles with electron microscopic studies of casein. Lehensm.- Wiss. u.-Technol., 29, 326-333. Lelievre, J.R., Shaker, R. and Taylor, M.W. (1990) The role of homogenization in the manufacture of Halloumi and Mozzarella cheese from recombined milk. J. Sot. Dairy Technol., 43, 2 l-29. Malin, E.L. and Brown, E.M. (1995). Influence of casein peptide conformations on textural properties of cheese. In C’hemi.rlr!, oj’Structure/Function Relationships in Cheese, eds E.L. Malin and M.H. Tunick. Plenum Press, New York, pp. 303-3 IO. Malin. E.L.. Tunick, M.H., Smith, P.W. and Holsinger, V.H. (1995). Inhibition of proteolysis in Mozzarella cheese prepared from homogenized milk. In Chemistry of’ Structure/Function Relationships in Cheese, eds E.L. Malin and M.H. Tunick. Plenum Press, New York. pp. 237-246. Masi, P. and Addeo, F. (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, 2 17 -229. McPherson. A.V.Dash, M.C.Kitchen. B.J. (1989) Isolation and composition of milk fat globule membrane material. II. From homogenized and ultra heat treated milks. J. Dairy Res., 51, 289-297. Paquet, A. and Kalab, M. (1988) Amino acid composition and structure of cheese baked as a pizza ingredient in conventional and microwave ovens. Food Microstructure, 7, 93.-103. Putnam, J.J. and Allshouse, J.E. (1994). Food Consumption, Prices, and Espenditures, 1970-93. Statistical Bulletin SBY15. US Government Printing Office, Washington, DC. Schmidt, D.G. (1982). Associations of caseins and casein micelle structure. In Developments in Daity Chemistr_yl, ed. P.F. Fox. Applied Science Publishers, Essex, pp. 61-93. Schmidt, D.G. and Buchheim, W. (1970) Elektronenmikroskopische untersuchung der feinstruktur von caseimicellen in kuhmilch. Mi/chwissensch#, 25, 596-600. Schmidt, D.G. and Buchheim, W. (1976) Particle size distribution in casein solutions. Neth. Milk Dairy J., 30, 17-28. Taneya, S., Izutsu, T., Kimura, T. and Shioya, T. (1992) Structure and rheology of string cheese. Food Struct., 11, 61-71. Taranto, M.V., Wan, P.J., Chen, S.L. and Rhee, K.C. (1979) Morphological, ultrastructural and rheological characterization of Cheddar and Mozzarella cheese. Scanning Electron Microsc., 1979, 273-278. Tunick, M.H. (1994) Effects of homogenization and proteolysis on free oil in Mozzarella cheese. J. Dairy Sci., 77, 2487-2493. Tunick, M.H., Mackey, K.L., Shieh, J.J., Smith, P.W., Cooke, P. and Malin, E.L. (1993a) Rheology and microstructure of low-fat Mozzarella cheese. int. Dairy J., 3, 649-662. Tunick. M.H.. Malin, E.L., Smith, P.W., Shieh, J.J., Sullivan, B.C.. Mackey, K.L. and Holsinger, V.H. (1993b) Proteolysis and rheology of low fat and full fat Mozzarella cheeses prepared from homogenized milk. J. Dairy Sri., 76, 36213628. Tunick, M.H., Malin, E.L.. Smith, P.W. and Holsinger, V.H. (1995) Effects of skim milk homogenization on proteolysis and rheology of Mozzarella cheese. Int. Duiry J., 4, 483491. Walstra, P. (1975) Effect of homogenization on the fat globule size distribution in milk. Neth. Mi/k Dairy J., 29, 279-294. Wood, E.J. (1990) Applying Fourier and associated transforms to pattern characterization in textiles. Textile Res. J., 60, 2 12-220.
ELSEVIER
IHI. Duirj~ Journtrl7 ( 1997) I49- I55 1997 Published by Elsevier Science Ltd Printed in Great 0958-6946:97/$17.00
SO9S8-6946(96)00057-X
Britain + 0.00
Reorganization of Casein Submicelles in Mozzarella Cheese During Storage Michael H. Tunick, Peter H. Cooke, Edyth L. Malin, Philip W. Smith and V. H. Holsinger United Stutes Department
of’ Agriculture, Agricultural
(Received
Research Serviw, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmnor. PA 19038, USA
20 October
1995; accepted
16 October
1996)
ABSTRACT To gain a better understanding of the factors involved in textural changes in Mozzarella cheese during storage, electron microscopic imaging was used to compare the ultrastructure of the protein matrix on the day of stretching (0 weeks) and after 6 weeks of storage at 4°C. Low-fat and full-fat cheeses were prepared from nonhomogenized milk and from milk homogenized at 10.3 and 17.2 MPa. Analysis of the stained electron density distribution in image transforms of the protein matrix revealed that spacing patterns of regular structures with the dimensions of casein submicelles changed during storage, in most cases forming larger aggregates at longer average spacings, but there were no significant changes in total electron density between fresh and stored cheeses. These effects were related to proteolysis. Submicelles in low-fat cheeses homogenized at 17.2 MPa reorganized differently, a likely result of decreased proteolysis. Submicelles formed part of a new membrane around fat globules in cheeses made from homogenized milk, as previously observed. Changes in submicelle size and distribution could explain some of the differences in products made from homogenized and texture properties between fresh and stored Mozzarella cheeses, and between nonhomogenized milk. 0 1997 Published by Elsevier Science Ltd
INTRODUCTION
indicated that casein micelles dissociate upon removal of calcium to form noncolloidal protein complexes called submicelles (Schmidt, 1982; Farrell, 1988; Holt, 1992). This phenomenon has been observed in cheese and has been attributed to conversion of dicalcium paracaseinate to monocalcium phosphate in the curd (Kimura et al., 1992). Theoretical structures of casein submicelles have been proposed (Kumosinski et al., 1994, 1996). Reports on studies of submicelles in cheese are scarce. Age-related changes of the submicelle structure of cheeses such as Domiati (Abd El-Salam and El-Shibiny, 1973), Camembert, Harzer, and Tilsit (Knoop and Buchheim, 1980) have been investigated by transmission electron microscopy (TEM). TEM was also used to examine the casein particles in fresh string cheese that was prepared in a manner similar to that of Mozzarella (Kimura et al., 1992; Taneya et al., 1992). The patterns in the spacing between casein submicelles lend themselves to analysis using Fourier transform techniques, which has many applications such as characterizing textile patterns (Wood, 1990), but apparently has not been used to examine cheese structure. An earlier study from this laboratory dealt with microstructure and ultrastructure of low-fat Mozzarella made from nonhomogenized milk (Cooke et al., 1995). The purpose of this investigation was to examine changes during storage in the submicelle structure of low-fat and full-fat Mozzarella made from nonhomogenized and homogenized milk and relate them to factors affecting the characteristics of the cheese.
Mozzarella cheese is being consumed in the US at the rate of 3.4kg per capita, more than any cheese except Cheddar (Putnam and Allshouse, 1994). An understanding of the ultrastructure of Mozzarella and the factors that affect it will help improve the quality of this popular product. Although Mozzarella is considered to be an unripened cheese, it does undergo changes during storage (Kindstedt, 1993). The action of residual coagulant hydrolyzes asIcasein, and the resulting ccsl-casein peptide can be observed by urea-polyacrylamide gel electrophoresis (urea-PAGE) (Kindstedt, 1993) and by SDS-PAGE (Kiely et al., 1993; Tunick rt al., 1993a, b, 1995; Malin et al., 1995). Proteolysis of casein allows fat globules, which are initially dispersed in the protein matrix, to coalesce when the cheese is heated, thus increasing meltability and free oil (Kiely et al., 1993; Tunick, 1994). Proteolysis has been correlated also with decreases in the values of texture parameters such as hardness and springiness (Tunick et al., 1993a,b, 1995). The protein matrix of Mozzarella can be visualized by scanning electron microscopy (SEM), as shown in several reports on the microstructure of Mozzarella (Kalab, 1977; Taranto et al., 1979; Masi and Addeo, 1986; Paquet and Kalab, 1988; Kiely et ul., 1992 1993; Tunick et al., 1993a). Only two of these studies (Kiely et al., 1993; Tunick et al., 1993a) described changes during storage. Alterations in the casein matrix also may be present at the micelle level. Physical chemistry and electron microscopy have 149
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MATERIALS AND METHODS Cheese preparation Two batches of cheese were prepared each week on different days over a period of several months. Milk (22.7 kg) was standardized with cream (35% fat) or skim milk (0.14% fat) to 3.5% milk fat for full-fat Mozzarella or 1.0% milk fat for low-fat Mozzarella. The milk was then pasteurized at 63°C for 30min and, if desired for homogenized-milk cheese, homogenized in a two-stage Manton-Gaulin’ (Everett, MA, USA) homogenizer at 63°C. The second stage pressure was 3430 kPa, and the first stage pressure was 6870 kPa for the 10.3 MPa cheeses and 13740 kPa for the 17.2 MPa cheeses. Mozzarella cheese was prepared according to procedures previously described (Tunick et al., 1995). Cheese milk was held at 32.4”C and inoculated by direct addition of 125mL of CR7 starter culture (MarschallRh6ne Poulenc, Madison, WI, USA), described by the manufacturer as 50% Streptococcus salivarius, subsp. thermophilus and 50% Lactobacillus delbrueckii, subsp. bulgaricus. After the pH decreased 0.1 unit, 4.4g of #01034 single strength calf rennet (Chr. Hansen’s Laboratory, Milwaukee, WI, USA) were added. The curd was cut after 35 min and held for another 15 min. Low-fat cheeses were stirred at 32.4”C for 10min and held for 90min. The whey (pH 6.36.4) was then drained and the curd rinsed, covered with 32.4”C water for 30min, then cut into slabs. Full-fat cheeses were treated similarly except that the curd was heated to 459°C for 45 min and held for 50 min before draining and cutting. The rinsing step was omitted. When the pH of both cheeses decreased to 5.2-5.3, the slabs were covered with cheesecloth and stored at -20°C overnight. The next day, the curd was divided into eight parts and stretched and kneaded multidirectionally by hand for 7min in 70-80°C water. The samples were pressed cups 224 mL polyethylene measuring into approximately 80mm in diameter and 55 mm high, cooled, removed from the cups, brined for 2h in 23% salt solution, blotted dry with paper towels, and stored in vacuum-sealed pouches at 4°C for 6 weeks. Two cheeses were prepared each week over a period of several months according to a 3x2 factorial arrangement in a completely random design. The design parameters included two fat levels (low-fat and full-fat) and three homogenization pressures (0, 10.3, and 17.2 MPa). There were at least two replicates of each type of cheese. Samples for ultrastructural analysis were taken on the day of stretching and after 6 weeks of storage. The full-fat cheeses contained 47.049.5% moisture and 47.2-51.3% fat in dry matter (FDM), and the low-fat cheeses contained 54.1-56.8% moisture and 21.3-24.6% FDM (Tunick et al., 1993b). All of the cheeses had a final pH value of 5.2-5.3.
in a solution of 1% glutaraldehyde in 0.1 M sodium cacodylate at pH 7.2. The samples were washed in 0.1 M sodium cacodylate buffer, soaked in 2.0 mL of 2% osmium tetroxide-0.1 M sodium cacodylate buffer for 2 h, and rinsed with distilled water. The samples were then dehydrated in a graded series of ethanol solutions (50, 80, 90, 100 and loo%), transferred to propylene oxide, infiltrated overnight with 50% propylene oxide-50% epoxy resin embedding medium (Electron Microscopy Sciences, Fort Washington, PA, USA), and embedded in 100% epoxy resin. Sections 70-90nm thick were cut with a microtome, stained with lead citrate and uranyl acetate, and imaged with a Philips model CM 12 transmission electron microscope (Philips Electronics, Mahwah, NJ, USA) in the bright field image mode. Images were recorded on 8.26x 10.16 cm Kodak type 4489 photographic film (Eastman Kodak Co., Rochester, NY, USA) at an instrumental magnification of 60,000 x . Selected areas of trans-illuminated photographic negatives, equivalent to 0.85pm square, were digitized at a resolution of 1.6nm/pixel and frame averaged using a series 68 television camera (DAGE-MTI, Michigan City, IN, USA), synchronized with a DT2853 frame grabber (Data Translation, Marlboro, MA, USA), and controlled with Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA) (Kumosinski et al., 1996). Ten-frame averages were processed to flatten the background, and brightness and contrast were adjusted to span 256 gray levels. Regular (reciprocal) spacings of electron dense regions were measured from peaks of intensity in radial distribution plots of Fast Fourier Transforms, computed from averaged images. Total areas of electron density were determined by integration of binary images, derived from gray level contours (Cooke et al., 1995). Average spacings of near-neighbor distances in electron density related to subinicelles were estimated from peaks of intensity in radial distribution profiles of the transforms.
RESULTS AND DISCUSSION The effect of the sequence of digital image processing steps on image features is illustrated in Fig. 1 (a low-fat nonhomogenized Mozzarella at 0 weeks) and Fig. 2 (the same sample at 6 weeks). The size and organization of discrete stained, electron dense phases in cheese samples remain proceeding essentially the same from (A) photographic prints of the electron microscope negatives to (B) digitized images to (C) flattened, brightness-enhanced and contrast-enhanced images to (D) binary images. Cheeses from nonhomogenized milk
TEM Cubes of approximately 5 mm sides were removed from the interior of the cheese with a razor and fixed ‘Mention of brand or firm name does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.
Electron dense regions measuring 8-20nm in diameter were observed in transmission electron micrographic images of thin sections of the matrix of all cheese samples obtained on the day of stretching (Figs 1, 3A and 4A). The size range of these regions indicates they are casein submicelles (Schmidt and Buchheim, 1970, 1976; Buchheim and Welsch, 1973),
Casein submicelles in Mozzarella cheese storage
Fig. 1. Sequential processing steps on images of low-fat nonhomogenized Mozzarella on day of stretching. (B) Digitized image of photographic negative. (C) Flattened, brightness-enhanced. and contrast-enhanced. gray level segmentation. Scale bar corresponds to IOOnm.
which have been observed previously in string cheeses at pH 4.9-5.5 (Kimura et al., 1992; Taneya et crl., 1992). A full-fat nonhomogenized Mozzarella at 0 weeks, shown in Fig. 3A, clearly revealed these electron dense regions. After 6 weeks of storage, numerous electron dense regions, measuring 3040nm in diameter, were found (Fig. 3B). The same regions were observed in low-fat nonhomogenized samples (Figs 1 and 2). These large, dense regions closely resembled objects observed in other cheeses coagulated by calf rennet (Knoop and Buchheim, 1980). In each case, the 0- and 6-week samples contain a different distribution of electron density, relating to a change in organization of stained material at dimensions corresponding to submicelles. The average spacing for regions related to submicelles was around 17 nm at 0 weeks and increased to around 40nm by 6 weeks (Fig. 5A). Similar changes in average size and spacing of submicelles at 0 and 6 weeks were observed in both low-fat and full-fat samples. The O-week sample exhibits a shoulder between I5 and 30nm-‘, with the top of the shoulder corresponding to gray level around 90. The spacing is approximately 20 nrn-’ After 6 weeks, the shoulder of intensity has shifted to 30_50nm-‘. Despite the apparent differences in size and spacing of submicellar structures between cheeses at 0 and 6 weeks, the total area of electron density was comparable (Table l), suggesting that submicelles were reorganized during storage.
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(A) Photographic print. (D) Binary image from
Proteolysis of x,~-casein in Mozzarella cheese results from the action of the coagulant, which can resist thermal inactivation if cooking and stretching conditions are extreme 1989). As not (Fox, previously reported (Tunick et al., 1993b), much of the a,,-casein in the low-fat and full-fat cheeses was degraded into the cc,,-I-casein peptide during the storage period. Although a,,-casein is primarily responsible for the structure of the protein matrix in cheese (Fox, 1989), over half the casein in Mozzarella includes CQ- and p-caseins, which are not as readily attacked by calf rennet in cheese (Fox et al., 1995). Our earlier studies showed that the percentages of c(,~- and /I-casein in low-fat and fullfat Mozzarella coagulated by calf rennet do not decrease significantly during storage (Tunick et al., 1993a,b, 1995). Therefore, the images suggest that the loss of significant amounts of x,,-casein and concurrent formation of peptides may facilitate rearrangement of the remaining submicelle particles into a pattern of clusters open spaces, possibly and through hydrophobic or electrostatic interactions. Molecular modeling of a,,-I-casein peptides ranging from 9 to 69 residues in length shows that they have a strong tendency to become more compact (Malin and Brown, 1995) which could also facilitate clustering. In addition, these compact peptides may replace fat globules in maintaining a looser density of the protein matrix.
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Fig. 2. Sequential processing steps on images of low-fat nonhomogenized Mozzarella after 6 weeks of refrigerated storage. (A) Photographic print. (B) Digitized image of photographic negative. (C) Flattened, brightness-enhanced, and contrast-enhanced. (D) Binary image from gray level segmentation. Scale bar corresponds to 100 nm.
Cheese made from homogenized milk The number and size of the fat droplets in the cheeses made from homogenized milk were related to homogenization pressure. The fresh cheeses made from milk homogenized at 10.3 MPa contained submicelles of the same diameter as observed with the nonhomogenized cheeses, along with fat droplets measuring 60400nm in diameter. The droplets were not spherical due to stretching of the curd. A low-fat 10.3 MPa Mozzarella at 0 weeks, containing a uniform distribution of regular submicellar regions, is shown in Fig. 3C. At 6 weeks, the average size of the regions increased (Fig. 3D), and the average spacing increased from 15 nm at 0 weeks to 35-40 nm at 6 weeks (Fig. 5B). The integrated areas occupied by submicelles in these projected images again remained constant during the storage period (Table 1). The membrane border was visible in many of the droplets. The diameter of fat globules increases to < 1 pm when milk is homogenized (Walstra, 197.5), and the surface area of the fat increases 4- to 6-fold as a result (McPherson et al., 1984). TEM micrographs demonstrate the partial layer of casein particles first reported by Henstra and Schmidt (1970) along with some of the original fat globule membrane first observed by Keenan et al. (1988). The clustering of particles at 6 weeks (Fig. 3D) and increase in average spacing (Fig. 4B) were again
evident and were apparently unaffected by homogenization. The full-fat cheeses made from milk homogenized at 17.2 MPa contained small regions of electron density that were of l&12nm diameter at 0 weeks (Fig. 4A) and 30-40nm at 6 weeks (Fig. 4B). The average spacing increased from around 18 nm at 0 weeks to approximately 40nm at 6 weeks (Fig. 6A). The total integrated areas identified with submicelles before and after storage were comparable (Table 1). The ultrastructure of low-fat cheeses homogenized at 17.2MPa resembled the other cheeses at 0 weeks with regular small electron dense regions around lo-20 nm diameter (Fig. 4C), but at 6 weeks the submicelles were not uniformly reorganized into large regions (Fig. 4D), and their distribution contained a broad range of spacings (Fig. 6B). These effects may be related to retardation of proteolysis in both low-fat and full-fat cheeses at higher homogenization pressures (Malin et effect of al., 1993, although the precise homogenization on cheese proteins is not yet understood. The fat droplets had diameters ranging from 60_280nm, and appeared to be coated by membranes.
Effects on physical properties Hardness and meltability of Mozzarella are strongly correlated with homogenization pressure (Tunick et al., 1993b), an indication that fat-casein interactions
Casein submicelles in Mozzarella cheese storage
Fig. 3. Digitized image areas of electron microscope negatives. Dense (black) areas represent electron dense clusters separated electron-lucent areas; larger lucent (white) circular areas represent fat droplets. (A) Full-fat nonhomogenized cheese on day stretching. (B) Same, after 6 weeks of refrigerated storage. (C) Low-fat Mozzarella cheese made from milk homogenized 10.3 MPa, on day of stretching. (D) Same, after 6 weeks of refrigerated storage. Ruled grating corresponds to a scaled spacing 16.7 nm.
Table 1. Total Integrated Areas of Electron Dense Regions in Mozzarella Cheeses During Refrigerated Storage. Average of Six Determinations Homogenization Pressure (MPa) 0
10.3 17.2
(% of total area) 0 Weeks
6 Weeks
44 48 47
48 52 48
take place in cheese made from homogenized milk. Homogenization breaks up fat globules into smaller entities and ruptures the fat globule membrane. The casein submicelles that become incorporated into a new membrane material surrounding fat globules in Mozzarella prepared from homogenized milk interact with the casein matrix, thereby allowing the fat to become part of the matrix (Lelievre et al., 1990). The insulation of the smaller fat droplets in the matrix prevents the fat from melting easily (Tunick, 1994), decreasing meltability. The protein-protein interactions between the new membrane and the matrix reduce flexibility of the casein, making the cheese harder. The ultrastructure of the casein submicelles in lowfat and full-fat Mozzarella cheeses made from
153
by of at of
nonhomogenized or homogenized milk was more porous after 6 weeks of storage than at the outset, which should lead to fewer physical interactions between casein micelles and result in a weaker casein matrix. This effect has been observed in other cheeses such as Domiati, in which TEM micrographs have shown that casein aggregates disintegrate with proteolysis into a loose structure, resulting in a cheese with a weak body (Abd El-Salam and ElShibiny, 1973). As mentioned previously, a significant decrease in the strength of the matrix would also allow the fat globules to coalesce, a phenomenon observed by SEM (Kiely et al., 1993; Tunick et al., 1993a). Such changes would make the cheese more compressible and meltable and less elastic. Texture profile studies have shown that hardness and springiness values in Mozzarella decrease during storage; other tests revealed that elastic and viscous moduli decrease and Schreiber meltability values increase. All these effects have been correlated with proteolysis of cr,t-casein (Tunick et al., 1993a,b, 1995). It therefore appears that the proteolytic breakdown of cr,,-casein in Mozzarella cheese leads to a reorganization and weakening of the protein matrix, resulting in a softer, less elastic, more meltable cheese. The protein matrix in cheese is probably a more fluid environment than is generally believed.
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250 1
B
40 20
10 nm
-
0 weeks
5
-l ------ 6 weeks
Fig. 5. Radial distribution profiles of reciprocal spacings in electron density in Fast Fourier Transforms of protein matrix images of Mozzarella cheeses made from nonhomogenized and homogenized milk after 0 and 6 weeks of storage. Relative intentsity is measured in gray levels. Spacings are measured in nm . (A) Nonhomogenized full-fat cheese. (B) Low-fat cheese homogenized at 10.3 MPa.
40 23
10 nm
Fig. 4. Digitized image areas of electron microscope tives. (A) Full-fat Mozzarella cheese made from milk genized at 17.2 MPa, on day of stretching. (B) Same, weeks of refrigerated storage. (C) Low-fat Mozzarella made from milk homogenized at 17.2 MPa, on day of ing. (D) Same, after 6 weeks of refrigerated storage. grating corresponds to a scaled spacing of 16.7 nm.
negahomoafter 6 cheese stretchRuled
-
Oweeks
5
-l ------ 6 weeks
Fig. 6. Radial distribution profiles of reciprocal spacings in electron density in Fast Fourier Transforms of protein matrix images of Mozzarella cheeses made from milk homogenized at 17.2MPa. (A) Full-fat cheese at 0 and 6 weeks. (B) Low-fat cheese at 0 and 6 weeks.
Cusein .suhmicelle.s in Mozxrella
ACKNOWLEDGEMENTS The authors thank Dr Harold M. Farrell, Jr. for helpful discussions, Joe Uknalis for TEM sample preparation, Melinda Yin for image analyses, and Peggy Greb for photographic services.
REFERENCES Abd El-Salam, M.H. and El-Shibiny, S. (1973) An electronmicroscope study of the structure of Domiati cheese. J. Dairv Res., 40, 113. I 15. Buchheim, W. and Welsch, U. (1973) Evidence for the submicellar composition of casein micelles on the basis of electron microscopical studies. Neth. Milk Da+), J., 27, 163-180. Cooke, P.H., Tunick, M.H., Malin, E.L., Smith, P.W. and Holsinger, V.H. (I 995). Electron density patterns in low-fat Mozzarella cheeses during refrigerated storage. In Chemistry ~f.Strll(.ture/Function Relationships in Cheese, eds E.L. Malin and M.H. Tunick. Plenum Press, New York, pp. 31 I-320. Farrell, H.M., Jr. (1988). Physical equilibria: proteins. In Fundumenta1.v oj’ Dairy Chemistry, ed. N.P. Wong. Van Nostrand Reinhold Co., New York, pp. 461-510. Fox, P.F. (1989) Proteolysis during cheese manufacture and ripening. J. Dairy Sri., 72, 1379-1400. Fox, P.F., Singh, T.K. and McSweeney, P.L.H. (1995). Biogenesis of flavour compounds in cheese. In Chemistry ?f‘ Struc.turelFut?~tion Relationships in Cheese, eds E. L. Malin & M.H. Tunick. Plenum Press. New York, pp. 5998. Henstra, S. and Schmidt, D.G. (1970) On the structure of the fat-protein complex in homogenized cow’s milk. Neth. Milk Dairy J., 24, 45-5 I. Holt. C. (1992) Structure and stability of bovine casein micelles. Adv. Prot. Chem., 43, 63-I 13. Kalab, M. (1977) Milk gel structure. VI. Cheese texture and microstructure. Milchwissenschqft, 32, 449458. Keenan, T.W.. Mather, I.H. and Dylewski, D.P. (1988). Physical equilibria: lipid phase. In Fundamentals of Da&y Chemistr,,, ed. N.P. Wong. Van Nostrand Reinhold Co., New York, pp. 51 l-582. Kiely. L.J., Kindstedt, P.S., Hendricks, G.M., Levis, J.E., Yun. J.J. and Barbano, D.M. (1992) Effect of draw pH on the development of curd structure during the manufacture of Mozzarella cheese. Food Struct., 11, 217-224. Kiely, L.J., Kindstedt, P.S., Hendricks, G.M., Levis, J.E.. Yun, J.J. and Barbano, D.M. (1993) Age related changes in the microstructure of Mozzarella cheese. Food Struct., 12, 13-20. Kimura, T., Sagara. Y., Fukushima, M. and Taneya, S. (1992) Effect of pH on submicroscopic structure of String cheese. Milch,cissenschqft. 47, 547-552. Kindstedt, P.S. (1993) Effect of manufacturing factors, composition, and proteolysis on the functional characteristics of Mozzarella cheese. Crit. Revs. Food Sci. Nutr., 33, 167-187. Knoop, A.-M. and Buchheim, W. (1980) The different development of the structure in Harzer-. Tilsit- and Camembert-cheese during ripening. Milch~~issenschqft, 35, 482-488. Kumosinski, T.F., King, G. and Farrell, H.M. Jr. (1994) An energy-minimized casein submicelle working model. J. Protein Chem., 13, 681-700. Kumosinski, T.F., Uknalis, J.J., Cooke, P.H. and Farrell,
cheese storage
155
H.M. Jr. ( 1996) Correlation of refined models for casein submicelles with electron microscopic studies of casein. Lehensm.- Wiss. u.-Technol., 29, 326-333. Lelievre, J.R., Shaker, R. and Taylor, M.W. (1990) The role of homogenization in the manufacture of Halloumi and Mozzarella cheese from recombined milk. J. Sot. Dairy Technol., 43, 2 l-29. Malin, E.L. and Brown, E.M. (1995). Influence of casein peptide conformations on textural properties of cheese. In C’hemi.rlr!, oj’Structure/Function Relationships in Cheese, eds E.L. Malin and M.H. Tunick. Plenum Press, New York, pp. 303-3 IO. Malin. E.L.. Tunick, M.H., Smith, P.W. and Holsinger, V.H. (1995). Inhibition of proteolysis in Mozzarella cheese prepared from homogenized milk. In Chemistry of’ Structure/Function Relationships in Cheese, eds E.L. Malin and M.H. Tunick. Plenum Press, New York. pp. 237-246. Masi, P. and Addeo, F. (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, 2 17 -229. McPherson. A.V.Dash, M.C.Kitchen. B.J. (1989) Isolation and composition of milk fat globule membrane material. II. From homogenized and ultra heat treated milks. J. Dairy Res., 51, 289-297. Paquet, A. and Kalab, M. (1988) Amino acid composition and structure of cheese baked as a pizza ingredient in conventional and microwave ovens. Food Microstructure, 7, 93.-103. Putnam, J.J. and Allshouse, J.E. (1994). Food Consumption, Prices, and Espenditures, 1970-93. Statistical Bulletin SBY15. US Government Printing Office, Washington, DC. Schmidt, D.G. (1982). Associations of caseins and casein micelle structure. In Developments in Daity Chemistr_yl, ed. P.F. Fox. Applied Science Publishers, Essex, pp. 61-93. Schmidt, D.G. and Buchheim, W. (1970) Elektronenmikroskopische untersuchung der feinstruktur von caseimicellen in kuhmilch. Mi/chwissensch#, 25, 596-600. Schmidt, D.G. and Buchheim, W. (1976) Particle size distribution in casein solutions. Neth. Milk Dairy J., 30, 17-28. Taneya, S., Izutsu, T., Kimura, T. and Shioya, T. (1992) Structure and rheology of string cheese. Food Struct., 11, 61-71. Taranto, M.V., Wan, P.J., Chen, S.L. and Rhee, K.C. (1979) Morphological, ultrastructural and rheological characterization of Cheddar and Mozzarella cheese. Scanning Electron Microsc., 1979, 273-278. Tunick, M.H. (1994) Effects of homogenization and proteolysis on free oil in Mozzarella cheese. J. Dairy Sci., 77, 2487-2493. Tunick, M.H., Mackey, K.L., Shieh, J.J., Smith, P.W., Cooke, P. and Malin, E.L. (1993a) Rheology and microstructure of low-fat Mozzarella cheese. int. Dairy J., 3, 649-662. Tunick. M.H.. Malin, E.L., Smith, P.W., Shieh, J.J., Sullivan, B.C.. Mackey, K.L. and Holsinger, V.H. (1993b) Proteolysis and rheology of low fat and full fat Mozzarella cheeses prepared from homogenized milk. J. Dairy Sri., 76, 36213628. Tunick, M.H., Malin, E.L.. Smith, P.W. and Holsinger, V.H. (1995) Effects of skim milk homogenization on proteolysis and rheology of Mozzarella cheese. Int. Duiry J., 4, 483491. Walstra, P. (1975) Effect of homogenization on the fat globule size distribution in milk. Neth. Mi/k Dairy J., 29, 279-294. Wood, E.J. (1990) Applying Fourier and associated transforms to pattern characterization in textiles. Textile Res. J., 60, 2 12-220.