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Microstructure of Dairy Foods. 2. Milk Products Based on Fat1
Microstructure of Dairy Foods. 2. Milk Products Based on Fat 1 M I L O S L A V KALAB
Food Research Institute Research Branch, Agriculture Canada Ottawa, Ontario, Canada K 1A 0C6 ABSTRACT
Cold-stage scanning electron microscopy and transmission electron microscopy of replicas of freeze-fractured samples have been suggested as the electron microscopic techniques best suited to study the microstructure of milk products based on fat. New developments in other techniques such as fixation of fat with imidazole-buffered osmium tetroxide and embedding in a resin also have been mentioned. Microstructure of various forms of cream, ice cream, cream cheese, cream cheese spread, and butter, established by the techniques mentioned, has been reviewed with respect to the specific properties of each of the fat-based milk products. INTRODUCTION
Milk products were arbitrarily divided into those based on protein and those based on fat (38). Products based on protein, such as yogurt and most cheeses, have a rigid protein matrix. If fat is present, it is dispersed in that' matrix. Microstructure o f some products in this group was reviewed earlier (38). This review deals with milk products based on fat. They have no rigid protein matrix and are in the form of emulsions either of the oil-in-water type (cream) or the water-in-oil type (butter). If protein is present, it is dispersed in the aqueous phase of the product. Milk products based on fat include various forms of cream such as coffee cream, sour cream, whipped cream, and ice cream, as well as cream cheese, butter, and dairy spreads. In spite of their high fat content, cheeses with a rigid protein matrix are viewed as milk products based on protein; cream cheese and cream cheese spreads, however,
Received April 5, 1985. 1This is part 2 of a review (38). 1985 J Dairy Sci 68:3234--3248
may be considered to be milk products based on fat. All milk products are made from milk, which consists of two major kinds of corpuscula r constituents, casein micelles and fat globules. Casein micelles are protein particles approximately 100 nm in diameter, and fat globules in fresh milk range from 500 nm to 10 gtm in diameter (48). The fat globules are visible under an optical (light) microscope (46, 53), but the dimensions o f the casein micelles are below the resolution limits o f light microscopes. Optical microscopy makes it possible to examine fat globules and air cells in the original product with the aqueous phase (moisture) present. Fluorescence microscopy is also well suited at this level of resolution; it was used in microstructural studies of cheese, particularly to establish the distribution of fat (78). However, casein micelles can be visualized only by electron microscopy, which provides considerably higher resolution than optical microscopy. Electron microscopy requires that the sample be examined in vacuo; therefore, reduction of the vapor tension is one of the most important prerequisites. The water vapor tension is reduced either by drying or freezing the sample. Reviews have recently been published on electron microscopic techniques used in food science in general and in dairy science in particular (19, 40, 41, 68) and also on the microstructure of milk products (11, 16, 38, 64). ELECTRON MICROSCOPY T E C H N I Q U E S
Because o f the nature o f fat, not all the
techniques described in the reviews are suitable to show the microstructure of milk products based on fat without introducing some kind of artifact. Conventional scanning electron microscopy (SEM), in which the sample is fixed and dehydrated (dried), is unsuitable with most fat-based milk products. However, most recent
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to embedding in Araldite. In a simpler version of this technique, the fat globule suspension is mixed with a warm agar sol and the mixture is cooled to form a gel, which is then treated as a solid sample. Products such as cream cheese, which disintegrate easily in aqueous fixatives, should also be encapsulated in agar gel to maintain their structure unchanged during the subsequent preparative steps (40). The encapsulation is accomplished by momentarily dipping small samples of the cream cheese into a warm (<40°C) agar sol. Fixation stabilizes the protein and lipid components of the sample and makes them resistant to dehydrating and embedding agents. Glutaraldehyde is used to fix proteins and, subsequently, osmium tetroxide is used as a so-called postfixative agent to fix lipids. Reaction of osmium tetroxide with double bonds in unsaturated lipids results in labile cyclic monoesters, which spontaneously hydrolyze and release osmium. In the presence of pyridine and other tertiary bases, osmium is bound permanently to the lipids (30). For this reason, osmium tetroxide gives considerably better results when used in an imidazole buffer (4) than in conventional buffers. Imidazole-buffered osmium tetroxide was adapted for both SEM and TEM of milk products (1). It makes possible a clear distinction between liquid (unsaturated) and crystalline (saturated) milk fat lipids (Figure 2). Recently, Brooker and Wells (12) etched sections of epoxy resin-embedded cheese curd, yogurt, whipped cream, and cream cheese. The exposed structures were coated with gold and examined by SEM. Although native milk fat globules were damaged by the etching process, homogenized milk fat was not adversely affected. One of the most suitable techniques to study the microstructure of high-fat milk products is to freeze a very small sample of less than 1 mm 3 rapidly, for example, in a jet of liquid propane cooled with liquid nitrogen or in nitrogen slush (73), fracture the frozen sample in vacuo, and replicate the fracture plane by evaporating platinum at an acute angle and reinforcing the replica with carbon evaporated at a 90 ° angle. The replica is cleaned and examined by TEM (19). This method is superior to any other method [although artifacts (10) may also develop], because the samples are best Journal of Dairy Science Vol. 68, No. 12, 1985
preserved by cryofixation, i.e., fixation by rapid freezing. Rapid freezing prevents the formation of ice crystals and distortion of the initial microstructure in the sample under study. This brief description of electron microscopical techniques most frequently used makes it easier to understand micrographs illustrating papers mentioned in this review. F A T G L O B U L E S IN M I L K
Fat is present in fresh milk in the form of globules, 500 nm to 10 /~m in diameter (48), encased in lipoprotein membranes. The properties of fat globules and their ultrastructure have been studied in great detail (13, 14, 16, 18, 23, 25, 27, 46, 48, 58, 64, 65, 75, 77), and most of the available knowledge has been compiled by Mulder and Walstra (53) in a comprehensive treatise. McPherson and Kitchen (52) reviewed the formation, composition, structure, and behavior of the bovine milk fat globule membrane and Darling (24) reviewed the destabilization of dairy emulsions. The microstructure of the fat flobule membrane varies and depends on the length of time elapsed since the fat globule had been secreted from the secretory cell in the mammary gland. Wooding (77) showed that the milk fat globule membrane immediately after separation of the fat globule from the secretory cell consists of a continuous unit membrane separated by a zone of dense material from the fat globule itself. This "initial" membrane is fragmented and is shed off during the passage of the fat globule from the secretory cell into the secretory alveole and into expressed milk. The process leaves a single dense ("secondary") membrane around the fat globule. The stability of fat globules in milk depends on the continuity of this secondary membrane. It is considerably more elastic than the initial membrane and readily corrugates. Buchheim (15) observed corrugations ("blebs") of the secondary membrane in freeze-fractured milk samples. The mechanism of blebbing was studied by Pinto da Silva et al. (58). The corrugations or convolutions were also clearly visible in thin sections, particularly when a fixative containing lead acetate was used (Figure 3) (37). In fresh milk, fat globules rapidly raise to the surface of milk because of their'buoyancy
MICROSTRUCTURE OF DAIRY FOODS: REVIEW developments (1, 2) make it possible to encapsulate cream in agar gel tubes using a modified Salyaev's technique (67), fix the protein and fat with imidazole-buffered osmium tetroxide (2, 4), impregnate the sample with ethanol, freezefracture it, dry the fragments, and examine them by conventional SEM (Figure 1). Another possibility is to examine freezefractured samples in an SEM equipped with a cold stage (40, 69). The sample, such as whipped cream (70), is rapidly frozen and freeze-fractured and the fragments are coated with a thin layer of carbon and are examined while frozen. It is also possible to shadow the freeze-fractured sample with gold, separate and clean the gold layer in the form of a replica, and examine the replica by SEM at ambient temperature (40, 45). A rapid SEM examination of freezefractured uncoated specimens is sometimes also possible. It is based on the presence of ions, such as sodium, calcium (Na +, Ca2+), etc., in the frozen aqueous phase of the milk product. The ions render the sample electrically conductive and suitable for electron microscopical examination using a low current electron beam at a low accelerating voltage. However, there is a high incidence of artifacts arising from
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changes in the topographic features of the fracture plane unprotected with a carbon or gold coating. The images produced are inferior as compared with gold-coated samples; a relatively small proportion of the aqueous phase containing ions in high-fat milk products is partially responsible for such problems (40). Transmission electron microscopy (TEM) offers a wider selection of suitable techniques: negative staining and metal-shadowing may be used with suspensions (emulsions) such as cream; however, these techniques have been used more frequently with casein micelles than with fat globules. Encapsulation of a small volume of a liquid specimen in an agar gel tube prior to embedding in a resin was suggested by Salyaev (67). The encapsulated specimen is subsequently fixed, dehydrated, embedded in a resin, and sectioned in the same way as a solid specimen. This procedure was used to examine liquid samples containing fat globules and casein micelles (35). Hobbs (36) prepared fat globules for thin-sectioning by fixing them with glutaraldehyde and centrifuging the mixture. Fat globules concentrated in the floating cream layer were then collected on a Nucleopore filter and were immobilized in an agar gel prior
Figure 1. Scanning electron microscopy of cultured high-fat cream (58%). A. Cream encapsulated in agar gel, fixed with glutaraldehyde, and postfixed with imidazole-buffered osmium tetroxide (1), dehydrated in ethanol, freeze-fractured, critical-point dried, and examined at ambient temperature. B. Cream rapidly frozen in Freon 12 at --150°C, freeze-fractured in liquid nitrogen, coated with gold while frozen using Katoh's procedure (45), and examined at 100°C. f = Fat globules; m = fat globule membrane fragments; p = protein. Journal of Dairy Science Vol. 68, No. 12, 1985
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Figure 2. Transmission electron microscopy of a fat globule cluster. The sample was fixed with glutaraldehyde, postfixed with imidazole-buffered osmium tetroxide (1), embedded in a resin, sectioned, and stained with uranyl acetate and lead citrate solutions. Dark arrows point to a membrane that covers the entire fat globule cluster. Light arrow points to fat crystals which contain saturated fatty acids, m = Coagulated casein micelles.
a n d f o r m a layer o f c r e a m t h e r e . To p r e v e n t this s e p a r a t i o n f r o m t a k i n g place, m i l k is homogenized, which means that the dimensions o f t h e fat g l o b u l e s are c o n s i d e r a b l y r e d u c e d . A s s o c i a t e d w i t h this r e d u c t i o n in size is a c o r r e s p o n d i n g increase in surface area b y a
f a c t o r o f 5 to 6 (25). T h e n e w l y f o r m e d fat surface is r a p i d l y c o a t e d w i t h surface-active m a t e r i a l (caseins a n d u n d e n a t u r e d w h e y prot e i n s ) f r o m t h e m i l k s e r u m (25). T h e comp o s i t i o n o f t h e n e w m e m b r a n e significantly c o n t r i b u t e s t o t h e s t a b i l i t y o f t h e e m u l s i o n . In Journal of Dairy Science Vol. 68, No. 12, 1985
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Figure 3. Transmission electron microscopy of fat globules. "Blebs" (arrows) are noticeable in fat globule membranes in heated milk postfixed with a fixative which contained lead acetate (37). f = Fat globules; m = casein micelles.
general, f r a g m e n t a t i o n o f t h e fat globules is p r o p o r t i o n a l to t h e h o m o g e n i z a t i o n pressure. H e n s t r a a n d S c h m i d t (34) f o u n d t h a t at a Journal of Dairy Science Vol. 68, No. 12, 1985
relatively l o w pressure o f 5.07 MPa (50 a t m ) , m o s t fat globules r e m a i n e d i n t a c t and were c o v e r e d w i t h p r o t e i n , b u t at 35.46 MPa ( 3 5 0
MICROSTRUCTURE OF DAIRY FOODS : REVIEW atm), the fat globules disintegrated into minute spheres and their conglomerates were cemented together by casein. CR EAM
Cream is a product obtained from milk by centrifugation and basically consists of all the milk components in which fat globules are dispersed at a high concentration. So-called "coffee cream" contains 10 to 18% fat, whipping cream contains 24 to 35% fat, and the fat content in plastic cream is 80 to 85% (51). Several authors (3, 25, 26, 31) studied the effects of homogenization on the microstructure of cream. When added to hot coffee, homogenized cream sometimes causes "feathering", which is the formation o f curd floating on the surface of the coffee. Experimental results have indicated (3) that feathering depends on m a n y factors, such as the homogenization temperature and pressure, the preheating temperature, and the fat-to-protein ratio in the cream. Cream that does not feather immediately after processing may do so after storage. This problem is particularly serious with homogenized ultra-high temperature, aseptically packed cream. Anderson et al. (3) studied the microstructure of the cream before and after feathering using an acetate buffer instead of coffee in model experiments. Before feathering, the fat globules were encased in uniform membranes, 4-nm thick, which consisted of a single electron-dense line. Casein micelles, 70 to 170 nm in diameter, were attached to the fat globules. Feathered cream was in the form o f a coagulum, in which the identification of individual casein micelles was more difficult. Fat globules were cemented together by a matrix containing a high concentration of nonmicellar casein. The fat globule membranes were almost twice as thick as in the original cream. Doan (26) showed that a limited increase in the total solids content in the cream reduced clumping and slightly reduced feathering. Addition of citrate or carbonate somewhat decreased the concentration of micellar c a s e i n most probably by depleting the micelles of calcium. This resulted in an almost complete absence of clustering. This concept was supported by low casein and calcium concentrations in the fat phase during the early stages of storage o f cream that contained additives.
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The adsorption of surface-active substances at the fat-serum interface during homogenization is equal to the concentration of these substances; protein adsorption depended on the temperature of the homogenization. When washed cream was homogenized, fat globule clusters were formed only in the presence of chelating agents or surfactants. A possible explanation of this phenomenon is that, at a relatively low concentration of the surface-active molecules, two adjacent fat globules share such molecules (53). Darling and Butcher (25) found that whey proteins adhering to fat globules following homogenization were easier to remove by washing before pasteurization than were the adhering casein components. After subsequent pasteurization and on storage, whey proteins were more tightly bound to the fat globules; the membrane consisted of protein composite material, which contained casein micelles, casein submicelles, and nonmicellar proteins. McCarthy and Headon (51) showed micrographs of membranous material, which was concentrated during the production of butter oil. Cream was pasteurized at 74°C and subsequently passed through a clarifier. A heavy (plastic) cream fraction, containing 80 to 85% fat and a membrane-rich skim milk fraction, were obtained. The latter fraction was compacted by ultracentrifugation at 100,000 × g for 60 min. Examination of the pellet by TEM showed accumulation of membranous material. Microstructure of frozen cream was studied by Knoop and Wortmann (49). Fat globule membrane disintegrated and the cream was destabilized during storage for a year at - 1 5 ° C . To r e e m u l s i f y this product, the authors suggested mixing it with skim milk or with whole milk whereby new membranes would be formed on the fat globule surfaces. Whipping incorporates a large volume o f air into the cream and produces a fat-rich foam (17, 33, 53, 70). Fat globules were found by cold-stage SEM (70) to be absorbed and densely packed at the air-serum interface of the air cells and to be partially protruding into the air ceils (Figure 4). In the lamellae between the air cells, membranes of some fat globules were ruptured, the fat globules were partially coalesced, and a three-dimensional network was formed.
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Figure 4. Whipped cream made from unhomogenized (A) and homogenized (B) cream. Both samples were whipped to maximum rigidity and examined by cold-stage scanning electron microscopy. Fat globules (light arrows) and air cells (a) are smaller in whipped homogenized cream than in whipped unhomogenized cream. Dark arrows point to a membrane surrounding the air cells (air-serum interface), partially penetrated by fat globules (arrowheads). Reproduced by courtesy of D. G. Schmidt and A.C.M. van Hooydonk (69) and SEM Inc., AMF O'Hare, Chicago, Illinois.
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MICROSTRUCTURE OF DAIRY FOODS : REVIEW In homogenized cream, the fat globules were considerably smaller than in unhomogenized cream and the differences were evident at all stages of whipping. Also, the air cells were smaller in whipped homogenized cream than in whipped unhomogenized cream. As whipping progressed, the dimensions of the air cells in both kinds o f whipping cream were gradually decreased. Maximum stability of the air cells in the homogenized cream was reached before maximum rigidity, whereas in t h e unhomogenized cream, maximum foam strength and overrun almost coincided in time. Fat globule membranes gradually disintegrated during whipping and the released liquid fat cemented the remaining fat globules. Overwhipping led to the disintegration of the air cells and the formation of butter granules similar to those formed during churning. The foam had collapsed. Longer whipping was needed to achieve similar changes in homogenized cream. Schmidt and van H o o y d o n k (70) concluded that in homogenized cream there is a lack of balance between the stabilization of the air cells and the destabilization of the fat globules. This balance is assumed to be responsible for the rigidity of the foam lamellae. ICE C R E A M
Similar to whipped cream, ice cream contains a large volume of air in the form of air cells. To retain its form during freezing and low-temperature storage, ice cream is formulated as a complex system of genuine solutions (sugars and salts), colloidal solutions (proteins and stabilizers), and suspensions and emulsions (fat and emulsifiers). As far as the microstructure ofice cream is concerned, the surface and interface build-up during manufacture are of a greater importance than the percentage of various components (54). The ratios between fat, nonfat milk solids, sugar, emulsifier-stabilizer, and overrun may vary, hut generally they divide the product into five groups: dessert ice (15% fat), ice cream (10%), ice milk (4%), sherbet (2%), and water ice (no fat). Dessert ice and ice cream contain 15 and 14% sugar, respectively, whereas water ice contains 22% sugar (54). Manufacture, structure, and texture of ice cream were reviewed in varying detail (5, 6, 7, 8, 9, 14, 23, 28, 54, 55, 57, 71, 72, 76). The ice cream mix
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is first pasteurized, homogenized, and cooled to 4°C. The main reason for cooling the mix is to supercool the fat in the emulsion and to induce crystallization of heterogeneously nucleated droplets. The emulsion is then aged for 2 to 3 h, during which time the stabilizers become fully hydrated and the adsorption of proteins to the fat globule surfaces is continued. Although electron microscopy and electrophoresis have established that casein is adsorbed on fat globules in homogenized milk and ice cream, there is a probability that denatured whey proteins associated with casein may also participate in this adsorption; the use of buttermilk in the mix introduces more membrane proteins and lipoproteins. Additional fat crystallizes during aging of the mix. Changes in the ice cream emulsion during aeration and freezing are numerous and lead to rupture of membranes on most fat globules; consequently, a part of the fat is released and is spread at the air bubble interfaces, whereas another part of the fat cements the agglomerates of the remaining fat globules. Dimensions of ice crystals in ice cream significantly affect texture of the product: if they exceed 40 to 50 /lm in length, the ice cream acquires a coarse texture. A smooth texture is characterized by ice crystals shorter than 20 /.tm. lce cream contains approximately 50% of its water in the form of ice at - 5 . 5 ° C , which is the temperature at which ice cream is consumed. However, for storage and handling, the ice content must be increased to approximately 80% of the total water content. Lactose crystals are found in ice cream, that contains a high concentration of nonfat milk or whey solids, particularly if the temperature of storage is allowed to fluctuate. Lactose crystals 16 to 30/am long lead to the feeling of sandiness. Fat globules, casein micelles, ice crystals, and phase interfaces were easily distinguished in ice cream by electron microscopy (14) (Figure 5). Fat crystallized inside fat globules was demonstrated at high magnifications. Freezefracturing was also used in studies of the microstructure of the ice cream mix, the microstructure of ice cream extruded at various temperatures, and the microstructure of ice •cream containing vegetable oil (6, 7, 8). The number of fat globules per unit area of the examined freeze-etched surface may serve Journal of Dairy Science Vol. 68, No. 12, 1985
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Figure 5. Chocolate ice cream, a = Part of an air cell; f = fat globules; w = aqueous phase; dark arrow points to casein micelles; irregular dark areas (asterisks) are residues of the sample retained in spite of cleaning the replica. The rnicrograph of a platinum-carbon replica is reproduced by courtesy of W. Buchheim.
as an indicator of destabilization of the ice cream mix; these globules are those that have remained intact while others either have disintegrated partially and formed large clusters or have disintegrated completely and contributed to the coating of the air cells. C R E A M CHEESE
American cream cheese contains a minimum of 33% fat; Neufchatel cheese, which has a similar microstructure, contains less fat (20 to 33%) and may contain more moisture (22). Both cheeses are spreadable. Two types of cream cheese are manufactured (22, 44, 50, 74): in the traditional system of manufacturing, the cream mix is pasteurized, homogenized, and ripened with a lactic bacterial culture until a pH value of approximately 4.6 is reached. The curd is heated to 52 to 63°C, after which it is drained. Journal of Dairy Science Vol. 68, No. 12, 1985
The curd is either cold-packed or hot-packed. In the newly formulated method of manufacturing, which is used to produce "cream cheese spread" (42), the amount of total solids in the cream mix is increased to correspond to the final desired composition of the cheese. Following pasteurization, homogenization, and cooling to incubation temperature of approximately 30°C, the mix is inoculated with a lactic bacterial culture and is incubated until the desired acidity (pH 4.6) is reached. The curd is homogenized and packed without cooling. This procedure does not require the draining of whey. Stirring and homogenization of the mix, which are the essential steps in the manufacture of cream cheese, disrupt the curd and produce a corpuscular microstructure in the finished product. There is no rigid protein matrix and its absence makes this cheese spreadable.
MICROSTRUCTURE OF DAIRY FOODS : REVIEW Each of the two systems of manufacture is associated with a distinct type of sensory attributes and microstructure (44). Cream cheese made in the traditional way of manufacture is firmer and rates higher in the typical cream cheese flavor, whereas the newly formulated cream cheese is easier to spread and has a lower adhesiveness. The former cheese was composed mostly of small (approximately 2 /am in diameter) fat globules with a relatively uniform distribution of protein. Similar results were obtained by Ohashi et al. (56). In some products, the fat globules were aggregated in clusters; approximately 20/am in diameter, and protein in micellar form was accumulated in the superficial layer of the fat globule clusters (44) (Figure 6). In newly formulated cream cheese, most fat globules were ruptured during manufacture and formed large fat particles, over 2 /am in diameter. Space between the fat particles consisted of relatively uniformly distributed protein (Figure 6). Neufchatel cheese, characterized by a lower fat content, resembled newly formulated cream cheese as far as microstructure was concerned. Recently, a cream cheese spread was developed by mixing cultured high-fat cream with coagulated whey protein (42) or milk curd (43) (Figure 6C). Buchheim and Thomasow (21) added emulsifying salts to cream cheese and found that without heating the casein-fat aggregates in the cheese disintegrated. Heating increased the disintegration into fat globules and nonmicellar casein and resulted in a very fine homogeneous fat distribution in the casein-whey protein matrix. There are also imitation cream cheeses on the market: the milk-based product (a mixture of cream cheese and cottage cheese) was easy to distinguish by electron microscopy (44) by the presence of casein micelles and small fat globules from the non-dairy imitation cream cheese. BUTTER
Butter differs from the preceding fat-based milk products by being a water-in-oil emulsion, which means that water (approximately 20% of the total mass of butter) is in the form of a very fine dispersion in semisolid milk fat; water droplets are smaller than 10 /am in diameter. Fat lamellae separate the water droplets from
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each other and prevent them from coalescing. This fine dispersion contributes to the keeping quality of butter. The microstructure of butter was described in many research papers as well as reviews (16, 32, 47, 48, 54, 59, 60, 61, 62, 63, 66). Sweet and microbiologically ripened (cultured) cream is the starting material for the production of butter. Churning at 7 to 18°C disrupts fat globule membranes in the cream and leads to the coalescence of the fat globules and the formation of butter grains which are then separated from buttermilk. A part of the disintegrated fat globule membranes is retained in butter and the other part is released in buttermilk where the fat globule membrane fragments can be detected by electron microscopy (39). Still intact fat globules are also embedded in the free butterfat (Figure 7). The higher the free milk fat content, the softer and more spreadable the butter. Buchheim and Precht (20) classified fat globules in butter into four main types. There may be a number of intermediate forms depending on the distribution of liquid and crystallized fat in the globules, as seen in replicas of freeze-fractured butter specimens. The fat globules may contain a very small amount of crystalline fat in the form of thin monomolecular layers (Type 1); they may have individual fat crystals and crystal aggregates of irregular shape in the interior and a relatively thin crystalline shell at the globule surface (Type 2); they may consist of liquid fat in the interior and a thick (up to 500 nm) crystalline shell consisting of parallel monomolecular layers at the surface (Type 3); or they may be composed of a thick crystalline shell and a high concentration of crystalline aggregates in the globule interior (Type 4). Fat globules that have remained intact during churning exhibited the same structural features in butter as in the cream. Only some fat globules were stable enough to withstand the shear forces of churning. Such fat globules possessed a thick shell (100 to 500 nm) of crystalized high-melting fat fractions and crystal aggregates varying in dimensions. Crystal aggregates were also found (20) in the continuous liquid fat phase in butter. The crystals, either isolated or in groups with a mostly parallel orientation, displayed slightly curved surfaces and were considered to represent Journal of Dairy Science Vol. 68, No. 12, 1985
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Figure 6. Cream cheese (A) and cream cheese spread (B and C). Transmission electron microscopy o f samples postfixed with o s m i u m tetroxide, e m b e d d e d in a resin, a n d stained with uranyl acetate and lead citrate. A. Traditional m a n u f a c t u r e . Small fat globules are aggregated (f) and protein particles (arrows) are concentrated at t h e surface of t h e aggregates, w = A q u e o u s phase. B. New formulation (44). Membranes (dark arrows) surr o u n d mostly only small fat globules and are n o t f o u n d on large fat particles (f). Protein (hollow arrows) is relatively u n i f o r m l y distributed in the cream cheese spread, w = A q u e o u s phase. C. New formulation based on North American Ricotta cheese (42). Small fat globules (f) and protein particles (arrows) are u n i f o r m l y distrib u t e d in the product. Journal of Dairy Science Vol. 68, No. 12, 1985
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Figure 7. Butter. Two micrographs of platinum-carbon replicas are reproduced by courtesy of W. Buchheim. Butter consists of fat globules (f) and a small number of water droplets (w) dispersed in free fat. A. General view. B. Detail of a water droplet in which casein micelles (dark arrow) are evident; the droplet is covered with a thin layer of fat crystals (hollow arrows). Sample residues in the replicas are marked with asterisks. fragments of disrupted fat globules. However, crystals that developed in butter during 10 d of storage were newly formed fat crystals and were probably involved in the setting and hardening of stored butter. Crystallization of milk fat was recently studied microscopically by Foley and Brady (29); the temperature at which milk fat crystallized influenced its firmness, crystalline conformation, and the proportion of solid fat at 5°C. CONCLUSIONS
Optical microscopy has been used in dairy science for a long time, but the use of electron microscopy is relatively new. The greatest attention has been paid to the basic constituents of m i l k such as casein micelles and fat globules. The development of new microscopical methods and the modification of methods used in other scientific disciplines have made it possible to study interactions of milk constituents with each other as well as with other substances. Changes in the nature of the constituents and
the interaction products affect the microstructure of resulting milk products. Milk products based on fat are the subject of this review. Their microstructure is related to other characteristics such as consistency in whipped cream, smoothness in ice cream, and spreadability in cream cheese and in butter, etc. Other effects are related to the conditions under which milk products are stored, for example, duration and temperature changes. The use of nondairy ingredients, such as soy protein and vegetable oils, in imitation milk products poses a challenge to detect such ingredients and understand their effects of the product. In addition to electron microscopy, fluorescence microscopy also has the potential of significantly contributing to microstructural studies of milk products based on fat. ACKNOWLEDGMENTS
The author thanks W. Buchheim (Bundesanstalt fiir Milchforschung, Kiel, Federal Republic of Germany) and D. G. Schmidt and A.C.M. Journal of Dairy Science Vol. 68, No. 12, 1985
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van H o o y d o n k ( N e d e r l a n d s I n s t i t u u t v o o r Z u i v e l o n d e r z o e k , Ede, t h e N e t h e r l a n d s ) f o r p e r m i s s i o n t o r e p r o d u c e m i c r o g r a p h s in Figures 4, 5, and 7. Technical assistance and assistance w i t h t h e m a n u s c r i p t p r o v i d e d b y Paula AllanWojtas is gratefully a c k n o w l e d g e d . A p p r e c i a t i o n is e x p r e s s e d t o D. B. E m m o n s and H. W. M o d l e r for useful suggestions and for reviewing t h e m a n u s c r i p t . E l e c t r o n M i c r o s c o p e Centre, Research Branch, Agriculture Canada, O t t a w a , p r o v i d e d facilities. This review is C o n t r i b u t i o n 638 f r o m t h e F o o d R e s e a r c h I n s t i t u t e , Agriculture Canada in O t t a w a . REFERENCES
1 Allan-Wojtas, P., and M. Kalab. 1984. Milk gel structure. XIV. Fixation of fat globules in wholemill~ yoghurt for electron microscopy. Milchwissenschaft 39(6): 323. 2 Allan-Wojtas, P., and M. Kalab. 1984. A simple procedure for the preparation of stirred yoghurt for scanning electron microscopy. Food Microstruct. 3(2):197. 3 Anderson, M., B. E. Brooker, T. E. Cawston, and G. C. Cheeseman. 1977. Changes during storage in stability and composition of ultra-heat-treated aseptically-packed cream of 18% fat content. J. Dairy Res. 44:111. 4 Angermtiller, S., and H. D. Fahimi. 1982. Imidazolebuffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy. Histochem. J. 14:823. 5 Arbuckle, W. S. 1977. Ice cream. 3rd ed. AVI Publ. Co., Inc., Westport, CT. 6 Berger, K. G. 1976. Ice cream. Pages 141-213 in Food emulsions. Stig Friberg, ed. Marcel Dekker, Inc., New York and Basel. 7 Berger, K. G., B. K. Builimore, G. W. White, and W. B. Wright. 1972. The structure of ice cream -- Part 1 and Part 2. Dairy Ind. 37:419, 493. 8 Berger, K. G., and G. W. White. 1971. An electron microscopical investigation of fat destabilization in ice cream. J. Food Technol. 6:285. 9 Bhandari, V., R. Balachandran, and D. N. Prasad. 1984. Influence of stabilizers and an emulsifier on the ultra-structure of spray-dried ice-cream mix. N.Z.J. Dairy Sci. Technol. 19:55. 10 B~hler, S. 1976. Artefacts and specimen preparation faults in freeze-etch technology. Balzers AG, Balzers, Liechtenstein. 11 Brooker, B. E. 1979. Milk and its products. Pages 273-331 in Food microscopy. J. G. Vaughan, ed. Academic Press, New York, NY. 12 Brooker, B. E., and K. Wells. 1984. Preparation of dairy products for scanning electron microscopy: etching of epoxy resin-embedded material. J. Dairy Res. 51(4):605. 13 Buchheim, W. 1970. Der Verlaufder Fettkristallisation in den Fettkiigelchen der Milch. Elektronenmikroskopische Untersuchungen mit Hilfe der
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Gefrier/itztechnik. Milchwissenschaft 25:65. 14 Buchheim, W., 1970. Elektronenmikroskopische Darstellung der Struktur yon Speiseeis. Siisswaren 16:763. 15 Buchheim, W. 1970. Zur Struktur der Hiille yon Milchfettkiigelchen. Naturwissenschaften 57:672. 16 Buchheim, W. 1971. Neuere Ergebnisse der elektronenmikroskopischen Strukturforschung yon Milch und Milchprodukten. Molkerei-Ztg. Welt der Milch 25:461. 17 Buchheim, W. 1974. Electron microscopical studies on the structure of whipped cream. 19th Int. Dairy Cong., New Delhi IE :245. 18 Buchheim, W. 1982. Paracrystalline arrays of milk fat globule membrane-associated proteins as revealed by freeze-fracture. Naturwissenschaften 69:505. 19 Buchheim, W. 1982. Aspects of sample preparation for freeze-fracture/freeze-etch studies of proteins and lipids. Food Microstruct. 1(2):189. 20 Buchheim, W., and D. Precht. 1979. Elektronenmikroskopische Untersuchung der Kristallisationsvorg//nge in den Fettkiigelchen wlihrend der Rahmreifung. Milchwissenschaft 34(11):657. 21 Buchheim, W., and J. Thomasow. 1984. Structural changes in cream cheese induced by thermal processing and emulsifying salts. North Eur. Dairy J. 50:38. 22 Cheese Varieties and Descriptions. 1953. US Dep. Agric., Agric. Handbook No. 54, Washington, DC. 23 Cremers, L. F., and W. S. Arbuckle. 1954. The identification of fat globules in the internal structure of ice cream. J. Dairy Sci. 37:642. 24 Darling, D. F. 1982. Recent advances in the destabilization of dairy emulsions. J. Dairy Res. 49:695. 25 Darling, D. F., and D. W. Butcher. 1978. Milk-fat globule membrane in homogenized cream. J. Dairy Res. 45:197. 26 Doan, F. J. 1931. The relation of feathering and heat stability of cream to fat clumping produced by homogenization. J. Dairy Sci. 14:527. 27 Eggmann, H. 1969. Elektronenmikroskopische Untersuchungen an Milch und Milchprodukten. 2. Anwendung der Gefrier~itztechnik. Milchwissenschaft 24:479. 28 Filchakova, N. N. 1974. Influence of air phase dispersity on hard ice-cream texture. In: Current studies on the thermophysical properties of foodstuffs. Bull. Inst. Int. Froid 3 (Suppl.):321. 29 Foley, J., and J. P. Brady. 1984. Temperatureinduced effects on crystallization behaviour, solid fat content and the firmness values of milk fat. J. Dairy Res. 51(4):579. 30 Geyer, G. 1977. Lipid fixation. Acta Histochem. Suppl. XIX:202. 31 Goulden, J.D.S., and Phipps L. W. 1964. Factors affecting the fat globule sizes during the homogenization of milk and cream. J. Dairy Res. 31:195. 32 Graf, E., and H. Bauer. 1976. Milk and milk products. Pages 295-383 in Food emulsions. Stig Friberg, ed. Marcel Dekker, Inc., New York and Basel. 33 Graf, E., and H. R. Miiller. 1965. Fine structure
M I C R O S T R U C T U R E OF DAIRY FOODS: REVIEW and whippability of sterilized cream. Milchwissenschaft 20: 302. 34 Henstra, S., and D. G. Schmidt. 1970. On the structure o f t h e fat-protein comlSlex in h o m o g e n i z e d cow's milk. Neth. Milk Dairy J. 2 4 : 4 5 . 35 Henstra, S., and D. G. Schmidt. 1970. Ultradtinnscbnitte aus Milch mit Hilfe der Mikrokapselmetbode. Naturwissenschaften 57:247. 36 Hobbs, D. G. 1979. An improved m e t h o d o f preparing bovine milk fat globules for electron microscopy. Milchwissenschaft 34:201. 37 Kalab, M. 1977. Milk gel structure. VII. Fixation of gels c o m p o s e d o f low-methoxyl pectin and milk. Milchwissenschaft 32:719. 38 Kalab, M. 1979. Microstructure o f diary foods. 1. Milk p r o d u c t s based on protein. J. Dairy Sci. 62:1352. 39 Kalab, M. 1980. Possibilities o f an electron-microscopic detection of buttermilk m a d e from sweet cream in adulterated skim milk. Scanning Electron Microsc. 1980/11I :645. 40 Kalab, M. 1981. Electron microscopy o f milk products: A review of techniques. Scanning Electron Mierosc. 1981/111:453. 41 Kalab, M. 1983. Electron microscopy of foods. Pages 4 3 - 1 0 4 in Physical properties of foods. M. Peleg and E. B. Bagley, ed. AVI Publ. Co., Inc., Westport, CT. 42 Kalab, M., and H. W. Modler. 1985. Milk gel structure. XV. Electron microscopy of w h e y protein-based cream cheese spread, Mitchwissenschaft 40(4): 193. 43 Kalab, M., and H. W. Modler. 1985. Development of mierostructure in a cream cheese based on Queso Blanco cheese. F o o d Mierostruct. 4(1):89. 44 Kalab, M., A. G. Sargant, and D. A. Froehlich. 1981. Electron microscopy and sensory evaluation of commercial cream cheese. Scanning Electron Microsc. 1981/1II:473. 45 Katoh, M. 1979. SEM replica t e c h n i q u e for butter and cheese. J. Electron Microsc. ;28:199. 46 King, N. 1955. Milk fat globule m e m b r a n e and some associated p h e n o m e n a . C o m m o n w . Agric. Bur., F a r n h a m Royal, Bucks., Erigl. 47 Knoop, A.-M. 1979. Milchforschung m i t d e m Elektronenmikroskop. Z. Lebensm. Unters. Forsch. 168:305. 48 Knoop, E. 1972. Strukturaufkl/irung durch elektronenmikroskopische U n t e r s u c h u n g e n an Eiweiss u n d Milchfett. Milchwissenschaft 27:364. 49 Knoop, E., and A. Wortmann. 1959. Mikroskopische Strukturaufkl~trungen in eigefrorenem Rahm. Milchwissenshaft 14:106. 50 Kosikowski, F. 1966. Pages 1 1 9 - 1 3 4 in Cheese and f e r m e n t e d milk foods. Kosikowski and Assoc., Brooktondale, NY. 51 McCarthy, M., and D. R. Headon. 1979. Lipid and protein composition of a membrane-rich fraction of butter oil. J. Dairy Res. 4 6 : 5 1 1 . 52 McPherson, A. V., a n d B. J. Kitchen. 1983. Reviews of the progress o f dairy science: The bovine milk fat globule m e m b r a n e - its formation, composition, structure and behaviour in milk and dairy products. J. Dairy Res. 50:107.
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53 Mulder, H., and P. Walstra. 1974. The milk fat globule. C o m m o n w . Agric. Bur., F a r n h a m Royal, Bucks., Engl., and PUDOC (Ctr. Agric. Publ. Doc.), Wageningen, Neth. 54 Nielsen, B. J. 1973. Building a n d formation of ice cream microstructure during processing. Mod. Dairy 52:10. 55 Nielsen, B. J. 1978. C o m b i n e d emulsifier/stabilizers for ice cream. Gordian 78:176. 56 Ohashi, T., S. Nagai, K. Masaoka, S. Haga, K. Yamauchi, and N. F. Olson. 1983. Physical properties and microstructure o f cream cheese. Nippon S h o k u h i n Kogyo Gakkaishi 30(5):303. 57 Olenev, Yu. A. 1982. Vliyanie kristallizatsii laktozy v m o r o z h e n o m pri khranenii na ego kachestvo. Kholodil'naya T e k h n i k a No. 5 : 39. 58 Pinto Da Silva, P., A. Peixoto De Menezes, and i. H. Matber. 1980. Structure a n d d y n a m i c s o f t h e bovine milk fat globule m e m b r a n e viewed by freeze fracture. Exp. Cell Res. 125:127. 59 Precht, D., and W. Buchheim. 1979. Elektronenmikroskopische U n t e r s u c h u n g e n fiber die physikalische Struktur yon Streichfetten. I. Die Mikrostruktur der Fettk/Jgelchen in Butter. Milchwissenschaft 34:745. 60 Precht, D., and W. Buchheim. 1979. Elektronenmikroskopische U n t e r s u c h u n g e n iiber die physikalische Struktur yon Streichfetten. 2. Die Mikrostruktur der zwischenglobul~iren Fettphase in Butter. Milchwissenschaft 35 (7): 393. 61 Precht, D., and W. Buchheim. 1980. Elektronenmikroskopische U n t e r s u c h u n g e n fiber die physikalische Struktur yon Streichfetten. 3. Die wgssrige Phase in der Butter. Milchwissenscb aft 35 (11 ) :684. 62 Precht, D., and K.-H. Peters. 1981. Die Konsistenz der Butter. I. Elektronenmikroskopische Unters u c h u n g e n z u m Einfluss unterschiedlicher R a h m reifungstemperaturen auf die H~iufigkeit b e s t i m m t e r Fettkiigelchentypen im R a h m . Milchwissenschaft 36(10):616. 63 Precht, D., and K.-H. Peters. 1981. Die Konsistenz der Butter. II. Zusammenhiinge zwischen der submikroskopischen Struktur yon R a h m f e t t kiigelchen sowie Butter u n d der Konsistenz in Abhgngigkeit yon speziellen physikalischen R a h m reifungsverfahren. Milchwissenschaft 36(11):673. 64 Resmini, P. 1979. Strnrtura e m i c r o s t r u t t u r a dei prodotti lartiero caseari. Ind. Latte 15:33. 65 Saito, Z. 1973. Electron micrographs o f fat globules and casein micelles in cow's milk. Bull. Fac. Agric., Hirosaki Univ. No. 20:1. 66 Saito, Z. 1975. [Oiling-off properties and microscopic structures o f butter, margarine, and soft-type margarine.] Bull. Fac. Agric. Hirosaki Univ. No. 25:1. (In Japanese.) 67 Salyaev, R. K. 1968. A m e t h o d of fixation and e m b e d d i n g of liquid and fragile materials in agar microcapsulae. Proc. 4 t h Eur. Reg. Conf. Electron Microsc., Rome, Italy, II:37. 68 Sehmidt, D. G. 1982. Electron microscopy o f milk and milk products: Problems and possibilities. F o o d Mierostruct. 1(2):151. 69 Schmidt, D. G., S. Henstra, and F. Thiel. 1979. A simple low-temperature technique for scanning
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electron microscopy o f cheese. Mikroskopie (Wien) 35:50. 70 Schmidt, D. G., and A.C.M. van H o o y d o n k . 1980. A scanning electron microscopical investigation of t h e whipping of cream. Scanning Electron Microsc. 1980/III :653. 71 Shaw, D. J. 1963. The physical structure of ice cream. Pages 1 2 5 - 1 3 1 in Rheology of emulsions. P. Sherman, ed. Pergamon Press, Lond. 72 Spiess, W.E.L., and R. Adam. 1974. Methods of quality evaluation o f ice-cream and storage behaviour o f vanilla ice-cream. Current studies on the thermophysical properties of foodstuffs. Bull. lnst. Int. Froid 3 (Suppl.):289.
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73 Umrath, W. 1974. Cooling bath for rapid freezing in electron microscopy. J. Microsc. 101(1):103. 74 Van Slyke, L. L., a n d W. V. Price. 1952. Cheese. Orange J u d d PubI. Co., Inc., New York, NY. 406-430. 75 Walstra, P. 1974. High-melting triglycerides in the fat globule m e m b r a n e : an artefact? Neth. Milk Dairy J. 28:3. 76 Wielinga, W. 1977. Some aspects of ice cream stabilization. Ice Cream Erozen Confect. 30(1):49. 77 Wooding, F.B.P. 1971. The structure of t h e milk fat globule m e m b r a n e . J. Ultrastruct. 37:388. 78 Yiu, S. H. 1985. A fluorescence microscopic study of cheese. Food Microstruct. 4(1):99.
Food Research Institute Research Branch, Agriculture Canada Ottawa, Ontario, Canada K 1A 0C6 ABSTRACT
Cold-stage scanning electron microscopy and transmission electron microscopy of replicas of freeze-fractured samples have been suggested as the electron microscopic techniques best suited to study the microstructure of milk products based on fat. New developments in other techniques such as fixation of fat with imidazole-buffered osmium tetroxide and embedding in a resin also have been mentioned. Microstructure of various forms of cream, ice cream, cream cheese, cream cheese spread, and butter, established by the techniques mentioned, has been reviewed with respect to the specific properties of each of the fat-based milk products. INTRODUCTION
Milk products were arbitrarily divided into those based on protein and those based on fat (38). Products based on protein, such as yogurt and most cheeses, have a rigid protein matrix. If fat is present, it is dispersed in that' matrix. Microstructure o f some products in this group was reviewed earlier (38). This review deals with milk products based on fat. They have no rigid protein matrix and are in the form of emulsions either of the oil-in-water type (cream) or the water-in-oil type (butter). If protein is present, it is dispersed in the aqueous phase of the product. Milk products based on fat include various forms of cream such as coffee cream, sour cream, whipped cream, and ice cream, as well as cream cheese, butter, and dairy spreads. In spite of their high fat content, cheeses with a rigid protein matrix are viewed as milk products based on protein; cream cheese and cream cheese spreads, however,
Received April 5, 1985. 1This is part 2 of a review (38). 1985 J Dairy Sci 68:3234--3248
may be considered to be milk products based on fat. All milk products are made from milk, which consists of two major kinds of corpuscula r constituents, casein micelles and fat globules. Casein micelles are protein particles approximately 100 nm in diameter, and fat globules in fresh milk range from 500 nm to 10 gtm in diameter (48). The fat globules are visible under an optical (light) microscope (46, 53), but the dimensions o f the casein micelles are below the resolution limits o f light microscopes. Optical microscopy makes it possible to examine fat globules and air cells in the original product with the aqueous phase (moisture) present. Fluorescence microscopy is also well suited at this level of resolution; it was used in microstructural studies of cheese, particularly to establish the distribution of fat (78). However, casein micelles can be visualized only by electron microscopy, which provides considerably higher resolution than optical microscopy. Electron microscopy requires that the sample be examined in vacuo; therefore, reduction of the vapor tension is one of the most important prerequisites. The water vapor tension is reduced either by drying or freezing the sample. Reviews have recently been published on electron microscopic techniques used in food science in general and in dairy science in particular (19, 40, 41, 68) and also on the microstructure of milk products (11, 16, 38, 64). ELECTRON MICROSCOPY T E C H N I Q U E S
Because o f the nature o f fat, not all the
techniques described in the reviews are suitable to show the microstructure of milk products based on fat without introducing some kind of artifact. Conventional scanning electron microscopy (SEM), in which the sample is fixed and dehydrated (dried), is unsuitable with most fat-based milk products. However, most recent
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to embedding in Araldite. In a simpler version of this technique, the fat globule suspension is mixed with a warm agar sol and the mixture is cooled to form a gel, which is then treated as a solid sample. Products such as cream cheese, which disintegrate easily in aqueous fixatives, should also be encapsulated in agar gel to maintain their structure unchanged during the subsequent preparative steps (40). The encapsulation is accomplished by momentarily dipping small samples of the cream cheese into a warm (<40°C) agar sol. Fixation stabilizes the protein and lipid components of the sample and makes them resistant to dehydrating and embedding agents. Glutaraldehyde is used to fix proteins and, subsequently, osmium tetroxide is used as a so-called postfixative agent to fix lipids. Reaction of osmium tetroxide with double bonds in unsaturated lipids results in labile cyclic monoesters, which spontaneously hydrolyze and release osmium. In the presence of pyridine and other tertiary bases, osmium is bound permanently to the lipids (30). For this reason, osmium tetroxide gives considerably better results when used in an imidazole buffer (4) than in conventional buffers. Imidazole-buffered osmium tetroxide was adapted for both SEM and TEM of milk products (1). It makes possible a clear distinction between liquid (unsaturated) and crystalline (saturated) milk fat lipids (Figure 2). Recently, Brooker and Wells (12) etched sections of epoxy resin-embedded cheese curd, yogurt, whipped cream, and cream cheese. The exposed structures were coated with gold and examined by SEM. Although native milk fat globules were damaged by the etching process, homogenized milk fat was not adversely affected. One of the most suitable techniques to study the microstructure of high-fat milk products is to freeze a very small sample of less than 1 mm 3 rapidly, for example, in a jet of liquid propane cooled with liquid nitrogen or in nitrogen slush (73), fracture the frozen sample in vacuo, and replicate the fracture plane by evaporating platinum at an acute angle and reinforcing the replica with carbon evaporated at a 90 ° angle. The replica is cleaned and examined by TEM (19). This method is superior to any other method [although artifacts (10) may also develop], because the samples are best Journal of Dairy Science Vol. 68, No. 12, 1985
preserved by cryofixation, i.e., fixation by rapid freezing. Rapid freezing prevents the formation of ice crystals and distortion of the initial microstructure in the sample under study. This brief description of electron microscopical techniques most frequently used makes it easier to understand micrographs illustrating papers mentioned in this review. F A T G L O B U L E S IN M I L K
Fat is present in fresh milk in the form of globules, 500 nm to 10 /~m in diameter (48), encased in lipoprotein membranes. The properties of fat globules and their ultrastructure have been studied in great detail (13, 14, 16, 18, 23, 25, 27, 46, 48, 58, 64, 65, 75, 77), and most of the available knowledge has been compiled by Mulder and Walstra (53) in a comprehensive treatise. McPherson and Kitchen (52) reviewed the formation, composition, structure, and behavior of the bovine milk fat globule membrane and Darling (24) reviewed the destabilization of dairy emulsions. The microstructure of the fat flobule membrane varies and depends on the length of time elapsed since the fat globule had been secreted from the secretory cell in the mammary gland. Wooding (77) showed that the milk fat globule membrane immediately after separation of the fat globule from the secretory cell consists of a continuous unit membrane separated by a zone of dense material from the fat globule itself. This "initial" membrane is fragmented and is shed off during the passage of the fat globule from the secretory cell into the secretory alveole and into expressed milk. The process leaves a single dense ("secondary") membrane around the fat globule. The stability of fat globules in milk depends on the continuity of this secondary membrane. It is considerably more elastic than the initial membrane and readily corrugates. Buchheim (15) observed corrugations ("blebs") of the secondary membrane in freeze-fractured milk samples. The mechanism of blebbing was studied by Pinto da Silva et al. (58). The corrugations or convolutions were also clearly visible in thin sections, particularly when a fixative containing lead acetate was used (Figure 3) (37). In fresh milk, fat globules rapidly raise to the surface of milk because of their'buoyancy
MICROSTRUCTURE OF DAIRY FOODS: REVIEW developments (1, 2) make it possible to encapsulate cream in agar gel tubes using a modified Salyaev's technique (67), fix the protein and fat with imidazole-buffered osmium tetroxide (2, 4), impregnate the sample with ethanol, freezefracture it, dry the fragments, and examine them by conventional SEM (Figure 1). Another possibility is to examine freezefractured samples in an SEM equipped with a cold stage (40, 69). The sample, such as whipped cream (70), is rapidly frozen and freeze-fractured and the fragments are coated with a thin layer of carbon and are examined while frozen. It is also possible to shadow the freeze-fractured sample with gold, separate and clean the gold layer in the form of a replica, and examine the replica by SEM at ambient temperature (40, 45). A rapid SEM examination of freezefractured uncoated specimens is sometimes also possible. It is based on the presence of ions, such as sodium, calcium (Na +, Ca2+), etc., in the frozen aqueous phase of the milk product. The ions render the sample electrically conductive and suitable for electron microscopical examination using a low current electron beam at a low accelerating voltage. However, there is a high incidence of artifacts arising from
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changes in the topographic features of the fracture plane unprotected with a carbon or gold coating. The images produced are inferior as compared with gold-coated samples; a relatively small proportion of the aqueous phase containing ions in high-fat milk products is partially responsible for such problems (40). Transmission electron microscopy (TEM) offers a wider selection of suitable techniques: negative staining and metal-shadowing may be used with suspensions (emulsions) such as cream; however, these techniques have been used more frequently with casein micelles than with fat globules. Encapsulation of a small volume of a liquid specimen in an agar gel tube prior to embedding in a resin was suggested by Salyaev (67). The encapsulated specimen is subsequently fixed, dehydrated, embedded in a resin, and sectioned in the same way as a solid specimen. This procedure was used to examine liquid samples containing fat globules and casein micelles (35). Hobbs (36) prepared fat globules for thin-sectioning by fixing them with glutaraldehyde and centrifuging the mixture. Fat globules concentrated in the floating cream layer were then collected on a Nucleopore filter and were immobilized in an agar gel prior
Figure 1. Scanning electron microscopy of cultured high-fat cream (58%). A. Cream encapsulated in agar gel, fixed with glutaraldehyde, and postfixed with imidazole-buffered osmium tetroxide (1), dehydrated in ethanol, freeze-fractured, critical-point dried, and examined at ambient temperature. B. Cream rapidly frozen in Freon 12 at --150°C, freeze-fractured in liquid nitrogen, coated with gold while frozen using Katoh's procedure (45), and examined at 100°C. f = Fat globules; m = fat globule membrane fragments; p = protein. Journal of Dairy Science Vol. 68, No. 12, 1985
MICROSTRUCTURE OF DAIRY FOODS: REVIEW
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Figure 2. Transmission electron microscopy of a fat globule cluster. The sample was fixed with glutaraldehyde, postfixed with imidazole-buffered osmium tetroxide (1), embedded in a resin, sectioned, and stained with uranyl acetate and lead citrate solutions. Dark arrows point to a membrane that covers the entire fat globule cluster. Light arrow points to fat crystals which contain saturated fatty acids, m = Coagulated casein micelles.
a n d f o r m a layer o f c r e a m t h e r e . To p r e v e n t this s e p a r a t i o n f r o m t a k i n g place, m i l k is homogenized, which means that the dimensions o f t h e fat g l o b u l e s are c o n s i d e r a b l y r e d u c e d . A s s o c i a t e d w i t h this r e d u c t i o n in size is a c o r r e s p o n d i n g increase in surface area b y a
f a c t o r o f 5 to 6 (25). T h e n e w l y f o r m e d fat surface is r a p i d l y c o a t e d w i t h surface-active m a t e r i a l (caseins a n d u n d e n a t u r e d w h e y prot e i n s ) f r o m t h e m i l k s e r u m (25). T h e comp o s i t i o n o f t h e n e w m e m b r a n e significantly c o n t r i b u t e s t o t h e s t a b i l i t y o f t h e e m u l s i o n . In Journal of Dairy Science Vol. 68, No. 12, 1985
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Figure 3. Transmission electron microscopy of fat globules. "Blebs" (arrows) are noticeable in fat globule membranes in heated milk postfixed with a fixative which contained lead acetate (37). f = Fat globules; m = casein micelles.
general, f r a g m e n t a t i o n o f t h e fat globules is p r o p o r t i o n a l to t h e h o m o g e n i z a t i o n pressure. H e n s t r a a n d S c h m i d t (34) f o u n d t h a t at a Journal of Dairy Science Vol. 68, No. 12, 1985
relatively l o w pressure o f 5.07 MPa (50 a t m ) , m o s t fat globules r e m a i n e d i n t a c t and were c o v e r e d w i t h p r o t e i n , b u t at 35.46 MPa ( 3 5 0
MICROSTRUCTURE OF DAIRY FOODS : REVIEW atm), the fat globules disintegrated into minute spheres and their conglomerates were cemented together by casein. CR EAM
Cream is a product obtained from milk by centrifugation and basically consists of all the milk components in which fat globules are dispersed at a high concentration. So-called "coffee cream" contains 10 to 18% fat, whipping cream contains 24 to 35% fat, and the fat content in plastic cream is 80 to 85% (51). Several authors (3, 25, 26, 31) studied the effects of homogenization on the microstructure of cream. When added to hot coffee, homogenized cream sometimes causes "feathering", which is the formation o f curd floating on the surface of the coffee. Experimental results have indicated (3) that feathering depends on m a n y factors, such as the homogenization temperature and pressure, the preheating temperature, and the fat-to-protein ratio in the cream. Cream that does not feather immediately after processing may do so after storage. This problem is particularly serious with homogenized ultra-high temperature, aseptically packed cream. Anderson et al. (3) studied the microstructure of the cream before and after feathering using an acetate buffer instead of coffee in model experiments. Before feathering, the fat globules were encased in uniform membranes, 4-nm thick, which consisted of a single electron-dense line. Casein micelles, 70 to 170 nm in diameter, were attached to the fat globules. Feathered cream was in the form o f a coagulum, in which the identification of individual casein micelles was more difficult. Fat globules were cemented together by a matrix containing a high concentration of nonmicellar casein. The fat globule membranes were almost twice as thick as in the original cream. Doan (26) showed that a limited increase in the total solids content in the cream reduced clumping and slightly reduced feathering. Addition of citrate or carbonate somewhat decreased the concentration of micellar c a s e i n most probably by depleting the micelles of calcium. This resulted in an almost complete absence of clustering. This concept was supported by low casein and calcium concentrations in the fat phase during the early stages of storage o f cream that contained additives.
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The adsorption of surface-active substances at the fat-serum interface during homogenization is equal to the concentration of these substances; protein adsorption depended on the temperature of the homogenization. When washed cream was homogenized, fat globule clusters were formed only in the presence of chelating agents or surfactants. A possible explanation of this phenomenon is that, at a relatively low concentration of the surface-active molecules, two adjacent fat globules share such molecules (53). Darling and Butcher (25) found that whey proteins adhering to fat globules following homogenization were easier to remove by washing before pasteurization than were the adhering casein components. After subsequent pasteurization and on storage, whey proteins were more tightly bound to the fat globules; the membrane consisted of protein composite material, which contained casein micelles, casein submicelles, and nonmicellar proteins. McCarthy and Headon (51) showed micrographs of membranous material, which was concentrated during the production of butter oil. Cream was pasteurized at 74°C and subsequently passed through a clarifier. A heavy (plastic) cream fraction, containing 80 to 85% fat and a membrane-rich skim milk fraction, were obtained. The latter fraction was compacted by ultracentrifugation at 100,000 × g for 60 min. Examination of the pellet by TEM showed accumulation of membranous material. Microstructure of frozen cream was studied by Knoop and Wortmann (49). Fat globule membrane disintegrated and the cream was destabilized during storage for a year at - 1 5 ° C . To r e e m u l s i f y this product, the authors suggested mixing it with skim milk or with whole milk whereby new membranes would be formed on the fat globule surfaces. Whipping incorporates a large volume o f air into the cream and produces a fat-rich foam (17, 33, 53, 70). Fat globules were found by cold-stage SEM (70) to be absorbed and densely packed at the air-serum interface of the air cells and to be partially protruding into the air ceils (Figure 4). In the lamellae between the air cells, membranes of some fat globules were ruptured, the fat globules were partially coalesced, and a three-dimensional network was formed.
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Figure 4. Whipped cream made from unhomogenized (A) and homogenized (B) cream. Both samples were whipped to maximum rigidity and examined by cold-stage scanning electron microscopy. Fat globules (light arrows) and air cells (a) are smaller in whipped homogenized cream than in whipped unhomogenized cream. Dark arrows point to a membrane surrounding the air cells (air-serum interface), partially penetrated by fat globules (arrowheads). Reproduced by courtesy of D. G. Schmidt and A.C.M. van Hooydonk (69) and SEM Inc., AMF O'Hare, Chicago, Illinois.
Journal of Dairy Science Vol. 68, No. 12, 1985
MICROSTRUCTURE OF DAIRY FOODS : REVIEW In homogenized cream, the fat globules were considerably smaller than in unhomogenized cream and the differences were evident at all stages of whipping. Also, the air cells were smaller in whipped homogenized cream than in whipped unhomogenized cream. As whipping progressed, the dimensions of the air cells in both kinds o f whipping cream were gradually decreased. Maximum stability of the air cells in the homogenized cream was reached before maximum rigidity, whereas in t h e unhomogenized cream, maximum foam strength and overrun almost coincided in time. Fat globule membranes gradually disintegrated during whipping and the released liquid fat cemented the remaining fat globules. Overwhipping led to the disintegration of the air cells and the formation of butter granules similar to those formed during churning. The foam had collapsed. Longer whipping was needed to achieve similar changes in homogenized cream. Schmidt and van H o o y d o n k (70) concluded that in homogenized cream there is a lack of balance between the stabilization of the air cells and the destabilization of the fat globules. This balance is assumed to be responsible for the rigidity of the foam lamellae. ICE C R E A M
Similar to whipped cream, ice cream contains a large volume of air in the form of air cells. To retain its form during freezing and low-temperature storage, ice cream is formulated as a complex system of genuine solutions (sugars and salts), colloidal solutions (proteins and stabilizers), and suspensions and emulsions (fat and emulsifiers). As far as the microstructure ofice cream is concerned, the surface and interface build-up during manufacture are of a greater importance than the percentage of various components (54). The ratios between fat, nonfat milk solids, sugar, emulsifier-stabilizer, and overrun may vary, hut generally they divide the product into five groups: dessert ice (15% fat), ice cream (10%), ice milk (4%), sherbet (2%), and water ice (no fat). Dessert ice and ice cream contain 15 and 14% sugar, respectively, whereas water ice contains 22% sugar (54). Manufacture, structure, and texture of ice cream were reviewed in varying detail (5, 6, 7, 8, 9, 14, 23, 28, 54, 55, 57, 71, 72, 76). The ice cream mix
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is first pasteurized, homogenized, and cooled to 4°C. The main reason for cooling the mix is to supercool the fat in the emulsion and to induce crystallization of heterogeneously nucleated droplets. The emulsion is then aged for 2 to 3 h, during which time the stabilizers become fully hydrated and the adsorption of proteins to the fat globule surfaces is continued. Although electron microscopy and electrophoresis have established that casein is adsorbed on fat globules in homogenized milk and ice cream, there is a probability that denatured whey proteins associated with casein may also participate in this adsorption; the use of buttermilk in the mix introduces more membrane proteins and lipoproteins. Additional fat crystallizes during aging of the mix. Changes in the ice cream emulsion during aeration and freezing are numerous and lead to rupture of membranes on most fat globules; consequently, a part of the fat is released and is spread at the air bubble interfaces, whereas another part of the fat cements the agglomerates of the remaining fat globules. Dimensions of ice crystals in ice cream significantly affect texture of the product: if they exceed 40 to 50 /lm in length, the ice cream acquires a coarse texture. A smooth texture is characterized by ice crystals shorter than 20 /.tm. lce cream contains approximately 50% of its water in the form of ice at - 5 . 5 ° C , which is the temperature at which ice cream is consumed. However, for storage and handling, the ice content must be increased to approximately 80% of the total water content. Lactose crystals are found in ice cream, that contains a high concentration of nonfat milk or whey solids, particularly if the temperature of storage is allowed to fluctuate. Lactose crystals 16 to 30/am long lead to the feeling of sandiness. Fat globules, casein micelles, ice crystals, and phase interfaces were easily distinguished in ice cream by electron microscopy (14) (Figure 5). Fat crystallized inside fat globules was demonstrated at high magnifications. Freezefracturing was also used in studies of the microstructure of the ice cream mix, the microstructure of ice cream extruded at various temperatures, and the microstructure of ice •cream containing vegetable oil (6, 7, 8). The number of fat globules per unit area of the examined freeze-etched surface may serve Journal of Dairy Science Vol. 68, No. 12, 1985
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Figure 5. Chocolate ice cream, a = Part of an air cell; f = fat globules; w = aqueous phase; dark arrow points to casein micelles; irregular dark areas (asterisks) are residues of the sample retained in spite of cleaning the replica. The rnicrograph of a platinum-carbon replica is reproduced by courtesy of W. Buchheim.
as an indicator of destabilization of the ice cream mix; these globules are those that have remained intact while others either have disintegrated partially and formed large clusters or have disintegrated completely and contributed to the coating of the air cells. C R E A M CHEESE
American cream cheese contains a minimum of 33% fat; Neufchatel cheese, which has a similar microstructure, contains less fat (20 to 33%) and may contain more moisture (22). Both cheeses are spreadable. Two types of cream cheese are manufactured (22, 44, 50, 74): in the traditional system of manufacturing, the cream mix is pasteurized, homogenized, and ripened with a lactic bacterial culture until a pH value of approximately 4.6 is reached. The curd is heated to 52 to 63°C, after which it is drained. Journal of Dairy Science Vol. 68, No. 12, 1985
The curd is either cold-packed or hot-packed. In the newly formulated method of manufacturing, which is used to produce "cream cheese spread" (42), the amount of total solids in the cream mix is increased to correspond to the final desired composition of the cheese. Following pasteurization, homogenization, and cooling to incubation temperature of approximately 30°C, the mix is inoculated with a lactic bacterial culture and is incubated until the desired acidity (pH 4.6) is reached. The curd is homogenized and packed without cooling. This procedure does not require the draining of whey. Stirring and homogenization of the mix, which are the essential steps in the manufacture of cream cheese, disrupt the curd and produce a corpuscular microstructure in the finished product. There is no rigid protein matrix and its absence makes this cheese spreadable.
MICROSTRUCTURE OF DAIRY FOODS : REVIEW Each of the two systems of manufacture is associated with a distinct type of sensory attributes and microstructure (44). Cream cheese made in the traditional way of manufacture is firmer and rates higher in the typical cream cheese flavor, whereas the newly formulated cream cheese is easier to spread and has a lower adhesiveness. The former cheese was composed mostly of small (approximately 2 /am in diameter) fat globules with a relatively uniform distribution of protein. Similar results were obtained by Ohashi et al. (56). In some products, the fat globules were aggregated in clusters; approximately 20/am in diameter, and protein in micellar form was accumulated in the superficial layer of the fat globule clusters (44) (Figure 6). In newly formulated cream cheese, most fat globules were ruptured during manufacture and formed large fat particles, over 2 /am in diameter. Space between the fat particles consisted of relatively uniformly distributed protein (Figure 6). Neufchatel cheese, characterized by a lower fat content, resembled newly formulated cream cheese as far as microstructure was concerned. Recently, a cream cheese spread was developed by mixing cultured high-fat cream with coagulated whey protein (42) or milk curd (43) (Figure 6C). Buchheim and Thomasow (21) added emulsifying salts to cream cheese and found that without heating the casein-fat aggregates in the cheese disintegrated. Heating increased the disintegration into fat globules and nonmicellar casein and resulted in a very fine homogeneous fat distribution in the casein-whey protein matrix. There are also imitation cream cheeses on the market: the milk-based product (a mixture of cream cheese and cottage cheese) was easy to distinguish by electron microscopy (44) by the presence of casein micelles and small fat globules from the non-dairy imitation cream cheese. BUTTER
Butter differs from the preceding fat-based milk products by being a water-in-oil emulsion, which means that water (approximately 20% of the total mass of butter) is in the form of a very fine dispersion in semisolid milk fat; water droplets are smaller than 10 /am in diameter. Fat lamellae separate the water droplets from
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each other and prevent them from coalescing. This fine dispersion contributes to the keeping quality of butter. The microstructure of butter was described in many research papers as well as reviews (16, 32, 47, 48, 54, 59, 60, 61, 62, 63, 66). Sweet and microbiologically ripened (cultured) cream is the starting material for the production of butter. Churning at 7 to 18°C disrupts fat globule membranes in the cream and leads to the coalescence of the fat globules and the formation of butter grains which are then separated from buttermilk. A part of the disintegrated fat globule membranes is retained in butter and the other part is released in buttermilk where the fat globule membrane fragments can be detected by electron microscopy (39). Still intact fat globules are also embedded in the free butterfat (Figure 7). The higher the free milk fat content, the softer and more spreadable the butter. Buchheim and Precht (20) classified fat globules in butter into four main types. There may be a number of intermediate forms depending on the distribution of liquid and crystallized fat in the globules, as seen in replicas of freeze-fractured butter specimens. The fat globules may contain a very small amount of crystalline fat in the form of thin monomolecular layers (Type 1); they may have individual fat crystals and crystal aggregates of irregular shape in the interior and a relatively thin crystalline shell at the globule surface (Type 2); they may consist of liquid fat in the interior and a thick (up to 500 nm) crystalline shell consisting of parallel monomolecular layers at the surface (Type 3); or they may be composed of a thick crystalline shell and a high concentration of crystalline aggregates in the globule interior (Type 4). Fat globules that have remained intact during churning exhibited the same structural features in butter as in the cream. Only some fat globules were stable enough to withstand the shear forces of churning. Such fat globules possessed a thick shell (100 to 500 nm) of crystalized high-melting fat fractions and crystal aggregates varying in dimensions. Crystal aggregates were also found (20) in the continuous liquid fat phase in butter. The crystals, either isolated or in groups with a mostly parallel orientation, displayed slightly curved surfaces and were considered to represent Journal of Dairy Science Vol. 68, No. 12, 1985
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Figure 6. Cream cheese (A) and cream cheese spread (B and C). Transmission electron microscopy o f samples postfixed with o s m i u m tetroxide, e m b e d d e d in a resin, a n d stained with uranyl acetate and lead citrate. A. Traditional m a n u f a c t u r e . Small fat globules are aggregated (f) and protein particles (arrows) are concentrated at t h e surface of t h e aggregates, w = A q u e o u s phase. B. New formulation (44). Membranes (dark arrows) surr o u n d mostly only small fat globules and are n o t f o u n d on large fat particles (f). Protein (hollow arrows) is relatively u n i f o r m l y distributed in the cream cheese spread, w = A q u e o u s phase. C. New formulation based on North American Ricotta cheese (42). Small fat globules (f) and protein particles (arrows) are u n i f o r m l y distrib u t e d in the product. Journal of Dairy Science Vol. 68, No. 12, 1985
MICROSTRUCTURE OF DAIRY FOODS: REVIEW
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Figure 7. Butter. Two micrographs of platinum-carbon replicas are reproduced by courtesy of W. Buchheim. Butter consists of fat globules (f) and a small number of water droplets (w) dispersed in free fat. A. General view. B. Detail of a water droplet in which casein micelles (dark arrow) are evident; the droplet is covered with a thin layer of fat crystals (hollow arrows). Sample residues in the replicas are marked with asterisks. fragments of disrupted fat globules. However, crystals that developed in butter during 10 d of storage were newly formed fat crystals and were probably involved in the setting and hardening of stored butter. Crystallization of milk fat was recently studied microscopically by Foley and Brady (29); the temperature at which milk fat crystallized influenced its firmness, crystalline conformation, and the proportion of solid fat at 5°C. CONCLUSIONS
Optical microscopy has been used in dairy science for a long time, but the use of electron microscopy is relatively new. The greatest attention has been paid to the basic constituents of m i l k such as casein micelles and fat globules. The development of new microscopical methods and the modification of methods used in other scientific disciplines have made it possible to study interactions of milk constituents with each other as well as with other substances. Changes in the nature of the constituents and
the interaction products affect the microstructure of resulting milk products. Milk products based on fat are the subject of this review. Their microstructure is related to other characteristics such as consistency in whipped cream, smoothness in ice cream, and spreadability in cream cheese and in butter, etc. Other effects are related to the conditions under which milk products are stored, for example, duration and temperature changes. The use of nondairy ingredients, such as soy protein and vegetable oils, in imitation milk products poses a challenge to detect such ingredients and understand their effects of the product. In addition to electron microscopy, fluorescence microscopy also has the potential of significantly contributing to microstructural studies of milk products based on fat. ACKNOWLEDGMENTS
The author thanks W. Buchheim (Bundesanstalt fiir Milchforschung, Kiel, Federal Republic of Germany) and D. G. Schmidt and A.C.M. Journal of Dairy Science Vol. 68, No. 12, 1985
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van H o o y d o n k ( N e d e r l a n d s I n s t i t u u t v o o r Z u i v e l o n d e r z o e k , Ede, t h e N e t h e r l a n d s ) f o r p e r m i s s i o n t o r e p r o d u c e m i c r o g r a p h s in Figures 4, 5, and 7. Technical assistance and assistance w i t h t h e m a n u s c r i p t p r o v i d e d b y Paula AllanWojtas is gratefully a c k n o w l e d g e d . A p p r e c i a t i o n is e x p r e s s e d t o D. B. E m m o n s and H. W. M o d l e r for useful suggestions and for reviewing t h e m a n u s c r i p t . E l e c t r o n M i c r o s c o p e Centre, Research Branch, Agriculture Canada, O t t a w a , p r o v i d e d facilities. This review is C o n t r i b u t i o n 638 f r o m t h e F o o d R e s e a r c h I n s t i t u t e , Agriculture Canada in O t t a w a . REFERENCES
1 Allan-Wojtas, P., and M. Kalab. 1984. Milk gel structure. XIV. Fixation of fat globules in wholemill~ yoghurt for electron microscopy. Milchwissenschaft 39(6): 323. 2 Allan-Wojtas, P., and M. Kalab. 1984. A simple procedure for the preparation of stirred yoghurt for scanning electron microscopy. Food Microstruct. 3(2):197. 3 Anderson, M., B. E. Brooker, T. E. Cawston, and G. C. Cheeseman. 1977. Changes during storage in stability and composition of ultra-heat-treated aseptically-packed cream of 18% fat content. J. Dairy Res. 44:111. 4 Angermtiller, S., and H. D. Fahimi. 1982. Imidazolebuffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy. Histochem. J. 14:823. 5 Arbuckle, W. S. 1977. Ice cream. 3rd ed. AVI Publ. Co., Inc., Westport, CT. 6 Berger, K. G. 1976. Ice cream. Pages 141-213 in Food emulsions. Stig Friberg, ed. Marcel Dekker, Inc., New York and Basel. 7 Berger, K. G., B. K. Builimore, G. W. White, and W. B. Wright. 1972. The structure of ice cream -- Part 1 and Part 2. Dairy Ind. 37:419, 493. 8 Berger, K. G., and G. W. White. 1971. An electron microscopical investigation of fat destabilization in ice cream. J. Food Technol. 6:285. 9 Bhandari, V., R. Balachandran, and D. N. Prasad. 1984. Influence of stabilizers and an emulsifier on the ultra-structure of spray-dried ice-cream mix. N.Z.J. Dairy Sci. Technol. 19:55. 10 B~hler, S. 1976. Artefacts and specimen preparation faults in freeze-etch technology. Balzers AG, Balzers, Liechtenstein. 11 Brooker, B. E. 1979. Milk and its products. Pages 273-331 in Food microscopy. J. G. Vaughan, ed. Academic Press, New York, NY. 12 Brooker, B. E., and K. Wells. 1984. Preparation of dairy products for scanning electron microscopy: etching of epoxy resin-embedded material. J. Dairy Res. 51(4):605. 13 Buchheim, W. 1970. Der Verlaufder Fettkristallisation in den Fettkiigelchen der Milch. Elektronenmikroskopische Untersuchungen mit Hilfe der
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