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Protein-Lipid Interactions and their Relation to the Physical-Chemical Stability of Concentrated Milk. A Review1,2
PROTEIN-LIPID INTERACTIONS A N D T I t E I R R E L A T I O N TO T H E PHYSICAL-CHEMICAI, STABILITY OF CONCENTRATED M I L K . A R E V I E W i.-' J. tl. BRUNNER Department of Food Science, Michigan State University, East Lansing Various aspects of chemical and physical intere, ctions involving lipids and protein have been the object of numerous detailed studies, especially with reference to blood and cellular lipoprotein complexes (4, 33). This p a p e r will emphasize (a) the niechanisms of lipid-lipid and lipid-protein interactions, (b) the nature of the lipoprotein complex constituting the socalled fat globule membrane of normal bovhm milk, and (c) stone of the effects of processing procedures on the characteristics of the f a t / plasma interfacial layer.
To develop a practical understanding of these interactions we must first consider the relationship of lipid molecules to proteins and to each other. This objective might best be served through a consideration of the types of forces involved (Figure 1). R
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LIPID-LIPID
AND
PROTEIN-LIPID
Iee
i
.J
Non-Polar
INTERACTIONS
There is little evidence to suggest that lipidlipid or protein-lipid interactions occur through covalent bonds (e.g., ester bonds). Rather, association appears to occur through interactions involving functional groups common to both of the reacting substances, such as occur between nonpolar fatty acid chains and similar residues constituting the side chains of amino acid residues in the peptide chain of a protein molecule. Also, interactions involving polar or charged groups may occur. Possibly, the binding of lipids to proteins or vice versa may depend on such factors as configuration, nmltiple attachment sites, a n d / o r the matching of polarity between opposing groups, in much the same manner as for the combination of enzyme and substrate. In understanding the nature of protein-lipid interactions, we must be careful not to draw the apparent conclusion that the bound lipid molecule associates itself only with receptive sites in the protein. For, if this were the ease, we could not justify the existence of low-density lipoproteins, such as the fl-lipoprotein of blood plasma, in which the lipid moiety constitutes the larger portion of the complex ( > 70% lipid). An individual lipoprotein particle contains many thousand lipid molecules, far more than could be expected to make direct contact with the larger protein components. Consequently, most of the lipid molecules find themselves associated with other lipid molecules.
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Fro, 1. S(.hematic representation of three types of bonding forces involved in lipid-lipid and protein lipid interactions.
Presented at the Fifth l~lilk Concentrates S~'mposium, University of Illinois, Urbana. October, 1961.. -"Michigan Journal Article no. 3004.
Non,polar. The arrangement of bonds around carbon atoms in a saturated hydrocarbon chain predisposes a symmetrical distribution of shared electron pairs. These structures, which are called nonpolar or van der Waal's forces, exert close-in attraction. They become important only when the interacting molecules or groups are in close fit. Polar. In bonds between atoms possessing wide differences in electronegativity, the shared pair of electrons is not distributed symmetrically. F o r example, the O-H bond is polarized by the attraction of 0 for the electron pair so that the 0 atom is negatively charged and the H atom becomes positive. This is a polar group and, as such, exerts attractive forces for oppositely charged polar groups. Tile functioning of the hydrogen bond which operates between the 0 of the O-H group and the peptide- or phospholipid-N represents the prin-
943
944
J.R. BRUNNER
cipal attracting force in many protehMipid interactions. Charged groups. Many of the polar groups in lipids and proteins behave as proton donors or aeceptors and, therefore, exist as charged groups. These are strong attractive forces that serve to orient polar molecules or groups over distances beyond the influence of other attractive forces. Charged group attraction could account for the general orientation of the lipoprotein complex and may determine the structure. Phospholipids, mono-diglycerides, cholesterol, and fatty acids constitute the polar lipids of milk, in that they possess polar groups oriented toward water or groups of opposite charge. These lipids have a tendency to orient at an oil/water interface in such manner that the nonpolar portion of the molecule is soluble in the oil while the polar portion is soluble in water. Structurally, they are balanced so that they are only moderately soluble in water (except for the water-soluble, short-chain fatty acids); thus, when added to an aqueous phase they tend to associate into sol-like micelles, with the polar heads reaching out for the water and the nonpolar tails extending into the micelte. Such structures tend to imbibe water in a bound form (40 to 60% of micellular weight) without passing into solution. Interfacially oriented polar ]ipids contribute a strong electrostatic field with somewhat elusive characteristics. Mutual repulsion by likecharged groups is reduced considerably by increasing the ionic strength in the aqueous phase. Under these conditions, oppositely charged groups move into the interracial area to compensate many of the repulsive forces and contribute to the instability of the fihn. I f repulsive forces of the charges borne by the interfacially oriented polar heads of the molecule are weak, net cohesive forces between these groups may be exerted. The packing of polar groups at an interface has been interpreted in terms of hydrogen bonding (1). Obviously, these polar lipids also possess nonpolar characteristics, a contribution of their long hydrocarbon structure, which adds to the cohesiveness of interfaeially oriented molecules. Long-chain saturated fatty acids, such as stearic, pack rather closely, whereas the distortion around the double bond of oleie acid precludes close p~eking. Triglyeerides, in general, form less cohesive films than their corresponding fatty acids. I t has been suggested that their ester structures do not enter into hydrogen bonding with the neighboring molecules (2).
The higher triglyecrides, cholesterol esters, fl-carotene, long-chain f a t t y acids and, to a lesser extent, cholesterol are examples of nonpolar lipids completely immiscible with water. These lipids can be stabilized through the adsorption of polar lipids or proteins carried in the aqueous phase. Cholesterol and cholesterol esters form closely packed solid films in which the hydrocarbon moieties interact strongly through nonpolar bonding (van der Waal's or London forces). Hydrogen bonding cannot be considered a significant factor in this type of interaction (2). Emulsions, such as the oil-in-water emulsion typical of milk, are formed when two immiscible phases are mixed. I f film-forming substances like the polar lipids a n d / o r proteins are present, the emulsion is more easily formed and stabilized by the tendency on the p a r t of these materials to orient at the oil/water interface. More stable enmlsions are formed when two or more fihn-forming substances are present in the system. F o r example, the presence of both phospholipids and proteins promotes the formation of a more stable enmlsion than either agent alone. The forces of interaction in mixed fihns are difficult to evaluate. Electrolytes tend to pronmte the floceulation of emulsion, but the fihn must be ruptured or penetrated before coalescence occurs. The factors working" for aggregation of the emulsion are mitigated by the repulsive forces exerted by the bound water in the interracial surface. What has already been said about the forces influencing the interaction and orientation of lipids is applicable also to proteins. The proteins of milk exist in both the fonn of micellular sols (casein) and true solutions (sermn proteins). Both physical forms are surface-active. We recognize the increased complexity of protein-protein a n d / o r protein-lipid interactions, since the proteins exist as large molecules and interacting molecular systems, nmch larger than the most complex of lipid molecules. Many of the fundamental concepts of protein interactions have been discussed in this symposium. However, let us consider a few properties of proteins oriented in fihns. Proteins spread at an interface are believed to occupy an area corresponding to the thickness of a single polypeptide chain rather than the thickness of the protein in its coiled native configuration in aqueous solutions. The initiation of the unfolding, sometimes irreversible (denaturation), may involve no more than an alignment of the polar or nonpolar groups with respect to the aqueous and nonaqueous plmses. In large mole-
STABILITY
OF
CONCENTRATED
cules it is conceivable that some of the nonpolar groups extend into the hostile aqueous phase, with a corresponding loss in free eneL~'y. Presumably, all of the polar groups available to the water phase bind water in a nmre or less stabilized form, amounting to ~ 10 to 20% of the weight of the protein. The loss or alteration of this bound water during freezing or dehydration causes undesirable changes in the physical and chemical properties of the protein or protein-lipid complex. Ions and ionic strengths affect the stability of this bound water in various ways. Gurd (13) has placed lipid-protein interactions in two categories: (1) lipid molecules bound to proteins through distinct reaction sites and (2) interactions characterized by the combination of proteins with lipid molecules in combination with other lipid molecules. We should expect to encounter both types of' interactions in milk and its products. As an example of the first type of interaction, we might select the classical example of long-chain fatty acid binding by serum albumin (11). The longer the chain, with some exceptions, the greater the tendency to form a complex. Palmitic and stearic are bound nearly identically. Linoleic binds less strongly than oleic or stearic. Several types of binding sites are involved in these interactions, which may reflect changes in the physical properties of the components: for example, caprylate-bound serum albumin is stabilized against denaturation by heat (5). The resurfacing of butter oil with plasma proteins at the time of homogenization in all probability represents a simple protein-lipid interaction. The fl-lipoprotein of human plasma and mierosomal lipoprotein complexes are considered to be examples of the second type of interaction. In some respects the native lipoprotein complex constituting the fat globule membrane falls in this category. Proteins that are bound to interfacially oriented lipids are displaced from their surface aligmuent by the application of external pressure only to reorient when the pressure is reduced. Such a phenomenon might partly explain the temperature-dependent orientation of the euglobulins and lipases at the f a t / p l a s m a interfacial surface. Possibly, the nature of the lipid component oriented at the surface exerts some influence on the adsorbability of the protein layer. The author acknowledges the excellent review of protein-lipid interactions by Gurd (13), from which much of the preceding discussion was drawn.
945
MILK
THE
FAT
GLOBULE
LIPOPNOTEIN
MEMBRAN'E
COMPLEX
tti freshly secreted, uncooled milk the lipoprotein complex of the so-called fat globule membrane represents tile principal proteinlipid interaction. King (23), in his excellent review, The Milk F a t Globule Membrane, depicts the membrane structure as a surface layer of polar phospholipides and other less polar lipids--eholesterol, vitamin A, etc.--which, in association with molecules of a high-melting triglyeeride, extends into the peripheral area of the fat droplet (Figure 2). Attached to the polar heads of the phospholipids, he showed a closely oriented layer of protein that seemed to extend into the plasma to an extent dependent upon the residual electrostatic charge. Although all of these components, and perhaps others, are known to be present at the f a t / plasma interface, his illustration may represent an over-sin~plification of the situation as it actually occurs. Whether these components are oriented, as King suggests, as neatly organized adsorption layers or as adsorbed, complex lipoprotein particles awaits the results of more definitive experimental evidence. A particulate-like stl~ucture in which the polar and nonpolar lipids associate and thus form insoluble micelles onto which proteins, or even a specific protein, are adsorbed seems worthy of consideration. Many thousands of these lipoprotein particles could form the membrane-like surface of the f a t droplet. Morton (29) has shown the similarity between adsorbed membrane materials and microsomes, a cellular lipoprotein complex. He based this conclusion on the enzyme complement identified with the °-
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VITAMIN A
FIG. 2. Structure of the milk fat globule membrnne as represented by N. King (23).
946
J ~. m~uxx~.:R
m e m b r a n e a n d on electron p h o t o m i e r o g r a p h i e data. H o w e v e r , these assays c a n n o t be considered extensive enough to constitute absolute evidence t h a t the f a t globule m e m b r a n e s are microsomes p e r se. H a n s s o n ' s (14) elect r o n p h o t o m i c r o g r a p h s of the d e f a t t e d memb r a n e s t r u c t u r e indicate the presence of definite s t r u c t u r a l characteristics. O t h e r concepts relative to the f o r m a t i o n of the m e m b r a n e include the suggestion of M u l d e r (30) t h a t the f a t surface was coated with prot o p l a s m i c m a t e r i a l d u r i n g its f o r m a t i o n . Somm e r (37) h y p o t h e s i z e d t h a t the f a t d r o p l e t s were covered b y a m o n o l a y e r of p h o s p h o l i p i d s , which a c t u a l l y t u r n e d o u t to be a hi-layer, a n d t h a t g l o b u l a r size was r e l a t e d to the a v a i l a b i l i t y of p h o s p h o l i p i d s . R e c e n t l y G r e e n b a n k a n d P a l l a n s e h (12) suggested t h a t the f a t s u r f a c e was coated w i t h a h i g h l y surfaee-aetive protein onto which l i p o p r o t e i n s were absorbed, n m e h as p r o p o s e d b y M o r t o n (29). D e s p i t e o u r i n a b i l i t y to decide on the g e o m e t r y or origin of the f a t globule m e m b r a n e , m a n y of us (3, 6, 16, 21, 34, 38) h a v e h a d an i n t e r e s t i n g time a t t e m p t i n g to d e t e r m i n e its composition. F r o m these d a t a we can l e a r n much a b o u t the n a t u r e of the m e m b r a n e a n d how it is influenced b y different e n v i r o n m e n t a l factors. ¢O~IPOSITION
OF T t t E
FAT
~¢IE~fBRANE
COMPLEX
Since the time of S t o r c h (38), m e m b r a n e p r e p a r a t i o n s h a v e been o b t a i n e d by c h u r n i n g plasma-free, w a s h e d cream. Despite the eritieisms aimed at this procedure, it r e p r e s e n t s the best of several possible methods a n d does give u s i n f o r m a t i o n relative to the most closely associated i n t e r f a e i a l substances. I f we are n o t concerned w i t h the loosely associated materials or with absolute d i s t r i b u t i o n data, we can j u s t i f y this e x p e r i m e n t a l a p p r o a c h . P r e p a r a tions o b t a i n e d f r o m a similar source of milk a n d isolated by identical p r o c e d u r e s show a r e m a r k a b l e degree of r e p l i c a t i o n with respect to composition a n d physical-chemical behavior. A s u m m a r i z a t i o n of o u r e x p e r i m e n t a l d a t a is p r e s e n t e d in Table 1. W h o l e - m e m b r a n e p r e p a r a t i o n s c o n t a i n e d f r o m 30 to 6 0 % p r o t e i n , d e p e n d i n g on p r e p a r a t i v e procedures. F r e e d f r o m the lipid moiety b y t r e a t m e n t with ethanol in ether, the p r o t e i n m a t e r i a l consisted of a p p r o x i m a t e l y equal p o r t i o n s of water-soluble a n d insoluble fractions. The insoluble f r a c t i o n was o b t a i n e d as a flesh-colored, m u c o i d a l - a p p e a r i n g p r o t e i n . I t was solubilized b y treatm e n t with sodium sulfide, peraeetic acid in c o m b i n a t i o n w i t h a m m o n i a water, sodium dodecyl sulfate, thioethanol, a n d sodium h y d r o x -
TABLE
1
Summary of the characteristics of the far globule membrane Characteristics of the total menlbrane substance Contains from 30 to 60% protein, depending upon the age of the milk and method employed in its isolation, consisting of two principal fractions. (1) A water-soluble fraction comprising about 45% of the totat protein and charaeterized by its carbohydrate moiety ( ~ 6.5%) has been classified tentatively as a glyeoprotein. Sedimentation coefficients (S~o) ranging between 8 and 17 have been reported for this protein and reflect the amount of contaminating lipid in the preparation. The more lipid in the protein fraction, the higher is the S~. The protein shows heterogeneous characteristics in freeboundary eleetropboresis. (2) A water-insoluble fraction, reddish-brown in color and comprising about 55% of the protein, which was solubilized by disulfide cleaving agents, has been classified tentatively as a pseudokeratin. The protein exists as a high molecular weight aggregate, too large to charaeterize by the usual physical methods. Contains from 40 to 70% lipid, which has been classified by silicic acid chromatography. Component Percentage of total lipid Carotenoids 0-0.5 Squalene 0-0.6 Cholesterol esters 0.6-0.8 Triglyeerides 50.0-53.4 Free f a t t y acids Traces-6.3 Cholesterol 3.6-5.2 Mono-diglycerides 12.8-17.0 Phospholipids 20.4-28.7 ~Iost of the triglyeerides consist of a high-melting fraction (50-530). Conceivably, the glyeerides do not constitute an integral part of the membrane ]ipoprotein complex. Lipid constituents of eentrifugMly fraetionated membrane lipoproteins (25,000 × G × 2 hr) F a t globule membrane substance was separated into a high-density pellet and a lower-density supernatant which contained approximately 10 and 60% of lipid, respectively. The lipid moieties were elassifled by silieie acid chromatography. Component Carotenolds Squalene Cholesterol esters Triglyeeride Fatty acids Cholesterol Mono-diglycerides Phospholipids
Percentage of total lipid Pellet Supernatant 0. 0.3 0. ] .0 0. 5.4 44.2 50.5 4.7 5.5 3.7 2.4 9.2 15.5 38.2 23.2
When considered on the basis of absolute yield, these two lipid fractions were quite similar, with the exception of the glyeeride components, which were higher in tlle supernatant fraetion. Conceivably, the phospholipids eonstitute the principal lipid of the membrane lipoprotein complex.
STABILITY
OF
CONCENTRATED
ide at p H 10-11, agents generally used to solubilize keratin- or pseudokeratin-like proteins. The soluble protein fraction varied from reddish-brown to white, depending' on the age of the milk and techniques employed in the isolation. This fraction, obtained as a fat-free protein, showed single eleetrophoretic and ultracentrifugal boundaries in phosphate or veronal buffers ( p H 6-8.6) and two or three electrophoretie boundaries at lower values of p i t (2-3). The protein contained 11 to 12% nitrogen, plus hexose, hexosamine, and sialie acid (N-aeetyl neuraminie) and was classified tentatively as a glyeoprotein (41). Palmer and Samuelsson (34) and, more recently, Jackson et al. (19) have reported that the membrane protein (soluble fraction) was highly antigenic and apparently different from any of the other proteins of milk. The latter investigator classified the protein as a mueoprotein because of its relatively high hexose content. Whether this protein differs from one of the proteins that make up the so-called proteose-peptone fraction of skimmilk remains to be deternfined. PreliminaD" data obtained in our laboratory by the Ouchterlony antibody-antigen diffusion technique suggest that these fractions might be biological identities. More work remains to be done before this implication can be recorded as a fact. The total lipid moiety, obtained from the alcohol-ether extract of the total membrane, when ehromatographed on a silicie acid colunm, was composed of about 50% triglyeerides and 20 to 30% phospholipids. Smaller quantities of cholesterol, fatty acids, and mono-diglyeerides and traees of cholesterol esters, carotene, and squalene-like substances were observed (43). Some of the supposedly peripherally bound lipid components were removed from the intact membrane by washing with ethyl ether before addition of alcohol (sqnalene, carotene), indicating that these materials did not constitute an integral p a r t of the membrane lipids. We have reported a classification of lipids constituting centrifugally separated (25,000 × G for 2 hr) lipoprotein fractions of the whole membrane material (7). The clear supernatant layer carried a low-density lipoprotcin containing about 60% lipids, whereas the highdensity lipoprotein constituting the sedimented pellet contained about 11% lipids. The a p p a r ent difference between the lipids of these two fractions was found in the triglyceride to phospholipid ratios, which for the pellet was about 1:1 and for the supernatant about 2:1. A slightly higher concentration of mono-diglye-
MILK
9~7
eride was noted in the supernatant fraction. Recently, Alexander and Lusena (3) reported lipid characteristics of various centrifugally eut membrane fractions. An interesting characteristic of the membrane lipids is the presence of relatively large amounts of phospholipids and triglycerides--presmnably the high-melting fraction reported by Palmer and his associates years ago (22). The highmelting glyceride fraction has been isolated from butteroil (35, 42) and is believed to constitute the total triglyeeride of the isolated membrane lipids (43). Conceivably, then, phosphotipids constitute the major lipid class found in the membrane-lipoprotein complex. These two lipoprotein fractions were suspended in veronal buffer and examined both eleetrophoretically and ultraeentrifugally. The pellet fraction showed one eleetrophoretie peak, but it was too large an aggregate to study in the ultracentrifuge. The supernatant fraction showed heterogeneous electrophoretie characteristics. The ultraeentrifugal diagrams possessed multi-boundary characteristics, suggesting the existence of several lipoprotein species of various states of aggregation. When lipid was removed from the complex, the remaining protein moiety showed ultraeentrifugal characteristics more indicative of a homogeneous component. Interestingly, the removal of lipid from the complex is accompanied by a corresponding reduction in the sedimentation eoeffteient, an observation reflecting the role played by the lipid in the state of aggregation of the lipoprotein. ~I:ODIFICATION OF T H E
RESYLTING
FRO~[
F A T G L O B U L E ~-~¢IEMBRA~E
PROCESSING
PROCEDURES
The fat membrane lipoprotehls are sensitive to the usual agents that tend to disorganize native proteins and are sensitized by agents promoting destruction of the lipid components. All processing manipulations are detrimental to the stability of the native membrane material. In practice, we must select processes or a combination of processes that exert minimal detrimental influences on the physicaI-chemieal equilibria at the membrane interface. Some of these ft~etors are considered below. Mill: supply. At the normal p H values of milk, the membrane exists in a relatively stable, hydrophilie state. An increase in the p H enhances this condition, while a decrease contributes to its instability (32). King (23) has discussed the destabilizing effect of incorporated air bubbles resulting from agitation of milk at temperature near the melting point of
948
J.R. ~m-xx~.:l~
the fat on the fat emulsion. Thurston (44), and more recently, Greenbank and Pallansch (12), have suggested that the membrane phospholipids are displaced into the plasma portion of milk as a result of agitation. Whether the phospholipids were displaced as unbound molecules or as membrane particulates was not clarified. The effect of agitating raw milk on the enhancement of induced lipolysis is well known and has been a contempora15" problem relative to pipe-line milking systems and farm tank storage of milk (9). The cooling and/or warming of milk influence the composition of fat membrane and, conceivably, the stability of the fat emulsion (24, 36). Separation of milk. Agitation effects encountered during the separation of milk cause the release of membrane materials, which seems to be aeeentuated at 90 F or above. Additional losses are encountered when quantities of incorporated air are present in the milk to contribute to the destabilization of the fat emulsion. I n fact, the result of such a condition is an excessive loss of small fat globules into the skimnfilk phase. Apparently, the cold-separation or clarification of milk (40 to 45 F), although somewhat inefficient, exerts a less destabilizing effect on the native membrane structure. At these temperatures the euglobulin fraction of the plasma proteins is associated with the globular surface (36). Frmu a practical consideration, I would conjecture that cold separation, provided the process was sufficiently efficient to remove small globules, would be more desirable than warm separation for skimmilk going into NFDM. I n this way the phospholipid content of skimmilk could be reduced, and hence its tendency to develop storage deterioration. Heating and vacuum processing. Lowenstein and Gould (28) showed that when milk was heated to 82 C (180 F ) for 15 rain there was a loss of recoverable membrane material and the loss of protein exceeded the loss of lipids. Studies conducted in our laboratory by Jackson (17) showed that the amount of membrane material recovered from homogenized milk heated to 1 7 5 F 30 rain was less than from a batch heated to 145 or 160 F. Apparently a mild heat treatment in the absence of violent agitation does not cause excessive losses of membrane material. However, high-velocity heating systems from which air has not been excluded could be a factor in the destabilization of the fat emulsion. Greenbank and Pallansch (12) observed that the agitation which accompanied concentration of milk in single-effect evaporators caused a migration of phospholipids into
the skimmilk phase. One might expect that single-pass evaporators would exert less influence on phospholipid migration. I t would be interesting to know whether homogenization at the temperatures of pasteurization would decrease the migration of membrane material into the skimmilk since, in practice, homogenization at this point (160 to 170 F) seems to enhance the stability of the fat emulsion. Homogenization. Although homogenization has been used to stabilize the fat emulsion in milk, some rather profound changes occur at the fat/plasma interface. Upon homogenization, the fat globules (average diameter = 4-5 tL) are broken into small globules (about 1 t~ in diameter) with an accompanying flvefold increase in surface area. Jackson and Brunner (18) have shown that this newly created surface ahsorbs casein as well as other plasma proteins. Interestingly, the absorbed casein appeared to be complexed with a lipid fraction, possibly a phospholipid. Fox et al. (10) and, more recently, Greenbank and Pallanseh (12) have reported the formation of a high-density, lipidcasein complex in honmgenized milk. Litman and Ashworth (27) described an insoluble scumlike material obtained from dlT whole milk for which they reported characteristics of a lipidprotein complex, possibly of phospholipidcasein origin. The resurfacing of the fat globule as a result of homogenization predisposes the lipid phase to enhanced lipolytie and photochemical activity (8, 46, 47). On the other hand, Thurston (44) and others (8, 40, 45) have demonstrated that homogenized nfilk is umre resistant to copper-catalyzed oxidative deterioration. Possibly, some of these changes in the characteristics of homogenized milk result frmn rearrangements involving the native membrane materials, for it is known that even honmgenized, washed cream shows hlcreased resistance to copper-induced oxidation. Possibly the membrane structure is extended in such a manner that copper is immobilized in a strongly chelated state, making it unable to serve oxidative pathways in its usual catalytic role. Tarassuk and Koops (40) have suggested that the increased resistance to copper-induced oxidation noted in homogenized milk resulted from a dilution of the susceptible reactive sites (phospholipids) on the increased membrane surface. Jackson and Pallansch (20) demonstrated that in butteroil-milk protein systems the interfacial tension was lowered significantly more by the readsorption of the soluble membrane protein (mueoprotein) than by any of the other fractions studied. Interestingly, the heat
STABILITY
OF C O N C E N T R A T E D
denaturation of fl-lactoglobulin increased its interfacial activity, a characteristic that might serve well in the resurfacing of the lipid phase in evaporated milk. Greenbank and Pallansch (12) presented evidence to suggest that highpressure homogenization (8,000 psi) resulted in a readsorption of the phospholipids at the fat/plasma interracial surface. It was not clear from their data whether the phospholipids were readsorbed or never left the interracial surface at these pressures. Rehomogenization of milk is effective in further reducing the size of fat globules and increasing the stability of the fat emulsion (26). Where practiced, the separation of homogenized products and rehomogenization of the separated portion would enhance the final stability of the fat emulsion. D~ying and freezing. Native lipoproteins, as well as induced lipid-protein interactions, are dissociated by sharp freezing and dehydration. Alexander and Lusena (3) and Thompson et al. (42) reported that the lipids of the fat membrane complex could be extracted with ethyl ether after freezing and/or freeze-drying. The mechanism of this destabilization seems to involve the state of the bound water in the complex. Browning reactions similar to the classical Maillard reaction between carbonyl groups of sugar and free amino acids have been reported for the interaction of phospholipids and sugars (15, 25). These sugar-amino condensations involve the addition of the amino groups to the aldehyde group of the sugar. Water is eliminated to produce N-substituted aldosylamines; then, through an Amadori-like rearrangement the N-substituted ketosylamine is formed, which degradates to colored melanoidin substances. Lipid-protein copolymerization reactions of the type described by Tappel (39) might conceivably play an important role in the shelflife of dry whole milk. This reaction involves the polymerization of peroxidized polyunsaturated fatty acids and the copolymerization of lipid oxidation products with proteins. These reactions could very well be catalyzed by the heine-containing eytochromes or other trace metal containing proteins of the membrane and milk plasma. The products of the reaction are insoluble and dark in color. In the same tone, Nishida and Kummerow (31) reported that the interaction of the hydroperoxide of methyl linoleate caused the destabilization of the lowdensity fl-lipoprotein of blood. Such a reaction in milk could contribute to the destabilization of the membrane lipoprotein. Despite the general conception that phospholipids contribute significantly to oxidative
949
MILK
deterioration, they do serve as synergistic antioxidants. The ionized phosphate group enters into the initial stages of the oxidative process, involving the unsaturated bonds in fatty acid molecules. At a time when these charged phosphate groups can no longer stabilize the hydroperoxides being formed, the system degenerates rapidly and rampant oxidative deterioration becomes apparent. Our studies with the so-called free fat of spray process whole milk powder demonstrated that this lipid fraction, extracted with a 50/50 mixture of ethyl and pet ether, was composed principally of triglycerides characteristic of the total milk triglycerides. Small quantities of phospholipids were observed, but were present in concentration below that found in the whole lipid fraction. Even though the quantity of free fat is low (~--5% of total lipid), it appears to be significant to the dispersibility of dry milk particles. The solvent rinsed powder is highly dispersible, indicating that the hydrophobic free fat interferes with the ability of water to reach the hydrophilic surface. In this respect, a surface layer of hydrophilic phospholipids covering the free-fat layer would be most beneficial. SUMMARY
We have considered the types of bonding forces responsible for lipid-lipid and proteinlipid interactions. These include (a) nonpolar bonds, which function at short distances and are usually referred to as van der Waal's forces ; (b) polar bonds, resulting from an unsystematical sharing of electrons, which are fairly strong forces and often function through the hydrogen bond; and (c) charged structures, which serve as proton donors or aeceptors and attract oppositely charged groups at considerable distances. The native membrane represents the principal lipid-protein complex in normal milk. It is composed of at least two proteins, phospholipids, a high-melting triglyceride fraction, cholesterol, free fatty acids, mono-diglycerides, cholesterol esters, carotene, and other minor lipid fractions. The normal membrane material is dislodged from its interracial surface by agitation, heat treatment, honmgenization, vacumn concentration, freezing, and drying. Its rearrangement at the interracial surface increases the susceptibility of milk to photo-activation and decreases its susceptibility to nletallic-catalyzed oxidative deterioration. Milk fat can be resurfaced with the proteins of the skimmilk phase, but they are less stable than the native emulsion. Oxi-
950
J, R. BRUNNER
dative deterioration of the lipids enhances the f o r m a t i o n o f insoluble lipid-lipid ~nd/or p r o tein complexes. REFERE:NCES (1) AL]~XA~D~, A. E. The Role of Hydrogen Bonds in Condensed Monolayers. Proc. Roy. Soe. (London), A 179: 470. 1941-42. (2) A ~ X A m ) ~ , A. E. A Proposed General Structure for Condensed Monomolecular Films. Proc. Roy. Soe. (London), A179: 486. 1941-1942. (3) ALEXA~n)ER,K. M., A~rO LUS~A, C. V. Fraetionation of the Lipoproteins of the F a t Globule Membrane from Cream. J. Dairy Sci., 4¢: 1414. 1961. (4) BALL, E. G., AND COOLER, 0. The Activity of Succinate Oxidase in I~elation to Phosphate and Phosphorus Compounds. J. Biol. Chem., 180: 113. 1949. (5) BOYFA~, P. D., BALLOU, G. A., A~D LUCK, 5. M. The Combination of F a t t y Acids and Related Compounds with Serum Albumin. II. Stabilization Against Urea and Guanidine Denaturation. J. Biol. Chem., 162: 199. 1946. (6) BI~L~N~NEI~,ft. R., DUNCAN, C. W., AND TROUT, G. 1~. The Fat-Globule Membrane of Nora homogenized and Homogenized Milk. I. The Isolation and Amino Acid Composition of the Fat-Membrane Proteins. Food Research, 18: 45¢. 1953. (7) BRUI'~NER, J. R., AND THOh[PS0h~, ~I. P. Characteristics of the Fat-Globule Membrane Lipoproteins. J. Dairy Sci., 44: 1170. 1961. (8) DOA~, F. J., Am) MY~a~s, C. It. Effect of Sunlight on Some Milk and Cream Produets. The Milk Dealer, 26 (1) : 893. 1936. (9) DUNK~¥, W. L., A~D S~ITg, L. M. Hydrolytic Rancidity in Milk. IV. Relation Between Tributyrinase and Lipo]ysis. J. Dairy Set., 34: 940. 1951. (10) Fox, K. K., HOLSINGFA~, VIRGINL~, CAttA, JEAN~, AN]) PM~LANSCH, M. J. Formation of a F a t Protein Complex in ~Iflk by Homogenization. J. Dairy Sci., 43: 1396. 1960. (11) GOOD~AN, E. D., AND SCHV~A~-, J. H. Molecular Interactions in Monolayers. I. Complex Formation. J. Colloid Sci., 8: 309. 1934. (12) G R ~ A N K , G. R., AND PALLANSCH, 5I. J. Migration of Phosphatides in Processing Dairy Products. J. Dairy Sci., 44: 1597. 1961. (13) GURD, R. R. N. I n Lipide Chemistry. D. J. ttanahan, Ed. pp. 208-325. John Wiley and Sons, Inc., New York-London. 1960. (14) HA~vSSO~, E. F a t Globules from Milk Studied in the Electron Microscope. Proc. 12th Intern. Dairy Congr., Stockholm, 2: 27. 1949.
(15) HODG~, J. E. Chemistry of Browning Reactions in Model Systems. J. Agr. Food Chem., 1: 928. 1953. (16) JACK, E. L., AXD BAttLE, C. D. The Electrokinetic Potential of Milk Fat. I I I . Relation to the F a t Globule Membrane. J. Dairy Set., 20: 637. 1937. (17) JACKS0.~~, R. H. Proteins of the Homogenized Milk Fat-Globule Membrane. Ph.D. thesis, Michigan State University. 1959. (18) JACKSO~~, R. H., A~D BRL~-NER, J. R. Characteristics of Protein Fractions Isolated from the F a t / P l a s m a Interface of Homogenized Milk. J. D~iry Sci., 43: 912. 1969. (19) JACKSO~~, R. H., CLARK, W. R., A~ND COULSON, E. J. Isolation and Some Properties of Mucoprotein from the F a t / P l a s m a Interface of Bovine Milk. Abstr. No. 64, Div. Biol. Chem., 140th meeting, Am. Chem. Soc. September, 1961. (20) JACKSON, R. H., AhFI) PALLANSCtI, M. 5. A Study of Factors Influencing the Energy of the Butteroil-Mi]k Serum Interface. Abstr. No. 10, Div. Agr. & Food Chem., 138th meeting Am. Chem. Soc. September, 1960. (21) J]~-N'ESS, R., Ah'D PALMER, L. S. Substances Adsorbed on the F a t Globules in Cream and Their Relation to Churning. V. Composition of the Membrane and Distribution of the Adsorbed Substances in Churning. J. Dairy Sci., 28: 611. 1945. (22) ffENNESS, R., AND PALMIER, L. S. Substances Adsorbed on the F a t Globules in Cream and Their Relation to Churning. VI. Relation of the High-Melting Triglyceride Fraction to B u t t e r f a t and the Membrane. J. Dairy Sci., 28: 653. 1945. (23) X~x-o, N. The Milk F a t Globule Membrane. 99 pp. Commonwealth Agr. Bureaux, Yarnham Royal, Bucks, England. 1955. (2~) KRUKOVSKY,V. N., AND SttARP, P. F. Effect of the Properties of the F a t and of the F a t Globule Surface on Lipolytie Activity in Milk. J. Dairy Sci., 23: 1109. 1940. (25) L ~ , C. H. Deteriorative Reactions Involving Phospholipids and Lipoproteins. J. Sci. Food Agr., 8 : 1 . 1957. (26) LEVITO~~, A., A-XD PALLAh.-SCH, M. J. Continuous Recycling in the Homogenization of Relatively Small Samples. J. Dairy Sci., 42: 20. 1959. (27) LIT.~L~N', I. I., .XX'D ASHWORTH, U. S. Insoluble Scum-like Materials on Reconstituted Whole Milk Powders. J. Dairy Sci., 40: 403. 1957. (28) LOWENSTEI~', M., A-N~D GOULD, I. A. The Effect of Heat on the Chemical Nature of the Material Adsorbed on the Milk F a t Globule. J. Dairy Set., 37: 644. 1954. (29) MORTOn, R. K. The Lipoprotein Particles in Cow's Milk. The Bioehem. J-., 57: 231. 1954.
STABILITY
OF
CONCENTRATED
(30) MURDER, H. The Physical Structure of Milk and Dairy Products. Prec. 121h Intern. Dairy Congr., Stockholm, 6: 64. 1949. (31) NISHm.% T., ~ ] ) KUM~F~OW, F. A. Interaction of Serum Lipoproteins with the Hydroperoxide of Methyl Linoleate. J. Lipid Research, 1: 450. 1960. (32) NUGE2CT, R. L. The Application of the Mudd Interfacial Techniques in the Study of Protective Protein Films in 0il-in-Water Emulsions. J. Phys. Chem., 36: 449. 1932. (33) ONCe'Z, J. L., Gum), F. i~. N., AND MEnIN, M. Preparation and Properties of Serum and Plasma Proteins. X X V . Composition and Properties of Human Serum fl-Lipoprotein. J. Am. Chem. See., 72:458. 1950. (3'4) PAnMEI~, L. S., AND SAMUELSS0iq, E. The Nature of the Substance Adsorbed on the Surface of th6 F a t Globules in Cow's Milk. Prec. Soc. Expt. Biol., New York, 21: 537. 1924. (35) PAT~O~¢, S., AND KFm~,~L P. The HighMelting Glyceride Fraction from Milk Fat. J. Dairy Sei., 41: 1288. 1958. (36) SHAr~e, P. F., AND KRUKOVSKY, V. N. Differences in Adsorption of Solid and Liquid F a t Globules as Influencing the Surface Tension and Creaming of Milk. J. Dairy Sei., 22: 743. 1939. (37) SOM~F/~, H. H. The F a t Emulsion in Milk from a Chemical Standpoint. The Milk Dealers, 41 (1) : 60. 1951. (38) STORC~, V. On Structure of " F a t Globu l e s " in Cow's Milk. (Translated and communicated by Faber, A.) Analyst, 22: 197. 1897. (39) TAPPEL, A. L. Studies of the Mechanism of Vitamin E Action. I I I . I n vitro Copolymerization of Oxidized Fats with Proteins. Arch. Bioehem. Biophys., 54: 266. 1955.
MILK
951
(40) TARASSUK, N. P., -~ND KOOFS, J. Inhibition of Oxidized Flavor in Homogenized Milk as Related to the Concentration of Copper and Phospholipids per Unit of F a t Globule Surface. J. Dairy Sci., 43': 93. 1960. (~1) THOMPSON, M. P., AND BEUNN~R, J. R. The Carbohydrates of Some G]ycoproteins of Bovine Milk. J. Dairy Sci., 42: 369. 1959. (42) THOMPSON, M. P., BaUNNF~, J. R., AND STnCE, C. M. Characteristics of High-Melting Triglyceride Fractions from the FatGlobule Membrane and Butter Oil of Bovine Milk. J. Dairy Sei., 42: 1651. 1959. (43) THOMPSON, M. P., BI%UNNEK, J. R., STINK, C. M., A~q) LL',q)QUIST, KAKIN. Lipid Components of the Fat-Globule Membrane. J. Dairy Sci., 44: 1589. 1961. (44) T~URSTO~r, L. M., BR~)WN, W. C., AND DUSTMAN, R. B. Oxidized Flavor in Milk. II. The Effect of Homogenization, Agitation and Freezing of Milk on Its Subsequent Susceptibility to Oxidized Flavor Development. J. Dairy Sci., 19: 671. 1936. (45) TROUT, G. M., AN1) GOULD, I. A. Homogenization as a Means of Stabilizing the Flavor of Milk. Michigan Agr. Expt. Sta., Quart. Bull., 21 (1) : 21. 1938. (46) T~OUT, G. M,, HALL0m~', C. P., ANt) GOULD, I. A. The Effects of Homogenization on Some of the Physical and Chemical Properties of Milk. Michigan Agr. Expt. Sta., Tech. Bull. 145. 34 pp. 1935. (47) WEINST~n¢, B. R., DUNCAN, C. W., AND TROU% G. M. The Solar-Activated Flavor of Y[omogenized Milk. IV. Isolation and Characterization of a Whey Constituent Capable of Producing the Solar-Activated Flavor. J. Dairy Sci., 34:570. 1951.
To develop a practical understanding of these interactions we must first consider the relationship of lipid molecules to proteins and to each other. This objective might best be served through a consideration of the types of forces involved (Figure 1). R
.
. I . °.
oa
.
oo
oo
H
H
H
! . . . . . . . . . . . .
LIPID-LIPID
AND
PROTEIN-LIPID
Iee
i
.J
Non-Polar
INTERACTIONS
There is little evidence to suggest that lipidlipid or protein-lipid interactions occur through covalent bonds (e.g., ester bonds). Rather, association appears to occur through interactions involving functional groups common to both of the reacting substances, such as occur between nonpolar fatty acid chains and similar residues constituting the side chains of amino acid residues in the peptide chain of a protein molecule. Also, interactions involving polar or charged groups may occur. Possibly, the binding of lipids to proteins or vice versa may depend on such factors as configuration, nmltiple attachment sites, a n d / o r the matching of polarity between opposing groups, in much the same manner as for the combination of enzyme and substrate. In understanding the nature of protein-lipid interactions, we must be careful not to draw the apparent conclusion that the bound lipid molecule associates itself only with receptive sites in the protein. For, if this were the ease, we could not justify the existence of low-density lipoproteins, such as the fl-lipoprotein of blood plasma, in which the lipid moiety constitutes the larger portion of the complex ( > 70% lipid). An individual lipoprotein particle contains many thousand lipid molecules, far more than could be expected to make direct contact with the larger protein components. Consequently, most of the lipid molecules find themselves associated with other lipid molecules.
H~
H I. . . . ool le Gle 0 ooi
.
~)'i
$~1
• I • Oi-R I
,8 el, o : .: H~
oo! C , '" ,O
ooL-
I • H i
.
.
.
.
.
H Polar
•
H:
N ~
;t
C oo
.
©It=in
Ohorgod Group
..e
G : O :
:o **.:.n
Fro, 1. S(.hematic representation of three types of bonding forces involved in lipid-lipid and protein lipid interactions.
Presented at the Fifth l~lilk Concentrates S~'mposium, University of Illinois, Urbana. October, 1961.. -"Michigan Journal Article no. 3004.
Non,polar. The arrangement of bonds around carbon atoms in a saturated hydrocarbon chain predisposes a symmetrical distribution of shared electron pairs. These structures, which are called nonpolar or van der Waal's forces, exert close-in attraction. They become important only when the interacting molecules or groups are in close fit. Polar. In bonds between atoms possessing wide differences in electronegativity, the shared pair of electrons is not distributed symmetrically. F o r example, the O-H bond is polarized by the attraction of 0 for the electron pair so that the 0 atom is negatively charged and the H atom becomes positive. This is a polar group and, as such, exerts attractive forces for oppositely charged polar groups. Tile functioning of the hydrogen bond which operates between the 0 of the O-H group and the peptide- or phospholipid-N represents the prin-
943
944
J.R. BRUNNER
cipal attracting force in many protehMipid interactions. Charged groups. Many of the polar groups in lipids and proteins behave as proton donors or aeceptors and, therefore, exist as charged groups. These are strong attractive forces that serve to orient polar molecules or groups over distances beyond the influence of other attractive forces. Charged group attraction could account for the general orientation of the lipoprotein complex and may determine the structure. Phospholipids, mono-diglycerides, cholesterol, and fatty acids constitute the polar lipids of milk, in that they possess polar groups oriented toward water or groups of opposite charge. These lipids have a tendency to orient at an oil/water interface in such manner that the nonpolar portion of the molecule is soluble in the oil while the polar portion is soluble in water. Structurally, they are balanced so that they are only moderately soluble in water (except for the water-soluble, short-chain fatty acids); thus, when added to an aqueous phase they tend to associate into sol-like micelles, with the polar heads reaching out for the water and the nonpolar tails extending into the micelte. Such structures tend to imbibe water in a bound form (40 to 60% of micellular weight) without passing into solution. Interfacially oriented polar ]ipids contribute a strong electrostatic field with somewhat elusive characteristics. Mutual repulsion by likecharged groups is reduced considerably by increasing the ionic strength in the aqueous phase. Under these conditions, oppositely charged groups move into the interracial area to compensate many of the repulsive forces and contribute to the instability of the fihn. I f repulsive forces of the charges borne by the interfacially oriented polar heads of the molecule are weak, net cohesive forces between these groups may be exerted. The packing of polar groups at an interface has been interpreted in terms of hydrogen bonding (1). Obviously, these polar lipids also possess nonpolar characteristics, a contribution of their long hydrocarbon structure, which adds to the cohesiveness of interfaeially oriented molecules. Long-chain saturated fatty acids, such as stearic, pack rather closely, whereas the distortion around the double bond of oleie acid precludes close p~eking. Triglyeerides, in general, form less cohesive films than their corresponding fatty acids. I t has been suggested that their ester structures do not enter into hydrogen bonding with the neighboring molecules (2).
The higher triglyecrides, cholesterol esters, fl-carotene, long-chain f a t t y acids and, to a lesser extent, cholesterol are examples of nonpolar lipids completely immiscible with water. These lipids can be stabilized through the adsorption of polar lipids or proteins carried in the aqueous phase. Cholesterol and cholesterol esters form closely packed solid films in which the hydrocarbon moieties interact strongly through nonpolar bonding (van der Waal's or London forces). Hydrogen bonding cannot be considered a significant factor in this type of interaction (2). Emulsions, such as the oil-in-water emulsion typical of milk, are formed when two immiscible phases are mixed. I f film-forming substances like the polar lipids a n d / o r proteins are present, the emulsion is more easily formed and stabilized by the tendency on the p a r t of these materials to orient at the oil/water interface. More stable enmlsions are formed when two or more fihn-forming substances are present in the system. F o r example, the presence of both phospholipids and proteins promotes the formation of a more stable enmlsion than either agent alone. The forces of interaction in mixed fihns are difficult to evaluate. Electrolytes tend to pronmte the floceulation of emulsion, but the fihn must be ruptured or penetrated before coalescence occurs. The factors working" for aggregation of the emulsion are mitigated by the repulsive forces exerted by the bound water in the interracial surface. What has already been said about the forces influencing the interaction and orientation of lipids is applicable also to proteins. The proteins of milk exist in both the fonn of micellular sols (casein) and true solutions (sermn proteins). Both physical forms are surface-active. We recognize the increased complexity of protein-protein a n d / o r protein-lipid interactions, since the proteins exist as large molecules and interacting molecular systems, nmch larger than the most complex of lipid molecules. Many of the fundamental concepts of protein interactions have been discussed in this symposium. However, let us consider a few properties of proteins oriented in fihns. Proteins spread at an interface are believed to occupy an area corresponding to the thickness of a single polypeptide chain rather than the thickness of the protein in its coiled native configuration in aqueous solutions. The initiation of the unfolding, sometimes irreversible (denaturation), may involve no more than an alignment of the polar or nonpolar groups with respect to the aqueous and nonaqueous plmses. In large mole-
STABILITY
OF
CONCENTRATED
cules it is conceivable that some of the nonpolar groups extend into the hostile aqueous phase, with a corresponding loss in free eneL~'y. Presumably, all of the polar groups available to the water phase bind water in a nmre or less stabilized form, amounting to ~ 10 to 20% of the weight of the protein. The loss or alteration of this bound water during freezing or dehydration causes undesirable changes in the physical and chemical properties of the protein or protein-lipid complex. Ions and ionic strengths affect the stability of this bound water in various ways. Gurd (13) has placed lipid-protein interactions in two categories: (1) lipid molecules bound to proteins through distinct reaction sites and (2) interactions characterized by the combination of proteins with lipid molecules in combination with other lipid molecules. We should expect to encounter both types of' interactions in milk and its products. As an example of the first type of interaction, we might select the classical example of long-chain fatty acid binding by serum albumin (11). The longer the chain, with some exceptions, the greater the tendency to form a complex. Palmitic and stearic are bound nearly identically. Linoleic binds less strongly than oleic or stearic. Several types of binding sites are involved in these interactions, which may reflect changes in the physical properties of the components: for example, caprylate-bound serum albumin is stabilized against denaturation by heat (5). The resurfacing of butter oil with plasma proteins at the time of homogenization in all probability represents a simple protein-lipid interaction. The fl-lipoprotein of human plasma and mierosomal lipoprotein complexes are considered to be examples of the second type of interaction. In some respects the native lipoprotein complex constituting the fat globule membrane falls in this category. Proteins that are bound to interfacially oriented lipids are displaced from their surface aligmuent by the application of external pressure only to reorient when the pressure is reduced. Such a phenomenon might partly explain the temperature-dependent orientation of the euglobulins and lipases at the f a t / p l a s m a interfacial surface. Possibly, the nature of the lipid component oriented at the surface exerts some influence on the adsorbability of the protein layer. The author acknowledges the excellent review of protein-lipid interactions by Gurd (13), from which much of the preceding discussion was drawn.
945
MILK
THE
FAT
GLOBULE
LIPOPNOTEIN
MEMBRAN'E
COMPLEX
tti freshly secreted, uncooled milk the lipoprotein complex of the so-called fat globule membrane represents tile principal proteinlipid interaction. King (23), in his excellent review, The Milk F a t Globule Membrane, depicts the membrane structure as a surface layer of polar phospholipides and other less polar lipids--eholesterol, vitamin A, etc.--which, in association with molecules of a high-melting triglyeeride, extends into the peripheral area of the fat droplet (Figure 2). Attached to the polar heads of the phospholipids, he showed a closely oriented layer of protein that seemed to extend into the plasma to an extent dependent upon the residual electrostatic charge. Although all of these components, and perhaps others, are known to be present at the f a t / plasma interface, his illustration may represent an over-sin~plification of the situation as it actually occurs. Whether these components are oriented, as King suggests, as neatly organized adsorption layers or as adsorbed, complex lipoprotein particles awaits the results of more definitive experimental evidence. A particulate-like stl~ucture in which the polar and nonpolar lipids associate and thus form insoluble micelles onto which proteins, or even a specific protein, are adsorbed seems worthy of consideration. Many thousands of these lipoprotein particles could form the membrane-like surface of the f a t droplet. Morton (29) has shown the similarity between adsorbed membrane materials and microsomes, a cellular lipoprotein complex. He based this conclusion on the enzyme complement identified with the °-
-----n--~:=:=~_:.~ ~-,
.,~ROTEIN
~ : BOUNDWATER CHOLESTERCL
/
_ _ .
_
VITAMIN A
FIG. 2. Structure of the milk fat globule membrnne as represented by N. King (23).
946
J ~. m~uxx~.:R
m e m b r a n e a n d on electron p h o t o m i e r o g r a p h i e data. H o w e v e r , these assays c a n n o t be considered extensive enough to constitute absolute evidence t h a t the f a t globule m e m b r a n e s are microsomes p e r se. H a n s s o n ' s (14) elect r o n p h o t o m i c r o g r a p h s of the d e f a t t e d memb r a n e s t r u c t u r e indicate the presence of definite s t r u c t u r a l characteristics. O t h e r concepts relative to the f o r m a t i o n of the m e m b r a n e include the suggestion of M u l d e r (30) t h a t the f a t surface was coated with prot o p l a s m i c m a t e r i a l d u r i n g its f o r m a t i o n . Somm e r (37) h y p o t h e s i z e d t h a t the f a t d r o p l e t s were covered b y a m o n o l a y e r of p h o s p h o l i p i d s , which a c t u a l l y t u r n e d o u t to be a hi-layer, a n d t h a t g l o b u l a r size was r e l a t e d to the a v a i l a b i l i t y of p h o s p h o l i p i d s . R e c e n t l y G r e e n b a n k a n d P a l l a n s e h (12) suggested t h a t the f a t s u r f a c e was coated w i t h a h i g h l y surfaee-aetive protein onto which l i p o p r o t e i n s were absorbed, n m e h as p r o p o s e d b y M o r t o n (29). D e s p i t e o u r i n a b i l i t y to decide on the g e o m e t r y or origin of the f a t globule m e m b r a n e , m a n y of us (3, 6, 16, 21, 34, 38) h a v e h a d an i n t e r e s t i n g time a t t e m p t i n g to d e t e r m i n e its composition. F r o m these d a t a we can l e a r n much a b o u t the n a t u r e of the m e m b r a n e a n d how it is influenced b y different e n v i r o n m e n t a l factors. ¢O~IPOSITION
OF T t t E
FAT
~¢IE~fBRANE
COMPLEX
Since the time of S t o r c h (38), m e m b r a n e p r e p a r a t i o n s h a v e been o b t a i n e d by c h u r n i n g plasma-free, w a s h e d cream. Despite the eritieisms aimed at this procedure, it r e p r e s e n t s the best of several possible methods a n d does give u s i n f o r m a t i o n relative to the most closely associated i n t e r f a e i a l substances. I f we are n o t concerned w i t h the loosely associated materials or with absolute d i s t r i b u t i o n data, we can j u s t i f y this e x p e r i m e n t a l a p p r o a c h . P r e p a r a tions o b t a i n e d f r o m a similar source of milk a n d isolated by identical p r o c e d u r e s show a r e m a r k a b l e degree of r e p l i c a t i o n with respect to composition a n d physical-chemical behavior. A s u m m a r i z a t i o n of o u r e x p e r i m e n t a l d a t a is p r e s e n t e d in Table 1. W h o l e - m e m b r a n e p r e p a r a t i o n s c o n t a i n e d f r o m 30 to 6 0 % p r o t e i n , d e p e n d i n g on p r e p a r a t i v e procedures. F r e e d f r o m the lipid moiety b y t r e a t m e n t with ethanol in ether, the p r o t e i n m a t e r i a l consisted of a p p r o x i m a t e l y equal p o r t i o n s of water-soluble a n d insoluble fractions. The insoluble f r a c t i o n was o b t a i n e d as a flesh-colored, m u c o i d a l - a p p e a r i n g p r o t e i n . I t was solubilized b y treatm e n t with sodium sulfide, peraeetic acid in c o m b i n a t i o n w i t h a m m o n i a water, sodium dodecyl sulfate, thioethanol, a n d sodium h y d r o x -
TABLE
1
Summary of the characteristics of the far globule membrane Characteristics of the total menlbrane substance Contains from 30 to 60% protein, depending upon the age of the milk and method employed in its isolation, consisting of two principal fractions. (1) A water-soluble fraction comprising about 45% of the totat protein and charaeterized by its carbohydrate moiety ( ~ 6.5%) has been classified tentatively as a glyeoprotein. Sedimentation coefficients (S~o) ranging between 8 and 17 have been reported for this protein and reflect the amount of contaminating lipid in the preparation. The more lipid in the protein fraction, the higher is the S~. The protein shows heterogeneous characteristics in freeboundary eleetropboresis. (2) A water-insoluble fraction, reddish-brown in color and comprising about 55% of the protein, which was solubilized by disulfide cleaving agents, has been classified tentatively as a pseudokeratin. The protein exists as a high molecular weight aggregate, too large to charaeterize by the usual physical methods. Contains from 40 to 70% lipid, which has been classified by silicic acid chromatography. Component Percentage of total lipid Carotenoids 0-0.5 Squalene 0-0.6 Cholesterol esters 0.6-0.8 Triglyeerides 50.0-53.4 Free f a t t y acids Traces-6.3 Cholesterol 3.6-5.2 Mono-diglycerides 12.8-17.0 Phospholipids 20.4-28.7 ~Iost of the triglyeerides consist of a high-melting fraction (50-530). Conceivably, the glyeerides do not constitute an integral part of the membrane ]ipoprotein complex. Lipid constituents of eentrifugMly fraetionated membrane lipoproteins (25,000 × G × 2 hr) F a t globule membrane substance was separated into a high-density pellet and a lower-density supernatant which contained approximately 10 and 60% of lipid, respectively. The lipid moieties were elassifled by silieie acid chromatography. Component Carotenolds Squalene Cholesterol esters Triglyeeride Fatty acids Cholesterol Mono-diglycerides Phospholipids
Percentage of total lipid Pellet Supernatant 0. 0.3 0. ] .0 0. 5.4 44.2 50.5 4.7 5.5 3.7 2.4 9.2 15.5 38.2 23.2
When considered on the basis of absolute yield, these two lipid fractions were quite similar, with the exception of the glyeeride components, which were higher in tlle supernatant fraetion. Conceivably, the phospholipids eonstitute the principal lipid of the membrane lipoprotein complex.
STABILITY
OF
CONCENTRATED
ide at p H 10-11, agents generally used to solubilize keratin- or pseudokeratin-like proteins. The soluble protein fraction varied from reddish-brown to white, depending' on the age of the milk and techniques employed in the isolation. This fraction, obtained as a fat-free protein, showed single eleetrophoretic and ultracentrifugal boundaries in phosphate or veronal buffers ( p H 6-8.6) and two or three electrophoretie boundaries at lower values of p i t (2-3). The protein contained 11 to 12% nitrogen, plus hexose, hexosamine, and sialie acid (N-aeetyl neuraminie) and was classified tentatively as a glyeoprotein (41). Palmer and Samuelsson (34) and, more recently, Jackson et al. (19) have reported that the membrane protein (soluble fraction) was highly antigenic and apparently different from any of the other proteins of milk. The latter investigator classified the protein as a mueoprotein because of its relatively high hexose content. Whether this protein differs from one of the proteins that make up the so-called proteose-peptone fraction of skimmilk remains to be deternfined. PreliminaD" data obtained in our laboratory by the Ouchterlony antibody-antigen diffusion technique suggest that these fractions might be biological identities. More work remains to be done before this implication can be recorded as a fact. The total lipid moiety, obtained from the alcohol-ether extract of the total membrane, when ehromatographed on a silicie acid colunm, was composed of about 50% triglyeerides and 20 to 30% phospholipids. Smaller quantities of cholesterol, fatty acids, and mono-diglyeerides and traees of cholesterol esters, carotene, and squalene-like substances were observed (43). Some of the supposedly peripherally bound lipid components were removed from the intact membrane by washing with ethyl ether before addition of alcohol (sqnalene, carotene), indicating that these materials did not constitute an integral p a r t of the membrane lipids. We have reported a classification of lipids constituting centrifugally separated (25,000 × G for 2 hr) lipoprotein fractions of the whole membrane material (7). The clear supernatant layer carried a low-density lipoprotcin containing about 60% lipids, whereas the highdensity lipoprotein constituting the sedimented pellet contained about 11% lipids. The a p p a r ent difference between the lipids of these two fractions was found in the triglyceride to phospholipid ratios, which for the pellet was about 1:1 and for the supernatant about 2:1. A slightly higher concentration of mono-diglye-
MILK
9~7
eride was noted in the supernatant fraction. Recently, Alexander and Lusena (3) reported lipid characteristics of various centrifugally eut membrane fractions. An interesting characteristic of the membrane lipids is the presence of relatively large amounts of phospholipids and triglycerides--presmnably the high-melting fraction reported by Palmer and his associates years ago (22). The highmelting glyceride fraction has been isolated from butteroil (35, 42) and is believed to constitute the total triglyeeride of the isolated membrane lipids (43). Conceivably, then, phosphotipids constitute the major lipid class found in the membrane-lipoprotein complex. These two lipoprotein fractions were suspended in veronal buffer and examined both eleetrophoretically and ultraeentrifugally. The pellet fraction showed one eleetrophoretie peak, but it was too large an aggregate to study in the ultracentrifuge. The supernatant fraction showed heterogeneous electrophoretie characteristics. The ultraeentrifugal diagrams possessed multi-boundary characteristics, suggesting the existence of several lipoprotein species of various states of aggregation. When lipid was removed from the complex, the remaining protein moiety showed ultraeentrifugal characteristics more indicative of a homogeneous component. Interestingly, the removal of lipid from the complex is accompanied by a corresponding reduction in the sedimentation eoeffteient, an observation reflecting the role played by the lipid in the state of aggregation of the lipoprotein. ~I:ODIFICATION OF T H E
RESYLTING
FRO~[
F A T G L O B U L E ~-~¢IEMBRA~E
PROCESSING
PROCEDURES
The fat membrane lipoprotehls are sensitive to the usual agents that tend to disorganize native proteins and are sensitized by agents promoting destruction of the lipid components. All processing manipulations are detrimental to the stability of the native membrane material. In practice, we must select processes or a combination of processes that exert minimal detrimental influences on the physicaI-chemieal equilibria at the membrane interface. Some of these ft~etors are considered below. Mill: supply. At the normal p H values of milk, the membrane exists in a relatively stable, hydrophilie state. An increase in the p H enhances this condition, while a decrease contributes to its instability (32). King (23) has discussed the destabilizing effect of incorporated air bubbles resulting from agitation of milk at temperature near the melting point of
948
J.R. ~m-xx~.:l~
the fat on the fat emulsion. Thurston (44), and more recently, Greenbank and Pallansch (12), have suggested that the membrane phospholipids are displaced into the plasma portion of milk as a result of agitation. Whether the phospholipids were displaced as unbound molecules or as membrane particulates was not clarified. The effect of agitating raw milk on the enhancement of induced lipolysis is well known and has been a contempora15" problem relative to pipe-line milking systems and farm tank storage of milk (9). The cooling and/or warming of milk influence the composition of fat membrane and, conceivably, the stability of the fat emulsion (24, 36). Separation of milk. Agitation effects encountered during the separation of milk cause the release of membrane materials, which seems to be aeeentuated at 90 F or above. Additional losses are encountered when quantities of incorporated air are present in the milk to contribute to the destabilization of the fat emulsion. I n fact, the result of such a condition is an excessive loss of small fat globules into the skimnfilk phase. Apparently, the cold-separation or clarification of milk (40 to 45 F), although somewhat inefficient, exerts a less destabilizing effect on the native membrane structure. At these temperatures the euglobulin fraction of the plasma proteins is associated with the globular surface (36). Frmu a practical consideration, I would conjecture that cold separation, provided the process was sufficiently efficient to remove small globules, would be more desirable than warm separation for skimmilk going into NFDM. I n this way the phospholipid content of skimmilk could be reduced, and hence its tendency to develop storage deterioration. Heating and vacuum processing. Lowenstein and Gould (28) showed that when milk was heated to 82 C (180 F ) for 15 rain there was a loss of recoverable membrane material and the loss of protein exceeded the loss of lipids. Studies conducted in our laboratory by Jackson (17) showed that the amount of membrane material recovered from homogenized milk heated to 1 7 5 F 30 rain was less than from a batch heated to 145 or 160 F. Apparently a mild heat treatment in the absence of violent agitation does not cause excessive losses of membrane material. However, high-velocity heating systems from which air has not been excluded could be a factor in the destabilization of the fat emulsion. Greenbank and Pallansch (12) observed that the agitation which accompanied concentration of milk in single-effect evaporators caused a migration of phospholipids into
the skimmilk phase. One might expect that single-pass evaporators would exert less influence on phospholipid migration. I t would be interesting to know whether homogenization at the temperatures of pasteurization would decrease the migration of membrane material into the skimmilk since, in practice, homogenization at this point (160 to 170 F) seems to enhance the stability of the fat emulsion. Homogenization. Although homogenization has been used to stabilize the fat emulsion in milk, some rather profound changes occur at the fat/plasma interface. Upon homogenization, the fat globules (average diameter = 4-5 tL) are broken into small globules (about 1 t~ in diameter) with an accompanying flvefold increase in surface area. Jackson and Brunner (18) have shown that this newly created surface ahsorbs casein as well as other plasma proteins. Interestingly, the absorbed casein appeared to be complexed with a lipid fraction, possibly a phospholipid. Fox et al. (10) and, more recently, Greenbank and Pallanseh (12) have reported the formation of a high-density, lipidcasein complex in honmgenized milk. Litman and Ashworth (27) described an insoluble scumlike material obtained from dlT whole milk for which they reported characteristics of a lipidprotein complex, possibly of phospholipidcasein origin. The resurfacing of the fat globule as a result of homogenization predisposes the lipid phase to enhanced lipolytie and photochemical activity (8, 46, 47). On the other hand, Thurston (44) and others (8, 40, 45) have demonstrated that homogenized nfilk is umre resistant to copper-catalyzed oxidative deterioration. Possibly, some of these changes in the characteristics of homogenized milk result frmn rearrangements involving the native membrane materials, for it is known that even honmgenized, washed cream shows hlcreased resistance to copper-induced oxidation. Possibly the membrane structure is extended in such a manner that copper is immobilized in a strongly chelated state, making it unable to serve oxidative pathways in its usual catalytic role. Tarassuk and Koops (40) have suggested that the increased resistance to copper-induced oxidation noted in homogenized milk resulted from a dilution of the susceptible reactive sites (phospholipids) on the increased membrane surface. Jackson and Pallansch (20) demonstrated that in butteroil-milk protein systems the interfacial tension was lowered significantly more by the readsorption of the soluble membrane protein (mueoprotein) than by any of the other fractions studied. Interestingly, the heat
STABILITY
OF C O N C E N T R A T E D
denaturation of fl-lactoglobulin increased its interfacial activity, a characteristic that might serve well in the resurfacing of the lipid phase in evaporated milk. Greenbank and Pallansch (12) presented evidence to suggest that highpressure homogenization (8,000 psi) resulted in a readsorption of the phospholipids at the fat/plasma interracial surface. It was not clear from their data whether the phospholipids were readsorbed or never left the interracial surface at these pressures. Rehomogenization of milk is effective in further reducing the size of fat globules and increasing the stability of the fat emulsion (26). Where practiced, the separation of homogenized products and rehomogenization of the separated portion would enhance the final stability of the fat emulsion. D~ying and freezing. Native lipoproteins, as well as induced lipid-protein interactions, are dissociated by sharp freezing and dehydration. Alexander and Lusena (3) and Thompson et al. (42) reported that the lipids of the fat membrane complex could be extracted with ethyl ether after freezing and/or freeze-drying. The mechanism of this destabilization seems to involve the state of the bound water in the complex. Browning reactions similar to the classical Maillard reaction between carbonyl groups of sugar and free amino acids have been reported for the interaction of phospholipids and sugars (15, 25). These sugar-amino condensations involve the addition of the amino groups to the aldehyde group of the sugar. Water is eliminated to produce N-substituted aldosylamines; then, through an Amadori-like rearrangement the N-substituted ketosylamine is formed, which degradates to colored melanoidin substances. Lipid-protein copolymerization reactions of the type described by Tappel (39) might conceivably play an important role in the shelflife of dry whole milk. This reaction involves the polymerization of peroxidized polyunsaturated fatty acids and the copolymerization of lipid oxidation products with proteins. These reactions could very well be catalyzed by the heine-containing eytochromes or other trace metal containing proteins of the membrane and milk plasma. The products of the reaction are insoluble and dark in color. In the same tone, Nishida and Kummerow (31) reported that the interaction of the hydroperoxide of methyl linoleate caused the destabilization of the lowdensity fl-lipoprotein of blood. Such a reaction in milk could contribute to the destabilization of the membrane lipoprotein. Despite the general conception that phospholipids contribute significantly to oxidative
949
MILK
deterioration, they do serve as synergistic antioxidants. The ionized phosphate group enters into the initial stages of the oxidative process, involving the unsaturated bonds in fatty acid molecules. At a time when these charged phosphate groups can no longer stabilize the hydroperoxides being formed, the system degenerates rapidly and rampant oxidative deterioration becomes apparent. Our studies with the so-called free fat of spray process whole milk powder demonstrated that this lipid fraction, extracted with a 50/50 mixture of ethyl and pet ether, was composed principally of triglycerides characteristic of the total milk triglycerides. Small quantities of phospholipids were observed, but were present in concentration below that found in the whole lipid fraction. Even though the quantity of free fat is low (~--5% of total lipid), it appears to be significant to the dispersibility of dry milk particles. The solvent rinsed powder is highly dispersible, indicating that the hydrophobic free fat interferes with the ability of water to reach the hydrophilic surface. In this respect, a surface layer of hydrophilic phospholipids covering the free-fat layer would be most beneficial. SUMMARY
We have considered the types of bonding forces responsible for lipid-lipid and proteinlipid interactions. These include (a) nonpolar bonds, which function at short distances and are usually referred to as van der Waal's forces ; (b) polar bonds, resulting from an unsystematical sharing of electrons, which are fairly strong forces and often function through the hydrogen bond; and (c) charged structures, which serve as proton donors or aeceptors and attract oppositely charged groups at considerable distances. The native membrane represents the principal lipid-protein complex in normal milk. It is composed of at least two proteins, phospholipids, a high-melting triglyceride fraction, cholesterol, free fatty acids, mono-diglycerides, cholesterol esters, carotene, and other minor lipid fractions. The normal membrane material is dislodged from its interracial surface by agitation, heat treatment, honmgenization, vacumn concentration, freezing, and drying. Its rearrangement at the interracial surface increases the susceptibility of milk to photo-activation and decreases its susceptibility to nletallic-catalyzed oxidative deterioration. Milk fat can be resurfaced with the proteins of the skimmilk phase, but they are less stable than the native emulsion. Oxi-
950
J, R. BRUNNER
dative deterioration of the lipids enhances the f o r m a t i o n o f insoluble lipid-lipid ~nd/or p r o tein complexes. REFERE:NCES (1) AL]~XA~D~, A. E. The Role of Hydrogen Bonds in Condensed Monolayers. Proc. Roy. Soe. (London), A 179: 470. 1941-42. (2) A ~ X A m ) ~ , A. E. A Proposed General Structure for Condensed Monomolecular Films. Proc. Roy. Soe. (London), A179: 486. 1941-1942. (3) ALEXA~n)ER,K. M., A~rO LUS~A, C. V. Fraetionation of the Lipoproteins of the F a t Globule Membrane from Cream. J. Dairy Sci., 4¢: 1414. 1961. (4) BALL, E. G., AND COOLER, 0. The Activity of Succinate Oxidase in I~elation to Phosphate and Phosphorus Compounds. J. Biol. Chem., 180: 113. 1949. (5) BOYFA~, P. D., BALLOU, G. A., A~D LUCK, 5. M. The Combination of F a t t y Acids and Related Compounds with Serum Albumin. II. Stabilization Against Urea and Guanidine Denaturation. J. Biol. Chem., 162: 199. 1946. (6) BI~L~N~NEI~,ft. R., DUNCAN, C. W., AND TROUT, G. 1~. The Fat-Globule Membrane of Nora homogenized and Homogenized Milk. I. The Isolation and Amino Acid Composition of the Fat-Membrane Proteins. Food Research, 18: 45¢. 1953. (7) BRUI'~NER, J. R., AND THOh[PS0h~, ~I. P. Characteristics of the Fat-Globule Membrane Lipoproteins. J. Dairy Sci., 44: 1170. 1961. (8) DOA~, F. J., Am) MY~a~s, C. It. Effect of Sunlight on Some Milk and Cream Produets. The Milk Dealer, 26 (1) : 893. 1936. (9) DUNK~¥, W. L., A~D S~ITg, L. M. Hydrolytic Rancidity in Milk. IV. Relation Between Tributyrinase and Lipo]ysis. J. Dairy Set., 34: 940. 1951. (10) Fox, K. K., HOLSINGFA~, VIRGINL~, CAttA, JEAN~, AN]) PM~LANSCH, M. J. Formation of a F a t Protein Complex in ~Iflk by Homogenization. J. Dairy Sci., 43: 1396. 1960. (11) GOOD~AN, E. D., AND SCHV~A~-, J. H. Molecular Interactions in Monolayers. I. Complex Formation. J. Colloid Sci., 8: 309. 1934. (12) G R ~ A N K , G. R., AND PALLANSCH, 5I. J. Migration of Phosphatides in Processing Dairy Products. J. Dairy Sci., 44: 1597. 1961. (13) GURD, R. R. N. I n Lipide Chemistry. D. J. ttanahan, Ed. pp. 208-325. John Wiley and Sons, Inc., New York-London. 1960. (14) HA~vSSO~, E. F a t Globules from Milk Studied in the Electron Microscope. Proc. 12th Intern. Dairy Congr., Stockholm, 2: 27. 1949.
(15) HODG~, J. E. Chemistry of Browning Reactions in Model Systems. J. Agr. Food Chem., 1: 928. 1953. (16) JACK, E. L., AXD BAttLE, C. D. The Electrokinetic Potential of Milk Fat. I I I . Relation to the F a t Globule Membrane. J. Dairy Set., 20: 637. 1937. (17) JACKS0.~~, R. H. Proteins of the Homogenized Milk Fat-Globule Membrane. Ph.D. thesis, Michigan State University. 1959. (18) JACKSO~~, R. H., A~D BRL~-NER, J. R. Characteristics of Protein Fractions Isolated from the F a t / P l a s m a Interface of Homogenized Milk. J. D~iry Sci., 43: 912. 1969. (19) JACKSO~~, R. H., CLARK, W. R., A~ND COULSON, E. J. Isolation and Some Properties of Mucoprotein from the F a t / P l a s m a Interface of Bovine Milk. Abstr. No. 64, Div. Biol. Chem., 140th meeting, Am. Chem. Soc. September, 1961. (20) JACKSON, R. H., AhFI) PALLANSCtI, M. 5. A Study of Factors Influencing the Energy of the Butteroil-Mi]k Serum Interface. Abstr. No. 10, Div. Agr. & Food Chem., 138th meeting Am. Chem. Soc. September, 1960. (21) J]~-N'ESS, R., Ah'D PALMER, L. S. Substances Adsorbed on the F a t Globules in Cream and Their Relation to Churning. V. Composition of the Membrane and Distribution of the Adsorbed Substances in Churning. J. Dairy Sci., 28: 611. 1945. (22) ffENNESS, R., AND PALMIER, L. S. Substances Adsorbed on the F a t Globules in Cream and Their Relation to Churning. VI. Relation of the High-Melting Triglyceride Fraction to B u t t e r f a t and the Membrane. J. Dairy Sci., 28: 653. 1945. (23) X~x-o, N. The Milk F a t Globule Membrane. 99 pp. Commonwealth Agr. Bureaux, Yarnham Royal, Bucks, England. 1955. (2~) KRUKOVSKY,V. N., AND SttARP, P. F. Effect of the Properties of the F a t and of the F a t Globule Surface on Lipolytie Activity in Milk. J. Dairy Sci., 23: 1109. 1940. (25) L ~ , C. H. Deteriorative Reactions Involving Phospholipids and Lipoproteins. J. Sci. Food Agr., 8 : 1 . 1957. (26) LEVITO~~, A., A-XD PALLAh.-SCH, M. J. Continuous Recycling in the Homogenization of Relatively Small Samples. J. Dairy Sci., 42: 20. 1959. (27) LIT.~L~N', I. I., .XX'D ASHWORTH, U. S. Insoluble Scum-like Materials on Reconstituted Whole Milk Powders. J. Dairy Sci., 40: 403. 1957. (28) LOWENSTEI~', M., A-N~D GOULD, I. A. The Effect of Heat on the Chemical Nature of the Material Adsorbed on the Milk F a t Globule. J. Dairy Set., 37: 644. 1954. (29) MORTOn, R. K. The Lipoprotein Particles in Cow's Milk. The Bioehem. J-., 57: 231. 1954.
STABILITY
OF
CONCENTRATED
(30) MURDER, H. The Physical Structure of Milk and Dairy Products. Prec. 121h Intern. Dairy Congr., Stockholm, 6: 64. 1949. (31) NISHm.% T., ~ ] ) KUM~F~OW, F. A. Interaction of Serum Lipoproteins with the Hydroperoxide of Methyl Linoleate. J. Lipid Research, 1: 450. 1960. (32) NUGE2CT, R. L. The Application of the Mudd Interfacial Techniques in the Study of Protective Protein Films in 0il-in-Water Emulsions. J. Phys. Chem., 36: 449. 1932. (33) ONCe'Z, J. L., Gum), F. i~. N., AND MEnIN, M. Preparation and Properties of Serum and Plasma Proteins. X X V . Composition and Properties of Human Serum fl-Lipoprotein. J. Am. Chem. See., 72:458. 1950. (3'4) PAnMEI~, L. S., AND SAMUELSS0iq, E. The Nature of the Substance Adsorbed on the Surface of th6 F a t Globules in Cow's Milk. Prec. Soc. Expt. Biol., New York, 21: 537. 1924. (35) PAT~O~¢, S., AND KFm~,~L P. The HighMelting Glyceride Fraction from Milk Fat. J. Dairy Sei., 41: 1288. 1958. (36) SHAr~e, P. F., AND KRUKOVSKY, V. N. Differences in Adsorption of Solid and Liquid F a t Globules as Influencing the Surface Tension and Creaming of Milk. J. Dairy Sei., 22: 743. 1939. (37) SOM~F/~, H. H. The F a t Emulsion in Milk from a Chemical Standpoint. The Milk Dealers, 41 (1) : 60. 1951. (38) STORC~, V. On Structure of " F a t Globu l e s " in Cow's Milk. (Translated and communicated by Faber, A.) Analyst, 22: 197. 1897. (39) TAPPEL, A. L. Studies of the Mechanism of Vitamin E Action. I I I . I n vitro Copolymerization of Oxidized Fats with Proteins. Arch. Bioehem. Biophys., 54: 266. 1955.
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951
(40) TARASSUK, N. P., -~ND KOOFS, J. Inhibition of Oxidized Flavor in Homogenized Milk as Related to the Concentration of Copper and Phospholipids per Unit of F a t Globule Surface. J. Dairy Sci., 43': 93. 1960. (~1) THOMPSON, M. P., AND BEUNN~R, J. R. The Carbohydrates of Some G]ycoproteins of Bovine Milk. J. Dairy Sci., 42: 369. 1959. (42) THOMPSON, M. P., BaUNNF~, J. R., AND STnCE, C. M. Characteristics of High-Melting Triglyceride Fractions from the FatGlobule Membrane and Butter Oil of Bovine Milk. J. Dairy Sei., 42: 1651. 1959. (43) THOMPSON, M. P., BI%UNNEK, J. R., STINK, C. M., A~q) LL',q)QUIST, KAKIN. Lipid Components of the Fat-Globule Membrane. J. Dairy Sci., 44: 1589. 1961. (44) T~URSTO~r, L. M., BR~)WN, W. C., AND DUSTMAN, R. B. Oxidized Flavor in Milk. II. The Effect of Homogenization, Agitation and Freezing of Milk on Its Subsequent Susceptibility to Oxidized Flavor Development. J. Dairy Sci., 19: 671. 1936. (45) TROUT, G. M., AN1) GOULD, I. A. Homogenization as a Means of Stabilizing the Flavor of Milk. Michigan Agr. Expt. Sta., Quart. Bull., 21 (1) : 21. 1938. (46) T~OUT, G. M,, HALL0m~', C. P., ANt) GOULD, I. A. The Effects of Homogenization on Some of the Physical and Chemical Properties of Milk. Michigan Agr. Expt. Sta., Tech. Bull. 145. 34 pp. 1935. (47) WEINST~n¢, B. R., DUNCAN, C. W., AND TROU% G. M. The Solar-Activated Flavor of Y[omogenized Milk. IV. Isolation and Characterization of a Whey Constituent Capable of Producing the Solar-Activated Flavor. J. Dairy Sci., 34:570. 1951.