Membrane phospholipids and sterols in microfiltered milk fat globules

Native milk fat globules of various mean diameters, ranging from d₄₃ = 2.3 µm to 8.0 µm, were obtained using microfiltration of raw whole milk. After milk fat globule washing, the milk fat globule membrane (MFGM) was separated by manual churning. After total lipid extraction and separation of polar lipids, their phospholipid (PL) and sterol composition was measured using thin‐layer chromatography, methyl ester analyses by gas chromatography, and gas chromatography coupled to mass spectrometry. The main PL species were phosphatidylethanolamine, phosphatidylcholine and sphingomyelin. The respective fatty acid composition of each PL species was measured. Many different minor bioactive sterols were detected in the MFGM, e.g. lanosterol, lathosterol, desmosterol, stigmasterol and β‐sitosterol. No significant differences in the PL and sterol profile were found between MFGM extracted from small and large milk fat globule fractions.     

The fatty acid composition of small and large naturally occurring milk fat globules

Native milk fat globules of various mean diameters, ranging from d₄₃ = 1.5 to 7.3 μm, were obtained using microfiltration of raw whole milk acquired in winter and spring. After total lipid extraction, fatty acid composition was characterized by methyl and butyl ester analysis using gas chromatography. The oleic and linoleic acid content of milk obtained in winter increased with fat globule size, whereas myristic and palmitic acid decreased. There was significantly more lauric, myristic and palmitoleic acid, and less stearic acid in small fat globules compared to large fat globules in milk obtained in both winter and spring. The relative content of oleic and linoleic acids were found to depend on fat globule size and season. Results are interpreted on the basis of the relative content of milk fat globule membrane depending on fat globule size, and on consequences of compositional variations on milk fat globule melting behavior.     

Milk fat globules: Fatty acid composition,size and in vivo regulation of fat liquidity

Populations of large and small milk fat globules were isolated and analyzed to determine differences in fatty acid composition. Globule samples were obtained by centrifugation from milks of a herd and of individual animals produced under both pasture and barn feeding. Triacylglycerols of total globule lipids were prepared by thin layer chromatography and analyzed for fatty acid composition by gas chromatography. Using content of the acids in large globules as 100%, small globules contained fewer short-chain acids, −5.9%, less stearic acid, −22.7%, and more oleic acids, +4.6%, mean values for five trials. These differences are consistent with alternative use of short-chain acids or oleic acid converted from stearic acid to maintain liquidity at body temperature of milk fat globules and their precursors, intracellular lipid droplets. Stearyl-CoA desaturase (EC 1.14.99.5), which maintains fluidity of cellular endoplasmic reticulum membrane, is suggested to play a key role in regulating globule fat liquidity. Possible origins of differences between individual globules in fatty acid composition of their triacylglycerols are discussed.     

Lipid domains in the milk fat globule membrane: Specific role of sphingomyelin

Lipids are secreted in milk in the form of unique colloidal assemblies, the milk fat globules, which are surrounded by a biological membrane, the milk fat globule membrane (MFGM). Recent confocal laser scanning microscopy experiments showed heterogeneities in the composition and structure of the MFGM. For the first time, we revealed the phase separation of polar lipids in the plane of the MFGM with the lateral segregation of sphingomyelin and cholesterol in rigid liquid‐ordered (L_o) domains surrounded by the fluid matrix of glycerophospholipids in the liquid‐disordered (L_d) phase. The MFGM is a unique and highly dynamic biophysical system, the functional and nutritional properties of which need to be further studied for health benefits.     

A comparative study of the lipids of globule membrane and fat core and of the milk serum of cows

Nine samples of fresh raw cow's milk were separated into fat globules and milk serum by centrifugation. After destabilization by freezing and thawing, the milk fat globules were resolved into membranes and fat cores. The lipid composition of these structures was compared to that of the surrounding milk serum. Of the total milk fat, 95–98% was in the fat cores, 0.5–1% in the globule membranes and the rest (1.5–4%) in the milk serum. The fat cores contained 88–93% triglyceride, 5.2–9.8% diglyceride, 1.5–7.3% free fatty acid and 0.2–0.4% cholesterol, but no phospholipid. The lipids of the membrane contained 21–44% phospholipid, made up of about equal proportions of phosphatidyl ethanolamine, phosphatidyl choline, and sphingomyelin. The other lipids of the membrane (56–79%) consisted of 83–88% triglyceride, 5.1–10.7% diglyceride, 1–5.1% free fatty acid and 0.4–1.9% cholesterol. The milk serum contained 30–45% phospholipid divided about equally among phosphatidyl ethanolamine, phosphatidyl choline and sphingomyelin. The rest (55–70%) of the milk serum lipids was made up of 71–83% triglycerides, 4.3–10.1% diglycerides, 8.7–15.7% free fatty acids, and 1.2–8.4% cholesterol. Corresponding phospholipid classes of milk serum and globule membranes had identical fatty acid compositions. The triglycerides and diglycerides of the globule membranes possessed increased proportions of palmitic and stearic acids in comparison to the glycerides of the fat cores. Taken in part from a PhD thesis submitted by T. C. Huang to Queen's University, Kingston, Canada in April, 1965. Presented in part at the 47th Canadian Chemical Conference and Exhibition held in Kingston, Canada, June 1–3, 1964.     

Structure of bovine milk fat triglycerides

The triglycerides of bovine milk fat globules were isolated and separated into short, medium and long chain lengths by thin-layer chromatography. The molecular weight distribution and the fatty acid composition of the component triglycerides was then separately determined by gas chromatography following argentation-thin-layer and preparative gas chromatography. Some 38 triglyceride types (28% of total), of which there could be up to 6 isomers, were specifically identified and quantitatively estimated. The quantitative estimates for the rest of the milk fat triglycerides were limited to much more complex glyceride groups. The results confirm the earlier claim that butyric and caproic acids occur in milk fat almost exclusively in combination with medium and long chain fatty acids. Presented in part at the AOCS Meeting, Philadelphia, October, 1966.     

Triglyceride composition of ewe,cow, and goat milk fat

The separation and identification of the components in milk fat, which are mainly triglycerides, is a challenge due to its complex composition. A reverse-phase high-performance liquid chromatography (HPLC) method with gradient elution and light-scattering detection is described in this paper for the triglyceride analysis in ewes’ milk fat. Triglyceride identification was carried out by combining HPLC, gas-liquid chromatography (GLC), and the calculated equivalent carbon numbers of several triglyceride standards. Quantitation of partially resolved peaks in the HPLC chromatogram was accomplished by applying a peak deconvolution program. Forty-four fatty acids were identified by GLC analysis, but only 19 were used for the following prediction of triglyceride molecular species; 181 triglycerides were identified, some of which were grouped at the same peak and needed application of the deconvolution program. Consequently, coefficients of variation were close to or lower than 5%. Moreover, the triglyceride composition of ewe, cow, and goat milk fat were compared by using these methods. These results show that ewe milk fat is richer in short- and medium-chain triglycerides, and cow milk fat is richer in long-chain and unsaturated triglycerides.     

Composition of milk fat globules with increased linoleic acid

Comparisons were made of the composition and distribution of the lipids in fat globules from conventional milks and polyunsaturated milks produced by cows fed protected lipid supplement. Washed creams were prepared from the milks of three individual cows fed a conventional ration, and three fed a protected sunflower-soybean supplement rich in linoleic acid. The washed creams were fractionated by treatment with sodium deoxycholate and centrifugation. Each washed cream and four fractions (designated as outer globule membrane, inner membrane, pellet, and globule core) were analyzed for protein, lipid, phospholipid, cholesterol, tocopherols, carotenoids, and fatty acid composition. The outer and inner membrane fractions were further fractionated into neutral and polar (phospholipid) lipid classes by thin layer chromatography. For both types of washed cream the approximate weight distribution of total solids was: outer membrane, 1%; inner membrane, 2%; pellet, 0.1%; and core, 96%. The percentages of protein, phospholipid, cholesterol, and carotenoids were all lower in the polyunsaturated than in the conventional creams. In the polyunsaturated creams, the percentages of both saturated and unsaturated C₁₈ acids were higher, and of acids of C₁₆ and shorter chain length lower, than in the conventional creams. The phospholipids in the outer and inner membranes from the polyunsaturated milks had larger proportions of linoleic acid than did the phospholipids from the conventional milks. However, this increase in unsaturation was less than that of the core neutral lipids. Pancreatic lipase hydrolysis of the core fractions showed that the increased linoleic acid was introduced preferentially at the 2-position of the triglycerides. In general, the observed changes in physical properties and in susceptibility of polyunsaturated milk to the development of oxidized flavor are consistent with the differences in the relative proportions of the various classes of lipids in the conventional and polyunsaturated milks. Data from thesis of D.H. Bianco submitted in partial fulfillment of the requirements for the M.S. Degree in Food Science, University of California, Davis. Presented in part at the AOCS Meeting, New Orleans, April, 1976.     

Dietary fat composition influences fatty acid composition of milk fat globule membrane in lactating cows

Milk fat globule membranes are derived directly from the apical plasma membrane of mammary epithelial cells. To evaluate the effect of dietary fat on mammary membranes, we determined the fatty acid composition of the milk fat globule membrane in lactating dairy cows fed diets supplemented with fats of different fatty acid composition, or infused intravenously with soy oil emulsion. A preliminary survey, using an abbreviated preparation procedure (membranes isolated at 48,000 x g-max for 15 min), yielded about 45% of the total membrane fatty acids that could be recovered by centrifuging at the same speed for 120 min, and showed that changes in fatty acid composition of membranes reflected dietary fatty acids to some extent. Dietary palmitic acid increased the content of 16:0 in the membranes. A high corn diet increased ruminal formation of t18:1, and its level increased to 12% of membrane fatty acids. Infusion of soy oil emulsion increased 18:2 membrane content, and decreased the levels of 18:1 and 20:4. All treatments decreased the ratio of unsaturated/saturated fatty acids as compared to controls, whereas the ratio of polyunsaturated/saturated fatty acids was increased by feeding a high corn diet or by infusing soy oil. The ratio of 18:2/c18:1 increased from 0.31 to 1.0 after infusing soy oil for 4 days. The fatty acids of membranes isolated upon 120-min centrifugation were slightly more saturated. The differences were not sufficiently large, however, to affect overall results significantly.     

Origin of the milk fat globule

Milk fat globules originate as fat droplets within the lactating mammary cell. These droplets are composed largely (>98%) of glycerides. Their constituent fatty acids are derived by lipolysis of very low density lipoproteins and chylomicrons of the blood and by the novo synthesis within the cell. Evidence of two principal routes for the synthesis of milk fat triglycerides has been presented: the so-called glycerolphosphate and monoglyceride pathways. Recent findings on these pathways are discussed. The nature of the milk triglycerides with their unique compliment and distribution of short chain fatty acids appears to depend upon a closely regulated relation between the soluble multienzyme complex that synthesizes the fatty acids and the glyceride synthetase that is bound to the endoplasmic reticulum. The resulting triglycerides appear to self-assemble into droplets from the surface of the endoplasmic reticulum. No special ultrastructures (transport particles, vesicles, etc.) have been detected in relation to this process. Milk fat droplets at the time of secretion average several microns in diameter, there being species variations. The basic secretion mechanism involves envelopment of the droplet in plasma membrane and expulsion of it from the cell. As a consequence there are at least two pools of polar lipids (cholesterol and phospholipids) associated with secreted milk fat globules, i.e., one from the plasma membrane and one entrained earlier from the endoplasmic reticulum at the time of triglyceride synthesis and accumulation. In all, the polar lipids do not make up more than 1–2% of the total lipids in milk and a substantial fraction of them has been identified recently with plasma membrane fragments occurring in the skim milk phase. Radiotracer and ultrastructural studies show that this membrane material does not result simply by shedding of surface from milk fat globules. This dispersed material and the lining around milk fat globules constitute valuable sources for the study of cell membranes. One of eight papers presented in the symposium “Milk Lipids,” AOCS Meeting, Ottawa, September 1972.     

The relationship of milk phospholipids to membranes of the secretory cell

Forty-two per cent of the lipid phosphorus in milk was found in skim milk lipoprotein; the other 58% occurs in the milk fat globule membrane (MFGM). Investigation of these two sources of lipid phosphorus revealed that they involve the same individual phospholipids, in essentially the same proportions with similar fatty acid compositions. Both contain sphingomyelin and cerebrosides in levels characteristic of those found in plasma membranes. Other points of resemblance between MFGM and skim milk lipoprotein, have been shown previously. Infusion of (¹⁴C) palmitate into the mammary gland of a lactating goat produced more extensive labeling of all the phospholipid classes in the skim milk lipoproteins than in those in the MFGM during the following 24 hr. When (¹⁴C) palmitate was infused into the jugular vein of a lactating goat, a precusor-product-type relationship was observed between specific activities of the skim milk and MFGM polar lipids. These results render the MFGM an unlikely origin of the skim milk lipoprotein. Other possible sources of this latter lipoprotein are Golgi vesicle membranes or plasma membrane of the lactating cell.     

A method for isolation of milk fat globules

The traditional procedure for isolating milk fat globules involves repeated cycles of centrifuging to obtain globules and redispersion of them in fresh buffer to eliminate other milk components. We have evaluated a simpler, less manipulative method whereby globules are centrifuged out of the milk and through an overlying buffer layer. Human milk samples ranging from 0.1 to 35 ml were centrifuged at 1500×g for 20 min after deposition under a suitable quantity of buffer. This yielded purified globules, in less time, which could be dispersed more satisfactorily than those by the traditional procedure. Protein, phospholipid and cholesterol contents of globules by the two methods were quite similar. A lower protein content (10.4 vs 13.2 mg/g of lipid) was characteristic of globules prepared by the multiple wash method. However, large differences could not be seen in gel electrophoresis patterns of the proteins. By using plastic centrifuge tubes, tube freezing and cleavage just below the globule layer enables clean separation of globule and nonglobule phases for analysis of milk component distributions. Macro (5 to 35 ml of sample) and micro (200 μl or less) versions of the method are described. Limited trials showed that the method can be applied satisfactorily to cow's and goat's milks, but for highly pure globules a deeper buffer column than that used with human milk is required because of their much higher casein content.     

Oxidative interactions of cholesterol in the milk fat globule membrane

The effects of oxidative interactions between cholesterol and milk fat globule membrane (MFGM) components,i.e., nonlipid fraction, total lipid, nonpolar lipid and polar lipid, on cholesterol oxidation were studied in the presence and absence of water. In the dry state, cholesterol natively present in MFGM appeared to be protected at 135°C. The nonpolar lipid and nonlipid fraction contributed to the protective effect of MFGM. Added cholesterol accelerated the oxidation of membrane lipid fractions. At 75°C, pure cholesterol and membrane lipid fractions did not show significant interaction. However, cholesterol and other lipids in MFGM were less stable than when these were heated separately. When cholesterol and membrane lipids were mixed in an aqueous medium at 75°C, each accelerated the oxidation of the other. The MFGM exhibited a high protective effect on cholesterol oxidation in an aqueous environment. The nonlipid fraction protected cholesterol against oxidation, whereas the lipid fraction was destructive. In the absence of water, the net balance between these two opposing factors was destructive. The presence of water reversed the balance in favor of protection. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Effect of milk fat fractions on fat bloom in dark chocolate

Anhydrous milk fat was dissolved in acetone (1∶4 wt/vol) and progressively fractionated at 5°C increments from 25 to 0°C. Six solid fractions and one 0°C liquid fraction were obtained. Melting point, melting profile, solid fat content (SFC), fatty acid and triglyceride profiles were measured for each milk fat fraction (MFF). In general, there was a trend of decreased melting point, melting profile, SFC, long-chain saturated fatty acids and large acyl carbonnumbered triglycerides with decreasing fractionation temperature. The MFFs were then added to dark chocolate at 2% (w/w) addition level. In addition, two control chocolates were made, one with 2% (w/w) full milk fat and the other with 2% (w/w) additional cocoa butter. The chocolate samples were evaluated for degree of temper, hardness and fat bloom. Fat bloom was induced with continuous temperature cycling between 26.7 and 15.7°C at 6-h intervals and monitored with a colorimeter. Chocolate hardness results showed softer chocolates with the 10°C solid fraction and low-melting fractions, and harder chocolates with high-melting fractions. Accelerated bloom tests indicated that the 10°C solid MFF and higher-melting fractions (25 to 15°C solid fractions) inhibited bloom, while the lowermelting MFFs (5 and 0°C solid fractions and 0°C liquid fraction) induced bloom compared to the control chocolates.     

Specific molecular and colloidal structures of milk fat affecting lipolysis,absorption and postprandial lipemia

In milk fat, fatty acids are located at specific positions on the triacylglycerol backbone. The sn‐2 position contains most saturated long‐chain fatty acids, while the sn‐3 position contains short‐chain fatty acids. Moreover, these triacylglycerols are structured as milk fat globules surrounded by their native membrane containing phospholipids. This native structure can be modified by the dairy processes to generate various possible colloidal structures with milk fat. The structure of triacylglycerols and the milk fat ultrastructure can impact on fatty acid digestion and absorption, which has a potential effect on cardiovascular risk factors linked to postprandial hypertriglyceridemia. The review points out the impact of the triacylglycerol structure and the ultrastructure of milk fat on these risk factors.     

Specific distribution of fatty acids in the milk fat triglycerides of goat and sheep

The triglycerides of the fat globules of sheep and goat milk were isolated and separated into short and long chain lengths by silicic acid column chromatography. The short chain lengths comprised major triglycerides with 34–44 acyl carbon atoms and accounted for nearly 50% of the total milk fat. The long chain lengths contained major triglycerides with 40–54 acyl carbons. Stereospecific analyses of the short chain triglyceride fraction showed that of the 20–23 moles per cent of C₄−C₈ fatty acids present, at least 95% were specifically attached to the glycerol molecule in the position corresponding to carbon 3 ofsn-glycerol. The distribution of the other fatty acids (C₁₀ or greater) did not show such marked specificity for either the 1 or the 2 position. Although individual triglycerides were not identified, the specific placement of the fatty acids could best the accounted for by assuming a common pool of long chain 1,2-diglycerides which served as precursors of the bulk of both short and long chain triglycerides during milk fat synthesis. Presented in part at the AOCS Meeting, New York, October 1968.     

The chemical and physical properties of interesterified milk fat fractions

Five fractions of anhydrous milk fat were obtained by fractional crystallization in the absence of solvent. The fractions were characterized, physically and chemically, before and after interesterification. Cholesterol tended to fractionate into the lowest-melting fraction. Slipping points of 38.5C, 32C, 28C, 22.5C, and 17C, respectively, were reflected in widely varying micropenetration curves, while differences in fatty acid composition were relatively small. The three higher-melting fractions, with very similar fatty acid compositions, had very similar physical properties after interesterification. Slipping points of 38C, 37C, 37C, 33C, and 32C, respectively, indicated that interesterification generally hardened the fractions. Interesterification altered the physical properties of lower-melting fractions more than the properties of higher-melting fractions. Interesterification caused some triglyceride degradation and some butyrate loss, but these factors could not fully explain the unequal physical property changes induced in the different fractions. Fractionation possibly tends to separate symmetrical triglycerides from their lower-melting, unsymmetrical isomers, yielding fractions with apparent differences in the degree of randomness of their triglyceride structure. Positioning of fatty acids within the triglyceride molecule seems to be a principal determinant of the physical properties of milk fat fractions. Published with approval of the Director of the Wisconsin Agricultural Experiment Station.     

Free fatty acid content of human milk: Physiologic significance and artifactual determinants

Analysis of human milk was conducted to determine if free fatty acids occur naturally or as a consequence of artifactual lipolysis after milk expression. Five mothers provided triplicate early morning milk samples on day 43 of lactation. Following extraction, lipid classes were separated by preparative thin layer chromatography and quantified by capillary gas liquid chromatography. Fresh milk samples collected with 20 volumes chloroform-methanol (1∶1, v/v) were analogous in total free fatty acid level and profile of fatty acids to a duplicate sample collected with 0.4M EDTA and immediately frozen at −10 C. Low milk levels of free fatty acids appear to exist naturally. During days 4–37 of lactation, four serial milk samples from 15 mothers were collected and frozen with 0.4M EDTA. The concentration of free fatty acids in colostrum (0.03–0.5%, w/w) was lower than for subsequent days (0.3–2.5%, w/w). Additional samples were collected with and without a lipase inhibitor (0.4M EDTA) and subjected to routine collection and storage procedures. Significantly different fatty acid profile and higher levels of free fatty acids in milk collected without a lipase inhibitor added indicate that domestic freezing and/or thawing ruptures the fat globule membrane, allowing sn-1-stereospecific serum stimulated lipoprotein lipase contact with its triglyceride substrate. Standard procedures for collection of human milk for gavage fed infants appear to stimulate artifactual lipolysis of milk triglyceride and subsequent release of free fatty acids. The proposed relationship between dietary free fatty acids and prolonged, unconjugated hyperbilirubinemia in the newborn is discussed with regard to the significance of preintestinal lipolysis.     

Triglyceride analysis by gas chromatography in assessment of authenticity of goat milk fat

The triglyceride (TG) composition of 35 samples of milk collected at different times of year from five herds of goats was analyzed using short capillary column-gas chromatography. The distribution of TG in goat milk fat was unimodal, peaking at C₄₀ (12.6%); the sum of TG from C₃₈ to C₄₄ accounted for about 50%, whereas the three classes of TG from C₄₈ to C₅₂ did not exceed 6% each. These results were compared with the corresponding data for cow milk fat. Significant differences between herds were observed, mainly in long-chain TG. To detect foreign fats in goat milk fat, two multiple regression equations based on TG content of the goat milk fat were proposed. Analysis of known mixtures of tallow, palm oil, and cow milk fat with goat milk fat have experimentally confirmed the accuracy of the equations.     

Cholesterol esters of milk and mammary tissue

The fatty acid composition and metabolic activity of cholesterol esters in milk and mammary tissue (cow, sow and goat) were investigated. Cholesterol esters of freshly secreted milks incubated at 40 C for 2 hr showed no change in fatty acid composition and no incorporation of 1-¹⁴C-palmitate. The fatty acid composition of cholesterol esters in the milk of all three species exhibited elevated levels of a unique group of fatty acids when compared to other milk ester lipid classes. This group was comprised of monounsaturated acids and acids with odd numbers of carbons. Tissue cholesterol esters contained lower levels of these acids. Evidence from experiments in which an odd carbon acid (C₁₅) was infused into the lactating mammary gland indicated that the group of unique acids is preferentially retained in the cholesterol ester fraction which is secreted with milk. These infusion experiments also provided evidence that cholesterol esters accumulate in developing milk fat globules in a manner paralleling triglyceride accumulation, and that acyl moieties of cholesterol esters may be desaturated in the form of the intact ester. Our results are compatible with the hypothesis that acyl moieties of cholesterol esters in lactating mammary tissue are turning over rapidly. Paper No. 3496 in the journal series of the Pennsylvania Agricultural Experiment Station.