Fat Globule Membrane Material Research Articles

Preparation of Liposomes from Milk Fat Globule Membrane Phospholipids Using a Microfluidizer

The isolation of milk fat globule membrane (MFGM) material from buttermilk on a commercial scale has provided a new ingredient rich in phospholipids and sphingolipids. An MFGM-derived phospholipid fraction was used to produce liposomes via a high-pressure homogenizer (Microfluidizer). This technique does not require the use of solvents or detergents, and is suitable for use in the food industry. The liposome dispersion had an average hydrodynamic diameter of 95nm, with a broad particle-size distribution. Increasing the number of passes through the Microfluidizer, increasing the pressure, or reducing the phospholipid concentration all resulted in a smaller average liposome diameter. Changing these variables did not have a significant effect on the polydispersity of the dispersion. Electron microscopy showed that the dispersions formed had a range of structures, including unilamellar, multilamellar, and multivesicular liposomes. The composition of the MFGM phospholipid material is different from that of the phospholipids usually used for liposome production in the pharmaceutical and cosmetic industries. The MFGM-derived fraction comprises approximately 25% sphingomyelin, and the fatty acids are primarily saturated and monounsaturated. These differences are likely to affect the properties of the liposomes produced from the phospholipid material, and it may be possible to exploit the unique composition of the MFGM phospholipid fraction in the delivery of bioactive ingredients in functional foods.

Adsorption of κ-casein onto native milk fat globule, latex particle, and emulsion surfaces

The adsorption of κ-casein onto three surfaces was examined: (1) native milk fat globules, (2) low charge density sulfated polystyrene latex particles coated with milk fat globule membrane (MFGM) material, and (3) MFGM-coated soy oil emulsions. Particle hydrodynamic diameter was measured using dynamic light scattering before and after addition of κ-casein, and also after subsequent addition of Mucor miehei rennet protease or trypsin. The thickness of MFGM coating on latex particles was 25 nm at pH <5.8 reducing to 10 nm at pH >6.4. There was no increase in diameter after adsorption of κ-casein onto MFGM-coated latex particles or soy oil globules. The radius of native fat globules decreased by 15–20 nm after κ-casein adsorption. Addition of rennet or trypsin to the latex or emulsion+MFGM+κ-casein system caused aggregation, providing indirect evidence of κ-casein adsorption. In most cases the aggregate size reached a plateau value within 12 min, except at pH 5.5 after addition of trypsin where aggregation continued for at least 25 min. A reduction in radius of around 10–20 nm occurred initially after the addition of rennet or trypsin to MFGM-coated latex particles at pH 6.7 and native fat globules at pH 5.5 and 6.0, both with an adsorbed secondary layer of κ-casein, providing evidence of steric stabilization by a κ-casein ‘hairy layer’. This did not occur for MFGM-coated soy oil globules with a κ-casein adsorbed layer.

Characterization of the interface of an oil-in-water emulsion stabilized by milk fat globule membrane material

A fraction derived from the membrane of milk fat globules (MFGM) was isolated from fresh raw cream. The adsorption of MFGM at the interface of oil-in-water emulsions was studied as well as the stability of the emulsions under different conditions (pH, temperature treatment) and the surface potential. The emulsions prepared with MFGM isolates were stable at neutral pH and were destabilized at low pH. By analysis of the protein adsorbed at the interface by polyacrylamide gel electrophoresis, we also determined that the MFGM that covered the surface of the oil-in-water emulsion could not be displaced by surfactants such as Tweens and Triton X-100 and was not affected by the presence of other proteins such as caseins and β-lactoglobulin added to the emulsion. A strong molecular interaction between the adsorbed MFGM, composed of phospholipid and protein, may exist at the interface, rendering the newly formed membrane very little affected by the presence of other surfactants. This MFGM-stabilized emulsion was characterized by a behaviour very different from that of emulsions stabilized by other milk proteins.

Promotion of intestinal drug absorption by milk fat globule membrane

A soybean oil emulsion was prepared by using milk fat globule membrane (MFGM) materials as an emulsifier. Its stability and effect on the intestinal absorption of vitamin D3, amphotericin B and kanamycin were studied. The MFGM emulsion was as highly stable as a Tween 80 emulsion. When the MFGM emulsion was sonicated with ultrasonic disrupter, the emulsion became more stable. Quantification of vitamin D3 in the intestinal lymph indicated that the MFGM emulsion enhanced lymphatic absorption of vitamin D3. The recovered percentage of vitamin D3 emulsified with MFGM in the lymph was 1.4 times that by Tween 80, and 2.5 times that of oil-in-water suspension. Statistically significant difference was found among them (p less than 0.05). The volumes of an oil phase in the emulsion affected the absorption of vitamin D3. The recovered percentage of vitamin D3 from the lymph increased with reducing the volume of the oil phase. Sonication of the emulsion did not affect the intestinal drug absorption. Furthermore, the MFGM emulsion had no effect on the intestinal absorption of amphotericin B and kanamycin.

Effect of Emulsifiers and Fortification Methods on Light Stability of Vitamin A in Milk

Lowfat milk is commercially fortified during processing by adding small quantities of oil containing an emulsifier and vitamin A. Added vitamin A is degraded more rapidly than indigenous vitamin A when milk is exposed to light. The objective of this research was to determine if added vitamin A could be made as stable to light as naturally occurring vitamin A by placing it in the more dilute milk fat environment with naturally occurring emulsifiers.Using HPLC to quantify retinyl palmitate, the effect of emulsifiers indigenous to milk versus a commercial emulsifier was first investigated. Butter serum, rich in fat globule membrane material, was compared with Durfax 80 in a 1% recombined milk system. No difference was found in vitamin A stability to light due to emulsifier type.A second experiment on 1% milk compared light stability of vitamin A added in concentrated milk fat emulsion, vitamin A added to all the milk fat, and indigenous vitamin A. Indigenous vitamin A and vitamin A added to all the fat were equally stable and more stable than vitamin A added in a concentrated milk fat emulsion.

Some comments on the isolation of fat globule membrane material

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Isolation and composition of milk fat globule membrane material

SummaryMilk fat globule membrane (MFGM) material was isolated from commercial pasteurized milks and pasteurized creams using a procedure specifically adapted for these products (McPhersonet al.1984a). Pasteurized cream membranes contained higher lipid levels while pasteurized milk membrane material had lower lipid contents than fresh raw MFGM. Electrophoretic analysis showed that membrane material from both commercial products contained, in addition to native MFGM polypeptides, significant amounts of β-lactoglobulin with lower levels of caseins and other skim-milk components. The incorporation of β-lactoglobulin was more pronounced in pasteurized cream membranes. Examination of the lipoprotein complexes present in these membrane preparations by linear sucrose density gradient centrifugation showed that pasteurized milk membrane material had a similar profile to fresh raw MFGM except that preferential binding of skim-milk components was found in some of the lipoprotein fractions. Pasteurized cream membranes showed considerably different density gradient patterns with only 2 lipoprotein fractions present, of which the major one was a low density complex.

Isolation and composition of milk fat globule membrane material

SummaryThe material surrounding the fat globules in commercial pasteurized homogenized milk was isolated by centrifugation of the freeze – thaw extract of the fat globules through a 52·5% (w/v) sucrose solution in simulated milk ultrafiltrate. The purified membrane material contained higher neutral lipid levels than fresh raw milk fat globule membrane (MFGM) material. Caseins and whey proteins, mainly β-lactoglobulin (β-lg) were found to be the major protein components of homogenized milk membrane material. Native MFGM polypeptides comprised only a small proportion of the total protein in these membranes. The total amount of membrane material isolated from homogenized milk was substantially higher than that from fresh raw milk. Analysis of homogenized milk membrane material by linear sucrose density gradient centrifugation showed a different pattern to that found with other milks, indicating the presence of new lipoprotein complexes. Commercial ultra heat treated (UHT) milks were also examined, but it was difficult to obtain isolated MFGM material free from core fat. However, fat globule fractions were isolated from these products by high speed centrifugation and these were analysed intact. Membrane material in these milks contained caseins and whey proteins (mainly β-1g). Natural MFGM polypeptides were present at extremely low levels. Phospholipid levels in UHT milk membranes were greatly reduced compared with membrane material isolated from other milks.

Isolation of bovine milk fat globule membrane material from cream without prior removal of caseins and whey proteins

SummaryBovine milk fat globule membrane (MFGM) material was isolated from cream by a new technique which did not involve removal of caseins and whey proteins before destabilization of the fat globules. These components were removed by centrifugation of the membrane material extract through a concentrated sucrose solution (52·5% (w/v) sucrose in 10 mM-Tris HCl buffer, pH 7·5). Membranes collected at the sample–sucrose interface, while whey proteins remained in the supernatant and caseins migrated into the concentrated sucrose solution. The yield of membrane material using this procedure was 25% less than that from conventional methods. This reduced yield was due mainly to lower levels of lipid material, in particular triglyceride. Electrophoretic analysis showed that the polypeptide composition of ‘unwashed’ membrane material was similar to that of conventionally prepared MFGM. This method is particularly suitable for the isolation of membrane material from milks and dairy products in which the fat globule stability is reduced or unknown.

Inhibition of Lipolysis by Milk Fat Globule Membrane Materials in Model Milk Fat Emulsion

The milk fat globule membrane (MFGM) enclosing fat droplets in bovine milk was isolated, and its effects on hydrolysis of milk fat by lipases were investigated by using a gum arabic-stabilized milk fat emulsion as substrate. The addition of isolated MFGM to the reaction mixture markedly inhibited hydrolysis by pancreatic and microbial (Rhizopus delemer) lipases. The inhibition was completely lost on tryptic digestion of MFGM, suggesting that the protein moiety of MFGM played a role in the inhibition. Soluble glycoprotein (SGP) which was isolated from delipidated MFGM produced marked inhibitory activity. The inhibition by SGP was dependent on substrate concentration, suggesting that the inhibition was at least partly due to coverage and blockage of the substrate surface by SGP.

Inhibition of lipolysis by milk fat globule membrane materials in model milk fat emulsion.

The milk fat globule membrane (MFGM) enclosing fat droplets in bovine milk was isolated, and its effects on hydrolysis of milk fat by lipases were investigated by using a gum arabic-stabilized milk fat emulsion as substrate. The addition of isolated MFGM to the reaction mixture markedly inhibited hydrolysis by pancreatic and microbial (Rhizopus delemer) lipases. The inhibition was completely lost on tryptic digestion of MFGM, suggesting that the protein moiety of MFGM played a role in the inhibition. Soluble glycoprotein (SGP) which was isolated from delipidated MFGM produced marked inhibitory activity. The inhibition by SGP was dependent on substrate concentration, suggesting that the inhibition was at least partly due to coverage and blockage of the substrate surface by SGP.

Crossed immunoelectrophoresis of bovine milk fat globule membrane protein solubilized with non-ionic detergent

Detergent solubilized bovine milk fat globule membrane material studied by crossed immunoelectrophoresis combined with histochemical techniques revealed four major protein complexes. All four were found to bind to concanavalin A and three were identified as sialoglycoproteins. Xanthine oxidase activity was associated with the non-sialoglycoprotein precipitate. Immunoabsorption with intact milk fat globules showed an internal location of the xanthine oxidase, whereas the three other main proteins plus Mg 2+-ATPase and 5′-nucleotidase were disposed on the outer membrane surface. The major proteins from milk fat globule membrane and membrane material isolated from skim milk showed immunochemical identity.

Oxidative Stability of Milk. I. The Antioxidative Effects of Trypsin Treatment and Aging

Trypsin treatment, known to retard trace metal-induced lipid peroxidation in milk, was shown also to inhibit oxidative degradation of vitamin A. The aging of milk prior to copper addition provided increased resistance to oxidation with an apparent increase in the antioxidative effectiveness of trypsin. Following both trypsin treatment and aging, total recoverable fat globule membrane material was decreased, but changes in the percent protein were only slight. Electrophoretic studies revealed that tryptic action produced marked hydrolysis of fat globule membrane proteins whereas aging produced only minor changes. Hydrolysis of skim milk proteins also was shown in milks treated with trypsin. Aging produced apparent aggregation of certain skim milk proteins. The antioxidative effect of tryptic action in milk is due primarily to an increase in metal chelating capacity. Spontaneous alteration of the fat globule membrane, coupled with increased chelation capacity in the skim milk, were probably responsible for the increased oxidative stability of aged milk. It was postulated that membrane material released from the fat globule contributed to the increase in chelation capacity and oxidative stability.

Molecular Weight Profile of Fat Globule Membrance Proteins

Fat globule membrane material, isolated by churning cream that had been washed four times, was extracted sequentially with .6M potassium chloride and centrifuged to yield pellet and supernatant fractions. Compositional data indicated that lipid components were removed preferentially into the supernatant fractions.Electropherograms of the pellet and supernatant fraction in 10% and 5% sodium dodecyl sulfate-containing polyacrylamide gels revealed that the protein species originally present in the membrane associated into higher molecular weight species concomitant with the removal of lipid moieties. Thus, a contribution of the lipid moiety to membrane integrity is suggested. Approximately 17 protein-stained zones, ranging in molecular weight from 13,500 to 208,000 daltons, were observed in gels of the membrane material.

Influence of Mastitis on Properties of Milk XI. Fat Globule Membrane

The effect of mastitis on the milk fat globule membrane of milk samples from individual quarters was investigated. Wisconsin Mastitis Test positive (>20mm) milk contained approximately 10% less milk fat globule membrane material than negative (<10mm) milk. The lipid portion of the milk fat globule membrane preparation from positive milk contained smaller amounts of phospholipids and larger amounts of cholesterol. Milk fat globule membrane preparations from positive milk had higher aldolase activity and lower xanthine oxidase activity. Dissociated membrane protein prepared from positive milk was resolved into five components by polyacrylamide gel electrophoresis while the membrane protein prepared from negative milk was resolved into three components.

Milk fat globule membrane material in skim-milk

SummaryIt has been claimed by Stewart, Puppione &amp; Patton (1972) that milk is a valuable source of cell membrane in a ‘relatively pure’ state originating from shed mammary secretory cell microvilli and golgi vesicles. The morphological basis for this has been re-examined in this paper. It is shown that the vesicular and elongated membranous structures found in skim-milk are identical to structures present as part of the initial milk fat globule membrane (MFGM) of alveolar and expressed milk, and that both structures survive extraction with chloroform–methanol. It is therefore suggested that a major part of the skim-milk membrane is material shed from the freshly secreted milk fat globule and consists of a specialised derivative of the secretory cell plasmalemma, the initial MFGM.

PHYSICOCHEMICAL AND BACTERIOLOGICAL ASPECTS OF PRESERVED MILK SAMPLES AND THEIR EFFECT ON FAT PERCENTAGE AS DETERMINED WITH THE MILKO-TESTER

Milko-Tester results decrease as a milk sample ages before analysis. Since no fat is physically removed, this phenomenon could be termed apparent fat loss. Reasons are advanced why the Milko-Tester shows lower results on stored samples. Bacteria counts, oiling-off determinations, and acidity titrations were made on preserved milk samples to show their possible relationship to apparent fat loss. It is believed that changes in fat globule membrane material are key factors that affect the optical properties utilized by the Milko-Tester and are responsible for depressed fat percentages. High initial bacteria count may be the major and primary factor responsible for apparent fat loss in preserved samples held at room temperature. Bacteria have been demonstrated to grow in the presence of potassium dichromate preservative. Under certain conditions preserved milk samples showed no apparent fat loss and could be tested accurately with the Milko-Tester up to 20 days.

Relationship of Milk Fat Globule Membrane Material to Flavor Development in Cheddar Cheese

The effect on ripening of varying the amount of fat globule membrane material in Cheddar cheese was investigated by altering the buttermilk content of the cheese milks. The stability of phospholipids during cheese ripening was also measured. The rate of lipolysis increased in cheeses made with skimmilk and butteroil (without buttermilk); but there was no significant flavor difference between cheeses made from whole milk, whole milk with additional buttermilk powder, or skimmilk and butteroil (homogenized at 352 102kg/m2). All had a typical mild flavor. Cheese made with skimmilk and butteroil homogenized at 422 103kg/ m2 were rancid after 6 mo ripening and contained very high amounts of free fatty acids. Cheese made with skimmilk and buttermilk powder contained only .5% fat and were hard and dry in texture with no typical flavor. Counts of starter streptococci were not affected by the presence or absence of buttermilk either during cheesemaking or ripening. Phospholipid in the cheeses decreased by 50 to 70% during the 6 mo ripening.

Ultrastructural Observations on the Milk Fat Globule Envelope of Cow's Milk

To further elucidate the complex fine structure of the milk fat globule envelope, fresh cream and isolated fat globule membrance material were examined electron microscopically by three different but complementary preparation techniques: Thin sectioning, negative staining, and freeze-etching. After thin sectioning most of the fat globules were surrounded only by a simple limiting membrance to which casein micelles may be adsorbed. A higher percentage of fat globules still enveloped by a unit membrance was found after freeze-etching, indicating that physical stabilization by rapid freezing is preferable to chemical fixation. Vesicle formation of isolated fat globule membrane material depends upon the purity of the membrane fractions. Based on these morphological observations a structural model of the milk fat globule envelope which may serve as a working hypothesis for further more detailed studies is proposed.

Lipid Composition of the Fat Globule Membrane of Human and Bovine Milk

Fat globule membrane material of human and cow's milk was isolated from the creams by successive ultracentrifugations. The polar and nonpolar lipids were then extracted and their constituents identified. Lipidic fractions of the membrane of both milks contained high-melting triglycerides and a large amount of polar lipids and intermediary compounds of their biosynthesis, such as phospholipids, sterols, alkanes and acyclic terpenes. Human milk contained more unsaponifiable matter and organic phosphorus, and slightly less triglyceride than cow's milk. The fatty acid composition of the triglycerides of both milks was similar, except for the remarkably small amount of arachidic acid and for the large amount of linoleic acid in human milk as compared to cow's milk. In both milks, the total amount of short-chain fatty acids was higher in the bulk of fat than in the membrane. Other differences in the lipids of human and cow's milk were also evidenced: these concerned mainly the degree of unsaturation, the positional distribution of fatty acids, cholesterol and 7-dehydrocholesterol content, the amount and type of phospholipids as well as their fatty acid distribution.