Size distribution of fat globules in human colostrum, breast milk, and infant formula

Only a few results are available on the size of human milk fat globules (MFG), despite its significance regarding fat digestion in the infant, and no data are available at <24 h postpartum (PP). We measured the MFG size distribution in colostrum and transitional human milk in comparison with fat globules of mature milk and infant formula. Colostrum and transitional milk samples from 18 mothers were collected regularly during 4 d PP and compared with mature milk samples of 17 different mothers and 4 infant formulas. The size distribution was measured by laser light scattering. For further characterization, the ζ-potential of some mature MFG was measured by laser Doppler electrophoresis. The MFG diameter decreased sigmoidally in the first days. At <12 h PP, the mode diameter was 8.9 ± 1.0 μm vs 2.8 ±0.3 μm at 96 h PP. Thus, the surface area of MFG increased from 1.1 ±0.3 to 5.4 ±0.7 m²/g between colostrum and transitional milk. In mature milk, the MFG diameter was 4 μm on average and increased with advancing lactation, whereas the droplets in infant formula measured 0.4 μm. The ζ potential of mature MFG was −7.8 ± 0.1 mV. The fat globules are larger in early colostrum than in transitional and mature human milk and in contrast with the small-sized fat droplets in infant formula. Human MFG also have a low negative surface charge compared with bovine globules. These structural differences can be of nutritional significance for the infant.     

Size distribution of casein globules

Summary Size distribution of casein particles in sheep's milk were determined by applying ultracentrifugation. The normal Gaussion distribution curve showed that the greatest proportion of particles had diameters ranging from 53.5 – 100.7 µm like in cow's milk; the average diameter of the particles was 82.7 µm.

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Zusammenfassung In der vorliegenden Untersuchung wurde die Größenverteilung von Caseinpartikeln in Schafsmilch unter Verwendung einer Ultrazentrifuge bestimmt. Die Gauss'sche Verteilungskurve ergab, ähnlich wie in Kuhmilch, daß der größte Teil der Partikel bei Schafmilch in einem Größenbereich von 53,5 – 100,7 µm lag; der durchschnittliche Durchmesser der Partikel betrug 81,7 µm.

     

Buffalo vs. cow milk fat globules: Size distribution,zeta-potential,compositions in total fatty acids and in polar lipids from the milk fat globule membrane

Although buffalo milk is the second most produced milk in the world, and of primary nutritional importance in various parts of the world, few studies have focused on the physicochemical properties of buffalo milk fat globules. This study is a comparative analysis of buffalo and cow milk fat globules. The larger size of buffalo fat globules, 5 vs. 3.5 μm, was related to the higher amount of fat in the buffalo milks: 73.4 ± 9.9 vs. 41.3 ± 3.7 g/kg for cow milk. Buffalo milks contained significantly lower amount of polar lipids expressed per gram of lipids (0.26% vs. 0.36%), but significantly higher amount of polar lipids per litre of milk (+26%). Buffalo and cow milk fat globule membranes contain the same classes of polar lipids; phosphatidylethanolamine, sphingomyelin (SM) and phosphatidylcholine (PC) being the main constituents. A significant higher percentage of PC and lower percentage of SM were found for buffalo milks. The fatty acid analysis revealed that saturated fatty acids, mainly palmitic acid, trans fatty acids, linolenic acid (ω3) and conjugated linolenic acid were higher in buffalo milk than in cow milk. Such results will contribute to the improvement of the quality of buffalo milk-based dairy products.     

Effect of nonthermal technologies on the native size distribution of fat globules in bovine cheese-making milk

Milk-fat globule membranes are susceptible to damage by mechanical and thermal processes. This damage is translated into alterations of milk fat structure and functionality of cheese-making milk. The objective of this work was to evaluate the effect of pulsed electrical fields (PEF), high hydrostatic pressure (HHP), and conventional thermal treatments on fat globule size distribution and ζ-potential. Milk was processed by HHP at 400 and 500 MPa for 0–20 min, and with PEF at 36 kV/cm and 42 kV/cm up to 64 pulses. The ζ-potential of HHP and PEF treated milk were − 15.47 mV and − 14.63 mV respectively. HHP treatments induced fat globules flocculation, increasing their mass moment mean diameter. Although PEF processing did not modify the true mean diameter of MFG, it induced small globules to clump together, causing an apparent increment in the population of larger milk-fat globules.

Industrial relevance

The market for traditional raw dairy products has increased in recent times in several regions of the world due to their unique flavor and texture attributes. However, the potential negative implications of consuming raw products limit the growth of this market segment. Manufacture of raw-like cheese from thermally pasteurized milk is not feasible, among other things, because of milk fat globule membrane damage caused by elevated temperatures. Nonthermal food preservation technologies offer the potential to produce milk technically suitable for the industrial manufacture of microbiologically safe raw-like dairy products.     

Structural changes of bovine milk fat globules during in vitro digestion

An in vitro digestion model that simulated gastric and intestinal fasting conditions was used to monitor the physical, chemical, and structural changes of fat globules from raw bovine milk. During in vitro gastric digestion, the fat globules were stable under low-acidic conditions. Some peptides and β-lactoglobulin were resistant to proteolysis by pepsin. Phospholipids, proteins, and peptides stabilized the globules in the stomach model. During in vitro intestinal digestion, most of the β-lactoglobulin and residual peptides were hydrolyzed by trypsin and chymotrypsin, and the lipolytic products, released from the hydrolysis of the triglyceride core of the globules, led to destabilization and coalescence of the globules. By accumulating at the surface of the fat globules, the lipolytic products formed a lamellar phase and their solubilization by bile salts resulted in the formation of disk-shaped micelles. This study brings new interesting insights on the digestion of bovine milk.     

Impact of cream washing on fat globules and milk fat globule membrane proteins

Milk proteins, contained within the aqueous phase surrounding fat globules, should be removed before analysis of the composition of the native milk fat globule membrane (MFGM). The effect of the conditions applied during washing of cream on MFGM integrity has not been fully studied, and factors potentially effecting a modification of MFGM structure have not been systematically assessed so far. In this study, a cream separator was used to investigate the impact of cream washing on milk fat globule stability and the corresponding loss of MFGM proteins. Flow velocity, fat content, and type of washing solution were varied. Particle size measurements and protein analyses were carried out after each washing step to determine fat globule coalescence, removal of skim milk proteins, and efficiency of MFGM isolation. Significant differences in fat globule stability and protein amount in the MFGM isolates were measured using different washing conditions.     

Composition and distribution of fatty acids in triglycerides from goat infant formulas with milk fat

Most infant formulas use vegetable oils in place of milk fat to provide an overall fatty acid profile similar to that of breast milk. Vegetable oils have 5 to 20% saturated fatty acids in the sn-2 position of triglycerides unless they are modified by interesterification. Interesterification is increasingly used for the fat for infant formulas to raise the level of saturated fatty acids in the sn-2 position to 40 to 60%. The objective of this study was to verify an alternative approach to providing the appropriate fatty acid profile, including in the sn-2 position, for a goat infant formula. In this method, 55% of total fat was made from goat milk fat and 45% from a mixture of unmodified high oleic sunflower, canola, and sunflower oils in a ratio of 44:30:26. The fatty acid profile was measured by gas-liquid chromatography and the relative percentage of fatty acids in the sn-2 position of triglycerides was measured via partial deacylation with Grignard reagent using trimethylsilyl derivatives of monoacylglycerols. Mixing goat milk fat with vegetable oils produced a formula with a profile of essential fatty acids and a ratio of linoleic:α-linolenic fatty acids within the required interval of 5 to 15:1 recommended for infant formula. The proportion of palmitic acid in the sn-2 position was 31%.     

Influence of enzymatic cross-linking on milk fat globules and emulsifying properties of milk proteins

The enzyme transglutaminase (TGase) can modify dairy protein functionality through cross-linking of proteins. This study examined the effects of TGase treatment on milk fat globules and the emulsifying properties of milk proteins. The extent of TGase-induced cross-linking of caseins increased with incubation time, with no differences between whole and skim milk. Extensive clustering of fat globules in extensively cross-linked raw whole milk occurred on homogenisation at 400 or 800 bar. Considerably less clustering of fat globules was observed when recombined milk (90 g fat L^–1) was prepared from TGase-treated skim milk and homogenised at 400 or 800 bar. TGase treatment did not affect fat globule size in cream, but prevented coalescence of fat globules therein, possibly through cross-linking of milk fat globule membrane components. TGase-induced cross-linking of milk proteins affected their emulsifying properties and may increase the stability of natural milk fat globules against coalescence.     

Hydrolysis of bovine milk fat globules by lipoprotein lipase: inhibition by proteins extracted from milk fat globule membrane

Extraction of membrane proteins from milk fat globules by GuHCl or by MgCl2 made the lipids more accessible to lipolysis by added lipoprotein lipase. The increase in lipolysis paralleled the loss of membrane proteins and was continuous up to 2.5 M GuHCl, which was the highest concentration used. About twice as much protein was extracted with 2.5 M GuHCl as with buffer only. The amount of protein lost was about 50% of total milk fat globule protein. Lipolysis of milk fat globules was inhibited by addition of the extracted protein. The extracted proteins also reduced lipolysis when added to whole milk. More protein was needed to inhibit lipolysis of milk fat globules treated with GuHCl compared with globules treated with buffer only. The inhibition by a given amount of protein decreased if more milk fat globules were used. Protein extracted with MgCl2 had similar effects as those extracted with GuHCl. The major components extracted with MgCl2 migrated in the 40 to 50-kdalton region on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By gel filtration chromatography, two protein fractions were obtained, which inhibited lipolysis more efficiently than the total extract. As has previously been found for inhibition of lipolysis by skim milk, the amount of extracted protein needed to inhibit lipolysis varied between preparations of milk fat globules. Milk with propensity to cold-induced ("spontaneous") lipolysis was normalized by addition of extracted proteins.     

Behaviour of homogenized fat globules during the spray drying of whole milk

The changes in milk fat globule size and fat globule surface proteins of both low-preheated and high-preheated concentrated milks, which were homogenized at low or high pressure prior to spray drying using a disc atomization drier, were examined. The average fat globule size (d₃₂) of the spray-dried milk powders was smaller than that of the corresponding concentrates, but a small proportion of very large globules (4–80 μm) was also formed during spray drying. As a consequence, total surface protein (mg protein g⁻¹ fat) increased due to the adsorption of casein micelles at the fat globule surface during spray drying. Confocal micrographs of the powders showed some apparent spreading of the fat on the surface of the powder particles, particularly when the concentrates were homogenized at low pressure. These results indicate disruption of the milk fat globules during spray drying, which consequently causes changes in the fat globule surface protein layer.     

Factors related to the formation of cytoplasmic crescents on milk fat globules

Cytoplasm from lactating cells is included with some milk fat globules at secretion. The objective was to search for factors causing this phenomenon. Globules bearing crescents of cytoplasm were selectively stained with the fluorescent dye, acridine orange, and their proportion in the globule population was obtained by counting from photomicrographs. Incidence of crescents on fat globules in milk samples of 50 human donors ranged from 1 to 29%, and the mean was 7.2%. Two bovine milk samples, both representing over 100 animals, contained 1% or less of globules with crescents. Globules in individual milkings of five beef cows showed the same low proportion of crescents. In addition to species, genetic and diurnal factors influenced numbers of crescents. Two sisters showed evidence within and between lactations of a persistent high proportion (greater than 25%) of globules with crescents. Samples collected in the a.m. contained a lower percentage of globules with crescents (6.5%) than those obtained in the p.m. (9.7%). Crescent incidence was not correlated with lipid or protein content of human milk, interval within a milking, days in lactation, or the donor's age. Evidence is presented to suggest that the concentration, distribution, and acylation of butyrophilin and xanthine oxidase, coat proteins of the apical plasma membrane, are important factors in globule crescent formation.     

Major proteins of the goat milk fat globule membrane

Fat is present in milk as droplets of triglycerides surrounded by a complex membrane derived from the mammary epithelial cell called milk fat globule membrane (MFGM). Although numerous studies have been published on human or bovine MFGM proteins, to date few studies exist on MFGM proteins from goat milk. The objective of this study was thus to investigate the protein composition of the goat MFGM. Milk fat globule membrane proteins from goat milk were separated by 6% and 10% sodium dodecyl sulfate-PAGE and were Coomassie or periodic acid-Schiff stained. Most of MFGM proteins [mucin-1, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin (MFG EGF-8, MFG-E8), and adipophilin] already described in cow milk were identified in goat milk using peptide mass fingerprinting. In addition, lectin staining provided a preliminary characterization of carbohydrate structures occurring on MFGM proteins from goat milk depending on α_S1-casein genotype and lactation stage. We provide here first evidence of the presence of O-glycans on fatty acid synthase and xanthine oxidase from goat milk. A prominent difference between the cow and the goat species was demonstrated for lactadherin. Indeed, whereas 2 polypeptide chains were easily identified by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight analysis within bovine MFGM proteins, lactadherin from goat milk consisted of a single polypeptide chain. Another striking observation was the presence of caseins associated with MFGM preparations from goat milk, whereas virtually no caseins were found in MFGM extracts from bovine milk. Taken together, these observations strongly support the existence of a singular secretion mode previously hypothesized in the goat.     

Effect of the fat globule membrane on in vitro digestion of milk fat globules with pancreatic lipase

The rate and extent of in vitro lipid digestion in raw and recombined milk were investigated by determining the release of fatty acids in simulated intestinal fluid containing pancreatic lipase. Changes in the globule size, surface charge and microstructure of fat globules during digestion were examined. In the absence of bile extract, the rate of lipid digestion was slower in raw milk than in recombined milk, suggesting that the composition of the milk fat globule membrane influences the rate of lipid hydrolysis in milk. Flocculation of fat globules occurred in the early stages of digestion; the globules then coalesced to form large particles, from within which triacylglycerols were removed. However, in the presence of bile extract, the changes in the size and microstructure of the fat globules during digestion were different. Bile extract may therefore affect physicochemical interactions of fat globules and hence alter the lipolysis during the digestion.     

Native vs. damaged milk fat globules: membrane properties affect the viscoelasticity of milk gels

The storage modulus G' of rennet and acid milk gels filled with milk fat globules was measured as a function of the fat globule surface composition (native milk fat globule membrane, caseins and whey proteins, or a mixture of the three due to mechanical treatments) and surface area (i.e., the fat globule size). By different technological procedures, it was possible to obtain fat globules of constant surface composition but various sizes, and vice-versa, which had never been done. For both rennet and acid gels, a critical fraction of the fat globule surface covered by caseins and whey proteins was identified (approximately 40%), beyond which G' increased. Below this threshold, the gel viscoelasticity was unaffected by mechanical treatments. When the diameter of native milk fat globules decreased, the G' of rennet gels increased slightly, whereas that of acid gels decreased sharply. For both types of gels, G' increased when the diameter of partially disrupted fat globules decreased. For recombined globules completely covered with caseins and few whey proteins, G' increased with fat globule surface area for rennet gels whereas it decreased for acid gels. With the help of confocal microscopy and in the light of general structural differences between rennet and acid gels, a mechanism is proposed for the effect of fat globule damage and diameter on G', depending on the interactions the globules can undergo with the casein network.     

Effects of goat milk or milk replacer diet on meat quality and fat composition of suckling goat kids

The effects of a diet with goat milk "GM" or milk replacer "MR" on the meat quality and fat composition of suckling Murciano-Granadina kids were studied. MR consisted of powdered skimmed milk, coconut oil and fat, and cereal products and by-products. Raw meat quality (moisture, protein, lipids, ash, collagen, cholesterol, haem pigments, CIELab colour, pH and water retention capacity), fatty acid "FA" composition and eating quality of cooked meat (odour, flavour and texture) were determined. Diet had only a slight effect on raw meat quality but had a pronounced effect on fatty acid composition and eating quality of cooked meat. MR diet increased the water/protein proportion in the muscle. The saturated/unsaturated FA ratio in GM and MR fat was 0.94 and 2.27, respectively. The major FA in GM and MR fat were C16:0 and C18:1, respectively. Short-chain C4-C12 hardly accumulated in the adipose tissue of suckling kid, increasing the relative percentages of C14-C20. This effect was more pronounced in MR fat, due to the fact that MR contained more short-chain fatty acids than GM. MR diet gave cooked meat a more intense characteristic goat meat odour and flavour, more tenderness and more juiciness than the natural suckling diet. This fact could be related to differences in meat and fat composition.     

Chemical and structural characterisation of almond oil bodies and bovine milk fat globules

Lipids in almonds are present as oil bodies in the nut. These oil bodies are surrounded by a membrane of proteins and phospholipids and are a delivery vehicle of energy in the form of triglycerides, similarly to the more studied bovine milk fat globule membrane. Chemical, physical and microscopic analyses revealed major differences in the composition and structure of almond oil bodies and bovine milk fat globules. The lipids of both natural emulsions differed in degree of unsaturation, chain length, and class. The almond oil body membrane does not contain any cholesterol or sphingomyelin unlike the bovine milk fat globule membrane. Therefore, the phospholipid distribution at the surface of the oil bodies did not present any liquid-ordered domains. The membranes, a monolayer around almond oil bodies and a trilayer around bovine fat globules, may affect the stability of the lipid droplets in a food matrix and the way the lipids are digested.     

Dynamic rheology of renneted milk gels containing fat globules stabilized with different surfactants

Anhydrous milk fat was emulsified with alpha s1-CN (casein), alpha s2-CN, beta-CN, kappa-CN, alpha-lactalbumin, beta-lactoglobulin, Tween 80, or phosphatidylcholine to produce a 30% fat cream in a 0.1 M imidazole pH 7 buffer. The creams were mixed with skim milk to yield a fat content of 3.4% and the viscoelastic properties of the recombined milks clotted with chymosin were measured. Recombined milk containing globules coated with the more amphipathic and phosphorylated alpha s2-CN and beta-CN clotted faster but gel firmness increased more slowly and weaker gels were formed. Gel firmness increased more rapidly for milks containing globules coated with of alpha s1-CN and kappa-CN that possess more uniformly distributed hydrophobic domains.     

Identification of ABCA1 and ABCG1 in milk fat globules and mammary cells--implications for milk cholesterol secretion

The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play an important role in cellular cholesterol homeostasis, but their function in mammary gland (MG) tissue remains elusive. A bovine MG model that allows repeated MG sampling in identical animals at different functional stages was used to test whether 1) ABCA1 and ABCG1 protein expression and subcellular localization in mammary epithelial cells (MEC) change during the pregnancy-lactation cycle, and 2) these 2 proteins were present in milk fat globules (MFG). Expression and localization in MEC were investigated in bovine MG tissues at the end of lactation, during the dry period (DP), and early lactation using immunohistochemical and immunofluorescence approaches. The presence of ABCA1 and ABCG1 in MFG isolated from fresh milk was determined by immunofluorescence. The ABCA1 protein expression in MEC, expressed as arbitrary units, was higher during the end of lactation (12.2 ± 0.24) and the DP (12.5 ± 0.22) as compared with during early lactation (10.2 ± 0.65). In contrast, no significant change in ABCG1 expression existed between the stages. Throughout the cycle, ABCA1 and ABCG1 were detected in the apical (41.9 ± 24.8 and 49.0 ± 4.96% of cows, respectively), basal (56.2 ± 28.1 and 54.6 ± 7.78% of cows, respectively), or entire cytoplasm (56.8 ± 13.4 and 61.6 ± 14.4% of cows, respectively) of MEC, or showed combined localization. Unlike ABCG1, ABCA1 was absent at the apical aspect of MEC during early lactation. Immunolabeling experiments revealed the presence of ABCA1 and ABCG1 in MFG membranes. Findings suggest a differential, functional stage-dependent role of ABCA1 and ABCG1 in cholesterol homeostasis of the MG epithelium. The presence of ABCA1 and ABCG1 in MFG membranes suggests that these proteins are involved in cholesterol exchange between MEC and alveolar milk.     

Effect of the size and interface composition of milk fat globules on their in vitro digestion by the human pancreatic lipase: Native versus homogenized milk fat globules

Although the bioavailability of dietary lipids is of primary importance in human nutrition and health, the mechanisms involved in lipid digestion are not fully understood and are of growing interest. The objective of this study was to determine the effect of the size of milk fat globules and of the composition of their interface on the activity of the human pancreatic lipase (PL). Native milk fat globules of various sizes covered by their biological membrane (MFGM) and homogenized fat globules of various sizes covered by milk proteins were prepared from whole milk and underwent lipolysis by the human PL with colipase and bile salts. A lag phase preceding the hydrolysis of milk TAG occurred with all native milk fat globules samples but not with homogenized milk samples. The kinetic parameters of human PL were determined by measuring the enzyme activity either after the lag phase for native milk fat globules samples or immediately after the addition of the enzyme for homogenized milk samples. The catalytic efficiency of human PL is 4.6-fold higher on small (1.8 μm) than large (6.7 μm) native milk fat globules, related to a 3.6-fold larger available surface. Despite the 25-fold larger available surface, milk TAG from homogenized milk are only 2-fold better hydrolyzed compared to native milk fat globules, as a possible result of a less favourable interface covered by milk proteins. The potential mechanisms involved in native vs. homogenized milk fat globules digestion by the human PL are discussed. Our study highlights the crucial role of the MFGM in the efficient digestion of milk fat globules and brings new insight for the design of dairy products and infant formulas.     

Digestive utilization of goat and cow milk fat in malabsorption syndrome.

We studied the effects of goat and cow milk fat on the digestive utilization of this nutrient and on some of the biochemical parameters that are related to the metabolisim of lipids, using rats with a resection of 50% of the distal small intestine and control animals (transected). The fat content in all the diets was 10% but the lipid quality was varied: the standard diet was based on olive oil, while the other two diets included fat obtained from lyophilized goat milk and cow milk, respectively. The digestive utilization of the fat was lower in the resected animals than in the transected ones for all three diets studied. In both resected and transected animals. the apparent digestibility coefficient (ADC) of the fat was greater with the standard diet (olive oil) than with diets whose fat content was provided by goat or cow milk. The digestive utilization of the fat was greater in the transected and resected rats receiving a diet of goat's milk (rich in medium-chain triglycerides) than those given a cow-milk-based diet and more closely approached the values obtained for olive oil. The consumption of goat milk reduced levels of cholesterol while levels of triglycerides, HDL, GOT and GPT remained with in the normal ranges, for both transected and resected animals. The advantageous effect of goat milk on the metabolisim of lipids with respect to cow milk suggests that the former should be included in the diet in eases of malabsorption snydrome.