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Milk fat globules (MFGs) are secreted from the mammalian gland and are composed of a triacylglycerol core surrounded by a triple membrane structure, the milk fat globule membrane (MFGM). The MFGM contains complex lipids and proteins reported to have nutritional, immunological, neurological and digestive functions. Human and ruminant milk are shown to share a similar MFG structure but with different size, profile and abundance of protein and polar lipids. This review summarizes the reported data on human, bovine, caprine and ovine MFG composition and concentration of bioactive components in different MFG-size fractions. A comprehensive understanding of compositional variations between milk from different species and MFG size fractions may help promote various milk sources as targeted supplements to improve human development and health. MFG size and MFGM composition are species-specific and affected by lactation, diet and breed (or maternal origin). Purification and enrichment methods for some bioactive proteins and lipids present in the MFGM have yet to be established or are not scaled sufficiently to be used to supplement human diets. To overcome this problem, MFG size selection through fractionation or herd selection may provide a convenient way to pre-enrich the MFG fraction with specific protein and lipid components to fulfill human dietary and health requirements.

Milk fat globule membrane (MFGM) fractions were isolated from fresh raw cream at different temperatures and pH values. The polar lipid (PL) composition of these fractions, and of commercially produced α-serum, β-serum, and buttermilk powder samples, was determined. Lower temperature and pH during churning increased the total level of PLs in the MFGM fractions. Phosphatidylethanolamine (31–36% of total PLs), phosphatidylcholine (31–39%), and sphingomyelin (13–24%) were the major PLs in all MFGM fractions; phosphatidylserine (2–7%) and phosphatidylinositol (2–4%) were the minor ones. The β-serum fraction had the highest total PL content of the commercial samples (2.9 mg g−1 fat); buttermilk powder contained the least amount of total PL amongst all fractions (0.99 mg g−1). Processing temperature and pH can therefore influence the PL composition, likely due to how the treatments impact on the different PLs that are located within different regions within the MFGM trilayer structure.

Pulsed electric field (PEF)-treated milk (25.7 kV/cm for 34 μs after preheating to 55 °C and holding for 24 s) was microbiologically stable for 21 days at 4 °C, and similar to thermally treated milk (63 °C for 30 min or 73 °C for 15 s). Alkaline phosphatase inactivation was comparable after PEF (preheating followed by PEF) and both thermal treatments. PEF treatment initially reduced xanthine oxidase (30%) and plasmin (7%) activities, but after 21 days of refrigerated storage these activities were similar to the initial untreated milk. During refrigerated storage of PEF (preheating followed by PEF) and thermally treated milk, lipolytic activity increased and pH levels decreased.

The protein composition, redox potential (Eh), and xanthine oxidase (XO) content and activity were determined for anhydrous milk fat emulsions containing milk fat globule membrane (MFGM) fractions derived from either buttermilk or commercial sources of bovine α-serum, β-serum, and buttermilk powder (BMP). Caseins were the dominant proteins in the MFGM fractions isolated from BMP and buttermilk, whereas fractions from α- and β-serum contained higher amounts of membrane proteins. The XO content and activity was >70-fold and >700-fold higher in α- and β-serum samples, respectively, compared with the BMP fraction. The Eh values of the recombined emulsions were highest for α-serum (196 mV), and β-serum (169 mV), followed by BMP (131 mV). These positive values contrasted with the highly negative Eh of the buttermilk emulsion (−580 mV). This study demonstrates how milk-processing methods can alter the composition and functionality of the MFGM.

Two green tea polyphenols, catechin and epigallocatechin gallate (EGCG), were encapsulated in soy lecithin liposomes before being incorporated into low-fat hard cheese that was ripened for 90 days at 8 °C. Fortification of low-fat cheese with liposome-encapsulated catechin or EGCG led to a significant increase ( p in vitro gastrointestinal digestion. There was no significant effect of encapsulation on cheese composition, pH, or cheese yield from milk. Catechin and EGCG were not detected in the cheese whey, indicating complete retention in the cheese structure. About half of the initial catechin and one-third of EGCG were recovered from the cheese digesta. The process of liposomal encapsulation of phenolic compounds enabled a high level of retention in low-fat cheese with release under gastrointestinal digestive conditions to increase the antioxidative functionality of cheese.

Six types of milk fat globule membrane (MFGM) fractions were isolated from fresh raw cream using various churning conditions at different temperature (10, 15 or 20 °C) and pH values (5.5 or 6.6) and characterised for protein composition, xanthine oxidase (XO) content and activity, and oxidation–reduction potential (E h ). A greater proportion of non-membrane proteins was found at higher temperatures and higher cream pH, and the XO content decreased significantly under these conditions. XO activity was greater at lower temperatures and pH, yielding a more positive E h value for the emulsions. Storage at 4 °C for 14 d significantly reduced the Eh of the emulsions to between −320 mV and −580 mV for low pH/low temperature, and high pH/high temperature emulsions, respectively. This study suggests that it is possible to obtain MFGM fractions with specific Eh reducing ability by altering churning conditions.

Green tea extract (GTE) was incorporated into full-fat cheeses at 250, 500, and 1000 ppm to investigate the effect of green tea catechins on antioxidant properties and microstructure of cheese, and recovery of catechins. Cheeses were ripened for 90 days at 8 °C followed by simulated gastrointestinal digestion. The composition on Day 0, and pH, total phenolic content (TPC), and antioxidant activity (AA) of the cheeses on Days 0, 30, and 90, were determined. The concentrations of the major GTE catechins in the curd and from the digesta of mature cheeses were determined using HPLC. Addition of GTE significantly (p ≤ 0.05) decreased the pH of whey and curd during cheese manufacture and ripening, however there was no significant (p > 0.05) effect on moisture, protein, or fat contents. Addition of GTE increased TPC and AA at all concentrations, but in a non-linear manner. Individual catechins were selectively retained in the curd, whereas different portions were recovered from the cheese digesta. Transmission electron microscopy showed that the ripened control cheese had a regular distribution of milk fat globules entrapped in a homogeneous structure of casein proteins, which was disrupted by GTE. Interactions between green-tea catechins and cheese fat were confirmed by Fourier transform infrared spectroscopy.

A new rapid isocratic HPLC method was developed to quantify xanthine oxidase activity in several dairy products by determining both substrate (xanthine) and product (urate) concentrations at short retention times (6.76 min and 4.92 min, respectively) with high values for linearity and reliability. The impact of added green tea catechins (1000 ppm) on xanthine oxidase activity was also examined in dairy products containing fat globules using the developed HPLC method. The addition of (+)-catechin, (−)-epigallocatechin gallate (EGCG), and green tea extract significantly ( p ≤ 0.05) decreased the xanthine oxidase activity of these products. The effect of EGCG on decreasing xanthine oxidase activity of milk fat globules was stronger than for (+)-catechin, and can be attributed to different structures of green tea polyphenols, such as different numbers of OH groups in catechins. The results of this study suggest possible associations between milk fat globule surfaces and green tea catechins.

(+)-Catechin or green tea extract were encapsulated in soy lecithin nanoliposomes and incorporated into a full-fat cheese, then ripened at 8 °C for 90 days. Cheese samples were subjected to simulated gastrointestinal digestion to measure total phenolic content (TPC) and antioxidant activity of the cheese digesta, and to determine the catechin recovery after digestion by high performance liquid chromatography (HPLC). Addition of catechin or green tea extract significantly (P ≤ 0.05) increased TPC and antioxidant activity (measured by ferric reducing antioxidant power and oxygen radical absorbance capacity) of the full-fat cheese without affecting pH or proximate composition. HPLC analysis confirmed retention of encapsulated catechins in the cheese curd; however, individual catechins were recovered in differing amounts (15-52%) from cheese digesta after 6 h of digestion. Transmission electron microscopy and Fourier transform infrared spectroscopy provided evidence for association of nanoliposomes with the surface of milk fat globules inside the cheese matrix. The study shows the potential for using cheese as a delivery vehicle for green tea antioxidants.

The proteolytic changes under in vitro digestive conditions were examined by urea-polyacrylamide gel electrophoresis, Kjeldahl determination of nitrogen, and fluorescamine analysis to evaluate the level of free amino terminal groups as an indicator of the degree of proteolysis. The protein and fat components of cheese digesta were visualized by confocal laser scanning microscopy. Cheese disintegration and subsequent release of proteins and polypeptides was quantified after each of three in vitro digestion phases: oral, gastric (pepsin, pH 2), and intestinal (pancreatic enzymes, pH 6.8). Most of the disintegration of the cheese protein matrix (˜75%), proteolysis, and protein release occurred during the intestinal digestion phase. Fat aggregate release from the cheese matrix mostly occurred in the gastric and intestinal digestion phases, and globules became more spherical after the gastric phase. Proteolysis took place primarily in the gastric and intestinal phases. These results show the importance of microstructure and the digestive environment on the release of cheese components.

(+)-Catechin, the representative catechin in green tea, was incorporated into a full-fat cheese (at 125–500 ppm) followed by ripening for 90 days at 8 °C and digesting for six hours. Determination of pH, proximate composition, total phenolic content (TPC) and antioxidant activity (AA) after manufacture and ripening demonstrated that the addition of (+)-catechin significantly (p ≤ 0.05) decreased the pH of both whey and curd during cheese manufacturing and ripening with no significant (p > 0.05) effect on the moisture, protein and fat contents. (+)-Catechin increased TPC, as well as AA, though the increase was not proportional with increasing the concentration of added (+)-catechin. About 57%–69% of (+)-catechin was retained in the cheese curd, whereas about 19%–39% (depending on the concentration) was recovered from the cheese digesta. Transmission electron micrographs showed that the ripened control cheese had a homogeneous pattern of milk fat globules with regular spacing entrapped in a homogenous structure of casein proteins, whereas the addition of (+)-catechin disrupted this homogenous structure. The apparent interaction between (+)-catechin and cheese fat globules was confirmed by Fourier transform infrared spectroscopy. These associations should be taken into account when incorporating antioxidants, such as (+)-catechin, to create functional dairy products, such as cheese.

Due to their well-known health benefits, green tea catechins have received recent attention as natural additives in foods such as dairy products. However, they may present some irreversible associations with milk components (e.g. protein and milk fat globules). To investigate the behaviour of two important green tea catechins, (+)-catechin (C) and (−)-epigallocatechin gallate (EGCG), in a standard whole milk system under the conditions of cheesemaking, 250 and 500 ppm of each catechin were added to whole milk (3.3% fat). Although both C and EGCG at either concentration increased both total phenolic content and total antioxidant capacity of the subnatants obtained from the milk system, there was a less linear increase when the concentration of the catechins was doubled, whereas C or EGCG were recovered (measured by HPLC) differently. Overall, these results suggest a degree of associations between green tea catechins with milk proteins and milk fat.

Molecular integrations between green tea catechins and milk fat globules in a cheese matrix were investigated using solid-state magic angle spinning nuclear magnetic resonance spectroscopy. Full-fat cheeses were manufactured containing free catechin or free green tea extract (GTE), and liposomal encapsulated catechin or liposomal encapsulated GTE. Molecular mobility of the carbon species in the cheeses was measured by a wide-line separation technique. The (1)H evolution frequency profile of the (13)C peak at 16ppm obtained for the control cheese and cheeses containing encapsulated polyphenols (catechin or GTE) were similar, however, the spectrum was narrower for cheeses containing free polyphenols. Differences in spectral width indicates changes in the molecular mobility of --CH3- or -C-C-PO4- species through hydrophobic and/or cation-π associations between green tea catechins and cheese fat components. However, the similar spectral profile suggests that encapsulation protects cheese fat from interaction with catechins.

Thermodynamics of milk components (milk fat, xanthine oxidase, caseins and whey proteins) in pulsed electric field (PEF)-treated milk were compared with thermally treated milk (63 °C for 30 min and 73 °C for 15s). PEF treatments were applied at 20 or 26 kV cm(-1) for 34 μs with or without pre-heating of milk (55 °C for 24s), using bipolar square wave pulses in a continuous mode of operation. PEF treatments did not affect the final temperatures of fat melting (Tmelting) or xanthine oxidase denaturation (Tdenaturation), whereas thermal treatments increased both the Tmelting of milk fat and the Tdenaturation for xanthine oxidase by 2-3 °C. Xanthine oxidase denaturation was ∼13% less after PEF treatments compared with the thermal treatments. The enthalpy change (ΔH of denaturation) of whey proteins decreased in the treated-milk, and denaturation increased with the treatment intensity. New endothermic peaks in the calorimetric thermograms of treated milk revealed the formation of complexes due to interactions between MFGM (milk fat globule membrane) proteins and skim milk proteins. Evidence for the adsorption of complexes onto the MFGM surface was obtained from the increase in surface hydrophobicity of proteins, revealing the presence of unfolded hydrophobic regions.

The determination of putative chemical interactions between the milk fat globule membrane and green tea catechins provided useful information about the role of milk fat globules (MFGs) in high-fat dairy systems, such as cheese, and containing bioactive compounds, such as tea catechins. Catechins from green tea (125-1,000 ppm), including (+)-catechin, (-)-epigallocatechin gallate, and green tea extract were added to washed MFGs to examine possible interactions. The addition of catechins gave a significant change in the size and ζ-potential of MFGs. The recovery of different catechins from the milk fat globule suspensions was found to vary, suggesting selective association with the milk fat globule membranes. The interactions were further investigated using transmission electron microscopy and Fourier transform infra-red spectroscopy. It is suggested that catechins are localised in association with milk fat globule membrane domains as they contain both hydrophobic and hydrophilic moieties with potential points of molecular interaction.