Effect of homogenization and heat treatment on the behavior of protein and fat globules during gastric digestion of milk

The effects of homogenization and heat treatment on the formation and the breakdown of clots during gastric digestion of whole milk were investigated using a human gastric simulator. Homogenization and heat treatment led to formation of coagula with fragmented and crumbled structures compared with the coagulum formed from raw whole milk, but a larger fraction of the protein and more fat globules were incorporated into the coagula induced by action of the milk-clotting enzyme pepsin. The fat globules in the whole milk appeared to be embedded in the clots as they formed. After formation of the clot, the greater numbers of pores in the structures of the clots formed with homogenized milk and heated whole milk led to greater rates of protein hydrolysis by pepsin, which resulted in faster release of fat globules from the clots into the digesta. Coalescence of fat globules occurred both in the digesta and within the protein clots no matter whether they were in homogenized or heated milk samples. The formation of clots with different structures and hence the changes in the rates of protein hydrolysis and the release of milk fat into the digesta in the stomach provide important information for understanding the gastric emptying of milk and the potential to use this knowledge to manipulate the bioavailability of fat and other fat-soluble nutrients in dairy products.     

Changes in the surface protein of the fat globules during homogenization and heat treatment of concentrated milk

The changes in milk fat globules and fat globule surface proteins of both low-preheated and high-preheated concentrated milks, which were homogenized at low or high pressure, were examined. The average fat globule size decreased with increasing homogenization pressure. The total surface protein (mg m-2) of concentrated milk increased after homogenization, the extent of the increase being dependent on the temperature and the pressure of homogenization, as well as on the preheat treatment. The concentrates obtained from high-preheated milks had higher surface protein concentration than the concentrates obtained from low-preheated milks after homogenization. Concentrated milks heat treated at 79 degrees C either before or after homogenization had greater amounts of fat globule surface protein than concentrated milks heat treated at 50 or 65 degrees C. This was attributed to the association of whey protein with the native MFGM (milk fat globule membrane) proteins and the adsorbed skim milk proteins. Also, at the same homogenization temperature and pressure, the amount of whey protein on the fat globule surface of the concentrated milk that was heated after homogenization was greater than that of the concentrated milk that was heated before homogenization. The amounts of the major native MFGM proteins did not change during homogenization, indicating that the skim milk proteins did not displace the native MFGM proteins but adsorbed on to the newly formed surface.     

Symposium review: Fat globules in milk and their structural modifications during gastrointestinal digestion

The fat globules in milk are unique oil droplets that are stabilized by a specific and structurally complex membrane, the milk fat globule membrane (MFGM). In the last decade, excellent progress has been made on studying the structure of the milk fat globules and the MFGM and how common processing treatments affect these structures to deliver dairy products with improved functional properties. Although the digestion of milk fat to deliver energy and lipid-soluble nutrients is essential for survival of the neonate, there is little understanding of the complex processes involved. The structural alterations to fat globules during gastrointestinal processing affect the way in which milk fat is digested, absorbed, and metabolized. The packaging of these globules within the MFGM or in other forms may affect the bioaccessibility of raw or processed milk fat globules; in turn, this may affect access of the gastrointestinal enzymes to the globules and, therefore, may influence the rate and extent of lipid digestion. This review focuses on recent advances in understanding milk fat globules during gastrointestinal digestion, including the effects of processing on their bioavailability and the kinetics of lipid digestion. Possible effects of the dairy matrix on lipid digestion and physiological responses are briefly described.     

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.     

Buffalo milk fat globules and their biological membrane: in situ structural investigations

Milk fat globules and their surrounding biological membrane (the MFGM) are not well understood despite the importance of these milk components in human nutrition and the role of fat globules in determining the properties of dairy products. The objectives of this study were to investigate these unique colloidal assemblies and the microstructure of the MFGM in buffalo milk, which is the second largest global source of dairy products. In-situ structural investigations were performed at room temperature using confocal microscopy with multiple fluorescent probes (Nile Red, Rh-DOPE, the lectin WGA-488). Microscopic observations showed cytoplasmic crescents around fat globules and the heterogeneous distribution of glycosylated molecules and polar lipids with the occurrence of lipid domains. The lipid domains in the buffalo MFGM appear to form by the segregation of lipids with a high phase transition temperature (e.g. sphingomyelin and saturated phosphatidylcholine molecular species) and cholesterol resulting in a gel phase or a Lo phase forming circular domains. The structure of the buffalo MFGM results from a non-random mixing of components, consistent with observations for other species. Structural heterogeneities of the MFGM could affect the processability of buffalo fat globules and the bioavailability of milk lipids.     

Microstructure of fat globules in whole milk after thermosonication treatment

The structure of fat globules in whole milk was studied after heat and thermosonication treatments to observe what happens during these processes at the microscopic level using scanning electron microscopy. Raw whole milk was thermosonicated in an ultrasonic processor-Hielscher UP400S (400 W, 24 kHz, 120 microm amplitude), using a 22-mm probe at 63 degrees C for 30 min. Heat treatment involved heating the milk at 63 degrees C for 30 min. Color and fat content were measured to correlate the images with analytical measurements. The results showed that the surface of the fat globule was completely roughened after thermosonication. Ultrasound waves were responsible for disintegrating the milk fat globule membrane (MFGM) by releasing the triacylglycerols. Furthermore, the overall structure of milk after sonication showed smaller fat globules (smaller than 1 microm) and a granular surface. This was due to the interaction between the disrupted MFGM and some casein micelles. Minor changes in the aspect of the globules between thermal and raw milks were detected. Color measurements showed higher L* values for sonicated samples. Sonicated milk was whiter (92.37 +/- 0.20) and generally showed a better degree of luminosity and homogenization compared to thermal treated milk (88.25 +/- 0.67) and raw milk (87.82 +/- 0.18). Fat content analysis yielded a higher value after sonication (4.24%) compared to untreated raw milk (4.04%) because fat extraction is more efficient after sonication. The advantages of thermosonicated milk are that it can be pasteurized and homogenized in just 1 step, it can be produced with important cost savings, and it has better characteristics, making thermosonication a potential processing method for milk and most other dairy products.     

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-based fractionation of native milk fat globules by two-stage centrifugal separation

A two-stage centrifugal separation method, at various separation temperatures and feed rates, was employed to fractionate milk and cream on the basis of fat globule size. It involved a modified and a conventional centrifugal separation in first and second stages, respectively. In the first stage, two streams of milk: one rich in larger fat globules and another rich in smaller fat globules, were obtained by fractionation in a modified cream separator. In the second stage, the two streams from the first stage were each further fractionated in a conventional cream separator. Depending on the temperature and feed rate of the first stage, this double separation method was able to create streams with mean fat globule size (D [4, 3]) as small as 1.35 μm and as large as 4.28 μm without affecting the droplet integrity. The developed method has potential for size-based fractionation of native fat globules in industrial scale.Industrial relevanceThe developed method has potential for size based fractionation of native fat globules in industrial scale.     

Proteolysis of milk fat globule membrane proteins during in vitro gastric digestion of milk

The influence of gastric proteolysis on the physicochemical characteristics of milk fat globules and the proteins of the milk fat globule membrane (MFGM) in raw milk and cream was examined in vitro in simulated gastric fluid (SGF) containing various pepsin concentrations at pH 1.6 for up to 2 h. Apparent flocculation of the milk fat globules occurred in raw milk samples incubated in SGF containing pepsin, but no coalescence was observed in either raw milk samples or cream samples. The changes in the particle size of the fat globules as a result of the flocculation were dependent on the pepsin concentration. Correspondingly, the physical characteristics of the fat globules and the composition of the MFGM proteins in raw milk changed during incubation in SGF containing pepsin. The major MFGM proteins were hydrolyzed at different rates by the pepsin in the SGF; butyrophilin was more resistant than xanthine oxidase, PAS 6, or PAS 7. Peptides with various molecular weights, which altered with the time of incubation and the pepsin concentration, were present at the surfaces of the fat globules.     

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.     

Fat globules selected from whole milk according to their size: Different compositions and structure of the biomembrane,revealing sphingomyelin-rich domains

Milk fat globules are unique delivery systems for biologically active molecules in the gastrointestinal tract. However, their properties have not yet been fully investigated. In this study, we performed a comparative analysis of the polar lipid and fatty acid compositions of milk fat globules as a function of their size and investigated the structure of the milk fat globule membrane (MFGM). An optimised process of microfiltration was used to select the small milk fat globule (SMFG; 1.6 μm) fractions and the large milk fat globule (LMFG; 6.6 μm) fractions from the same initial whole milks (4.2 μm). The SMFG-fractions contained significantly (i) higher amounts of polar lipids, 8.9 ± 0.9 vs 2.7 ± 0.3 mg/g fat for LMFG-fractions and 6.3 ± 0.5 mg/g fat for whole milks, (ii) lower relative proportions of phosphatidylcholine and sphingomyelin in the MFGM, (iii) higher amounts of C12:0, C14:0, C16:0, C18:1 trans, C18:2 c9 tr11, and lower amounts of C18:0 and C18:1 c9 than did LMFG-fractions and whole milks. Whatever the size of native milk fat globules, the biophysical characterisation performed in-situ, using confocal laser scanning microscopy, showed heterogeneities in the MFGM. The lateral segregation of sphingomyelin in rigid liquid-ordered domains, surrounded by the fluid matrix of glycerophospholipids in the liquid-disordered phase, was revealed. The heterogeneous distribution of glycolipids and glycoproteins was also observed in the MFGM. A new model for the structure of the MFGM is proposed and discussed. The physical, chemical and biological consequences, (i) of the differences in milk fat globule compositions according to their size and (ii) of the specific structure of the MFGM due to sphingomyelin remain to be elucidated.     

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.     

Disruption of fat globules during concentration of whole milk in a pilot scale multiple-effect evaporator

The effects of heat treatment by direct steam injection (DSI) and concentration of whole milk using a pilot scale multiple-effect evaporator on fat globule size and the total milk fat globule membrane (MFGM) proteins were examined. In both nonpreheated and preheated whole milk, the size of milk fat globules decreased while the surface protein concentration of the fat globules increased as the milk passed through each effect of the evaporator. These results indicate that the fat globules were disrupted during DSI heating and evaporation and the proteins from the skim milk were adsorbed onto the fat globule surface.     

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.     

Changes in the surface protein of the fat globules during ultra-high pressure homogenisation and conventional treatments of milk

Disruption of fat globules upon homogenisation provokes a reduction of their size and a concomitant increase in their specific surface area. In order to overcome this phenomenon, the milk fat globule membrane (MFGM) adsorbs non-native MFGM proteins. The aim of the present study was to examine the effects of UHPH conditions (temperature and pressure) on the milk fat globule and the surface proteins by comparison with conventional treatments applied in the dairy industry. Transmission electron microscopy and SDS-PAGE revealed major differences. In UHPH-treated milk, casein micelles were found to be adsorbed on the MFGM in a lesser extent than in conventional homogenisation–pasteurisation. However, greater adsorption of directly bonded casein molecules, released by UHPH through the partial disruption of casein micelles, was observed especially at high UHPH pressures. Adsorption of whey proteins on the MFGM of conventionally homogenised–pasteurised milk was mainly through intermolecular disulfide bonds with MFGM material, whereas in UHPH-treated milk, disulfide bonding with both indirectly and directly adsorbed caseins was also involved.     

The Effect of Milk Processing on the Microstructure of the Milk Fat Globule and Rennet Induced Gel Observed Using Confocal Laser Scanning Microscopy

ABSTRACT: Confocal laser scanning microscopy (CLSM) was successfully used to observe the effect of milk processing on the size and the morphology of the milk fat globule in raw milk, raw ultrafiltered milk, and standardized and pasteurized milk prepared for cheese manufacture (cheese-milk) and commercial pasteurized and homogenized milk. Fat globule size distributions for the milk preparations were analyzed using both image analysis and light scattering and both measurements produced similar data trends. Changes to the native milk fat globule membrane (MFGM) were tracked using a MFGM specific fluorescent stain that allowed MFGM proteins and adsorbed proteins to be differentiated on the fat globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the identity of native MFGM proteins isolated from the surface of fat globules within raw, UF retentate, and cheese-milk preparations, whereas only casein was detected on the surface of fat globules in homogenized milk. The microstructure, porosity, and gel strength of the rennet induced gel made from raw milk and cheese-milk was also found to be comparable and significantly different to that made from homogenized milk. Our results highlight the potential use of CLSM as a tool to observe the structural details of the fat globule and associated membrane close to its native environment.     

Development of the milk fat microstructure during the manufacture and ripening of Emmental cheese observed by confocal laser scanning microscopy

Changes in the physico-chemical properties and microstructure of milk fat globules were investigated during the manufacture and ripening of Emmental cheese. The measurement of fat globule size and apparent zeta-potential showed that they were slightly affected during cheese milk preparation, i.e. storage of cheese milk overnight at 4 °C and pasteurisation. After rennet-induced coagulation and heating of curd grains, coalescence caused the formation of large fat globules (i.e.>10 μm). The structure of fat in Emmental cheese was characterised in situ using confocal laser scanning microscopy (CLSM). The rennet-induced coagulation lead to the formation of a continuous network of casein strands in which fat globules of various sizes were entrapped. Heating of curd grains induced the formation of fat globule aggregates. Pressing of the curd grains resulted in the greatest disruption of milk fat globules, their coalescence, the formation of non-globular fat (free fat) and the release of the milk fat globule membrane (MFGM) material. This study showed that milk fat exists in three main forms in ripened Emmental cheese: (i) small fat globules enveloped by the MFGM; (ii) aggregates of partially disrupted fat globules and (iii) free fat, resulting from the disruption of the MFGM and allowing free triacylglycerols to fill voids in the protein matrix. The curd grain junctions formed in Emmental cheese were also characterised using CLSM: they are compact structures, rich in protein and devoid of fat globules.     

Ultrasonication retains more milk fat globule membrane proteins compared to equivalent shear-homogenization

Ultrasonication, like common shear homogenization, can reduce the milk fat globule size and may change the milk fat globule membrane (MFGM). This work compared the effect of ultrasonication to equivalent shear homogenization on MFGM proteins and lipid-derived volatile components. Results showed that treating milk with ultrasound at 35 kJ/L would realize a similar size distribution of the milk fat globules as shear-homogenization at 20 MPa. Proteomics analysis revealed that in total 192 MFGM proteins were identified and quantified and a number of these proteins were lost after both treatments; however, more MFGM proteins remained after ultrasonication than after shear-homogenization. SDS-PAGE results showed that milk plasma proteins, and especially caseins, were absorbed on the milk fat globules after both treatments. In addition, the amount of the volatile free fatty acids increased after both treatments.Industrial relevance: Ultrasonication, as an innovative food processing technology, in comparison to traditional homogenization, was shown to equally efficiently decrease the MFG size, but lead to less damage to native MFGM proteins, which may be due to its longer homogenization time window. These results increased knowledge on the biochemical changes of milk fat globules after their size reduction and showed that ultrasonication could be used as a novel approach to improve dairy product quality.     

Effect of washing conditions on the recovery of milk fat globule membrane proteins during the isolation of milk fat globule membrane from milk

During the isolation of milk fat globule membrane (MFGM) from milk, washing is considered the most critical stage in which loss of MFGM components occurs. In this study, using a cream separator, the influence of washing on the recovery of MFGM proteins was investigated. The residue of non-MFGM proteins in the MFGM material obtained after washing was quantitatively determined using densitometric analysis of one-dimensional sodium dodecyl sulfate-PAGE after silver staining of the gel. Using deionized water as the washing solution did not increase the loss of MFGM proteins compared with other common salt solutions in terms of recovery of MFGM proteins and contamination with non-MFGM proteins. The increase in wash temperature from 38 to 46°C did not show a significant decrease in yield of MFGM proteins because of variation between the experimental replicates. Coalescence of fat globules occurs during isolation. To increase MFGM purity while maintaining a high MFGM protein recovery, using larger volumes of wash solution is more advisable rather than increasing the number of washings from 2 to 3.     

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.