Native Milk Fat Globules Research Articles

Protein fortification of model cheese matrices using whey protein-enriched double emulsions

Whey proteins represent 20% of the protein content of milk and are an underutilised by-product of cheese manufacturing. This study was aimed at encapsulating whey proteins in the fat content of cheese using double emulsions. A two-stage power ultrasound (20 kHz) emulsification process was used to produce double emulsions with an internal aqueous phase enriched with high concentrations of whey proteins contained within droplets of sunflower oil. Primary water-in-oil (W1/O) nanoemulsions were successfully formed at an applied ultrasonic power of 1.35 W/mL, using 20% w/w and 30% w/w whey protein concentrate (WPC) solutions in the internal phase. The inner water droplets were stabilised by a combination of food grade lipophilic emulsifiers included in the sunflower oil at minimum concentrations of 1% w/w lecithin and 3% w/w PGPR. The secondary oil emulsions were formed by emulsifying the primary W1/O emulsions in 5% w/w WPC solutions, with the whey proteins serving as the emulsifying agent. The encapsulation loading rate of whey proteins within the double emulsion droplets was investigated in relation to ultrasound parameters and formulation loading rates. A very high encapsulation loading rate of ∼45 gwhey protein/Ldouble emulsion was achieved using 0.81 W/mL of ultrasound, with oil droplets of comparable diameter to native milk fat globules (∼10 μm). These double emulsions were successfully incorporated into renneted and cooked curd systems to enable the retention of whey protein in cheese matrices. This study demonstrates the potential of ultrasound emulsification to form whey protein-enriched double emulsions with minimum food-grade emulsifiers to fortify the protein content of cheese and other food products.

Effect of substituting whey cream for sweet cream on the textural and rheological properties of cream cheese

In the manufacture of cream cheese, sweet cream and milk are blended to prepare the cream cheese mix, although other ingredients such as condensed skim milk and skim milk powder may also be included. Whey cream (WC) is an underutilized fat source, which has smaller fat droplets and slightly different chemical composition than sweet cream. This study investigated the rheological and textural properties of cream cheeses manufactured by substituting sweet cream with various levels of WC. Three different cream cheese mixes were prepared: control mix (CC; 0% WC), cream cheese mixes containing 25% WC (25WC; i.e., 75% sweet cream), and cream cheese mixes with 75% WC (75WC; i.e., 25% sweet cream). The CC, 25WC, and 75WC mixes were then used to manufacture cream cheeses. We also studied the effect of WC on the initial step in cream cheese manufacture (i.e., the acid gelation process monitored using dynamic small amplitude rheology). Acid gels were also prepared with added denatured whey proteins or membrane proteins/phospholipids (PL) to evaluate how these components affected gel properties. The rheological, textural, and sensory properties of cream cheeses were also measured. The WC samples had significantly higher levels of PL and insoluble protein compared with sweet cream. An increase in the level of WC reduced the rate of acid gel development, similar to the effect of whey phospholipid concentrate added to mixes. In cream cheese, an increase in the level of added WC resulted in significantly lower storage modulus values at temperatures <20°C. Texture results, obtained from instrumental and sensory analyses, showed that high level of WC resulted in significantly lower firmness or hardness values and higher stickiness compared with cream cheeses made with 25WC or CC cream cheeses. The softer, less elastic gels or cheeses resulting from the use of high levels of WC are likely due to the presence of components such as PL and proteins from the native milk fat globule membrane. The use of low levels of WC in cream cheese did not alter the texture, whereas high levels of WC could be used if manufacturers want to produce more spreadable products.

Whipping properties of recombined, additive-free creams

There is increasing industrial interest in the use of the milkfat globule membrane as a food ingredient. The objective of this research was to determine whether the aerosol whipping performance of cream separated into butter and buttermilk, and then recombined, would perform in a manner similar to untreated cream. Churning of cream tempered to different solid fat contents was used to separate butter from buttermilk, which were then recombined at the same ratios as the initial extraction yield, or with 25% extra buttermilk. Differences in milkfat globule size distributions among the recombined creams were apparent; however, their whipping behavior and overrun were similar. Importantly, all recombined creams did not yield properties similar to the original cream, indicating that the unique native milkfat globule membrane structure plays a role in cream performance well beyond its simple presence.

Effects of pulsed electric field on fat globule structure, lipase activity, and fatty acid composition in raw milk and milk with different fat globule sizes

To modify the structure of milk fat globules (MFGs), raw milk (RM) with native MFGs, and milk with small (MS) and large (ML) MFGs, were subjected to moderate pulsed electric field (PEF) treatment at 9 and 16 kV·cm−1 for 30 μs. The changes caused by PEF on MFG structure leading to MFG-MFG and MFG-protein aggregation were found to depend on the size, composition and/or previous damage caused by the separation and mixing processes during production of MS and ML, in addition to the intensity of the PEF treatment. Although, PEF treatment had a more pronounced effect on MFGM proteins of MS, these MFG were less affected by PEF due to significant adsorption of milk proteins. The structural changes caused by PEF were accompanied by changes in lipase activity and fatty acid (FA) profiles, i.e. increase in the proportion of C6:0, C8:0, C10 and some long-chain FAs and a decrease in medium and other long chain FAs. Industrial relevancePEF is gaining attention in the dairy industry. However, information regarding the effect of PEF processing on milk components and functionality is still limited. The present investigation contributes to understand the potential of PEF at moderate field intensities to modify the MFGs and consequences for milk stability, lipolysis and composition of FAs. The new insight is relevant in development of new products and ingredients containing milk fat.

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 relevance The developed method has potential for size based fractionation of native fat globules in industrial scale.

Relationships between milks differentiated on native milk fat globule characteristics and fat, protein and calcium compositions

Many studies have shown that milk fat globule (MFG) diameter varies in dairy cows in relation to diet and/or breed. However, the mechanisms governing the size of the fat globules remain hypothetical. Our objective was to determine the variable biochemical characteristics (fat, protein, fatty acids (FA), casein and calcium (Ca) contents) between individual milk which differed in both MFG diameter and membrane content, in order to speculate about the links between milk synthesis and MFG secretion. With this aim, we built five databases of individual milk samples from 21 experiments performed between 2003 and 2011. Three of them grouped data from trials dealing with breed/diet effects and included information about: (i) MFG size/membrane, fat and protein contents (n=982), (ii) previous parameters plus FA profile (n=529) and (iii) previous parameters plus true protein composition and calcium contents (n=101). A hierarchical clustering analysis performed on these three databases yielded four groups differing in the MFG characteristics. We observed significant differences among groups for the following parameters: (i) fat content and fat : protein ratio; (ii) de novo and polyunsaturated FA contents; (iii) Ca contents. These relationships could result from potential process regulating the synthesis and secretion of MFG: (i) the apical membrane turnover for MFG secretion and (ii) cytoplasmic lipid droplet formation in the lactocyte during its migration from the basal to the apical pole. The two other databases grouped data from trials dealing with milking frequency (n=211), milking kinetics and milk type (residual v. cisternal) (n=224). They were used to study the relationships between the size of the MFG and milk composition for high native fat contents (from 60 up to 100 g/kg in residual milks). We observed curvilinear relationships between the size of the MFG and fat content, as well as with the fat : protein ratio. This result suggests that MFG diameter reaches a threshold but mechanisms are still unknown.

Native milk fat globule size and its influence on whipping properties

The objective of this study was to investigate the influence of native milk fat globule size on the aeration of high fat dairy products with regard to maximum firmness time, gas inclusion and foam stability. The results showed that whipping time to maximum firmness was inversely proportional to mean fat globule size for both unhomogenised and slightly homogenised (2 MPa) creams. Additionally, increasing native mean fat globule size of the creams resulted in increased overrun. No significant differences in serum drainage were found between creams with different native milk fat globule size. Furthermore, when creams with native large mean fat globules were homogenised, the results showed that the maximum firmness time was in accordance with the mean fat globule size of non-aggregated creams. In the present study, cream fractionation was achieved by creaming or in a cost effective and fast manner using a modified centrifugal separator.

Quantification of MFGM proteins in buttermilk and butter serum by means of a stain free SDS-PAGE method

In comparison to skim milk and whey, buttermilk and butter serum contain high amounts of functional milk fat globule membrane (MFGM) proteins. Quantification of MFGM proteins is difficult because no analytical standard material is available. By means of conventional SDS-PAGE with subsequent staining, MFGM proteins are analytically separated. However, due to various protein structures (e.g., glycosylation) the affinity to the dye is inconsistent. A direct correlation between measured band intensity and protein concentration and, therefore, absolute protein quantification is not possible. The purpose of this study was to apply a novel stain free SDS-PAGE technique that relies on measuring UV light fluorescence of tryptophan in order to quantify MFGM proteins. Thus, proteins can be quantified without the need for analytical standards, provided that the relative tryptophan content relative to the molecular weight is known. In this study, buttermilk and butter serum obtained from raw cream were examined and the three most abundant membrane proteins were absolutely quantified. Higher amounts of peripheral MFGM proteins were determined in buttermilk (0.9g/L) and butter serum (2.7g/L), in comparison to integral MFGM proteins (0.3–0.5g/L). Conclusions were drawn regarding the absolute protein amount of MFGM material surrounding native milk fat globules.

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.

The structure of infant formulas impacts their lipolysis, proteolysis and disintegration during in vitro gastric digestion

Milk lipids supply most of the calories necessary for newborn growth in maternal milk or infant formulas. The chemical composition of infant formulas has been optimized but not the structure of the emulsion. There is still a major difference between the native emulsions of milk fat globules and processed submicronic emulsions in infant formulas. This difference may modify the kinetics of digestion of emulsions in newborns and influence lipid metabolism. To check this, semi-dynamic gastric in vitro digestions were conducted on three matrices: a standardized milk emulsion containing native milk fat globules referred to as minimally-processed emulsion and two processed model infant formulas (homogenized or homogenized/pasteurized). Gastric conditions mimicked those reported in newborns. The minimally-processed emulsion was lipolyzed and proteolyzed slower than processed formulas. The difference in initial structure persisted during digestion. The surface of the droplets was the key parameter to control gastric lipolysis kinetics, the pattern of released fatty acids and proteolysis by faster hydrolysis of adsorbed proteins.

Natural variation of bovine milk fat globule size within a herd

Native milk fat globule (MFG) size, pH, total fat, protein, and lactose composition in milk from individual cows was examined in autumn and spring 2012 and autumn 2013. Mean MFG diameters ranged between 2.5 and 5.7µm. Some cows were observed to consistently produce small or large MFG throughout the sampling period, though trends were not consistent across all cows. Small-MFG milk contained more total polar lipids; however, the relative proportion of individual polar lipids did not differ with MFG size, with the exception of phosphatidylcholine, which was greater in small-MFG milk. No significant correlation between MFG size and proximate composition, including calculated fat yield or pH, was found. This work revealed a natural cow-to-cow variation in MFG size exists, which could potentially be exploited to improve the functionality of milk for manufacturing and some products.

Phospholipid Architecture of the Bovine Milk Fat Globule Membrane Using Giant Unilamellar Vesicles as a Model

Giant unilamellar vesicles (GUVs) were constructed using an electroformation technique to mimic the morphology of the native milk fat globule membrane (MFGM) for the purpose of structural investigation. Bovine milk derived phospholipids were selected to manufacture GUVs which were characterized by confocal laser scanning microscopy after fluorescent staining. Circular nonfluorescent dark regions were observed in a 3/7 (mol/mol) surface mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine. Linear shaped dark lipid domains were found in GUVs containing sphingomyelin (SM) in the absence of cholesterol. The dark regions were interpreted as a gel phase formed by a high gel-liquid phase transition temperature (Tm) of DPPC and SM. This study provides a strategy for investigating the lipid structural organization within the native MFGM using a model lipid bilayer system and reveals that a SM and cholesterol association network is not the only requirement for nonfluorescent lipid domain formation and that PE is preferably located in the inner leaflet of the phospholipid bilayer.

Cryo-SEM images of native milk fat globule indicate small casein micelles are constituents of the membrane

Schematic representation of the native milk fat globule membrane: casein micelles and whey proteins on the surface of the membrane.

Milk fat globule membrane isolate induces apoptosis in HT-29 human colon cancer cells

A native milk fat globule membrane (MFGM) isolate obtained from raw milk was assessed for its anticarcinogenic capacity using a colon cancer cell line (HT-29). To prevent microbial contamination and eliminate the presence of lipopolysaccharide (LPS) in the milk used for MFGM isolation, the milk was obtained from the mammary glands of cows using a catheter. Cell proliferation assays demonstrated a reduction of exponentially growing cancer cells of up to 53%, expressed as DNA synthesis (BrdU test), after 72 h stimulation with 100 μg of MFGM protein per mL. Using a similar MFGM concentration, the sulforhodamine B assay resulted in 57% reduction of cell density after 48 h incubation. This bioactivity was comparable to that of known anticancer drugs, 0.1 mM melphalan and 20 μM C2-ceramide, which achieved a cell division reduction of 25 and 40%, respectively, under the same experimental conditions. The toxic effect of the MFGM extracts on HT-29 cells was confirmed by the significant reduction in lactate dehydrogenase enzyme (LDH) by the residual viable cells. An increase of caspase-3 activity (up to 26%) led to the conclusion that MFGM has an apoptotic effect on HT-29 cancer cells.

Hydrolysis of native milk fat globules by microbial lipases: Mechanisms and modulation of interfacial quality

The hydrolysis of native milk fat globules by three microbial lipase preparations, two sn-1,3 specific and one non-specific, exhibiting variable level of an annex phospholipase activity was investigated. The reaction kinetics, the evolution of supramolecular structure and the interfacial properties of hydrolyzed fat globules were monitored. The hydrolytic efficiency of the three enzymes preparations was lower for the natural emulsion than for a model emulsion based on homogenized milk fat. Therefore, it indicated a limited accessibility of the enzymes to the natural emulsion. The hydrolysis was preceded by a lag time, the duration of which was negatively correlated to the annex phospholipase activity. Pre-hydrolysis of membrane components with protease or phospholipase modulated the observed lag times. The sn-1,3 specific lipases triggered an accumulation of sn-2 monoglycerides with a limited destabilization of the natural emulsion structure while the non specific lipase, which generated fatty acids as major hydrolytic products, induced a significant destabilization of the emulsion. Our results suggest that the specificity of lipase contributes to diversify the structure and interfacial reactivity of natural milk fat-based emulsions, and consequently their functional and nutritional values.

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.

Surface Characterization of Bovine Milk Phospholipid Monolayers by Langmuir Isotherms and Microscopic Techniques

Monolayers were prepared from phospholipids extracted from bovine milk and used as a model system to mimic the native milk fat globule membrane (MFGM) surface structure in various microscopic experiments. The natural complex mixtures of phospholipids were isolated from bovine raw milk, raw cream, processed whole milk, and buttermilk powder by total lipid extraction and solid-phase extraction. A Langmuir film balance mounted on an epifluorescence microscope was used to analyze the physical behavior of the monolayer films and the phase coexistence resulting from the formation of phospholipid microdomains within these films. Atomic force microscopy was used for nanometer-scale topographic resolution of the microdomains. This study allowed comparison of the behavior of phospholipid monolayers from dairy products at different stages of processing, analysis of the formation of microdomains, and the study of the effect of milk processing on lipid-lipid interactions and phase coexistence. It was observed that milk processing changes the physical behavior of phospholipid monolayers by altering the phospholipid profile and the fatty acid distribution.

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.

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

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.

Functionality of smaller vs control native milk fat globules in Emmental cheeses manufactured with adapted technologies

Abstract Emmental cheeses were produced with control native milk fat globules (CFG) or smaller ones (SFG) selected from the same milk by microfiltration. Either the same regular technology was used for both cheeses (so-called SFGreg and CFGreg), or two different adapted technologies to obtain cheeses with the same moisture (so-called SFGadapt and CFGadapt). SFGreg cheeses were more humid and presented lower firmness and longness than CFGreg cheeses. Yellow index was greater for SFGadapt than CFGadapt cheeses while melting coefficient and extrusion force were similar. SFGadapt cheeses exhibited improved sensory characteristics. Stretching and elasticity increase were always greater for SFG than for the corresponding CFG cheeses; eyes were always smaller in SFG cheeses. Confocal micrographs revealed larger inclusions of non-globular fat in CFG cheeses; there were more fat globules and aggregates in SFG cheeses. The ultrastructure of milk fat played a role in the functional and sensory properties of Emmental cheese and the use of smaller native globules may be advantageous.