Interactions of fat globule surface proteins during concentration of whole milk in a pilot-scale multiple-effect evaporator

The changes in milk fat globules and fat globule surface proteins during concentration of whole milk using a pilot-scale multiple-effect evaporator were examined. The effects of heat treatment of milk at 95 degrees C for 20 s, prior to evaporation, on fat globule size and the milk fat globule membrane (MFGM) proteins were also determined. In both non-preheated and preheated whole milk, the size of milk fat globules decreased while the amount of total surface proteins at the fat globules increased as the milk passed through each effect of the evaporator. In non-preheated samples, the amount of caseins at the surface of fat globules increased markedly during evaporation with a relatively small increase in whey proteins. In preheated samples, both caseins and whey proteins were observed at the surface of fat globules and the amounts of these proteins increased during subsequent steps of evaporation. The major original MFGM proteins, xanthine oxidase, butyrophilin, PAS 6 and PAS 7, did not change during evaporation, however, PAS 6 and PAS 7 decreased during preheating. These results indicate that the proteins from the skim milk were adsorbed onto the fat globule surface when the milk fat globules were disrupted during evaporation.     

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.     

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.     

Effect of fat content and homogenization under conventional or ultra-high-pressure conditions on interactions between proteins in rennet curds

The objective of this study was to investigate the influence of conventional and ultra-high-pressure homogenization on interactions between proteins within drained rennet curds. The effect of fat content of milk (0.0, 1.8, or 3.6%) and homogenization treatment on dissociation of proteins by different chemical agents was thus studied. Increasing the fat content of raw milk increased levels of unbound whey proteins and calcium-bonded caseins in curds; in contrast, hydrophobic interactions and hydrogen bonds were inhibited. Both homogenization treatments triggered the incorporation of unbound whey proteins in the curd, and of caseins through ionic bonds involving calcium salts. Conventional homogenization-pasteurization enhanced interactions between caseins through hydrogen bonds and hydrophobic interactions. In contrast, ultra-high-pressure homogenization impaired hydrogen bonding, led to the incorporation of both whey proteins and caseins through hydrophobic interactions and increased the amount of unbound caseins. Thus, both homogenization treatments provoked changes in the protein interactions within rennet curds; however, the nature of the changes depended on the homogenization conditions.     

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.     

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.     

Emulsifying properties of fractions prepared from commercial buttermilk by microfiltration

A novel method for the separation of milk-fat globule membrane (MFGM) isolate by microfiltration in the presence of citrate was applied to prepare a fraction to be used to stabilize oil-in-water emulsions. The emulsifying properties of this fraction, containing high amounts of MFGM, were compared with a buttermilk concentrate (BMC) prepared in a similar manner but still containing the original ratio of proteins (caseins, whey proteins, and MFGM). The objective of this work was to determine if the isolation procedure would result in an ingredient with different functionality when compared with BMC. These fractions were incorporated into oil-in-water emulsions at various isolate and oil concentrations. At low concentrations of isolate, MFGM emulsions showed better creaming stability and smaller oil droplet size distribution than whole buttermilk concentrate samples. The difference in stability was attributed to the compositional difference between the 2 ingredients prepared. A selective concentration of MFGM in buttermilk by microfiltration has the potential for the development of ingredients that differ substantially from the ingredients deriving from milk or whey.     

Protein profile of dairy products: Simultaneous quantification of twenty bovine milk proteins

While proteomic techniques allow the identification and relative quantification of thousands of proteins in a single run, methods for absolute quantification remain laborious. In this study, a newly developed multiple reaction monitoring (MRM) method using liquid chromatography mass spectrometry (LC-MS) that enables the simultaneous quantification of twenty key milk proteins is presented. The selected proteins comprise all individual caseins, the major whey proteins and most well-known milk fat globule membrane (MFGM) proteins. For validation, the twenty milk proteins in raw milk, raw cream, raw milk Emmental cheese and whey, were quantified as well as in eighteen commercial heat-treated dairy products. The method presented is ideally suited for various applications, for example, the comparison of the protein patterns in raw milk of cows at different stages of lactation or of different breeds.     

High-pressure-induced interactions between milk fat globule membrane proteins and skim milk proteins in whole milk

The association of beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) with milk fat globule membrane (MFGM), when whole milk was treated by high pressure in the range 100 to 800 MPa, was investigated using sodium dodecyl sulfate (SDS)-PAGE under reducing and nonreducing conditions. In SDS-PAGE under reducing conditions, beta-LG was observed in the MFGM material isolated from milk treated at 100 to 800 MPa for 30 min, and small amounts of alpha-LA and kappa-casein were also observed at pressures >600 MPa for 30 min. However, these proteins were not observed in SDS-PAGE under nonreducing conditions. These results indicate that beta-LG and alpha-LA associated with MFGM proteins via disulfide bonds during the high-pressure treatment of whole milk. The amount of beta-LG associated with the MFGM increased with an increase in pressure up to 800 MPa and with increasing time of pressure treatment. The maximum value for beta-LG association with the MFGM was approximately 0.75 mg/g of fat. Of the major original MFGM proteins, no change in butyrophilin was observed during the high-pressure treatment of whole milk, whereas xanthine oxidase was reduced to some extent beyond 400 MPa. In contrast to the behavior during heat treatment, PAS 6 and PAS 7 were stable during high-pressure treatment, and they remained associated with the MFGM.     

Comparison of emulsifying properties of milk fat globule membrane materials isolated from different dairy by-products

Emulsifying properties of milk fat globule membrane (MFGM) materials isolated from reconstituted buttermilk (BM; i.e., BM-MFGM) and BM whey (i.e., whey-MFGM), individually or in mixtures with BM powder (BMP) were compared with those of a commercial dairy ingredient (Lacprodan PL-20; Arla Foods Ingredients Group P/S, Viby, Denmark), a material rich in milk polar lipids and proteins. The particle size distribution, viscosity, interfacial protein, and polar lipids load of oil-in-water emulsions prepared using soybean oil were examined. Pronounced droplet aggregation was observed with emulsions stabilized with whey-MFGM or with a mixture of whey-MFGM and BMP. No aggregation was observed for emulsions stabilized with BM-MFGM, Lacprodan PL-20, or a mixture of BM-MFGM and BMP. The surface protein load and polar lipids load were lowest in emulsions with BM-MFGM. The highest protein load and polar lipids load were observed for emulsions made with a mixture of whey-MFGM and BMP. The differences in composition of MFGM materials, such as in whey proteins, caseins, MFGM-specific proteins, polar lipids, minerals, and especially their possible interactions determine their emulsifying properties.     

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.     

Microfiltration of buttermilk and washed cream buttermilk for concentration of milk fat globule membrane components

Buttermilk, the by-product from butter manufacture, has gained much attention lately because of the application potential of its milk fat globule membrane (MFGM) components as health ingredients. Microfiltration (MF) has been studied for buttermilk fractionation because of its ability to separate particles from dissolved solutes. However, the presence in this by-product of skim milk solids, especially casein micelles, restricts concentration of MFGM. The use of cream washed with skim milk ultrafiltrate to produce buttermilk with lower casein content was studied as well as fractionation of this buttermilk by MF. Results have shown that washing the cream prior to churning yields buttermilk with 74% less protein than normal cream buttermilk. Analysis of the protein profile of washed cream buttermilk revealed that caseins and whey proteins were the main classes of proteins removed. The MF of washed cream buttermilk resulted in permeation fluxes 2-fold higher than with normal cream buttermilk. The second separation of the cream induced high losses of phospholipids in the skim phase. However, retention of remaining phospholipids in washed cream buttermilk by the MF membrane was higher resulting in a phospholipids concentration factor 66% higher than that of normal cream buttermilk. The results presented in this study highlight the impact of casein micelles on the separation of MFGM components as well as their effect on permeation flux during MF.     

Changes in structure of the bovine milk fat globule membrane on heating whole milk.

The effects of heat-induced interactions between milk fat globule membrane components and skim milk proteins in whole milk on the structure of the membrane were examined by isopycnic sucrose density gradient centrifugation and by using Triton X-100 as a membrane probe. Skim milk components were incorporated into all the lipoprotein fractions separated by density gradient centrifugation. High density complexes, higher in density than those found in the natural milk fat globule membrane, were formed during the heat treatment. Losses of natural membrane polypeptides from the medium and low density lipoproteins were observed on heating. Heating whole milk also altered the rate of release of membrane components by detergent, with decreases in protein released and an increase in phospholipid constituents released. Studies on washed cream indicated that some of the changes in the membrane on heating whole milk occurred due to the heat treatment alone, independent of the interactions with skim milk proteins.     

Aggregation and Dissociation of Milk Protein Complexes in Heated Reconstituted Concentrated Skim Milks

Heating milk at 120°C at pH 6.55 or pH 6.85 caused the denaturation of whey proteins and increased their association with the casein micelles. The dissociation of K -, β-, and α_s-caseins (in that order by extent) from the casein micelles increased with severity of heat treatment. The effect was greater at higher pH. Gel filtration chromatography followed by gel electrophoresis of fractions showed the dissociated protein was composed of disulfide-linked k -casein/β-lactoglobulin complexes of varying composition, casein aggregates of varying sizes and some monomeric protein. When reconstituted concentrate was prepared from NFDM made from heated milk the non-sedimentable (88,000 ± g for 90 min) caseins or whey proteins/heating time profiles were altered and the rate of aggregation, as measured by turbidity of heated milks, was significantly reduced.     

Casein precipitation by acid and rennet coagulation of buttermilk: Impact of pH and temperature on the isolation of milk fat globule membrane proteins

Milk fat globule membrane (MFGM) proteins make up only 2–3% of the total protein content in buttermilk. Prior isolation of buttermilk MFGM material is therefore required for further investigation of its functional properties and application in food systems. Rennet induced coagulation has been applied to remove caseins, but 30–70% of MFGM proteins are entrapped in the casein curd under classical cheese making conditions. Therefore, factors influencing the rennet as well as acid induced coagulation of buttermilk with regard to a reduction of MFGM protein losses into the casein curd, i.e., pH, temperature and pre-heat treatment of buttermilk, were examined. Complete casein removal from buttermilk was reached using both coagulation methods, whereas retention of MFGM proteins in the serum phase was higher during renneting. Rennet induced coagulation at higher pH and lower temperatures resulted in weaker gel structures, which increased the yield of MFGM proteins in the buttermilk whey.     

Isolation of milk fat globule membrane (MFGM) material by coagulation and diafiltration of buttermilk

Due to the functional potential of milk fat globule membrane (MFGM), its isolation from buttermilk is receiving increasing attention by the food industry. However, extraction of MFGM proteins from buttermilk is challenging because of the high levels of serum proteins. In this study, a two-step approach was applied to obtain a MFGM isolate. First, native casein micelles from buttermilk were removed by rennet-induced coagulation. Next, the buttermilk whey obtained was filtered to remove the residual whey proteins. Purified MFGM isolate was collected after six diafiltration steps. The yield of MFGM proteins in the isolates was determined using sodium dodecylsulphate-polyacrylamide gel electrophoresis. In the MFGM isolates, 70% of peripheral membrane proteins were obtained, which is much more effective in comparison with recent isolation methods like cream washing or filtration of buttermilk. Further investigations on casein coagulation conditions of buttermilk may reduce losses, especially of integral MFGM proteins during renneting.     

Filtration of milk fat globule membrane fragments from acid buttermilk cheese whey

The proteins and polar lipids present in milk fat globule membrane (MFGM) fragments are gaining attention for their technological and nutritional properties. These MFGM fragments are preferentially enriched in side streams of the dairy industry, like butter serum, buttermilk, and whey. The objective of this study was to recover MFGM fragments from whey by tangential filtration techniques. Acid buttermilk cheese whey was chosen as a source for purification by tangential membrane filtration because it is relatively rich in MFGM-fragments and because casein micelles are absent. Polyethersulfone and cellulose acetate membranes of different pore sizes were evaluated on polar lipid and MFGM-protein retention upon filtration at 40°C. All fractions were analyzed for dry matter, ash, lipids, proteins, reducing sugars, polar lipid content by HPLC, and for the presence of MFGM proteins by sodium dodecyl sulfate-PAGE. A fouling coefficient was calculated. It was found that a thermocalcic aggregation whey pretreatment was very effective in the clarification of the whey, but resulted in low permeate fluxes and high retention of ash and whey proteins. By means of an experimental design, the influence of pH and temperature on the fouling and the retention of polar lipids (and thus MFGM fragments), proteins, and total lipids upon microfiltration with 0.15 μM cellulose acetate membrane was investigated. All models were highly significant, and no outliers were observed. By increasing the pH from 4.6 to 7.5, polar lipid retention at 50°C increased from 64 to 98%, whereas fouling of the filtration membrane was minimized. A 3-step diafiltration of acid whey under these conditions resulted in a polar lipid concentration of 6.79 g/100 g of dry matter. As such, this study shows that tangential filtration techniques are suited for the purification of MFGM fragments.     

Effect of heat and transglutaminase on solubility of goat milk protein‐based films

The effect of heat, transglutaminase and combination of heat and transglutaminase treatments on the solubility of films prepared from goat milk casein, goat milk whey proteins and whole goat milk proteins was investigated. Goat milk casein films were less soluble when treated with transglutaminase and combination of heat with transglutaminase compare with heat‐treated caseins alone. Heat treatment was more effective at decreasing the solubility of whey protein films. SDS‐PAGE patterns demonstrated that goat milk caseins were better cross‐linked by transglutaminase, whereas whey proteins were better cross‐linked by heat. The extent of cross‐linking was further enhanced when a combination of heat and transglutaminase was used.     

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.