Milk Fat Globule Membrane Isolates Research Articles

Buttermilk treatment strategies to obtain MFGM-enriched isolates and comprehensive characterization of their lipid and protein components

The extraction of Milk Fat Globule Membrane (MFGM) from dairy by-products, particularly buttermilk, involves a highly intricate procedures to obtain a bioactive isolate with multiple health benefits. This study delves into a laboratory ultrafiltration-based method enabling the acquisition of a concentrated MFGM isolate. Pre-ultrafiltration treatments, designed to clarify the sample, such as casein coagulation and the addition of sodium citrate, were examined to evaluate their impact on the lipid and protein components of the MFGM. Treatments combining rennet coagulation with centrifugation steps, as well as those combining rennet with sodium citrate, yielded highly pure MFGM isolates exhibiting a substantial concentration of polar lipids and a rich content of MFGM proteins. This laboratory investigation offers valuable insights into the potential industrial-scale applications of MFGM isolation and purification.

The affinity of milk fat globule membrane fragments and buttermilk proteins to hydroxyapatite

Buttermilk differs from skim milk by the presence of milk fat globule membrane (MFGM) fragments that are released during cream churning. MFGM is rich in health-promoting components, such as phospholipids and membrane proteins, but these compounds have a negative impact on buttermilk techno-functional properties in dairy applications. The isolation of MFGM from buttermilk improved its functionality while also recovering the MFGM bioactive components. Hydroxyapatite (HA) can be used to extract MFGM by adsorption via charged site interactions. However, the affinity of HA to MFGM or the main buttermilk proteins (casein micelles (CM), β-lactoglobulin (β-lg) and α-lactalbumin (α-lac)) is not known. The influence of important physicochemical parameters such as pH and temperature on these interactions is also unclear. For each buttermilk component, a quartz crystal microbalance diffusion analysis was performed to determine the maximum adsorption time and the attached mass density on HA-coated gold sensors. The influence of pH, ionic strength (IS), and temperature (T) on the affinity of each buttermilk component for HA particles was assessed using a 3-levels and 3-factors Box-Behnken design. The absorption rate was highest for the CM, followed by β-lg and α-lac, and then by the MFGM. Nevertheless, the final maximal attached mass densities to the HA were similar for the MFGM and CM, and 2.5 times higher than for β-lg and α-lac. This difference can be explained by the higher number of binding sites found in CM and their heavier mass. The model obtained by the Box-Behnken design plan showed that the adsorption of the CM changed with T, pH and IS. These results suggest that the techno-functional properties of buttermilk may be restored by specifically extracting MFGM with HA. Experiments are ongoing to determine conditions for fractionating MFGM directly from buttermilk.

On the use on hydroxyapatite suspensions for the separation of milk fat globule membrane components from buttermilk

The milk fat globule membrane (MFGM), which surrounds and stabilizes the fat globules, is released in buttermilk during cream churning. MFGM has many health benefits due to its composition rich in phospholipids and membrane proteins. Many techniques have been tried to separate the MFGM from the remaining milk solids non-fat, but they are challenging to carry out at an industrial scale. This research proposes a new approach to separating MFGM from buttermilk. This paper assessed the efficacy of hydroxyapatite (HA) cristal in interacting with MFGM isolates obtained from either raw or pasteurized cream. Different HA to MFGM ratios were used (10:1 and 20:1) to determine the impact of HA concentration on the adsorption. The results showed a very high affinity of the MFGM for HA and suggested the potential for its separation from buttermilk to improve its valorization.

In Vitro and In Vivo Anti-inflammatory Activity of Bovine Milkfat Globule (MFGM)-derived Complex Lipid Fractions.

Numerous health related properties have been reported for bovine milk fat globule membrane (MFGM) and its components. Here we present novel data on the in vitro and in vivo anti-inflammatory activity of various MFGM preparations which confirm and extend the concept of MFGM as a dietary anti-inflammatory agent. Cell-based assays were used to test the ability of MFGM preparations to modulate levels of the inflammatory mediators IL-1β, nitric oxide, superoxide anion, cyclo-oxygenase-2, and neutrophil elastase. In rat models of arthritis, using MFGM fractions as dietary interventions, the phospholipid-enriched MFGM isolates were effective in reducing adjuvant-induced paw swelling while there was a tendency for the ganglioside-enriched isolate to reduce carrageenan-induced rat paw oedema. These results indicate that the anti-inflammatory activity of MFGM, rather than residing in a single component, is contributed to by an array of components acting in concert against various inflammatory targets. This confirms the potential of MFGM as a nutritional intervention for the mitigation of chronic and acute inflammatory conditions.

Microfiltration of raw milk for production of high-purity milk fat globule membrane material

Commercial ingredients containing milk fat globule membrane (MFGM) material are currently isolated from heavily processed dairy streams. The aim of this study was to achieve a more gentle isolation of MFGM material by means of ceramic dia-microfiltration of raw whole milk to separate fat globules from casein micelles and whey proteins prior to MFGM extraction. A pilot-scale experiment with 1.4 μm pore size (membrane surface area 1.05 m2) resulted in an optimal outcome of low permeation of fat (2.5% permeation) and high permeation of proteins (97% permeation). This yielded an MFGM isolate with 7% w/w polar lipids and 30% w/w proteins, where contamination of non-MFGM proteins was only 25% of total protein content. Furthermore, mild pasteurization (72 °C, 15 s) introduced either before or after microfiltration had no impact on filtration efficiency or MFGM yield and composition. The work describes an industrially relevant production method for a less-processed MFGM material of high purity with potential for further separation and valorisation of protein-rich permeate streams.

Comprehensive characterization of neutral and polar lipids of buttermilk from different sources and its milk fat globule membrane isolates

Buttermilk (BM) has recently received much attention as a source of milk fat globule membrane (MFGM) due to its high polar lipid (PL) content, which gives it functional properties and health benefits. Nevertheless BM can be obtained from two different technological processes, i) the BM from the butter oil industry (BM1) and ii) BM from the butter manufacture (BM2). Neutral lipids (NL) and PL characterization from both BM and their MFGM isolated fractions have been qualitatively and quantitatively characterized including the individual phospho- and sphingolipids by HPLC-ELSD as well as triacylglycerols (TAG) and cholesterol and FAME by GC-FID. The results revealed that while BM (either from BM1 or BM2) had a PL fraction of 12–16 %, the MFGM isolated fraction contained about 40 % of PL with major presence of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM). Besides, MFGM showed a significant increase in the medium molecular weight TAG and cholesterol and almost two fold amount of PUFA content than BM due mainly to linoleic acid. The results of this study provide deeper information on the lipid composition of BM and MFGM isolated fractions of great importance for the design of dietary supplements with potential beneficial effects on human health.

Thermal effects on IgM-milk fat globule-mediated agglutination.

The process of agglutination causes firm cream layers in bovine milk, and a functioning agglutination mechanism is paramount to the quality of non-homogenized milks. The phenomenon is not well-described, but it is believed to occur due to interactions between immunoglobulins (Ig) and milk fat globules. For the first time, this paper demonstrates how the process of agglutination can be visualized using confocal laser scanning microscopy, rhodamine red and a fluoresceinisothiocynat-conjugated immunoglobulin M antibody. The method was used to illustrate the effect on agglutination of storage temperature and pasteurization temperature. Storage at 5 °C resulted in clearly visible agglutination which, however, was markedly reduced at 15 °C. Increasing storage temperature to 20 or 37 °C cancelled any detectable interaction between IgM and milk fat globules, whereby the occurrence of cold agglutination was documented. Increasing 20 s pasteurization temperatures from 69 °C to 71 °C and further to 73 °C lead to progressively higher inactivation of IgM and, hence, reduction of agglutination. Furthermore, 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that changes in storage temperature caused a redistribution of Ig-related proteins in milk fat globule membrane isolates. Poly-immunoglobulin G receptor was present in milk fat globule preparations stored at cold (4 °C) conditions, but absent at storage at higher temperature (25 °C). The findings provide valuable knowledge to dairy producers of non-homogenized milk in deciding the right pasteurization temperature to retain the crucial agglutination mechanism.

Placing pasteurisation before or after microfiltration impacts the protein composition of milk fat globule membrane material

Milk fat globule membrane (MFGM) is a value-added ingredient with numerous applications. During industrial isolation, the choice of processing steps is known to influence the intactness and bioactivity of the resulting MFGM-containing material. The present study demonstrates that pasteurisation at 80, 85 or 88 °C had a different impact on the protein composition of MFGM material, depending on whether it was performed before or after microfiltration of raw milk. MFGM isolates from milk pasteurised before microfiltration had a significantly higher content of β-lactoglobulin, but a lower level of periodic acid Schiff base 6 and 7, compared with MFGM isolated from milk pasteurised after microfiltration. The β-lactoglobulin content in MFGM material increased progressively with pasteurisation temperatures. Pasteurisation after filtration did not alter the protein composition of MFGM material significantly compared with an unpasteurised control. The results point towards improved process requirements for a more gentle isolation process for MFGM-enriched fractions.

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.

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 antiproliferative properties of the milk fat globule membrane are affected by extensive heating

The milk fat globule membrane (MFGM), the material surrounding milk fat globules, is not only interesting from a technological standpoint but it also shows great potential as a health ingredient, as it exerts cytotoxic and apoptotic effects against colon cancer cells. Although the effects of milk processing on the MFGM composition and functionality are well documented, less is understood on how processing may affect its bioactivity. This study aimed to determine if heating can affect the antiproliferative capacity of the MFGM. To do so, MFGM was extracted from milk heated at 80 °C for 10 min, as this temperature/time regime is known to cause extensive protein-protein interactions with changes in the processing functionality of milk. Two cell lines, whose morphological features are representative of two different stages of colon carcinogenesis (HT-29 and Caco-2), were used to test the antiproliferative capacity of MFGM isolates obtained either from untreated or heated milk. Cell proliferation analysis showed a similar dose-dependent decrease of DNA synthesis in both cell lines exposed to 6.25–200 μg of MFGM protein.mL−1, isolated from unheated milk. The heat treatment diminished the efficacy of the MFGM isolates, as only the highest concentrations of MFGM tested following heating showed a significant effect on cell proliferation. The decreased ability of MFGM isolates to affect the carcinoma cell proliferation was attributed to changes in composition, mainly phospholipid losses. Changes to the supramolecular structure of the MFGM caused by heating may also have played a role. This work demonstrates the importance of processing history in assessing the biological functionality of MFGM.

Modulation of immune function by milk fat globule membrane isolates

The nutritional value and characterization of minor milk components on mammalian immune function are not fully understood. The aim of this research was to test the ability of a milk fat globule membrane (MFGM) isolate to modulate murine immune function in vitro, by studying its effects on splenocyte proliferation, apoptosis, and cytokine production. Proliferation of spleen cells was not affected by the MFGM isolate; however, in the presence of polyclonal activators, the MFGM isolate suppressed cell proliferation. Results obtained by flow cytometry did not support programmed cell death as the cause of the MFGM immune-modulating capacity. A mode of suppression on the splenocyte activation process was suggested from a marked decrease in the production of IFN-γ and tumor necrosis factor-α cytokines, typical indicators of immune cell activation. The effect of MFGM on IL-4 secretion was significantly less than that for the other 2 cytokines. The activity exerted by the MFGM over concanavalin A-stimulated cells differed from that observed in cells treated with lipopolysaccharide, suggesting a different mode of action depending on the activator used. These results indicate the potential of MFGM extracts as functional ingredients with bioactive modulating capacity.

Distribution and isolation of milk fat globule membrane proteins during dairy processing as revealed by proteomic analysis

The milk fat globule membrane (MFGM) has received great attention due to health-beneficial properties, highlighting the potential to use MFGM isolates in manufacturing nutraceutical and functional foods. A proteomic approach employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to evaluate a procedure to isolate MFGM from milk and to determine the distribution of MFGM proteins during dairy processing. In total, 225 MFGM-associated proteins were identified. Among the newly found MFGM-associated proteins, 25 were detected with greater than five peptide matches. The distribution of MFGM proteins into buttermilk and butter during churning of cream was different, especially for minor MFGM proteins. Microfiltration of reconstituted industrial buttermilk after dissociating the casein micelles resulted in a change of protein prevalence in the retentate, leading to a greater number of minor MFGM proteins detected using LC-MS/MS.

Effect of processing on the composition and microstructure of buttermilk and its milk fat globule membranes

The effect of cream pasteurization on the composition and microstructure of buttermilk after pasteurization, evaporation and spray-drying was studied. The composition of milk fat globule membrane (MFGM) isolated from buttermilk samples was also characterized. Pasteurization of cream resulted in higher lipid recovery in the buttermilk. Spray-drying of buttermilk had a significant effect on phospholipid content and composition. After spray-drying, the phospholipid content decreased by 38.2% and 40.6%, respectively in buttermilk from raw or pasteurized cream when compared with initial buttermilks. Pasteurization of cream resulted in the highest increase in whey protein recovery in MFGM isolates compared with all other processing steps applied on buttermilk. A reduction in phospholipid content was also observed in MFGM isolates following spray-drying. Transmission electron microscopy of the microstructure of buttermilks revealed extremely heterogeneous microstructures but failed to reveal any effect of the treatments.

Production of a Novel Ingredient from Buttermilk

The presence of material derived from the milk fat globule membrane (MFGM) makes buttermilk (the byproduct of butter making) distinct from any other dairy product. Membrane filtration of commercial buttermilk was carried out to obtain isolates rich in MFGM material. The separation of MFGM from the skim milk proteins present in commercial buttermilk was carried out by the addition of sodium citrate followed by microfiltration through a membrane of 0.1-μm nominal pore size. The sodium citrate caused the dissociation of casein micelles and allowed permeation of a large proportion of the skim-milk derived proteins through the membrane. This process successfully concentrated MFGM material in the retentate, and demonstrated that membrane filtration can be employed to produce MFGM fractions from commercial buttermilk. The utilization of MFGM isolates from buttermilk is of increasing importance in light of recent studies suggesting the role of phospholipids in many health-related functions: buttermilk is an untapped resource of these functional components.

Effect of Heating of Cream on the Properties of Milk Fat Globule Membrane Isolates

Buttermilk contains large amounts of proteins derived from skim milk and from the milk fat globule membrane (MFGM). Heating the cream before churning, even at temperatures as low as 65 °C, strongly affected the functional properties of the membrane fraction and caused whey proteins to associate with the MFGM. The extent of this interaction depended on temperature. Pasteurization temperature of the cream had no effect on the emulsifying properties of whole buttermilk isolates, the casein content of which masked changes in the MFGM fraction. The stability of oil-in-water emulsions made using isolated MFGM material depended on the heat treatment of the cream. The solubility, color, iron content, amount of whey protein present in the MFGM isloate, and emulsifying properties of this material were all affected in parallel by the heat treatment. Keywords: Heating of cream; emulsifying properties; milk fat globule membrane isolate; buttermilk

Isolates from Industrial Buttermilk: Emulsifying Properties of Materials Derived from the Milk Fat Globule Membrane

Two isolates were produced from commercial buttermilk: (i) an isolate containing whey proteins, caseins, and proteins of the milk fat globule membrane (MFGM); (ii) an isolate of MFGM only. The MFGM isolate contained a large quantity of membrane fragments from the milk fat globule. The MFGM fraction was a poor emulsifier, compared to the whole isolate. Emulsions prepared with 10% soybean oil needed a much larger amount of MFGM isolate to produce a droplet size distribution similar to that found for the emulsion prepared using 1−2% (w/v) whole isolate. Heat treatment of the cream and the industrial churning process appeared to affect the emulsifying behavior of MFGM proteins. A large amount of β-lactoglobulin was present in the MFGM isolate as a result of heat-induced interactions during pasteurization of the cream. Keywords: Buttermilk; food emulsions; food proteins; protein adsorption

Studies on the susceptibility of membrane-derived proteins to proteolysis as related to changes in their emulsifying properties

Milk fat globule membrane (MFGM) isolates obtained from fresh unheated cream and industrial buttermilk were hydrolyzed using trypsin and chymotrypsin. The protein composition of these hydrolyzed samples was determined by gel electrophoresis. MFGM proteins obtained from industrial buttermilk were fully hydrolyzed after a short incubation time. In contrast, the MFGM isolates from unheated cream did not undergo complete hydrolysis, and intact proteins were still observed after 3 h of incubation. Thus, heat treatment of the cream caused profound changes in the composition and structure of the MFGM, so that, after the heating it became more susceptible to hydrolysis by trypsin and chymotrypsin. When proteolysis was carried out on emulsions prepared with MFGM isolates, neither the rate of hydrolysis nor the composition of the residual MFGM protein changed from that of MFGM isolates treated with enzymes in solution. The emulsifying properties of the MFGM isolates improved with proteolysis, as estimated by the size distribution of the emulsion droplets. A similar improvement in the droplet size distribution of emulsions was also observed when the hydrolysis was carried out after homogenization. In this case, the large particles formed during homogenization disappeared after proteolysis, suggesting that MFGM isolates, when present in less than saturating amounts, caused bridging flocculation of the emulsion. The improvement in the emulsifying properties resulting from proteolysis was substantial for MFGM isolates from unheated cream, and even greater for isolates derived from industrial buttermilk.