Concentration of polar MFGM lipids from buttermilk by microfiltration and supercritical fluid extraction

Buttermilk contains the milk fat globule membrane (MFGM), a material that possesses many complex lipids that function as nutritionally valuable molecules. Milk-derived sphingolipids and phospholipids affect numerous cell functions, including regulating growth and development, molecular transport systems, stress responses, cross membrane trafficking, and absorption processes. We developed a two-step method to produce buttermilk derivative ingredients containing increased concentrations of the polar MFGM lipids by microfiltration and supercritical fluid extraction (SFE). These processes offer environmentally benign alternatives to conventional lipid fractionation methods that rely on toxic solvents. Firstly, using a ceramic tubular membrane with 0.8-micron pore size, we evaluated the cross flow microfiltration system that maximally concentrated the polar MFGM lipids using a 2n factorial design; the experimental factors were buttermilk source (fresh, or reconstituted from powder) and temperature (50 degrees C, and 4 degrees C). Secondly, a SFE process using supercritical carbon dioxide removed exclusively nonpolar lipid material from the microfiltered buttermilk product. Lipid analysis showed that after SFE, the product contained a significantly reduced concentration of nonpolar lipids, and a significantly increased concentration of polar lipids derived from the MFGM. Particle size analysis revealed an impact of SFE on the product structure. The efficiency of the SFE system using the microfiltration-processed powder was compared much more favorably to using buttermilk powder.     

Combined effects of milk fat globule membrane polar lipids and protein concentrate on the stability of oil-in-water emulsions

Milk fat globule membrane (MFGM) material isolated from reconstituted buttermilk by microfiltration (i.e., whole MFGM) contains two major fractions, namely proteins (consisting mainly of MFGM-specific proteins and serum proteins) and lipids (original membrane polar lipids and contaminating triglycerides from the globule core). In this study, MFGM proteins and polar lipid (PL) concentrate were separated from whole MFGM material using solvent extraction. The particle size distribution, stability, surface protein and polar lipid load of oil-in-water emulsions prepared with protein or PL concentrate, individually or in combination, at various concentrations were examined. At low emulsifier concentrations (<2.3%), there was an interacting effect between proteins and PLs on the droplet size. The phase separation of emulsions prepared with a combination of 0.3% proteins and 0.3% PLs was similar to that of emulsions containing 0.3% proteins. The proteins were more preferentially adsorbed at the emulsion droplet surface compared with PLs.     

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.     

The influence of temperature and pressure factors in supercritical fluid extraction for optimizing nonpolar lipid extraction from buttermilk powder

The milk fat globule membrane, readily available in buttermilk, contains complex lipids claimed to be beneficial to humans. Phospholipids, including sphingolipids, exhibit antioxidative, anticarcinogenic, and antiatherogenic properties and have essential roles in numerous cell functions. Microfiltration coupled with supercritical fluid extraction (SFE) may provide a method for removing triacylglycerols while concentrating these nutritionally valuable lipids into a novel ingredient. Therefore, SFE as a method for phospholipid concentration needs to be optimized for triacylglycerol removal in buttermilk. The SFE conditions were assessed using a general full factorial design; the experimental factors were pressure (15, 25, and 35 MPa) and temperature (40, 50, and 60°C). Particularly interesting is that only triacylglycerols were removed from buttermilk powder. Little to no protein loss or aggregation was observed compared with the untreated buttermilk powder. Calculated theoretical values showed a linear increase for lipid solubility as pressure, temperature, or both were increased; however, experimental values showed nonlinearity, as an effect of temperature. In addition, the particular SFE parameters of 35 MPa and 50°C displayed enhanced extraction efficiency (70% total lipid reduction).     

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-microm 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.     

Combination of homogenization and cross-flow microfiltration to remove microorganisms from industrial buttermilks with an efficient permeation of proteins and lipids

Buttermilk is a source of interesting nutritional and functional components, e.g. polar lipids and proteins. However, it is still considered as a low-value by-product of the dairy industry with high variations in biochemical composition and bacterial contaminations. The objective of this study was to develop a process based on microfiltration, permitting the removal of microorganisms to ensure the safety of buttermilk components for human nutrition. Industrial buttermilks and the products collected during microfiltration were characterized using particle size measurements, biochemical and microbiological analysis. The combination of homogenization at 80 MPa and cross-flow microfiltration successfully removed bacteria from skimmed buttermilk: bacterial reduction > 4.8 log₁₀ with 0 cfu/ml in the permeate using the 0.8 μm pore size membrane and 1 cfu/ml with 1.4 μm membrane. Chemical analysis revealed the efficient permeation of proteins, total lipids and polar lipids. Polar lipid classes permeated equally the membrane. This work will contribute in improving the safety of buttermilk-based ingredients.Industrial relevanceThis work describes the development of an innovative process combining homogenization and cross-flow microfiltration for the selective removal of bacteria from industrial buttermilks. This process is an alternative to heat treatments that alter the nutritional and organoleptic properties of food products. The safety of buttermilk-based ingredients containing milk polar lipids of interest will contribute in their economic valorization for human nutrition.     

Flux and transmission of β-casein during cold microfiltration of skim milk subjected to different heat treatments

Raw skim milk was subjected to different heat treatments: thermization (65°C, 20 s), pasteurization (72°C, 15 s), and no heat treatment (milk was microfiltered using 1.4-µm membranes at 50°C for bacteria removal; 1.4 MF). The milk (thermized, pasteurized, and 1.4 MF) was cooled and stored at 2°C until processing (at least 24 h) with cold (～6°C) microfiltration using a benchtop crossflow pilot unit (Pall Membralox XLAB 5, Pall Corp., Port Washington, NY) equipped with 0.1-µm nominal pore diameter ceramic Membralox membrane (ET1-070, α-alumina, Pall Corp.). The flux was monitored during the process, and β-casein transmission and removal were calculated. The study aimed to indicate the conditions that should be applied to maximize β-casein passage through the membrane during cold microfiltration (5.6 ± 0.4°C) of skim milk. The proper selection of heat treatment parameters (temperature, time) of the feed before the cold microfiltration process will increase β-casein removal. It is not clear whether the difference in β-casein transmission between 1.4 MF, thermized, and pasteurized milk results from the effect of heat treatment conditions on β-casein dissociation from the casein micelles or on passage of β-casein through the membrane. The values of the major parameters (permeation flux and tangential flow velocity, through the wall shear stress) responsible for a proper membrane separation process were considerably lower than the critical values. It seems that the viscosity of the retentate has a great effect on the performance of the microfiltration membranes for protein separation at refrigerated temperatures.     

Improved isolation of bioactive components of bovine colostrum using cross‐flow microfiltration

Bovine colostrum contains bioactive components such as growth factors, immunoglobulins and antimicrobial factors. As conventional heat treatment methods inactivate these valuable components, cross‐flow microfiltration (MF) seems to be a promising option for the processing of bovine colostrum. A series of cross‐flow MF experiments with tubular ceramic membranes of various pore sizes and geometries were conducted. MF with pore sizes of 0.8 and 1.4 μm resulted in a 5.4‐ and 3.5‐log reduction of the microbial content, respectively. Applying 0.14‐ and 0.2‐μm membranes lead to a permeate that was almost free from micro‐organisms and casein. However, the maximum transmission of whey protein into the permeate was only 33%.     

Short communication: Evaluation of supercritical fluid extraction aids for optimum extraction of nonpolar lipids from buttermilk powder

The milk fat globule membrane, present in buttermilk, contains complex lipids such as phospholipids. Microfiltration coupled with supercritical fluid extraction (SFE) may provide a method of enriching these nutritionally valuable lipids into a novel ingredient. Therefore, SFE as a method for phospholipid enrichment needs to be optimized for lipid removal effectiveness. The role of matrix additions to the buttermilk powder for extraction efficiency was evaluated. Diatomaceous earth (biosilicates), Teflon beads, and physical vibration were tested and shown to reduce total lipid by 86, 78, and 70%, respectively. Four consecutive treatments were shown to exhaust the system; however, similar extraction efficiencies were noted for 1 treatment with biosilicate addition, 2 treatments with physical vibration, or 3 treatments with added Teflon beads. The extracted lipid material consisted of the nonpolar fraction, and protein concentration was observed to increase slightly compared with the control. Although higher lipid extraction was achieved from the powder with addition of diatomaceous earth, a removable aid is ideal for an edible product.     

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.     

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.     

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 heating whey proteins in the presence of milk fat globule membrane extract or phospholipids from buttermilk

The objective of this study was to determine the contribution of phospholipids from buttermilk as a nucleus in the heat-induced aggregation of whey proteins. Solutions of whey proteins (5%, w/v) were adjusted to pH 4.6 or 6.8 and then heated at 65 or 80 °C for 25 min with or without 1% (w/v) of milk fat globule membrane (MFGM) extract or phospholipid powder. The aggregation mechanisms were characterised using analysis with Ellman's reagent, one-dimensional gel electrophoresis, thin-layer chromatography, and three-dimensional confocal laser-scanning microscopy. Three-dimensional images showed protein/phospholipid interactions in the presence of MFGM extract or phospholipids, and thin-layer chromatography plates showed no trace of free phospholipids after 20 min at pH 4.6. Overall, the results demonstrate that phospholipids from buttermilk were involved in the formation of protein aggregates through the MFGM fragments at a low temperature, whereas phospholipids could interact directly with the proteins at a higher temperature (80 °C).     

An investigation of the fractionation of whey proteins by two microfiltration membranes with nominal pore size of 0.1 μm

There is growing interest in the fractionation of whey proteins because of the specific properties of individual whey proteins. The objective of this work was to assess the efficiency of two membranes to obtain two fractions, one rich in β‐lactoglobulin (β‐Lg) and the other rich in α‐lactalbumin (α‐La) from a whey‐protein concentrate using microfiltration (MF). Two MF membranes were tested for the fractionation: a flat‐sheet membrane VCWP and a spiral membrane MF‐7002, both with nominal pore sizes of 0.1 μm. The VCWP retained 78% of the proteins in solution, and this membrane was shown to be permeable to both proteins, β‐Lg and α‐La. The retention of protein by MF‐7002 was 65%, and there was a partial retention of lactose. The permeate collected in the MF‐7002 in the concentration stage was over 50%α‐La; although this was present in lower concentrations, it was passed preferentially to the permeate. The results indicated that some aggregation of the proteins may have occurred under the experimental conditions because there was only a partial separation of the proteins under study.     

Pilot-scale crossflow-microfiltration and pasteurization to remove spores of Bacillus anthracis (Sterne) from milk

High-temperature, short-time pasteurization of milk is ineffective against spore-forming bacteria such as Bacillus anthracis (BA), but is lethal to its vegetative cells. Crossflow microfiltration (MF) using ceramic membranes with a pore size of 1.4 μm has been shown to reject most microorganisms from skim milk; and, in combination with pasteurization, has been shown to extend its shelf life. The objectives of this study were to evaluate MF for its efficiency in removing spores of the attenuated Sterne strain of BA from milk; to evaluate the combined efficiency of MF using a 0.8-μm ceramic membrane, followed by pasteurization (72°C, 18.6 s); and to monitor any residual BA in the permeates when stored at temperatures of 4, 10, and 25°C for up to 28 d. In each trial, 95 L of raw skim milk was inoculated with about 6.5 log₁₀ BA spores/mL of milk. It was then microfiltered in total recycle mode at 50°C using ceramic membranes with pore sizes of either 0.8 μm or 1.4 μm, at crossflow velocity of 6.2 m/s and transmembrane pressure of 127.6 kPa, conditions selected to exploit the selectivity of the membrane. Microfiltration using the 0.8-μm membrane removed 5.91 ± 0.05 log₁₀ BA spores/mL of milk and the 1.4-μm membrane removed 4.50 ± 0.35 log₁₀ BA spores/mL of milk. The 0.8-μm membrane showed efficient removal of the native microflora and both membranes showed near complete transmission of the casein proteins. Spore germination was evident in the permeates obtained at 10, 30, and 120 min of MF time (0.8-μm membrane) but when stored at 4 or 10°C, spore levels were decreased to below detection levels (≤0.3 log₁₀ spores/mL) by d 7 or 3 of storage, respectively. Permeates stored at 25°C showed coagulation and were not evaluated further. Pasteurization of the permeate samples immediately after MF resulted in additional spore germination that was related to the length of MF time. Pasteurized permeates obtained at 10 min of MF and stored at 4 or 10°C showed no growth of BA by d 7 and 3, respectively. Pasteurization of permeates obtained at 30 and 120 min of MF resulted in spore germination of up to 2.42 log₁₀ BA spores/mL. Spore levels decreased over the length of the storage period at 4 or 10°C for the samples obtained at 30 min of MF but not for the samples obtained at 120 min of MF. This study confirms that MF using a 0.8-μm membrane before high-temperature, short-time pasteurization may improve the safety and quality of the fluid milk supply; however, the duration of MF should be limited to prevent spore germination following pasteurization.     

Microfiltration of butter serum upon casein micelle destabilization

The gross composition of butter serum, the aqueous phase of butter, is comparable to that of buttermilk, except that it has a higher content of material derived from the milk fat globule membrane (MFGM). As such, butter serum is a good source for further purification of MFGM material. The purified fraction could be of interest for its emulsifying and nutritional properties. The effect of sodium citrate and ethanol on the dissociation of butter serum casein micelles, and their effect on casein retention upon tangential microfiltration were investigated. Optimal conditions of casein micelle dissociation were assessed by using an experimental design (response surface full central composite orthogonal design) with temperature and ethanol or sodium citrate concentration as design variables and the Hunter L* value as response variable. For both dissociating agents, a highly significant reduced quadratic model was fit to the data. Microfiltration tests were performed on pure butter serum, and on butter serum in the presence of sodium citrate, under optimal dissociation conditions (50°C, 80 mM). A cellulose acetate membrane with a pore size of 0.15 μm was used. From the filtration curves and fouling coefficients it was clear that the addition of sodium citrate improved the permeation flux, and minimized fouling. All fractions were analyzed for dry matter, protein, lactose, lipid, and polar lipid contents. The protein fraction was further characterized by sodium dodecyl sulfate-PAGE. It was shown that sodium citrate greatly enhanced casein transmission through the membrane, but at the expense of substantial losses of polar lipids.     

Effect of cream treatment on phospholipids and protein recovery in butter-making process

A simple approach is proposed to recover native protein and phospholipid fractions from butter‐making process using equipments available in dairy‐processing plant. A washing treatment was used to remove protein from the cream and increase the phospholipids purity in buttermilk. Cream from a first separation was diluted with milk ultrafitration permeate and separated a second time. A 10X dilution factor reduced protein concentration in the cream from 1.6 ± 0.2 to 0.52 ± 0.03%. As a result, the phospholipids to protein ratio in buttermilk increased from 53 ± 10 to 172 ± 7 mg g^?1. In butter‐making process, an important portion of total phospholipids (～26%) is retained in butter. Butter serum made from washed cream could then be used to produce phospholipid concentrates with phospholipids to protein ratio of 473 ± 3 mg g^?1. Interestingly, the extracts from butter serum are characterised by a higher proportion of sphingomyelin compared with those from buttermilk.     

Chemical Pretreatment and Microfiltration for Making Delipidized Whey Protein Concentrate

Chemical pretreatment, microfiltration (MF) and ultrafiltration (UF) were applied to produce delipidized whey protein concentrates (WPC). Processes including both chemical pretreatment and MF resulted in WPC with <0.5% lipids. Low-pH UF and isoelectric point (PI) precipitation were more effective for lipid removal than chemical pretreatment by thermocalcic aggregation. Protein permeation ratios in MF processes were improved by UF preconcentration of whey. Protein permeation and flux were different between the two MF membranes used. Isoelectric point precipitation increased β-Lg contents, but not α-La, in the resulting WPC (B). Minor proteins exhibited lower concentrations in WPC B and MF WPC products.     

Thermocalcic aggregation of milk fat globule membrane fragments from acid buttermilk cheese whey

Fragments originating from the milk fat globule membrane (MFGM), which is rich in polar lipids and membrane-specific proteins, are gaining interest for their functional and nutritional properties. Acid buttermilk cheese whey was used as a source for MFGM purification, because its MFGM content is more than 5 times higher than that of standard rennet whey. Because polar lipids are the main constituent of the MFGM and only occur in membranous structures, the polar lipid content was taken as a parameter for the total MFGM fragment content. The process of thermocalcic aggregation was evaluated on its recovery of MFGM fragments in the pellet. This method, originally intended for whey clarification and defatting, is a combination of calcium addition, a pH increase, and a thermal treatment. The influence of pH (6.5 to 8), temperature (40 to 70°C), and calcium concentration (0.1 to 0.24 g/100 g) on the pellet mass and dry matter (DM) content and on recovery of protein and polar lipids (and thus indirectly on MFGM fragments) was investigated by means of a response surface Box-Behnken orthogonal design. Reduced quadratic models were fit to the experimental data and were found to be highly significant. No outliers were observed. The recovery of MFGM fragments was found to be highly dependent on the pH, and less dependent on temperature and calcium addition. Next to MFGM proteins, whey proteins were also found to be involved in the formation of aggregates. Optimal conditions were found at 55°C, pH 7.7, and 0.205 g of calcium/L of whey. Under these conditions, 91.0% of the whey polar lipids were recovered in a firm and compact pellet of only 7.86% of the original whey mass, with a polar lipid concentration of 8.34% on pellet DM. Washing with water and centrifugation of the pellet was successful because after one washing step, virtually all sugars were removed, whereas 75.9% of the whey polar lipids could still be recovered. As such, the polar lipid content of the washed pellet increased to 10.70% on a DM basis. However, a second washing step resulted in serious losses of MFGM material.