Article

A comparison of composition and emulsifyingproperties of MFGM materials prepared fromdifferent dairy sources by microfiltration

Jelena Miocinovic1, Thien Trung Le2, Eveline Fredrick3,Paul Van der Meeren4, Predrag Pudja1 and Koen Dewettinck3

AbstractMilk fat globule membrane (MFGM), due to its specific nature and composition, is known as material pos-sessing advantageous nutritional as well as technological properties. In this study MFGM materials wereproduced from several dairy sources such as buttermilk (BM), butter serum (BS) and buttermilk whey (BMW)by microfiltration (MF). The obtained materials, depending on the sources, were called BM-MFGM, BS-MFGMand BMW-MFGM, respectively. The compositions of starting materials and the isolated MFGM materials aswell as their emulsifying properties were analyzed and compared. As expected, the MF resulted in enrichmentof polar lipids (PLs), major components of MFGM. On dry matter basis, BM-MFGM and BS-MFGM were about2.5 times higher in PLs compared to their beginning materials while BMW-MFGM was about 8.3 timescompared to buttermilk powder (BMP). Sodium dodecyl sulfate polyacrylamide gel electrophoresisshowed that the microfiltered products still contained a high amount of non-MFGM proteins such as caseins,b-lactoglobulin, and a-lactalbumin. Emulsions of 35% soya oil in water were prepared with the mentionedmaterials using a homogenizer at various pressures. Generally, emulsions prepared with BMP and butterserum powder had significantly higher particle sizes than those prepared with the MFGM materials. This resultalong with microscopy observation and viscosity measurement indicated the presence of aggregated par-ticles in the former emulsions, probably as a result of lack of surface-active components. The differences incomposition, especially in content of PLs and proteins of the materials were the main reasons for the differ-ences in their emulsifying behaviors.

KeywordsMilk fat globule membrane, buttermilk, butter serum, buttermilk whey, emulsion, microfiltration

Date received: 28 September 2012; accepted: 8 April 2013

INTRODUCTION

Milk fat globule membrane (MFGM) is a biologicalmaterial that surrounds milk fat droplets. This mem-brane, about 10–20 nm thick in cross section, acts as anatural emulsifier to stabilize fat globules from coales-cence and protect the core fat against enzymatic deg-radation (Dewettinck et al., 2008). The native MFGM(in fresh milk) comprises approximately 60%

1Faculty of Agriculture, Institute of Food Technology andBiochemistry, University of Belgrade, Belgrade, Serbia2Faculty of Food Science and Technology, Nong Lam University,Ho Chi Minh, Vietnam3Laboratory of Food Technology and Engineering, Department ofFood Safety and Food Quality, Faculty of Bioscience Engineering,Ghent University, Gent, Belgium4Particle and Interfacial Technology Group, Faculty of BioscienceEngineering, Ghent University, Gent, Belgium

Corresponding author:Jelena Branko Miocinovic, Faculty of Agriculture, University ofBelgrade, Nemanjina 6, Belgrade 11000, Serbia.Email: jmiocin@agrif.bg.ac.rs

Food Science and Technology International 20(6) 441–451! The Author(s) 2013 Reprints and permissions:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/1082013213489566fst.sagepub.com

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membrane specific proteins, mainly glycoproteins, and40% lipids which comprises mainly phospho andsphingolipids (Fox and McSweeney, 1998). The com-position of MFGM is influenced by the diet of cows.Lopez et al. (2008) found that MFGM in milk fromcows fed with diet rich in polyunsaturated fatty acidsresulted in a higher amount of phospholipids, sphingo-myelin, stearic acid, etc. No significant differences inthe polar lipids (PLs) and sterol profile were foundbetween MFGM extracted from small and large milkfat globule fractions (Fauquant et al., 2007).

For the past two decades, a great deal of knowledgeon health beneficial effects of MFGM components havebeen accumulated (Dewettinck et al., 2008; Spitsberg,2005).

Technological and potential health properties of theMFGM have led to an interest in concentrating andisolating this material from dairy sources.

Some treatments in milk processing, such as churn-ing and agitation, disrupt the MFGM and release theminto the serum phase. Hence, buttermilk (BM), aqueousphase derived from the churning during butter makingprocess, and butter serum (BS), aqueous phase ofbutter obtained after melting and centrifugation ofbutter (Rombaut et al., 2006b), are among the dairyby-products which are considered as good sources ofMFGM material (Rombaut et al., 2006a, 2006b; Sodiniet al., 2006). The acid BM cheese whey was also con-sidered as a material with high MFGM content,more than five times than that of standard rennetwhey (Rombaut and Dewettinck, 2007). These mater-ials are characterized by a high content of PLs andMFGM proteins, but differ in the casein content(Dewettinck et al., 2008; Rombaut et al., 2006b;Sodini et al., 2006).

Most of attempts for isolating MFGM fragmentshave been the use of micro and ultrafiltration of regularBM (Corredig et al., 2003; Morin et al., 2004, 2008;Roesch et al., 2004; Sachdeva and Buchheim, 1997),acid whey and buttermilk whey (BMW; Rombautet al., 2007). If BM is used, the major restriction inmembrane filtration processes is the presence ofcasein micelles, which are of similar size with MFGMfragments, which in turn results in limited concentra-tion efficiency. Other approaches such as adding cit-rates to dissociate casein micelles (Corredig et al.,2003; Roesch et al., 2004, 2006a), rennet or acid coagu-lation (Sachdeva and Buchheim, 1997), as well as wash-ing of cream prior to the churning (Morin et al., 2007),could be employed to remove casein micelles from BMbefore filtration process. Fauquant et al. (2005) usedthe microfiltration (MF) for separation of milk fatglobules with different size and then by manual churn-ing and ultracentrifugation (50,000� g/2 h) to obtainan MFGM pellet.

MFGM materials are considered to be an efficientnatural emulsifier, due to a high content of PLs whichare surface-active materials due to their amphiphilicnature (Corredig and Dalgleish, 1997, 1998; Kanno,1989; Kanno et al., 1991; Roesch et al., 2004;Sachdeva and Buchheim, 1997). Roesch et al. (2004)found that emulsions made with MFGM isolatesobtained by MF of BM had shown a better stabilitydetermined by both, particle size distribution and lowerrate of flocculation, than emulsions made with butter-milk powder (BMP). However, literature data show thehuge differences in the functionality of MFGM mater-ials which are a result of various factors such as typeof raw material used for MFGM isolation, pre-treatments, especially heat treatments (Corredig andDalgleish, 1997), isolation procedure, compositionand concentration of isolates (Kanno, 1989; Kannoet al., 1991), as well as a procedure of emulsions prep-aration (Astaire et al., 2003; Roesch et al., 2004;Rombaut et al., 2006a). Despite numerous studies,information on the emulsifying properties of MFGMmaterials obtained from different dairy sources, espe-cially BS and acid BMW, by using MF is still limited.

The major objectives of this study was first to evalu-ate the possibility of using microfiltration to concen-trate MFGM materials from different sources andthen to study their emulsifying properties. MFGMmaterials were obtained from reconstituted BM andBS, after adding sodium citrate to dissociate caseinmicelles, and BMW using microfiltration. Chemicalanalyses and sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) were carried out todetermine the changes in composition from the startingmaterials to corresponding MFGM materials. Thebeginning materials, BMP and butter serum powder(BSP), all obtained microfiltered MFGM materialswere used in making emulsions and their capacity asemulsifiers were investigated. Results obtained in thiswork could help in better understanding of emulsifyingproperties of MFGM materials that could be markedas a base for the ingredients with enhanced nutritionaland technological properties.

MATERIALS AND METHODS

Chemical and materials

BMP and BSP were obtained from Frieslandfoods,(Lummen, Belgium). BM was obtained after churningof pasteurized cream, while BS was obtained as aque-ous phase after melting and centrifugation of butter(Rombaut et al., 2006b). Both materials were evapo-rated and spray dried to produce powders.Chloroform, methanol, diethyl ether and petroleumether used for lipid and PL extractions were of>99% purity and bought from Chem-Lab NV

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(Zedelgem, Belgium). Solvents of high-performanceliquid chromatography (HPLC) quality for HPLC ana-lysis were delivered from Biosolve (Valkenswaard, TheNetherlands). A milk PL standard was supplied bySpectral Service GmbH (D-50996, Koln, Germany).

Sample preparation

BM and BS were reconstituted from BMP and BSP,respectively, at 4% (wt/wt) total solid levels using deio-nized water under agitation (ratio of 1:4wt/wt totalBMP and BSP solids), with the addition of 1%(wt/wt) tri sodium citrate (38mM). The reconstitutedBM and BS were stored overnight at 4 �C in order toensure a full hydration. The concentrated materials(retentate or so-called MFGM materials) after theMF of BM and BS were called BM-MFGM andBS-MFGM, respectively.

For making acid BMW, BMwas reconstituted at 8%of total solids from BMP in a similar way as describedabove but without addition of sodium citrate. Thereconstituted BM was acidified to pH 4.6 at room tem-perature using citric acid 1M and casein coagulate wasremoved by filtering with a cheese cloth. pH of theBMW was adjusted back to pH 7.6 with KOH 1Mbefore it was introduced to the MF. The obtainedmaterial after MF of BMW was called BMW-MFGM.MF experiment was repeated twice or three times.

Microfiltration

Instruments and conditions for MF were described pre-viously (Le et al., 2011b). The MF pilot unit consistedof a Millipore lab scale plate-and-frame filtration frameequipped with a Millipore Pellicon 2 hydrophillizedpolyvinylnilfluoride multi-flat-sheet membrane with apore diameter of 0.22mm and area surface of 0.5m2

(Millipore, Brussels, Belgium). The process was drivenby an Almatec diaphragm pump connected to a pulsedampener (Kamp Lintfort, Germany). The feed flowwas set at 160L/h and permeate flow was controlledat 12.5 L/h.

Before filtration membrane was flushed with 35L ofdeionized water. Twenty liters of the sample solutionswere filtered each time. MF was carried out at 45 �Cand pH 7.6 in combination with three steps of diafiltra-tion with deionized water. The final retentates, namelyBM-MFGM, BS-MFGM and BMW-MFGM, wereconcentrated to about 10% solids and stored at�20 �C for future analysis and emulsion making.

Compositional analysis

Dry matter (DM) analysis was performed by gravimet-ric method (IDF, 1987b). Total lipids (TL) content was

determined using the Rose Gottlieb extraction method(IDF, 1987a). For MFGM samples, 3mL of CaCl210% (w/v) were added before the lipid extraction toincrease the ionic strength of the solution and henceimprove the phase separation (Le et al., 2010). Ashcontent was determined gravimetrically after ashing ina furnace at 550 �C. Lactose content of the materialswas determined by difference. The total protein (TP)content was obtained using Kjeldahl method (IDF,2002) with 6.38 as the protein conversion factor. PLswere extracted using chloroform/methanol mixturesand analyzed using HPLC-ELSD (evaporative lightscattering detector) method (Le et al., 2011a).

Protein profiling by SDS-PAGE. The separation systemand all reagents were obtained from Invitrogen(Merelbeke, Belgium). Six hundred fifty microliters ofsample was mixed with 250 mL of NuPAGE�LDS(Lithium dodecyl sulfate) sample buffer and 100 mL ofNuPAGE� reducing agent (contains 500mM dithio-threitol) and then heated at 70

�

C for 10min. (Leet al., 2009). Five microliter of each prepared sample(approximately 16 mg of TP) was loaded into aNuPAGE� Novex� Bis–tris 4–12% polyacrylamidegel (Bis 2-hydroxyethyl imino-tris hydroxymethylmethane-HCl). NuPAGE� MOPS (3-N-morpholino-propane-sulfonic acid) SDS buffer was used as the run-ning and loading buffers as according to the protocol ofInvitrogen. The same amount of molecular weight(MW) standard was used. Electrophoresis was per-formed in 50min at 200V and 125mA/gel. After sep-aration, the gel was stained with a Coomassie Blue stain(Simply blueTM SafeStain, Invitrogen). Scanning of thewet gel was done at 400 dpi using a high-resolutiontransmission scanner (UMAX Powerlook III, Taiwan).

Emulsions preparation

Soy oil-in-water emulsions at a concentration of 35%w/w oil were prepared with five different emulsifyingmaterials; BMP, BSP, BM-MFGM, BS-MFGM andBMW-MFGM. The supplemented concentrations ofthese materials were adjusted to have a standardizedprotein content of 2.30% (wt/wt) in the final formulae,which represents the average protein content of 35%dairy cream (van Lent et al., 2008). In the case ofBMW-MFGM, which contained very low TP content,BMP was used to obtain the fixed protein content in theemulsion called BMW-MFGM/BMP. Compositions ofthe emulsions are shown in Table 1.

Powders were diluted in water. The mixtures wereprepared under gentle stirring at room temperatureand stored overnight at 4 �C to allow complete hydra-tion. After adding soybean oil, the samples were pre-homogenized by vortex at 13,000 r/min for 2min at

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50 �C using Ultra turax rotator (T25, Ika, Werke) andthen homogenized using a laboratory scale two-stagehomogenizer (APV Lab 2000) at the same temperature.Two pressure combinations, namely 0/10 bar and 30/20 bar, were used. After that the emulsions werecooled and stored at 4 �C with addition of sodiumazide (0.02%) to prevent microbial growth.

Characterization of emulsion properties

Particle size distribution. The particle size distributionwas determined using a Mastersizer S (MalvernInstruments, Malvern, UK) equipped with a 300RFlens. The refractive index values for oil and water werefixed at 1.4600 and 1.3300, respectively, whereas theimaginary refractive index of the dispersed phase wasset to 0.01. Measurements on emulsions were carriedout after the emulsions were diluted in water and also

in a solution containing 1% SDS (Roesch et al., 2004).SDS was used to break potential emulsions dropletsaggregates. The Sauter diameter d3.2 was used to indi-cate and compare the emulsion droplet sizes.

Viscosity measurements. Viscosity of emulsions wasmeasured with a Rheometer (AR 2000, TAInstruments, UK) after one day of storage. Cylinderwas used as the measuring geometry and the tempera-ture was set at 20 �C. The apparent viscosity was rec-orded as a function of shear rate, ranging from 0.07 to400 s�1. The mean values of three replicates werepresented.

Microscopy analysis. Observations were carried out atroom temperature, 24 h after emulsions making, usingan optical microscope (Leitz Diaplan, Wetzlar, UK)with a 10� objective magnification. Each sample wasdiluted 10 times in distilled water and a droplet wasplaced on a glass slide and covered with a coverslip.Pictures were taken using an in-built colorview IIcamera (Olympus, Aartselaar, Belgium).

Statistical analysis. All statistical analyseswere performed by analysis of variance tests withTukey’s pairwise comparison using Statistica6.0 (Statistica Inc., PA, USA). Results were con-sidered statistically different if p-values were smallerthan 0.05.

RESULTS AND DISCUSSION

Composition of materials

The composition of all starting dairy by-products andtheir corresponding MFGM materials are given inTable 2.

Table 1. Composition of emulsions prepared with differ-ent materials

Emulsions/emulsifyingmaterial

Oil(%)

Emulsifyingmaterial (%)

Water(%)

BMP 35 7.2 57.8

BSP 35 7.9 57.1

BM-MFGM 35 30 35

BS-MFGM 35 30 35

BMW-MFGM/BMP 35 45þ 4.9 BMPa 13.7

BM: buttermilk; BS: butter serum; BMP: buttermilk powder; BSP:butter serum powder; BMW: buttermilk whey; MFGM: milk fatglobule membrane.aEmulsions BMW-MFGM/BMP were made with addition of 4.9%BMP due to low content of protein in BMW-MFGM.

Table 2. Composition of experimented materials.a

Dry matter(%)

Total protein(% in drymatter)

Total lipids(% in drymatter)

Ash(% in drymatter)

Lactose(% in drymatter)

Polar lipidsb

(% in totallipids)

Polar lipids(% in drymatter)

BMP 96.04� 0.14 33.43� 0.51 9.97� 0.66 7.50� 0.28 49.10� 0.24 31.90� 0.29 3.18� 0.24

BSP 95.40� 0.05 30.14� 0.14 14.05� 0.07 7.74� 0.03 48.07� 0.24 66.38� 1.61 9.32� 0.00

BMW 5.63� 0.01 7.01� 0.86 8.26� 0.90 9.86� 0.15 74.87� 0.19 30.28� 3.22 2.49� 0.01

BM-MFGM 11.92� 0.45 63.77� 2.96 24.84� 0.23 2.37� 0.92 9.02� 2.27 32.40� 2.41 8.05� 0.67

BS- MFGM 12.93� 2.51 60.70� 2.38 31.80� 3.22 4.90� 0.58 2.61� 0.26 79.92� 11.05 23.31� 1.79

BMW-MFGM 6.61� 0.20 24.43� 0.23 59.91� 0.08 12.20� 1.54 3.46� 1.39 35.00� 1.89 20.97� 1.16

BM: buttermilk; BS: butter serum; BMP: buttermilk powder; BSP: butter serum powder; BMW: buttermilk whey; MFGM: milk fat globulemembrane.aData of BMP and BSP are expressed as average�SD of duplicate analysis. Data of the BM-MFGM, BS-MFGM, BMW and BMW-MFGMare expressed as average�SD of three production batches.bTotal polar lipids consisted of glucosylceramides glucosylceramides, lactosylceramides, phosphatidyl inositol, phosphatidyl ethanola-mine, phosphatidyl serine, phosphatidyl choline and sphingomyelin.

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The gross composition of BMP was comparable withthat of BSP, except for PLs. The BMW was character-ized with low dry matter and protein content, due toremoval of caseins by coagulation and filtering(Table 2). The TP/DM and TL/DM of BM-MFGMwere increased 1.9 and 2.5 times compared to those ofBMP, respectively. Similar ratios have been found bycomparing BSP and BS-MFGM. In both the MFGMsamples, BM-MFGM and BS-MFGM, the PL/DMwas increased about 2.5 times compared to that ofthe starting materials, BMP and BSP. BS-MFGM dif-fered by almost three times higher PL/TL and PL/DMthan BM-MFGM as consequence of high content ofPLs in the initial BS. BMW-MFGM, contrary toBM-MFGM and BS-MFGM, was significantly lower(p< 0.05) in content of TP/DM, and higher in contentof TL/DM due to different composition of the initialmaterial (BMW). The PL/TL and PL/DM content ofBMW-MFGM, compared with the BMW, were signifi-cantly increased (p< 0.05), about 1.2 and 8.3 times,respectively. The successful concentration of PLsobtained by MF of BMW indicated that this materialalso can be used as a suitable source for isolation ofMFGM (Rombaut et al., 2007). Similar PL compos-ition of the MFGM material extracted from fractionsof fat globules of different sizes using MF of raw milkwas found by Fauquant et al. (2007). They found thatthe proportion of PL in the TL was higher in theMFGM of large fat globules than in that of small oruntreated fat globules, probably due to their the higherchurning ability.

SDS-PAGE shows that MFGM proteins were pre-sent in considerable concentrations in samples obtainedby MF (Figure 1). However, despite the addition of cit-rate, significant concentrations of caseins and whey pro-teins in MFGM materials were still observed. MFGMisolated from raw milk which mainly contained specificMFGM proteins, while the MFGM derived from BMPcontained high ratios of non-MFGM proteins, such ascaseins and whey proteins (Le et al., 2010). However,the level of caseins after MF of BM, which includedconcentration and two diafilitration steps, was reducedcompared to the other proteins present in the retentate,but still represented about 30% of TPs (Corredig et al.,2003). The presence of high whey protein content andcaseins in different MFGM materials is probably con-sequence of self-aggregation or their binding to theMFGM proteins due to historical, especially heat treat-ments during production of the initial materials (Cano-Ruiz and Richter, 1997; Corredig and Dalgleish, 1997;Ye et al., 2004). The SDS PAGE gel (Figure 1) showedthat MFGM proteins were concentrated during MF ofBMW, but the obtained BMW-MFGM also containedsignificant content of caseins and whey proteins. Thisactually confirmed that these caseins were alreadyattached to the MFGM due to historical treatmentsduring production of the powders. To further decreasethe concentrations of caseins and whey proteins, a mem-brane with higher pore sizes or increase level of diafil-tration can be tested. However, an increase inmembrane pore sizes is expected to result in a loweryield of MFGM fragments in the retentate.

14MW 1 2 3 4 5 6 7 8 9 10 11 12

Caseins

21

31

36

55

66 PAS 6/7ADPH

CD36

MUC1XOPAS III

BTN

97

116

200

Lactoferrin

α-Lactalbumin

β-Lactoglobulin

BSA

Figure 1. The SDS-PAGE of different materials. MW: molecular weight standard; Lane 1: skim milk powder; Lane 2: BMP;Lane 3: BSP; Lanes 4,5: BMW; Lanes 6–8: BM-MFGM; Lanes 9, 10: BS-MFGM; Lanes 11, 12: BMW-MFGM. The gel wasstained with Coomassie blue. SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; BM: buttermilk;BMP: buttermilk powder; BSP: butter serum powder; BMW: buttermilk whey; MFGM: Milk fat globule membrane.

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Emulsions properties

Particle size distribution. The particle size distribu-tions of the emulsions homogenized at 30/20 bar and0/10 bar with BMP, BM-MFGM, BMW-MFGM incombination with BMP (BMW-MFGM/BMP), BSPand BS-MFGM after dilution in water or 1% SDSsolutions are shown in Figures 2–4. The average par-ticle sizes expressed as Sauter mean diameter (d3.2)of emulsions made at higher pressure are shown inTable 3.

Particle size distributions of emulsions made withBMP, BSP and BMW MFGM/BMP at higher hom-ogenization pressure (30/20 bar) were quite differentafter incubation with SDS solution compared withwater before measuring (Figures 2–4.). These emulsionshad a smaller particle size during measurements afterdilution in SDS solution then in water. This indicatedthat the emulsions made with commercial BMP andBSP probably did not have enough surface-activematerials and that bridging between the droplets dueto the sharing of the surface-active componentsbetween droplets when the emulsions were made atthe higher pressure. The better emulsifying propertiesof MFGM isolates compared to BMP concentrate waspreviously found (Roesch et al., 2004). BMW-MFGM/

BMP did not show superior emulsifying properties asexpected due to the high content of surface-activematerials, mainly phospholipids. Explanation for thisis probably the use of significant amounts of BMP inthe preparation of emulsions which contributed about1.5 times more in dry matter than BMW-MFGM.

However, emulsions made with MFGM materials atthe both measurements, in water and SDS solution,were characterized by smaller particle size expressedas Sauter mean diameter (Table 3). Corredig andDalgleish (1997) found that the increase in MFGM iso-late concentration in making soybean oil emulsions canresult in a decrease of average droplet sizes (d3.2).

All emulsions prepared under the low homogeniza-tion pressure (0/10 bar) with different materials did notshow shift in particle size distributions after incubationwith SDS (Figures 2–4) indicated that there was noaggregation of droplets. However, the large particlesize indicated on the relatively poor stability of emul-sion as a result of low applied pressure. In furtherresearch, it is necessary to do more experiments withdifferent homogenization pressure, and analyze the sta-bility of emulsion depending on the material used.

However, to date, what components of MFGM,whether proteins or phospholipids or both, as well astheir interactions, are responsible for emulsifying

0

2

4

6

Vol

ume

(%)

8

10

0.01 0.1 1 10

Diameter (μm)

100 1000

Figure 2. Particle size distributions of emulsions prepared with BMP (*) and BM-MFGM (¨) at 30/20 bar and diluted inwater; BMP («) and BM-MFGM (#) at 30/20 bar diluted in SDS solution; BMP (*) and BM-MFGM (�) at 0/10 bar diluted inwater; BMP (�) and BM-MFGM (~) at 0/10 bar diluted in SDS solution.BM: buttermilk; BMP: buttermilk powder; MFGM: milk fat globule membrane; SDS: sodium dodecyl sulfate polyacryl-amide gel electrophoresis.

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activity is still unclear. Although proteins containhydrophilic and hydrophobic groups in their primarystructure, there are no clearly defined hydrophilic headand hydrophobic tail as found in low molecule weight

surface-active components such are phospholipids(Miura et al., 2006). The emulsifying properties ofMFGM materials are strongly dependent on the char-acteristics of the materials used, especially in regard to

0.00

2.00

4.00

6.00

Vol

ume

(%)

8.00

10.00

0.01 0.1 1 10

Diameter (μm)

100 1000

Figure 4. Particle size distribution of emulsions prepared with BMW-MFGM/BMP («,#) at 30/20 bar diluted in water andSDS, respectively; BMW-MFGM/BMP (*,�) at 0/10 bar diluted in water and SDS, respectively.BMW: buttermilk whey; BMP: buttermilk powder; MFGM: Milk fat globule membrane; SDS: sodium dodecyl sulfate.

0

2

4

6V

olum

e (%

)

8

0.01 0.1 1 10

Diameter (μm)

100 1000

Figure 3. Particle size distribution of emulsions prepared with BSP (*) and BS-MFGM (¨) at 30/20 bar and diluted inwater; BSP (�) and BS-MFGM (~) at 30/20 bar diluted in SDS solution; BSP (*) and BS-MFGM (�) at 0/10 bar diluted inwater; BSP («) and BS-MFGM (#) at 0/10 bar diluted in SDS solution.BS: butter serum; BSP: butter serum powder; MFGM: Milk fat globule membrane; SDS: sodium dodecyl sulfate.

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the composition of phospholipids and proteins, meth-ods of their isolation and preparation, the history ofraw materials and concentration used (Corredig andDalgleish, 1997; Corredig et al., 2003; Roesch et al.,2004).

Flow behavior. Results of viscosity measurements ofdifferent emulsions prepared at 30/20 bar are shown inFigure 5. Using of different emulsifying materialsresulted in a difference in rheological behavior.Emulsions made with BMP and BSP showed a moreshear thinning behavior during the shearing. Theapparent viscosity decreased with increasing shearrates, which could be a consequence of disruption ofaggregates. van Lent et al. (2008) reported that all sam-ples of reconstituted dairy creams made with skim milkpowder had a much higher apparent viscosity com-pared to samples of fresh cream and cream preparedwith phospholipids-rich cream residue powder. Theyfound that these skim milk-stabilized recombinedcreams were characterized by formation of clusters,probably as a consequence of low amount of free pro-tein because the fat droplets were covered with thicklayer of proteins. Similarly, creams formulated withsweet BM, butter-derived aqueous phase (2-3 timesmore lipids then BM) and low-melting butter oil werelower in viscosity compared to emulsions made withskim milk (Scott et al., 2003). It is known that the com-position of emulsions, such as dry matter, fat and pro-tein content, as well as concentration of emulsifyingmaterials, strongly influence on their rheological prop-erties (Euston and Hirst, 2000; Prentice, 1972).

Table 3. Sauter mean diameters of emulsions preparedwith different emulsifying materials at 30/20 bar measuredafter dilution in water and 1% SDS solution

Emulsifyingmaterials

d3.2 (mm)after dilutionin water

d3.2 (mm)after dilutionin SDS solution

BMP 1.81� 0.22 aA* 1.02� 0.07 aB

BM-MFGM 0.70� 0.02 bA 0.71� 0.07 bA

BSP 1.52� 0.06 acA 0.99� 0.18 aB

BS-MFGM 0.65� 0.03 bA 0.66� 0.03 bA

BMW-MFGM/BMP 1.22� 0.21 cA 0.70� 0.02 bB

BM: buttermilk; BS: butter serum; BMP: buttermilk powder; BSP:butter serum powder; BMW: buttermilk whey; MFGM: milk fatglobule membrane; SDS: sodium dodecyl sulfate.*Values in the same column which share a common regular super-script are not significantly different (at a¼ 5%); values in the samerow which share a common capital superscript are not significantlydifferent (at a¼ 5%).

0

1

2

3

Vis

cosi

ty (

Pas

)

6

5

4

0.01 0.1 1 10

Diameter (μm)

100 1000

Figure 5. Apparent viscosity of emulsions made at 30/20 bar in function of shear rates: with BMP (#), BSP (�), BM MFGM(«) and BS MFGM (*) as emulsifying materials.BM: buttermilk; BMP: buttermilk powder; BSP: butter serum powder; MFGM: Milk fat globule membrane; BS: butterserum.

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The different behavior of emulsions made with differentmaterials in this study were probably consequence ofdifferent composition, especially emulsifying compo-nents, such as PLs (Table 2). Similar results were pre-sented in the study of Scott et al. (2003). The increase inthe fluidity of emulsions in this study is probably resultof the higher phospholipids and unsaturated fatty acid

content (palmitoleic, oleic and linoleic acid) of above-mentioned materials (Scott et al., 2003).

Microscopy analysis. Microscopy observation of emul-sions made at 30/20 bar is shown in Figure 6.Observation of emulsion microstructure enables thedetermination of differences in the appearance and

(a) (b)

(e)

(c) (d)

Figure 6. Microscopy images of emulsions prepared at 30/20 bars with different emulsifying materials: (a) BMP; (b) BM-MFGM; (c) BSP; (d) BS-MFGM; (e) BMW-MFGM/BMP. The bars indicate 200 mm.BM: buttermilk; BMP: buttermilk powder; BSP: butter serum powder; MFGM: milk fat globule membrane; BS: butterserum; BMW: buttermilk whey.

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distribution of particles among emulsions made with dif-ferent materials. The images indicate that emulsionsmade with BMP, BSP and BMW-MFGM/BMP con-tained large aggregates pointing out an extensive floccu-lation. This observation is in agreement with thepreviously shown data (Figures 2–4 and Table 3).Emulsions prepared with BM-MFGM and BS-MFGMshowed individual droplets with no sign of droplet floc-culation. Emulsions made with BM-MFGM at muchhigher homogenization pressures (150/50 bar or 70/20 bar) did not show presence of aggregated droplets,while those made with BMP and BSP resulted in forma-tion a bigger aggregates, probably due to shortage ofsurface-active materials (data not shown). In thisstudy, all emulsionsmade at lower homogenization pres-sure (0/10 bar) did not show any differences influencedby materials used, nor the presence of aggregates. Thesedata confirmed our previously described results obtainedby light scattering analysis of particle size distribution.

CONCLUSION

Isolation of MFGM fragments from different dairyproducts can be done by application of MF processwith the addition of sodium citrate. Significantlyhigher content of MFGM components, especiallyphospholipids, in the isolated materials was found.However, the retention of proteins was still high despitethe addition of citrate before MF process.

Use of different emulsifying materials, namely BM,BS and their derived MFGM materials resulted in dif-ferences in particle sizes, viscosity and microstructureof the prepared emulsions. Emulsions prepared withMFGM materials showed smaller particle sizes com-pared to them prepared with BM and BS. The latteremulsions prepared at 30/20 bar showed presence ofdroplet aggregates while this was not the case for theformer emulsions. Significant difference between prop-erties of emulsions made with BM-MFGM andBS-MFGM was not found. The combination ofBMW-MFGM and BMP did not show expected emul-sifying properties, due to a high ratio of the BMP drymatter over than BMW-MFGM in emulsions prepar-ation. Generally, it can be concluded that emulsionsprepared with various material MFGM materialsshowed different emulsifying behavior than commer-cials powders. Selection of dairy materials forMFGM isolation is very important and depends onthe desired properties and composition as well as poten-tial uses of the obtained products.

ACKNOWLEDGMENTS

The authors would like to thank FrieslandCampinaProfessional (Lummen, Belgium) for providing the samplesof buttermilk and butter serum powder.

CONFLICT OF INTEREST

None declared.

FUNDING

The research stay of Jelena Miocinovic on the Ghent

University was funded by BASILEUS PROJECT-EM ECWProgramme approved by European Commission.

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