Food Research International 91 (2017) 26–37

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Lipid droplets coated with milk fat globule membrane fragments:

Microstructure and functional properties as a function of pH
Christelle Lopez ⁎, Chantal Cauty, Florence Rousseau, Marielle Blot, Antinéa Margolis, Marie-Hélène Famelart
STLO, INRA, Agrocampus Ouest, 35000 Rennes, France

a r t i c l e i n f o a b s t r a c t

Article history: Processed lipid droplets coated by milk fat globule membrane (MFGM) material are of primary interest to mimic
Received 23 August 2016 the specific functions provided by the fat globules in milk and dairy products. The objectives were to investigate,
Received in revised form 12 November 2016 as a function of pH, the properties and microstructure of MFGM-coated lipid droplets prepared with an ingredient
Accepted 16 November 2016
rich in MFGM containing polar lipids and proteins. The samples were prepared in water and in milk ultrafiltrate.
Available online 20 November 2016
The combination of microscopy techniques, zeta potential and particle size measurements, and rheological deter-
Keywords:
minations was used. We showed that all the components of the ingredient were highly sensitive to pH. Both the
Milk polar lipid polar lipids and proteins contributed to the isoelectric point of the MFGM-rich ingredient at pI = 4.2. Lipid drop-
Sphingomyelin lets were coated with MFGM fragments both adsorbed at the surface of fat and protruding in the aqueous phase.
Milk fat globule membrane Below pH 5.5 the microstructure of the emulsions was affected by aggregation of the lipid droplets and formation
Emulsion of a gel. The emulsions prepared in water did not show coalescence upon 30 days storage, while those prepared in
milk ultrafiltrate showed coalescence for pH below 5.5. This study demonstrates that the MFGM-rich ingredient
has excellent emulsifying properties and will contribute in the development of emulsions containing MFGM-
coated lipid droplets for techno-functional, nutritional and health benefits (e.g. in infant formulas).
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Oil in water emulsions are widely consumed in various processed
food products, e.g. desserts, mayonnaise, dressing, dairy creams.
Among these food emulsions, milk is a natural oil in water emulsion
where the lipid droplets, called milk fat globules, are biological entities
secreted by the mammary epithelial cells (Wooding, 1971). The milk
fat globules have a core of triacylglycerols (TAG) enveloped by a biolog-
ical membrane, known as the milk fat globule membrane (MFGM). The
unique composition and organization of the MFGM provides specific
technological, nutritional and biological functions to milk fat globules.
Preparing processed food emulsions containing lipid droplets covered
by MFGM components is therefore a challenge attracting many atten-
tions of food scientists and dairy companies.
The MFGM supply bioactive molecules, i.e. proteins and lipids (Jensen
& Newburg, 1995; Fong, Norris, & MacGibbon, 2007), organized with a
specific architecture corresponding to a trilayer of polar lipids integrating
membrane-specific proteins (Dewettinck et al., 2008; Lopez, 2011). The
main MFGM proteins are xanthine oxidase, adipophilin, fatty acid binding
proteins and glycoproteins such as butyrophilin, lactadherin, CD36, MUC-
1 and MUC-15 that are rich in sialic acid (Fong et al., 2007). The MFGM
⁎ Corresponding author at: INRA, STLO, 65 rue de Saint-Brieuc, 35000 Rennes, France.
E-mail address: Christelle.Lopez@inra.fr (C. Lopez).
contains cholesterol as the main milk sterol. Among the polar lipids, the
MFGM contains glycerophospholipids (phosphatidylcholine, PC; phos-
phatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylserine,
PS) and sphingolipids such as milk sphingomyelin (MSM) and
glycosphingolipids (cerebrosides, gangliosides) (Lopez, 2011; Garcia et
al., 2012).
The MFGM has gained interest as a functional element and as a bio-
logically active milk fraction with positive health effects. The MFGM is a
natural emulsifying agent that assures the physical stability of fat glob-
ules in milk (Lopez, 2011). It is involved in the interfacial mechanisms,
such as milk lipid digestion by the lipolytic enzymes (Garcia, Antona,
Robert, Lopez, & Armand, 2014; Berton et al., 2012). Recent studies
showed in humans that milk fat surrounded by the intact MFGM does
not impair the plasma lipoprotein protein profile nor the cholesterol
metabolism, in contrast to milk fat without MFGM, i.e. butter oil
(Rosqvist et al., 2015). Many beneficial functions are also attributed to
individual MFGM components i.e. lipids, proteins (see reviews
Spitsberg, 2005; Dewettinck et al., 2008; Lopez, 2011), and to MFGM-
rich products such as buttermilk (Conway, Couture, Gauthier, Pouliot,
& Lamarche, 2013). Some MFGM glycoproteins have shown activities
that might protect immature infants against gut-derived pathogen in-
fections (Zavaleta et al., 2011; Liu & Newburg, 2013; Timby et al.,
2015). Glycosphingolipids have been shown to contribute to the devel-
opment of the brain and gut immune system of infants. Milk
sphingomyelin and its metabolites play a role in gut maturation, in

http://dx.doi.org/10.1016/j.foodres.2016.11.025
0963-9969/© 2016 Elsevier Ltd. All rights reserved.
 C. Lopez et al. / Food Research International 91 (2017) 26–37
myelination of the developing central nervous system in the newborn
and inhibit intestinal absorption of cholesterol (Oshida, Shimizu,
Takase, Tamura, & Yamashiro, 2003; Motouri et al., 2003; Noh & Koo,
2004; Tanaka et al., 2013).
Milk fat globules enveloped by their biological membrane, i.e. the
MFGM, are far from being always consumed in dairy products since
technological processes used in the food industry lead to the formation
of processed lipid droplets devoid of the MFGM (Lopez, Cauty, &
Guyomarc'h, 2015). Breast-fed infants and infants receiving milk from
human milk bank ingest secreted milk fat globules (4 to 5 μm diameter)
covered by the MFGM (Lopez & Ménard, 2011; Gallier et al., 2015).
However, most of infant milk formulas contain processed lipid droplets
(0.5–1 μm diameter) mainly covered by milk proteins and soya lecithin
and does not contain the MFGM (Lopez et al., 2015; Gallier et al., 2015).
Infant milk formulas could be improved by the addition of bovine
MFGM material adsorbed at the surface of processed lipid droplets to
be closer to the human milk fat globule interface (concept of biomimetic
fat globules, Lopez et al., 2015). Recent studies revealed the benefits of
infant milk formulas supplemented with a bovine MFGM ingredient,
for example an improved cognitive development (Timby, Domellöf,
Hernell, Lönnerdal, & Domellöf, 2014a) and serum lipid status (Timby,
Lönnerdal, Hernell, & Domellöf, 2014b) of infants compared to a stan-
dard formula. Also, processed milk fat globules mainly from bovine ori-
gin are commercially available lipid entities that are consumed by
infants and adults under various forms such as fluid milk, cream and
cheeses (Lopez et al., 2015). The technological processes involved in
the manufacture of dairy products, mainly homogenization, disrupt
the MFGM and lead to the adsorption of milk proteins, i.e. caseins and
whey proteins, at the surface of fat droplets. Authors demonstrated
the importance of the quality of the interface, in terms of composition
and architecture (MFGM vs. milk proteins), on the mechanisms of fat
globule digestion (Garcia et al., 2014; Berton et al., 2012) and on the mi-
crostructure of the emulsion at acidic pH values (Garcia et al., 2014;
Lopez et al., 2015).
Given all these beneficial functions of the MFGM, the development
of MFGM-coated processed lipid droplets close to the milk lipid biolog-
ical assemblies is therefore of primary interest for food scientists, indus-
try and for health benefits of the consumers. A recent study provided a
proof of concept that the large size and the presence of MFGM compo-
nents at the surface of processed lipid droplets that are consumed in in-
fant milk formulas early in the life of infants reduce fat accumulation
and improve metabolic health later in their life, and then that the struc-
ture and interfacial composition of lipids contribute to metabolic pro-
gramming (Oosting et al., 2012).
The opportunities for the use of added-value ingredients rich in
MFGM, recycled from by-products of butter and anhydrous milk fat pro-
duction and tailored for specific applications, exist and should be further
considered for making healthier foods in a more sustainable manner
(van der Goot et al., 2016). In this respect, the use of MFGM-rich ingre-
dients in the manufacture of products, such as new concept of infant
milk formulas containing MFGM-coated lipid droplets (Oosting et al.,
2012; Gallier et al., 2015), processed creams and cheeses, needs further
understanding of their functional properties. Few research groups have
investigated the emulsifying properties of MFGM-rich ingredients
(Corredig & Dalgleish, 1997; Corredig & Dalgleish, 1998; Roesch,
Rincon, & Corredig, 2004). Furthermore, emulsion droplets are submit-
ted to changes in pH during the digestion and in various dairy products
such as cheeses, yoghurts or commercial creams. Hence, knowing the
properties of MFGM-coated lipid droplets as a function of pH is of pri-
mary interest.
The objectives of this study were therefore (i) to investigate the
properties of a MFGM-rich ingredient prepared from butter serum
with a process detailed in Gassi et al. (2016), (ii) to evaluate the oppor-
tunity to prepare food emulsions with this ingredient, and (iii) to char-
acterize the microstructure and rheological properties of emulsions and
physical stability of the MFGM-coated lipid droplets, as a function of pH.
27
2. Materials and methods
2.1. Materials
Anhydrous milk fat (AMF; melting point at 32 °C determined by
NMR) was provided by Corman (Corman, Limbourg, Belgium). The in-
gredient rich in milk fat globule membrane (MFGM) used in this
study was obtained from industrial butter serum using a multi-step pro-
cess previously detailed in Gassi et al. (2016). The chemical composition
of this MFGM-enriched ingredient was detailed in Gassi et al. (2016).
Briefly, this ingredient contained 661.2 ± 23.3 g of total fat with
310.1 ± 14.9 g of milk polar lipids per kg powder. The relative propor-
tions of milk polar lipids concentrated in the MFGM-rich ingredient
was 36.5 ± 0.7% milk sphingomyelin (MSM), 26.8 ± 0.3% phosphatidyl-
choline (PC), 20.9 ± 0.2% phosphatidylethanolamine (PE), 8.6 ± 0.2%
phosphatidylserine (PS) and 7.2 ± 0.2% phosphatidylinositol (PI). The
ingredient contained 280.9 ± 9.8 g of milk proteins per kg powder, i.e.
caseins, whey proteins and proteins from the MFGM (Butyrophilin, xan-
thine oxidase, adipophilin, lactadherin PAS6/7). The lipids contained in
the MFGM-rich ingredient, i.e. polar lipids (PC, PE, PI, PS and MSM) and
TAG, were separated from the proteins, by using the cold extraction pro-
cedure developed by Folch, Lees, and Stanley (1957). The protein frac-
tion was freeze-dried and both extracts, i.e. lipids and proteins, were
stored at −20 °C until further analysis. To separate the aqueous phase
of milk from other components (lipids, proteins), skimmed milk was
ultrafiltrated at 50 °C using pilot (TAMI/Tech Sep, Nyons, France)
equipped with 2 × 6.65 m2 of membrane (molecular weight cut-
off = 8 kDa, Tami Industries, Nyons, France). Recovered milk ultrafil-
trate was stored at 4 °C in sterile bottles before use.
2.2. Methods
2.2.1. Preparation of emulsions and their acidification
The powder of the MFGM-rich ingredient was dissolved in milliQ
water or in milk ultrafiltrate under agitation using a magnetic stirrer
during at least 12 h at 19 ± 1 °C for complete rehydration. The concen-
tration of the dispersion was 40 g/kg. Sodium azide was added at 0.2 g/L
to prevent the growth of bacteria. The pH was adjusted to pH 6.7 by
adding 1 M NaOH. To prepare emulsions, AMF was heated at 60 °C to en-
sure its full melting and then added at the concentration of 200 g/kg to
the dispersion of the ingredient previously heated at 50 °C. The mixture
was emulsified using a Polytron PT 3100 (Kinematica AG, Littau, Swit-
zerland) operating at 13000 rpm for 2 min. The emulsion was then ho-
mogenized at 10 MPa by recirculating 5 times at 50 °C, using Emulsiflex
C3 (Avestin, Ottawa, Canada). The emulsions were stored at 19 ± 1 °C
instead of 4–7 °C in the fridge, to avoid physical instability of the lipid
droplets induced by TAG crystallization (on cooling from the melt, the
temperature of milk fat crystallization is about 15 °C). The emulsions
were prepared in triplicate.
The same day of their preparation, the emulsions at pH 6.7 were
acidified by using glucono delta lactone (GDL, Sigma-Aldrich, Lyon,
France). The GDL powder was added to the emulsion to decrease the
pH at the desired value in about 12 h. The pH of the emulsions was mea-
sured with a Mettler Toledo delta 320 pH meter (Mettler, San Francisco,
USA).
2.2.2. Lipid droplet size measurements
The size distribution of the emulsion droplets was determined by
laser light scattering, using a Mastersizer 2000 (Malvern Instruments,
Malvern, U.K.). The refractive indexes of milk fat were set at 1.46 (at
466 nm) and 1.458 (at 633 nm) and the refractive index was set at
1.33 for water. The experiments were performed at room temperature
(19 ± 1 °C). The emulsions were dispersed in SDS solution at 1 g/L be-
fore measurement of particle size distribution. Aliquots of approximate-
ly 70 μL of emulsion were further diluted into 100 mL of water, in order
to reach 8% obscuration. One mL of a 35 mM EDTA/NaOH pH 7.0 buffer
28 C. Lopez et al. / Food Research International 91 (2017) 26–37
(N98% disodium salt dihydrate, Prolabo, Fortenay-sous-Bois, France)
was added to the measurement cell to disrupt the casein micelles. The
diameter of the lipid droplets was calculated by the software.
2.2.3. Zeta potential
The effect of pH on the apparent zeta-potential was investigated, by
preparing several samples: i) the MFGM-rich suspension prepared in
water or milk ultrafiltrate (40 g/kg), ii) the lipid extract of the ingredient
dispersed in water using ultrasonication to form tiny emulsion droplets
covered by polar lipids (13 g/kg), iii) the protein extract of the ingredi-
ent (13 g/kg), and iv) the MFGM-rich emulsions. The samples were
acidified at different pH values using HCl. The electrophorectic mobility
of samples was measured by electrophoretic light scattering using a
Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). The apparent
zeta-potential was calculated as follows: the zeta-potential ζ of a parti-
cle is calculated from its electrophoretic mobility μ, according to Henry's
equation: ζ = (μ·6 π η/ε)/f(κa), where η and ε are the viscosity and di-
electric constant of the solution respectively, at the temperature of the
measurement. “1/κ” is the Debye length and “a” is the radius of the par-
ticle. The Smoluchowski approximation, assuming f(κa) = 1.5 was
used. The experiments were performed at 20 °C, with ε = 79, and
η = 1.0 mPa.s in water and η = 1.187 mPa.s in milk ultrafiltrate. The ap-
plied voltage was adjusted to the sample conductivity: 150 V from 0 to
5 mS/cm−2 (i.e. samples in water) and 50 V above 5 mS/cm−2 (i.e. sam-
ples in milk ultrafiltrate). The pH of the samples was measured after
electrophoretic mobility measurements. All analyses were performed
at least three times for each sample.
2.2.4. Physical stability of the dispersions as a function of pH
The physical stability of the dispersions containing either the
MFGM-rich ingredient, the lipid extract or the proteins from the ingre-
dient were investigated as a function of pH. The dispersions adjusted at
different pH values were centrifuged at 20 °C, at 2400g for 30 min using
a Firlabo centrifuge (SV11TH, Firlabo A.A., Meyzieu, France). The absor-
bance of the supernatant was measured against water at 500 nm in a
Safas UVmc2 model spectrophotometer (Safas, Monaco, France), as re-
ported in Damodaran (2011). All experiments were carried out in
triplicate.
2.2.5. Microstructural analysis
2.2.5.1. Transmission Electron Microscopy. Samples of the MFGM-rich in-
gredient dispersed in water, emulsions at pH 6.7 and raw whole bovine
milk provided by a local dairy plant (Entremont, Montauban de Bre-
tagne, France) were characterized by transmission electron microscopy
(TEM). The samples were collected (v/v) from the agar 1.5% (w/v) after
solidified agar was cut into 1 mm piece then fixed overnight at room
temperature with 25 g/kg glutaraldehyde in 0.1 M Na cacodylate follow-
ed by 4-fold 15 min wash with the Na cacodylate 0.1 M pH 7.2 and then
a final wash with distilled water performed 5-fold for 5 min. The sam-
ples were post-fixed in 1% osmium tetroxyde in 0.1 M Na cacodylate
for 1 h at room temperature followed by 4-fold 15 min wash with the
Na cacodylate 0.1 M pH 7.2 and then washed 5-fold for 5 min with dis-
tilled water. Samples were dehydrated in a graded series of ethyl alco-
hol concentration, 50° for 30 min, 70° for 1 h, 80° for 1 h, 95° for 1 h,
100° 3 times for 30 min, then stored overnight at room temperature
in 100° and infiltrated with Epon (Electron Microscopy Sciences Epon
Kit 14120) in hydroxyl-propyl methacrylate (HPMA) and embedded
in Epon with 2.5% v/v BDMA resin mixture, then finally polymerized
at 60 °C for 24 h. Ultra-thin sections with a thickness of 90 nm were
cut using a Leica ultra-microtome and stained with 4% uranyl acetate
for 1 h. Sections were observed with a JEOL 1400 Transmission Electron
Microscope operating at 120 kV and images were recorded on camera
Gatan Orius SC 1000.
2.2.5.2. Confocal laser scanning microscopy. An inverted microscope
NIKON Eclipse-TE2000-C1si (NIKON, Champigny sur Marne, France)
was used for the confocal laser scanning microscopy (CLSM) experi-
ments. The microstructural analyses were performed using an argon
laser operating at an excitation wavelength of 488 nm with emission
detected between 500 nm and 530 nm, a He-Ne laser operating at
543 nm wavelength excitation and emission detected between
565 nm and 615 nm, and a diode operating at 633 nm with emission de-
tected with a long pass filter N650 nm. The observations were per-
formed using a 100 × (numerical aperture NA 1.4) oil immersion
objective. The staining protocols followed previously described
methods (Lopez, Madec, & Jimenez-Flores, 2010; Lopez & Ménard,
2011). Briefly, Nile Red (5H-Benzo α-phenoxazine-5-one, 9-
diethylamino, supplied by Sigma-Aldrich, St. Louis, USA) was used to
stain the triacylglycerol core of the fat globules, Fast Green FCF
(Sigma-Aldrich, St. Louis, USA) was used to stain proteins, N-(Lissamine
rhodamine B sulfonyl) dioleoylphosphatidyl ethanolamine (Rh-DOPE,
concentration of 1 mg/mL in chloroform; Avanti polar lipids Inc., Bir-
mingham, England) was used to label the phospholipids, wheat germ
agglutinin Alexa fluor 488 (WGA488, Invitrogen, Cergy Pontoise,
France) was used to label the glycosylated molecules from glycopro-
teins and glycolipids of the MFGM, i.e. carbohydrate moieties containing
N-acetylglucosamine and N-acetyl neuraminic acid (sialic acid) resi-
dues. Single (e.g. Rh-DOPE) and multiple labelling (e.g. Nile red with
Fast green, Nile Red with WGA, Rh-DOPE with WGA) of emulsion com-
ponents were performed. The stained samples were kept at room tem-
perature for at least 30 min prior to observation by CLSM. The
microstructural analyses were performed at room temperature (19 ±
1 °C). The two dimensional images had a resolution of 512 × 512 pixels
and the pixel scale values were converted into micrometers using a scal-
ing factor.
2.2.6. Rheological properties
Flow measurements were applied on emulsions at 20 °C with a
MCR301 rheometer (Anton Paar France SAS, Les Ulis, France) using a
cone-plan geometry to avoid disturbing the overall sample structure.
After a 5 min storage of the sample at 20 °C without shearing, the strain
rate was increased from 1 to 200 s−1 in a logarithm progression, and
then decreased the same way. Dynamic viscosity was deduced from
the stress measurements. Low amplitude oscillation rheology was ap-
plied on emulsions at 20 °C at different pH values. A stress sweep
from 0.01 to 200 Pa at 1 Hz was applied on each sample to determine
the optimum stress value, sufficiently high to get significant oscillation,
but within the linear viscoelastic domain. A frequency sweep from 10 to
0.01 Hz was finally applied at 20 °C by applying this optimal stress value.
3. Results and discussion
3.1. Microstructure of the ingredient and physical stability as a function of
pH
3.1.1. Microstructural analysis of the MFGM-rich ingredient
TEM images showed the presence of MFGM fragments, proteins and
polar lipid vesicles in the fully hydrated ingredient (Fig. 1 A–E). After
disruption of milk fat globules upon churning, the MFGM may corre-
spond to a surface. The ultra-thin sections of samples prepared for
TEM experiments showed elongated structures corresponding to thin
sections of MFGM that were related to MFGM fragments. The electron
dense material in MFGM fragments corresponds to membrane proteins
and unsaturated fatty acids of milk polar lipids stained by osmium. Pro-
teins attached to the MFGM fragments protruded in the aqueous phase
and could therefore correspond to glycoproteins that are the main pro-
teins of the MFGM known to form a glycocalix around fat globules (Fig.
1-C). The butyrophilin which is the main transmembrane glycoprotein
of the MFGM could actively participate in the physical stability of
MFGM fragments. Proteins were also observed to connect two MFGM
 C. Lopez et al. / Food Research International 91 (2017) 26–37 29

Fig. 1. Transmission electron microscopy images of the dispersion of the milk fat globule membrane rich ingredient prepared in water at the concentration of 40 g/kg (top), of the
emulsions prepared with the ingredient (bottom) and of the surface of a bovine milk fat globule (bottom right). Large black arrows show the MFGM around a fat globule in bovine
milk. Large white arrows show MFGM material attached at the surface of the emulsion droplets and protruding in the aqueous phase. The scales bars are indicated in the figures.

fragments (Fig. 1-C). The heat treatments applied to the cream before proteins. The morphology of the MFGM fragments contained in the in-
the manufacture of butter and applied to industrial butter serums dur- gredient was in agreement with previous TEM observations performed
ing the preparation of the ingredient (92 °C, 3 min; Gassi et al., 2016) in buttermilks (Corredig & Dalgleish, 1997; Morin, Jiménez-Flores, &
might have favored interactions between MFGM proteins and milk Pouliot, 2007; Lopez et al., 2015) and in industrial butter serums
30 C. Lopez et al. / Food Research International 91 (2017) 26–37

(Lopez et al., 2015; Gallier et al., 2015; Lambert et al., 2016). TEM images
revealed the presence of spherical particles corresponding to vesicles
with a diameter about 50 to 600 nm in which milk polar lipids originat-
ing from the MFGM are organized as bilayers (Fig. 1-B). Vesicles formed
by MFGM fragments have also been observed in Gallier et al. (2015).
Very few casein micelles were observed in the MFGM-rich ingredient,
which is different from what was reported in buttermilk and butter
serum (Lambert et al., 2016; Gallier et al., 2015). This is due to the
acid gelation used in the process to remove casein micelles and concen-
trate the MFGM fragments in the acid serum (Gassi et al., 2016).
3.1.2. Physical stability of the MFGM-rich ingredient as a function of pH
The physical stability of the MFGM-rich ingredient and of the lipid
and protein extracts of the ingredient was investigated as a function of
pH, by performing observations of the samples after centrifugation
combined with measurements of absorbance of the supernatants
(Fig. 2). Changes in the absorbance of the MFGM-rich ingredient were
observed as a function of pH (Fig. 2-A). For pH values ranging from 7
to 5.5, the absorbance of the ingredient samples remained constant.
The absorbance increased from pH 5.5 to 4.6, which may correspond
to an increase in the size of the particles due to aggregation but not

Fig. 2. Physical stability of the milk fat globule membrane (MFGM) rich ingredient and of the lipid and proteins extracts of the ingredient investigated as a function of pH. A: Images of the
tubes after centrifugation at 2400 g for 30 min of the MFGM ingredient in water adjusted at different pH values, combined with measurements of the absorbance of the supernatant
measured at 500 nm. B: Images of the tubes containing the lipid extract of the ingredient in water adjusted at different pH values taken after centrifugation, C: Images of the tubes
containing the protein extract of the ingredient in water adjusted at different pH values taken after centrifugation.
 C. Lopez et al. / Food Research International 91 (2017) 26–37 31

sufficiently large to induce particle sedimentation. In water, the absor-
bance of the supernatant was low between pH 4.5 and 3.5 with a mini-
mum measured at pH 4, in accordance with the observation of
sedimentation after centrifugation of the samples (Fig. 2-A). For pH
below 3.5, the turbidity of the ingredient samples may be related to a
solubilization of components in water. In milk ultrafiltrate, we observed
a high increase of the turbidity of the samples for 3.5 ≤ pH ≤ 4.5 which
may be due to an aggregation between the components with an in-
crease in size (Fig. 2-A). The sedimentation of the sample was not
observed when the MFGM-rich ingredient was dispersed in milk ultra-
filtrate (not shown). The same experimental procedure was applied to
the lipid extract of the ingredient dispersed in water (Fig. 2-B). Phase
separation with sedimentation of lipid vesicles was observed for
pH = 2. The absorbance of the supernatant of the lipid extract samples
remained constant from pH 6.7 to pH 3, decreased at pH 2 in agreement
with sedimentation and then re-increased at pH b 2 (results not
shown). The high absorbance observed for pH = 1.5 was related to
the solubilization of lipid particles (not shown). The sedimentation of

Fig. 3. Changes in the zeta potential and microstructure of the milk fat globule membrane rich ingredient and of the lipid and protein fractions contained in this ingredient, investigated as a
function of pH. A: Zeta potential as a function of pH. Bars give the standard deviation (n = 3 experiments). B, C, D: Microstructure as observed by confocal laser scanning microscopy. The
proteins were stained with fast green FCF (green), the lipids were stained with nile red (red). The pH values of the samples and the scale bars are indicated in the figures. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)
32 C. Lopez et al. / Food Research International 91 (2017) 26–37
the proteins contained in the ingredient (i.e. MFGM proteins, caseins,
whey proteins; Gassi et al., 2016) occurred below pH 5.5 (Fig. 2-C).
3.1.3. Zeta potential as a function of pH combined with microstructural
analysis
Zeta potential values of the dispersion of the MFGM-rich ingredient,
and of the lipid and protein extracts contained in the ingredient were
characterized as a function of pH (Fig. 3). The measurements of zeta po-
tential as a function of pH were combined with microstructural analysis
performed by confocal microscopy.
Zeta potential of the MFGM-rich ingredient was negative at pH 6.7,
i.e. at milk pH, which means that the overall charge of the components
was negative (Fig. 3-A). The decrease in pH decreased the net charge of
the ingredient. This was interpreted as the increase in the proton
amount that acted on the negative charges brought by the ingredient
components, i.e. proteins and polar lipids, and to the regression of ioni-
zation of negative groups. The isoelectric point of the MFGM-rich ingre-
dient was pI = 4.2 with no significant difference between water and
milk ultrafiltrate as solvent phase (Fig. 3-A). This value is in agreement
with previous studies reporting pI ~ 4 to 5 for the MFGM (Kanno & Kim,
1990; Corredig & Dalgleish, 1998; Malik, Danthine, Paul, & Blecker,
2015). The zeta potential of the MFGM-rich ingredient was negative
for pH higher than pI and positive for pH lower than pI (Fig. 3-A). Micro-
structural analyses revealed the formation of complexes between lipids
and proteins for pH around the isoelectric point of the ingredient (Fig. 3-
D). The aqueous phase of the samples was non-fluorescent, meaning the
absence of lipids and proteins. Hence, lipid-protein complexes could
correspond to the aggregation of MFGM fragments and milk proteins
through protein-protein interactions. These experiments demonstrated
that MFGM components have minimum solubility in aqueous phase at
pH corresponding to their pI, which explains their aggregation and sed-
imentation in water for pH ranging from 4.2 to 3.5 (Figs. 3 and 2). For pH
below 3.5, the CLSM images showed a homogeneous distribution of
lipids with some aggregates of proteins (Fig. 3-D), in accordance with
the absorbance measured (Fig. 2A).
The zeta potential of the lipid extract was negative at pH 6.7 (Fig. 3-
A), which is due to the anionic polar lipids PS and PI, and decreased in
absolute value when pH decreased. The isoelectric point determined
for the lipid extract of the ingredient was pI = 2. CLSM images showed
that the spatial distribution of lipid vesicles was homogeneous for
pH 6.7 and below (Fig. 3-C). Near the isoelectric point of milk polar
lipids, i.e. pH 2, the lipid particles aggregated due to a lack in electrostat-
ic repulsions (Fig. 3-C), and sedimented as observed in Fig. 2B.
The decrease in pH induced a decrease in the absolute value of the
zeta potential of the proteins (Fig. 3A). The isoelectric point determined
for the proteins was pI = 5. CLSM images showed an aggregation for
pH = 5 and pH = 4, which is close to the pI of the proteins contained
in the ingredient (Fig. 3-B). This aggregation of the proteins explained
the sedimentation observed for pH values below 5.5 (Fig. 2C). Authors
reported that isolated MFGM proteins have isoelectric points ranging
from pH 3.5 to 7.6 (Mather, Tamplin, & Irving, 1980; Jack & Mather,
1990; Kim, Kanno, & Mizokami, 1992). Butyrophilin, one of the most
abundant MFGM proteins, is characterized by an isoelectric point of
4.96 (Jack & Mather, 1990) and could explain the pI observed for the
proteins contained in the ingredient.
The isoelectric point characterized for the MFGM-rich ingredient, i.e.
pI = 4.2, is intermediate between those determined for the polar lipids
(PC, PE, PI, PS, MSM) and the proteins (MFGM proteins and milk pro-
teins), showing that all these components affect the physical stability
of the ingredient with pH. The observed pI value for the MFGM-rich in-
gredient is in agreement with the hypothesis put forward by Kanno and
Kim (1990) of an isoelectric point for the whole MFGM of pI ~ 4.8. These
results showed that the MFGM-rich ingredient is sensitive to pH and
that its physical stability results from both polar lipids and proteins be-
haviors as a function of pH. Whatever the medium of dispersion, i.e.
water or milk ultrafiltrate, the physical instability of the ingredient
was observed at 3.5 ≤ pH ≤ 4.5.
3.2. Emulsions prepared with a MFGM-rich ingredient: microstructure and
rheology as a function of pH
3.2.1. Microstructure of the emulsions at pH 6.7
The size distribution of the emulsion droplets prepared at pH 6.7 in
water or in milk ultrafiltrate, under a homogenization pressure of
10 MPa, ranged from 0.03 μm to 3 μm with a modal diameter of
0.83 ± 0.01 μm (Fig. S1-A). The lipid droplets were not aggregated.
The combination of microscopy techniques, i.e. TEM and CLSM with
various fluorescent probes, was used to investigate the microstructure
of the surface of the lipid droplets (Figs. 1, 4, 5). TEM images showed
the presence of a thin layer of dark dense material around the TAG
core of the lipid droplets prepared with the MFGM-rich ingredient
(Fig. 1 F–I). This thin layer resembles the MFGM present around bovine
milk fat globules (Fig. 1) suggesting similar organization and is consis-
tent with pioneering TEM images showing the structure of the MFGM
(Wooding, 1971). We interpreted the dense line as MFGM material
adsorbed at the surface of the lipid droplets. The thickness of MFGM ma-
terial surrounding lipid droplets (Fig. 1 F–H) was homogeneous. More-
over, TEM images revealed that MFGM fragments were attached at the
surface of the lipid droplets and protruded in the aqueous phase.
MFGM fragments were also detected in the aqueous phase of emulsion
(arrows in Fig. 1 G–I). This structural analysis showed that the MFGM
fragments present in the ingredient were not disrupted during the ho-
mogenization process. This is in agreement with structural analysis re-
cently performed on MFGM-covered lipid droplets aiming at
mimicking the milk fat globule in infant milk formulas (Gallier et al.,
2015). CLSM images showed that polar lipids, proteins and glycosylated
molecules (glycoproteins and glycolipids from the MFGM) were located
at the surface of lipid droplets, around the TAG core (Fig. 4). Glycosylat-
ed molecules were heterogeneously distributed around the lipid drop-
lets and protruded in the aqueous phase of the emulsion (Fig. 4 A–D).
Some lipid droplets were covered both by polar lipids and glycosylated
molecules and interpreted as MFGM-coated lipid droplets while others
were only covered by polar lipids (Fig. 4 E–G). CLSM images revealed
the heterogeneity that existed between individual lipid droplets and
the inhomogeneity of the emulsion. The combination of TEM and
CLSM images revealed the presence of MFGM fragments at the surface
of lipid droplets (Figs. 1 & 4), as reported at the surface of milk fat glob-
ules (Lopez et al., 2010; Lopez & Ménard, 2011).
3.2.2. Microstructure of the emulsions as a function of pH
Particle size measurements revealed an aggregation of the lipid
droplets below pH 5.5 in water (Fig. S1) and in milk ultrafiltrate (results
not shown). After dissociation of the aggregates with SDS, the size of the
lipid droplets was not significantly different between the emulsions,
meaning that the decrease in pH did not lead to coalescence either in
water (Fig. S1-F) or in milk ultrafiltrate (results not shown).
CLSM investigations showed that the decrease in pH affected the mi-
crostructure of the emulsions (Fig. 5). Aggregation of lipid droplets was
observed for pH b 5, both in water (Fig. 5 A–F) and in milk ultrafiltrate
(Fig. 5 G–I). For pH 4.6, the formation of a network of connected lipid
droplets was observed, leading to a heterogeneous distribution of the
lipid droplets in the volume of the emulsion (Fig. 5 E–F). Some lipid
droplets were covered by a thick layer of proteins in connection with
other proteins and were involved in the formation of the network
(Fig. 5 E). CLSM images showed lipid droplets dispersed in the emulsion
and not covered by a thick layer of proteins (highlighted by arrows in
Fig. 5 F). These lipid droplets may be covered mainly by milk polar
lipids. The microstructure of the network formed below pH 5 appeared
to be different in water and in milk ultrafiltrate. A higher connectivity of
the lipid droplets to form a network through protein-protein interac-
tions was observed in milk ultrafiltrate (Fig. 5 H–I).
 C. Lopez et al. / Food Research International 91 (2017) 26–37 33

Fig. 4. Confocal laser scanning microscopy images of emulsions prepared with a milk fat globule membrane rich ingredient. A–D: Co-localisation of triacylglycerols in the core of the
emulsion droplets with glycosylated components, i.e. glycoproteins, E: Overlay of images showing the co-localisation of glycosylated components (F) and polar lipids (G). H–J: Polar
lipids located at the surface of the emulsion droplets. The triacylglycerols were stained with nile red fluorescent dye, the glycosylated molecules were stained with wheat germ
agglutinin 488, polar lipids were stained with Rhodamine-DOPE. The scale bars are indicated in the figures.

Changes in the microstructure of the emulsions as a function of pH
resulted from interfacial phenomena. The zeta potential of the emul-
sions prepared with the MFGM-rich ingredient was investigated as a
function of pH (Fig. 6A) and combined with structural observations
(Fig. 6B and C). In water, the zeta potential of the emulsion was –
61 ± 1 mV for pH 6.7, meaning that the overall charge of the compo-
nents was negative. In milk ultrafiltrate, the zeta potential of the emul-
sion was − 16 ± 1 mV for pH 6.7, showing the effect of ions on the
interfacial properties of the emulsion droplets. The decrease in pH in-
duced a decrease in the absolute value of the zeta potential measured
for the emulsions. The isoelectric points determined for the emulsions
were pI = 4.3 in water and pI = 4.1 in milk ultrafiltrate (Fig. 6A).
Below pI the overall charge of the emulsions was positive. Our results
are in agreement with Corredig and Dalgleish (1998) who reported an
isoelectric point pI ~4 to 5 for emulsions containing soybean lipid drop-
lets covered by a MFGM isolate. The pI values determined for the emul-
sions were close to the pI values obtained for the MFGM-rich ingredient
(Fig. 3 A). CLSM observations performed in the same samples than those
used for zeta potential measurements showed that i) for pH 4.6 and
above the MFGM-coated lipid droplets were homogeneously distribut-
ed in the volume of the emulsion, ii) that aggregation of the lipid drop-
lets occurred for pI = 4.3 (Fig. 6 B). In water, phase separation was
observed for pH ~ 4 with a concentration of lipid droplets at the top of
the sample (Fig. 6 C). The aggregated lipid droplets separated out of
solution and the lower density of TAG as compared to water explains
their localization at the top of the sample, in contrary to the dispersion
of the ingredient that sedimented for pH 4 (Fig. 3). These results showed
that the lipid droplets covered by the MFGM-rich ingredient had a min-
imum solubility in water at pH values close to their pI. In milk ultrafil-
trate, we did not observe any phase separation as a function of pH
(results not shown).
Decreasing the pH of the emulsions decreased the overall negative
charge on the lipid droplet surface, which decreased the repulsive elec-
trostatic interactions between droplets and favored their aggregation
for pH below 5.5. This is in agreement with the aggregation of MFGM
coated lipid droplets below pH 5 reported by Corredig and Dalgleish
(1998). Then, the pH affects the interfacial properties of the lipid drop-
lets covered by MFGM-rich ingredient and alters their physical stability
with phase separation but absence of coalescence.
3.2.3. Rheological properties of the emulsions as a function of pH
The rheological properties of the emulsions were investigated as a
function of pH (Fig. 7). For pH values ranging from 6.6 to 5.5, the emul-
sions corresponded to visco-elastic liquids, as revealed by the changes
in the storage modulus G′ and loss modulus G″ as a function of the fre-
quency (Fig. 7 A–C). Below an oscillatory frequency of 0.1 Hz, the vis-
cous component of the emulsions dominated, while for higher
frequency values the rheological behavior of the emulsions was more
34 C. Lopez et al. / Food Research International 91 (2017) 26–37

Fig. 5. Microstructure of the emulsions prepared with a milk fat globule membrane rich ingredient investigated as a function of pH. Confocal laser scanning microscopy images showing the
lipids stained in red and the proteins stained in green. A–F: Emulsions prepared in water, G–I: Emulsions prepared in milk ultrafiltrate. The scale bars are indicated in the figures. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

elastic. For pH ≤ 5.2, the storage modulus was higher than the loss mod- ultrafiltrate as compared to those prepared in water. The viscosity of
ulus whatever the oscillatory frequency, indicating the presence of a gel the emulsions increased for pH b 5.5 and was significantly higher in
(Fig. 7 D–E). The moduli were higher in emulsions prepared in milk the emulsions prepared with milk ultrafiltrate as compared to those
 C. Lopez et al. / Food Research International 91 (2017) 26–37 35

Fig. 6. Changes in the zeta potential (A) and microstructure (B) of the MFGM-coated lipid droplets as a function of pH. (C) Phase separation observed as a function of pH.

prepared in water (Fig. 7 F). As a conclusion, both the pH and medium,
i.e. water vs. milk ultrafiltrate, affected the rheological properties of the
emulsions containing MFGM-coated lipid droplets.
3.2.4. Physical stability of the emulsions upon storage
The formation of lipid droplet aggregates for pH b 5.5 in both water
(Fig. S1) and milk ultrafiltrate (not shown) induced a phase separation
in the emulsions during storage, with a concentrated layer of MFGM-
coated lipid droplets at the top and an aqueous phase at the bottom of
the tubes (results not shown).
The physical stability of the emulsions prepared with the MFGM-
rich ingredient either in water or in milk ultrafiltrate and adjusted to
different pH values ranging from pH 6.7 to pH 4.6 was investigated as
a function of time upon storage at room temperature (Fig. S2). In
water, the mean diameter of the lipid droplets did not evolve signifi-
cantly whatever the pH (Fig. S2-A). These results showed an absence
of coalescence of the lipid droplets as a function of time. The decrease
of the negative charges of the lipid droplets (Fig. 6), and their aggrega-
tion below pH 5.4 (Fig. S2), did not alter the physical stability of the
MFGM-coated lipid droplets upon storage. The glycoproteins located
at the surface of lipid droplets and the MFGM fragments attached to
the lipid droplets and protruding in the aqueous phase (Figs. 1 & 4)
may contribute in the physical stability of the emulsion by providing
steric repulsion between the lipid droplets. In milk ultrafiltrate, an
36 C. Lopez et al. / Food Research International 91 (2017) 26–37

Fig. 7. Rheological properties of the emulsions prepared with a milk fat globule membrane rich ingredient. A–E: viscoelastic properties characterized as a function of pH in water (● storage
modulus G′; ○ loss modulus G″) and in milk ultrafiltrate (▲ storage modulus G′; Δ loss modulus G″). F: Viscosity of the emulsions determined at 10 s−1 (● emulsions prepared in water; ▲
emulsions prepared in milk ultrafiltrate).

increase in the size of the lipid droplets was observed as a function of
time, mainly for pH ≤ 5 (Fig. S2-B). This destabilization of the lipid
droplets could be explained by the lower negative charge of the
lipid droplets in milk ultrafiltrate as compared to water (Fig. 6) and
then to a lack of electrostatic repulsions between lipid droplets. We
can conclude that the minerals present in milk ultrafiltrate in addi-
tion with low pH favored the physical destabilization of the emulsion
by coalescence.
4. Conclusions
The preparation of emulsions containing MFGM-coated lipid drop-
lets is of primary interest to mimic the interfacial properties of milk
fat globules and to supply bioactive components for health benefits. In
this study, we showed that the MFGM-rich ingredient used and de-
scribed in Gassi et al. (2016) has excellent emulsifying capacity and
that the properties of the emulsions are affected by pH. Microstructural
investigations revealed the presence of MFGM fragments attached at
the surface of the lipid droplets, as well as polar lipids and glycoproteins.
This work demonstrated that the MFGM components are highly sensi-
tive to pH and that the behavior of the MFGM-rich ingredient results
both from the lipid and protein fractions. Changes in pH affected the mi-
crostructure of the emulsions and their rheological properties. The
emulsions behave as viscoelastic liquids at pH 6.7 and underwent aggre-
gation below pH 5 with the formation of gel and an increase in viscosity.
Such changes in the microstructure and rheology of emulsions contain-
ing MFGM-coated lipid droplets below pH 5 could have functional prop-
erties, e.g. could affect the mechanisms of lipid digestion by limiting the
accessibility of the digestive gastric lipase to the TAG core of the lipid
droplets. This study increased knowledge about the interfacial proper-
ties and microstructure of MFGM-coated lipid droplets as a function of
pH and will undoubtedly contribute in the development of food emul-
sions prepared with MFGM-rich ingredients (e.g. infant milk formulas,
dairy emulsions and spreads).
 C. Lopez et al. / Food Research International 91 (2017) 26–37
Acknowledgements
This study was funded by the French National Agency for Research
(ANR) under the ALID program (project Valobab; ANR-11-ALID-007)
and by CNIEL (French Dairy Interbranch Organization, Paris, France).
The authors thank the scientific and industrial partners of Valobab pro-
ject for valuable discussions. J.Y. Gassi and F. Gaucheron (INRA, STLO,
Rennes, France) are warmly acknowledged for the preparation of the
MFGM-rich ingredient, as well as the other members of the team that
actively participated in the project (B. Robert, B. Camier, E. Beaucher,
N. Leconte). The Microscopy Rennes Imaging Center (MRic, University
Rennes 1, France) platform is acknowledged.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.foodres.2016.11.025.
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