Food Hydrocolloids xxx (2016) 1e9

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Nature's complex emulsion: The fat globules of milk

Harjinder Singh*, Sophie Gallier 1
Riddet Institute and Massey Institute of Food Science and Technology, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand

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

Article history: The milk fat globule in milk represents a unique emulsion system designed by Nature to deliver energy,
Received 2 August 2016 essential fatty acids and lipid-soluble nutrients to the neonate. The fat globules range from 0.1 to 15 mm
Received in revised form in diameter and are stabilised by milk fat globule membrane (MFGM) which is composed of phospho-
3 October 2016
lipids, various glycoproteins, enzymes and cholesterol. Extensive knowledge on the intracellular origin,
Accepted 5 October 2016
composition and structure of fat globules and MFGM has been accumulated over the last 30 years.
Available online xxx
Recently, it has been speculated that the MFGM has profound effects on the accessibility of the tri-
glycerides for lipase-catalysed digestion. This has initiated a large number of studies on the digestion of
Keywords:
Fat globules
milk fat globules in various dairy systems involving both in vitro and in vivo human digestion models. In
Milk fat globule membrane addition, the identication of health-benecial components of MFGM has led to increasing interest in
Fat globule digestion developing MFGM as a food ingredient with unique functional properties and health benets. This re-
MFGM proteins view focuses on recent knowledge on composition and structure of fat globules, the MFGM, and the
MFGM ingredients behaviour of fat globules during gastro-intestinal digestion. MFGM ingredients and their applications are
Emulsions briey discussed.
2016 Elsevier Ltd. All rights reserved.

1. Introduction cholesterol are considered to be connected with the risk of car-

Milk is a complex biological uid that has evolved as the main
source of nutrition and immunological protection for the neonate.
The concentrations of the principal constituents of milk vary widely
among species: lipids 2e55%, proteins 1e20% and lactose 0e10%,
reecting the energy requirements and the growth rate of the
neonate. The lipid component of milk is very complex chemically
and exists as a unique emulsion, in the form of spherical droplets,
commonly known as fat globules. In bovine milk, lipids represent
approximately 3.5e5.2% of the total milk composition and are
predominantly composed of triglycerides, accounting for more
than 98% of the total milk lipids. The rest of the milk lipids (~2%) are
subdivided into various smaller classes, specically the diac-
ylglycerols (diglycerides), monoacylglycerols (monoglycerides),
free fatty acids, phospholipids and cholesterol. Milk fat is an
important carrier of lipid-soluble constituents, such as carotenoids,
liposoluble vitamins (A, D, E and K) and several volatile avour
compounds. The role of milk fat in human health is somewhat
controversial (Mozaffarian, 2016); the saturated fatty acids and
* Corresponding author.
E-mail address: H.Singh@massey.ac.nz (H. Singh).
1 progress on understanding how the MFGM and the physical
Present address: Fonterra Research and Development Centre, Palmerston North
4442, New Zealand.
diovascular disease, whereas some milk lipids such as conjugated
linoleic acid, sphingomyelin and butyric acid are shown to exhibit
anti-cancer activities (Parodi, 2001).
The fat globule in milk is stabilised by a complex interfacial layer
known as the milk fat globule membrane (MFGM). The structure of
the bovine milk fat globule and its membrane in its native state was
a subject of great interest in the 1970s (Anderson & Brooker, 1975;
Patton & Keenan, 1975; Pintodasilva, Peixotodemenezes,& Mather,
1980; Wooding, 1971), with a recent renewal of interest that has
been encouraged by new techniques of investigation (Gallier,
Gragson, Jimenez-Flores, & Everett, 2010; Lopez, 2011; Vander-
ghem et al., 2011). Major advances have been made in under-
standing the synthesis of milk fat globules, the composition and the
structure of the MFGM and how fat globules are inuenced by
various processing operations used in the dairy industry. The
impact of fat globule modications on the physical and chemical
properties of dairy products has been extensively studied (Gallier,
Acton, Garg, & Singh, 2016; Lopez, Cauty, & Guyomarc'h, 2015).
Interestingly, in recent years, the physical and biochemical sta-
bility of milk emulsions after consumption has generated a great
deal of research interest (Bourlieu & Michalski, 2015; Gallier et al.,
2010; Gallier, Gragson, Jime & Everett, 2012). There has been some
structures of fat globules are modied during gastrointestinal

http://dx.doi.org/10.1016/j.foodhyd.2016.10.011
0268-005X/ 2016 Elsevier Ltd. All rights reserved.

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dx.doi.org/10.1016/j.foodhyd.2016.10.011
2 H. Singh, S. Gallier / Food Hydrocolloids xxx (2016) 1e9
digestion and how they inuence the rates of lipid digestion.
Knowledge of complex interactions between the fat globules, the
MFGM and the physiological components, such as mucin, gastric
and intestinal enzymes (e.g. pepsin, trypsin and lipases) and bile
salts, is key to understanding the physiological behaviour of milk
emulsions during their transit through the gastrointestinal tract. In
this review, we discuss current advances in our understanding of
the structures and the stability of fat globules, from both physico-
chemical and digestion viewpoints.
2. Fat globules: formation, structure and stability
In bovine milk, the diameter of the fat globule ranges from 0.1 to
15 mm, with a mean diameter of 3e4 mm. The fat globule size dis-
tribution varies with the breed of cow, the stage of lactation,
feeding etc. For instance, the fat globules from the milk of Jersey
cows are normally larger than the fat globules from the milk of
Friesian and Brown Swiss cows and there is a decrease in the
average fat globule size as lactation progresses in bovine milk.
Although there are considerable variations between cows of the
same breed, the average fat globule diameter tends to increase with
an increase in the amount of fat in the diet (Huppertz & Kelly, 2006;
Walstra, 1995; Martini, Salary, & Altomonte, 2016).
The processes involved in the synthesis of fat globules in the
secretory cells of the mammary gland are complex and not fully
understood (Argov, Lamay, & German, 2008; Singh, 2006). The
general consensus is that the triglycerides in the core of fat globules
are synthesised at the rough endoplasmic reticulum, accumulate
into triglyceride-rich domains and are then released as discrete
droplets into the cytoplasm, coated with polar lipids and proteins
derived from the endoplasmic reticulum (Heid & Keenan, 2005;
Keenan & Dylewski, 1995). These intracellular droplets grow in
volume by fusion with each other to form cytoplasmic lipid drop-
lets of various sizes, which are then transported to the apical pole of
the epithelial cell and are secreted from the cell. During secretion,
the droplets are completely coated with the cellular bilayer mem-
branes of the epithelial cells of the mammary gland. It appears that
the cell membrane is rearranged and that some proteins and lipids
are excluded from the membrane.
It remains unclear why fat globules of various sizes are produced
and whether or not they have a special role in addition to simply
delivering energy in the form of lipids to the neonate (Bourlieu &
Michalski, 2015). After secretion into the milk, some of the milk
fat globules may fuse with one another, resulting in an increase in
globule diameter. However, it is not known whether this is directed
by cellular processes or whether the globules spontaneously self-
assemble depending on the composition of the globule surfaces.
Moreover, the globule size distribution might be altered at different
stages of lactation (Michalski, Briard, Michel, Tasson, & Poulain,
2005), feeding and nutrition (Couvreur, Hurtaud, Lopez, Delaby, &
Peyraud, 2006).
From an emulsion science perspective, the fat globules in milk
are clearly not simple oil-in-water emulsions, as the MFGM sur-
rounding the globules is not a simple monolayer of surface-active
material. Instead, the MFGM has a unique structure and a unique
composition that reect the fat globule biosynthesis process in the
mammary secretory cells. The MFGM is relatively thin, about
8e10 nm, and contains multilayers containing phospholipids
and proteins; a dense protein layer is located between the mono-
layer of phospholipids and proteins in contact with the triglyceride
core and the inner face of the outer layer (Fig. 1A), which consists of
a bilayer of phospholipids (Heid & Keenan, 2005). The outer
phospholipid bilayer contains various glycoproteins, enzymes,
phosphoproteins and cholesterol (Dewettinck et al., 2008). The
most abundant phospholipids are phosphatidylcholine,
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phosphatidylethanolamine and sphingomyelin whereas phospha-
tidylserine and phosphatidylinositol are present in lower amounts.
The phospholipids are segregated between liquid-ordered domains
(particularly rich in sphingomyelin and cholesterol and also called
lipid rafts; Fig. 2) and liquid-disordered phases (Gallier et al., 2010;
Lopez, 2011). The MFGM also contains two major neutral glyco-
sphingolipids, glucosylceramide (35%) and lactosylceramide (65%)
(Christie, Noble, & Davies, 1987). Gangliosides are glyco-
sphingolipids that are composed of a ceramide and an oligosac-
charide chain attached to one or more sialic acids and several
sugars.
The MFGM contains over 40 proteins, consisting of several gly-
cosylated proteins, including mucin 1 (MUC1), butyrophilin (BTN),
mucin 15 (MUC15), periodic acid Schiff 6/7 (PAS 6/7) and cluster of
differentiation 36 (CD36) (Mather, 2000). These proteins are mostly
transmembrane proteins (MUC1, MUC15, BTN and CD36), whereas
PAS 6/7 is not anchored into the membrane but is loosely adsorbed
at the surface (Dewettinck et al., 2008; Vanderghem et al., 2011).
The other three major proteins are xanthine dehydrogenase/oxi-
dase (XDH/XO), adipophilin (ADPH) and fatty acid binding protein
(FABP). XDH/XO is located in the dense proteinaceous layer of the
MFGM, whereas ADPH is principally located in the inner face of the
polar lipid bilayer and FABP is located in the monolayer close to the
lipid core (Vanderghem et al., 2011). The MFGM also contains about
25 enzymes and several other minor proteins (Dewettinck et al.,
2008; Singh, 2006; Spitsberg, 2005). Certain enzymes that are
intrinsic to the membrane have their active site accessible from one
but not both sides of the membrane.
The MFGM acts as a natural emulsifying agent, preventing the
occulation and coalescence of the fat globules in milk and pro-
tecting the fat against enzymatic action (Keenan & Dylewski, 1995).
However, because of the relatively wide distribution of sizes, the fat
globules in fresh milk tend to undergo creaming during storage.
Creaming of the fat globules occurs by the action of gravitational
force because of the difference in density between the fat phase and
the milk serum phase. Fat globules do not normally occulate,
because of electrostatic and steric repulsion. The zeta potential of
fat globules is about e 10 mV and some of the glycoproteins in the
MFGM cause sufcient steric repulsion. However, in cold raw milk,
occulation may occur by a phenomenon called cold agglutination.
It involves immunoglobulins M, also known as the cryoagglutinins,
and the skim milk membrane (SMM) fragments that are naturally
present in the milk serum phase. The cryoagglutinins induce oc-
culation by bridging the milk fat globules and the SMM together
(Huppertz & Kelly, 2006). Cold agglutination is prevented by
heating milk (>62
C) to inactivate the cryoagglutinins or by dis-
rupting the SMM fragments by homogenisation (Huppertz & Kelly,
2006; Walstra, 1995).
The fat globules may also undergo partial coalescence upon the
storage of milk at low temperatures, because of the formation of fat
crystals, which can protrude from the globule surface and puncture
the MFGM of an adjacent globule (Huppertz, & Kelly., 2006;
Walstra, 1995). Partial coalescence gives rise to large irregularly
shaped granules or a continuous network of clumped fat that tends
to cream rapidly. The partially coalesced fat globules can them-
selves coalesce upon an increase in temperature and the melting of
the fat crystals. During partial coalescence, some membrane com-
ponents may be released into the milk serum.
The fat globules are vulnerable to disruption during normal
dairy processing operations (Singh, 2006). These processing treat-
ments can also considerably modify the composition of the MFGM,
which will consequently inuence the surface properties and the
interactions of the fat globules. Rapid beating of air into milk,
intense turbulence, as in a high-pressure homogeniser, and a very
high velocity gradient all cause fat globule disruption. During these
 H. Singh, S. Gallier / Food Hydrocolloids xxx (2016) 1e9 3

Fig. 1. Schematic diagram illustrating the interfacial changes occurring at the surface of the milk fat globule during gastric and intestinal digestion. (A) Native milk fat globule
membrane (MFGM) structure. A phospholipid trilayer embedding proteins. (B) Milk fat globule in gastric environment. Pepsin hydrolyzes MFGM proteins at different rates and
some MFGM glycoproteins are resistant to proteolysis; the resulting peptides are surface active enough to remain at the interface. Gastric lipase hydrolyzes the triglyceride core and
the released free fatty acids accumulate at the surface of the globules. (C) Beginning of the intestinal digestion. The bile salts displace the phospholipids, proteins, and peptides and
adsorb onto the interface. The colipase adsorbs on to the bile-salt-rich interface and the pancreatic lipase forms a complex with the colipase. (D) Trypsin and chymotrypsin hy-
drolyze some of the MFGM proteins and peptides into amino acids, which further absorb through the intestinal wall; most of the mucins and part of periodic acid-Schiff (PAS) 6/7
are resistant to proteolysis. The pancreatic lipase hydrolyzes the triglyceride core, and lipolytic products accumulate at the oil-water interface and are then solubilised into mixed
phospholipid-bile salt micelles for their transport through the intestinal wall. Cholesterol molecules are similarly transported. CD36 cluster of differentiation 36;
BTN butyrophilin; ADPH adipophilin; XO xanthine oxidase. Not to scale. Reproduced from (Gallier, Laubscher, & Jimenez-Flores, 2014) with permission from Elsevier.

processes, the MFGM is substantially damaged, increasing the
susceptibility of the milk fat to oxidation, hydrolytic rancidity,
coalescence and partial coalescence.
Homogenisation causes a marked reduction in the fat globule
size, depending on the intensity of the homogenisation pressure.
This induces a simultaneous increase in the globule surface area
and a break-up of the intact MFGM. As a result, there is insufcient
MFGM material to fully cover this additional fat globule surface and
skim milk proteins readily adsorb at the fat/water interface. The
casein micelles, rather than the whey proteins, adsorb preferen-
tially at the globule surface (Walstra & Oortwijn, 1982). Therefore,
the fat globule surface after the homogenisation of milk is stabilised
by both MFGM material and skim milk proteins and the rate of
creaming of the globules is markedly reduced. Sometimes, fat
globule clustering may occur during homogenisation, but this can
be eliminated by a two-stage homogenisation.
Temperature has a signicant impact on the stability and the
integrity of the MFGM. Heating milk above 60
C induces the
denaturation of the MFGM proteins and their association with the
whey proteins (Corredig & Dalgleish, 1996; Kim & Jimenez-Flores,
1995; Ye, Singh, Oldeld, & Anema, 2004). It has been suggested
that b-lactoglobulin may associate with the MFGM via sulphy-
dryledisulphide interchange reactions (Houlihan, Goddard,
Kitchen, & Masters, 1992) or that it may displace the original
MFGM material, either by directly competing or because the MFGM
may break down during heating, leaving gaps through which the
whey proteins may adsorb to the newly exposed fat surface
(Dalgleish & Banks, 1991). Ye et al. (2004) further proposed that the
thioledisulphide interchange reactions are initiated by free thiol
groups of the MFGM proteins, which become available for inter-
action at much lower temperatures than the denaturation tem-
perature of b-lactoglobulin. The specic protein components of the
MFGM that interact preferentially with b-lactoglobulin during heat
treatment have not yet been identied. Ye et al. (2004) also
examined the behaviour of the MFGM proteins during heat treat-
ments; it appears that xanthine oxidase and butyrophilin aggregate
at lower heating temperatures (starting from 60
C, 10 min) than
PAS 6/7, which begins to aggregate at 80
C (Ye, Singh, Taylor, &
Anema, 2002). Most of the major MFGM proteins (e.g. xanthine
oxidase and butyrophilin) remain anchored into the membrane,
but PAS 6/7 appears to migrate into the serum phase during
heating.

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4 H. Singh, S. Gallier / Food Hydrocolloids xxx (2016) 1e9
Fig. 2. Confocal laser scanning microscopic images of milk fat globules from raw milk
stained with Rd-DOPE, a uorescent phospholipid analog known to partition within
liquid-disordered phase and commonly used to study phase separation between
liquid-ordered and liquid-disordered phases in phospholipid layers (arrows pointing at
liquid-ordered domains). Scale bar 10 mm. Reproduced from Gallier, Gordon & Singh
(2013) with permission from Elsevier.
Heating also causes the release of membrane phospholipids into
the serum phase (Houlihan et al., 1992). Some of the MFGM ma-
terial, particularly the phospholipids (20%), is also released into the
milk serum upon the cooling of milk (Walstra, Wouters, & Geurts,
2006). Freezing and thawing cause signicant damage to the
MFGM and destabilization of the fat globules. The rapid incorpo-
ration of air can also destabilise the MFGM, which is the basic
mechanism for the manufacture of whipped cream and butter.
Any kind of processing of raw milk has an impact on the size of
the fat globules and/or the structure and composition of the globule
interface. This has an impact on the functionality of the processed
fat globules but likely also on the gastrointestinal processing of the
milk fat globules.
3. Behaviour of fat globules during gastrointestinal digestion
The human gastrointestinal tract consists of a number of inter-
active units, including the mouth, stomach, small intestine and
large intestine. All these units play important and different roles in
the overall digestion and absorption of food. Several studies have
attempted to understand the digestion of milk fat globules in the
gastrointestinal tract (Gallier, Ye, & Singh, 2012; Ye, Cui, & Singh,
2011). Aspects of the gastrointestinal digestion of emulsions, in
general, have been reviewed elsewhere (Sarkar, Goh, & Singh,
2009; Singh & Gallier, 2014). As lipid digestion is essentially an
interfacial process, the size of the fat globules and the structure of
the MFGM play a key role in the digestion and absorption of milk
fat.
The impact of oral processing on the milk fat globule structure
and its stability is likely to be minimal when milk is consumed
because of its short residence time in the mouth. In the stomach,
the main effects of the gastric environment on the structure of the
milk fat globules (see Fig. 1) are (1) the presence of enzymes
(pepsin is able to digest some of the MFGM proteins and the gastric
lipase initiates the digestion of the triglyceride core by releasing
free fatty acids from the sn-3 position) and (2) the pH drop causing
aggregation of the globules, especially in the presence of caseins in
the bulk phase, aggregating at pHs close to their isoelectric point of
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4.6 (Gallier, Cui, et al., 2013). The aggregation of the globules is also
assisted by the digestion of some of the MFGM proteins and the
interfacial accumulation of free fatty acids from lipolysis (Fig. 3),
both affecting the MFGM structure and subsequently the repulsive
forces between globules (Gallier, Ye, et al., 2012; Ye et al., 2011).
Recently, Ye, Cui, Dalgleish, and Singh (2016) studied the
behaviour of the milk fat globules in whole milk during gastric
digestion using a human gastric simulator. The action of pepsin
caused coagulation of the casein micelles, resulting in the forma-
tion of a closely knit network (clot), with the fat globules embedded
in the casein matrix. Some fat globules appeared to have coalesced
within the matrix whereas other fat globules were present in the
aqueous phase, possibly indicating some micro-phase separation
during the formation of the clot. With an increasing digestion time,
the knitted network became denser and the fat globules within the
matrix tended to aggregate and coalesce, so that large pools of fat
could be observed, especially after long digestion times. Interest-
ingly, the release of fat globules from the clots was linearly
dependent on the breakdown of the protein in the clots.
The small intestine is the main site for the digestion and release
of nutrients and their conversion into an absorbable form. The
pancreatic secretion induces a major change in pH, between 5 and
7, and also contains many enzymes, such as proteases and pepti-
dases (trypsin, chymotrypsin, carboxypeptidases etc.), lipases and
esterases (pancreatic lipase, cholesterol esterase, phospholipase A2
etc.) and pancreatic amylases (Singh & Gallier, 2014). Lipids are
mainly digested by pancreatic lipase (forming a complex with co-
lipase) and bile salts also play an important role in lipid digestion.
The conditions in the small intestine cause drastic changes in
both the internal structure and the external structure of milk fat
globules (Fig. 1). Pancreatic lipase hydrolyses the sn-1 and sn-3
positions of triglycerides to generate monoglycerides and the cor-
responding fatty acids. Some of these lipolytic products (e.g. fatty
acids, diglycerides and monoglycerides) can migrate and precipi-
tate at the surface of globules. Bile salts, which are highly surface
active, are able to displace the MFGM (Gallier, Laubscher, et al.,
2014; Gallier, Rutherfurd, Moughan, & Singh, 2014; Patton,
Borgstrom, Stemberger, & Welsch, 1986) from the surface and to
solubilise the interfacial free fatty acids to allow the co-lipase/
pancreatic lipase to anchor at the interface and to progress lipid
digestion (Borgstrom & Erlanson-Albertsson, 1982). Pancreatic
proteases are also able to digest MFGM proteins further. Phos-
pholipase A2 hydrolyses glycerophospholipids (Borgstrom &
Erlanson-Albertsson, 1982), whereas sphingomyelinase hydroly-
ses sphingomyelin (Kuchta, Kelly, Stanton, & Devery, 2012).
Confocal laser scanning microscopy studies (Gallier, Ye, et al.,
2012; Gallier, Zhu, et al., 2013) on the structure of the milk fat
globules during intestinal digestion preceded by gastric digestion
showed that phospholipids and a few glycoproteins were still
detected at the surface of the globules. A liquidcrystalline lamellar
phase was formed at the surface of some fat globules; this was
probably associated with the accumulation of lipolytic products
and the formation of vesicles carrying the lipolytic products for
later transport from the interface of the globules to the aqueous
phase and through the intestinal wall (Gallier, Ye, et al., 2012;
Gallier, Zhu, et al., 2013). Some large coalesced fat globules
appeared and microparticles of various sizes, probably containing
amphiphiles, were present in the aqueous phase.
Recently, Gallier et al. (Gallier, Cui, et al., 2013; Gallier, Zhu, et al.,
2013) studied the in vivo gastrointestinal digestion of fat globules in
rats (Figs. 3 and 4). The study showed increases in the size of the fat
globules and hydrolysis of the MFGM proteins after gastric diges-
tion. Spherical protrusions rich in lipolytic products were observed
at the surface of fat globules in the gastric chime (Fig. 3). During
intestinal digestion, the fat globules retained the size acquired in
 H. Singh, S. Gallier / Food Hydrocolloids xxx (2016) 1e9 5

Fig. 3. Confocal laser scanning microscopy (30 and 120 min) and differential interference contrast (180 min) images of the gastric chyme collected from rats 30, 120 and 180 min
after gavaging with cream derived from raw milk. The lipids were stained with Nile Red (red) and the proteins were stained with Fast Green FCF (blue). The black arrows are
pointing at spherical amorphous lipid protrusions. Scale bars 50 mm (30 min), 75 mm (120 min) and 25 mm (180 min). Adapted from Gallier, Cui, et al. (2013) with permission from
Elsevier. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

the stomach, but they were surrounded by a liquidcrystalline
lamellar phase, formed by the accumulation of lipolytic products,
calcium and possibly bile salts (Fig. 4). This lamellar phase was then
solubilised as multilamellar vesicles and, in contact with bile salts,
the vesicles formed smaller mixed micelles. The lamellar shell
surrounding the fat globules eventually cracked or solubilised,
releasing the undigested triglyceride core and leaving the empty
crystalline shell behind. These studies bring new interesting in-
sights into the digestion of bovine milk fat globules. Interestingly,
these observations are qualitatively similar to those observed in
in vitro model systems (Patton & Carey, 1979).
Gastrointestinal digestion is a complex process involving pro-
teases and lipases for efcient hydrolysis of the components of the
MFGM, the milk fat globule core and the surrounding or adsorbed
milk serum proteins.
4. Lipid digestion rates
As lipolysis occurs at the interface, the composition of the
MFGM (i.e. proteins and lipids in competition or interaction with
lipases and bile salts) and the size (i.e. the available surface area) of
the fat globules determine the kinetics of lipid digestion (Bourlieu
& Michalski, 2015; Gallier, Shaw et al., 2013; Gallier, Shaw et al.,
2014). As milk fat globules range from 0.1 to 15 mm, the digestion
kinetics of milk fat globules will vary as a function of size. In
addition, compared with large fat globules, smaller fat globules are

Fig. 4. Transmission electron microscopic images of upper (A, B) and lower (C, D) small intestinal digesta of rats gavaged with cream prepared from raw milk. Scale bars 2000 nm
(A), 500 nm (D) and 1000 nm (B and C). Formation of multilayers of lipolytic products and liquid-crystalline phases could be seen. Also the multilamellar and unilamellar vesicles
(50e200 nm) transporting lipolytic products could be seen. Reproduced from Gallier, Zhu, et al. (2013) with permission from Elsevier.

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6 H. Singh, S. Gallier / Food Hydrocolloids xxx (2016) 1e9
richer in medium-chain fatty acids and myristic acid and poorer in
long-chain fatty acids, and their MFGM phospholipids contain less
phosphatidylcholine and sphingomyelin (Michalski, Leconte, et al.,
2006). Therefore, considering the selective release of short- and
medium-chain fatty acids because of the stereospecicity of the
gastric lipase (Sams, Paume, Giallo, & Carriere, 2016) and the effect
of the interfacial composition on the adsorption of gastric lipase
(Bourlieu et al., 2016), it can be expected that the digestion rate and
the fatty acid release prole of small fat globules will differ from
those of large fat globules. The increase in surface area available for
lipase anchoring likely contributes to faster digestion of small fat
globules.
Homogenisation reduces the milk fat globule sizes, which re-
sults in a faster digestion rate as demonstrated in vitro (Berton,
Sebban-Kreuzer, Rouvellac, Lopez, & Crenon, 2009; Bourlieu,
Me
nard, et al., 2015; Ye, Cui, & Singh, 2010). The rate of the ag-
gregation of proteins, trapping milk fat globules, was also faster for
homogenised fat globules than for raw milk fat globules under
in vitro (Bourlieu, Me
nard, et al., 2015) and in vivo (Gallier, Cui,
et al., 2013) gastric conditions. The initial size of the milk fat
globules increases during gastric digestion but homogenised fat
globules remain smaller than raw milk fat globules throughout
digestion, as observed in vitro (Bourlieu, Me
nard, et al., 2015) and
in vivo (Armand et al., 1996; Armand et al., 1999; Gallier, Cui, et al.,
2013; Gallier, Zhu, et al., 2013).
Homogenisation and heat treatment change the interface of
milk fat globules by increasing the amount of milk proteins
adsorbed at the interface or interacting with the MFGM proteins (Ye
et al., 2002; Ye, Singh, Taylor, & Anema, 2004). The difference in the
interfacial composition of homogenised and native milk fat glob-
ules probably results in a different digestion rate, as observed in
rats gavaged with fat droplets with differing interface and size
(Borel et al., 1994; Michalski, Soares, et al., 2006). The different rate
and extent of aggregation under gastric conditions (Bourlieu,
Me
nard, et al., 2015; Gallier, Cui, et al., 2013) probably has an
impact on gastric emptying. This is the case for human milk fat
droplets emptying faster than infant formula fat droplets, which are
covered by proteins and smaller in size (Cavell, 1981). However, this
was demonstrated in infants who have a higher intragastric pH and
lower motility and enzymatic activity than adults. In adult humans
(Marciani, Wickham, et al., 2007) and in the growing rats (Gallier,
Rutherfurd, et al., 2014) the gastric emptying rate of acid-stable
emulsions was slower than that of acid-unstable emulsions.
Therefore, it can be hypothesized that raw milk empties from the
adult stomach more slowly than processed milk due to a faster
destabilization of the latter under acidic conditions.
5. Lipid digestion in infants
The process of digestion in infants differs from that in adults
because of the immaturity of the digestive tract, with lower enzyme
activity, gastric and pancreatic juice secretion, bile salt concentra-
tion and gastric motility at birth (Bourlieu et al., 2014). The gastric
and intestinal environments in infants are quite favourable for
lipolysis by gastric lipase. Human gastric lipase has a central role in
the lipid digestion in infants, fed either human milk or infant for-
mula. It is active at pHs from 2 to 7, with optimal activity at pH 5.6,
and can hydrolyze milk fat globules and other fat droplets in the
presence of bile salts and interfacial phospholipids in the stomach
and the small intestine (Sams et al., 2016). Infants have a high
intragastric pH and milk has a buffering capacity, which means that
the pH of the milk chyme is close to the pH of optimal activity of
gastric lipase (Bourlieu et al., 2014) during the 90e120 min resi-
dence time of the milk in the stomach (Billeaud, Guillet, & Sandler,
1990; Cavell, 1981). The digestion of lipids in infants is also carried
Please cite this article in press as: Singh, H., & Gallier, S., Nature's complex emulsion: The fat globules of milk, Food Hydrocolloids (2016), http://
dx.doi.org/10.1016/j.foodhyd.2016.10.011
out by milk endogenous lipases, pancreatic carboxyl ester hydro-
lase and pancreatic lipase-related protein 2 (Lindquist & Hernell,
2010).
After expression, human milk may be kept frozen until con-
sumption after thawing. Freezing and thawing alter the structure of
the human milk fat globules, as milk fat will crystallise and the
membrane will be ruptured in cold temperatures (Sousa,
Delgadillo, & Saraiva, 2016). Holder pasteurisation of human milk
is commonly carried out in human milk banks or at home to pre-
serve freshly expressed human milk for a longer period (Sousa
et al., 2016). It consists of heating the milk at 62.5
C for 30 min.
However, this process inactivates the endogenous bile-salt-
stimulated lipase (leading to reduced fat digestion), reduces the
nutritive content of human milk and affects the structure of some
milk nutrients, such as the MFGM (Sousa et al., 2016). High-
pressure processing is now considered to be an alternative to
Holder pasteurisation to preserve the quality and the quantity of
human milk nutrients.
The effect of Holder pasteurisation on term (de Oliveira,
Deglaire, et al., 2016) and preterm (de Oliveira, Bourlieu, et al.,
2016) human milk digestion was investigated using an in vitro
term and preterm digestion model. Under term conditions, gastric
and intestinal lipolysis of pasteurised term human milk was lower
than that of native term human milk and pasteurisation led to
protein-induced aggregation of the fat globules in the gastric phase.
Under preterm conditions, Holder pasteurisation of preterm milk
slowed intestinal lipolysis and induced aggregation of the fat
droplets in the gastric phase and the formation of larger aggregates
in the intestinal phase. The formation of protein-induced aggre-
gates in the stomach may lead to phase separation and the slowing
down of gastric emptying (de Oliveira, Bourlieu, et al., 2016).
In the gastric compartment of infants, human milk fat globules
do not tend to aggregate, because of the resistance of MFGM gly-
cosylated proteins and lipids to digestion, being able to provide
repulsion between droplets (Armand et al., 1996). The difference in
gastric stability and emptying, combined with the difference in
lipolysis extent due to inactivation of the bile-salt stimulated lipase,
may be responsible for the difference in postprandial lipid and
protein handling in breastfed and formula-fed infants.
6. MFGM as an ingredient and its role in human nutrition
In the past, MFGM-containing products, such as buttermilk
collected during butter production, were discarded or were spray
dried for animal feed (Lambert et al., 2016). However, increasing
knowledge of the complex structure and the nutritional and health
attributes of the MFGM has led researchers and companies to look
for ways to extract and develop MFGM-enriched dairy streams for
added-value products. Several dairy companies such as Fonterra
Co-operative Group Ltd, Arla Foods, Lecico GmbH and Megmilk
Snow Brand are currently selling ingredients that are rich in MFGM
or complex milk lipids for a diverse range of technological, func-
tional and nutritional benets. Academic groups are also investi-
gating ways to improve the efciency of the recovery of MFGM
from buttermilk, butter serum and whey buttermilk streams (Gassi
et al., 2016; Holzmller & Kulozik, 2016; Lambert et al., 2016).
Because of its original function in stabilising the fat globules in
whole milk, MFGM material isolated from buttermilk or cream is
considered to be an efcient natural surface-active material, with
high emulsifying capacity (Corredig & Dalgleish, 1998; Kanno,
Shimomura, & Takano, 1991). However, the method of separation,
the type of raw material and the pre-treatment of the cream or
buttermilk signicantly affect the composition of MFGM isolates
and hence their emulsication properties. It is not known with
certainty which components of the MFGM, whether phospholipids
 H. Singh, S. Gallier / Food Hydrocolloids xxx (2016) 1e9
or proteins or both, are responsible for the emulsication activity.
MFGM-derived phospholipids can be used to prepare liposomes
(Thompson & Singh, 2006). As the composition of the MFGM
phospholipid material is very different from that of the commonly
used soy- or egg-derived phospholipids, there are considerable
differences in the structure and properties of the liposomes pro-
duced from MFGM phospholipid material (Thompson, Haisman, &
Singh, 2006). It should be possible to exploit the unique composi-
tion of the MFGM phospholipids in the delivery and protection of
sensitive compounds, through the formation of liposomes with
enhanced functional properties.
With respect to nutritional and health benets, MFGM complex
milk lipids have been shown to improve the memory of ageing rats
(Guan, MacGibbon, Zhang, et al., 2015). Long-term supplementa-
tion of dietary milk phospholipids has also been shown to improve
the skin elasticity of human adults (Higurashi, Haruta-Ono, Ura-
zono, Kobayashi, & Kadooka, 2015). MFGM supplementation, in
combination with exercise, has been shown to improve the physical
performance of healthy middle-aged adults (Ota, Soga, Hase, &
Shimotoyodome, 2015; Soga, Ota, & Shimotoyodome, 2015).
Emulsication of fat droplets with milk phospholipids had a posi-
tive effect on lipid digestion and postprandial lipid metabolism in
rats (Lecomte et al., 2015). In addition to the ability to reduce
cholesterol absorption, MFGM supplementation has also been
associated with the prevention of coronary heart disease and
improved metabolic health (Rueda, 2014).
For decades, infant formulae were based on the composition of
human milk. There is now increasing interest in simulating the
structure of human milk in infant formulae because of the
improved knowledge on the effect of food structure on the diges-
tion and absorption of lipids (Gallier et al., 2015). Several research
groups and infant formula manufacturers have investigated the
addition of ingredients rich in MFGM or complex lipids into infant
formulae to improve several growth and development outcomes in
infants (Billeaud et al., 2016; Guan, MacGibbon, Fong, et al., 2015;
Hernell, Timby, Domello f, & Lo
nnerdal, 2016; Oosting et al., 2014;
Reis et al., 2016; Timby et al., 2015). Mead Johnson Nutrition Inc.
(Enspire), Fonterra Co-operative Group Ltd (Anmum) and
Semper AB (Baby Semp) are currently selling, in different markets,
infant formulae containing MFGM or complex milk lipids alone or
in combination with other bioactive nutrients and backed by
research evidence on the benet for brain development and
cognition (Guan, MacGibbon, Fong, et al., 2015; Mudd et al., 2016;
f, Hernell, Lo
nnerdal, & Domello f,
Reis et al., 2016; Timby, Domello
2014). Supplementation of dietary MFGM in infants (Timby et al.,
2015) and in children (Veereman-Wauters et al., 2012) and of
complex milk lipids in toddlers (Poppitt et al., 2014) may decrease
the risk of infections, with glycosylated proteins and lipids being
the most likely candidates for this protective effect, acting as a
decoy for pathogens and viruses (Hamosh et al., 1999).
7. Conclusions
The milk fat globule remains an intriguing emulsion with a
complex interfacial composition and structure. During the last
decade, signicant advances have been made in understanding the
secretion, structure and composition of the globules and MFGM.
However, much more remains to be discovered. For example, the
formation of lipid rafts, rich in sphingomyelin and cholesterol in
MFGM, was recently observed using microscopy but their biological
signicance and functions have not yet been understood. The
complexity of the MFGM glycocalyx and its role in the neonate gut
maturation is another current key research interest. Excellent
progress has also been made on the post secretion modications of
fat globules and characterization of complex interactions of MFGM
Please cite this article in press as: Singh, H., & Gallier, S., Nature's complex emulsion: The fat globules of milk, Food Hydrocolloids (2016), http://
dx.doi.org/10.1016/j.foodhyd.2016.10.011
7
during dairy processing. Several products containing different
fractions of MFGM are now produced by the dairy industry for
various nutritional and functional applications. Further studies on
the role of MFGM fractions in human health are needed. Recently,
there has been interest in understanding how the MFGM and fat
globules are modied during gastrointestinal digestion and how
these change inuence lipid digestion. Our understanding of
complex interactions between the fat globules, the MFGM and the
physiological components is incomplete. This knowledge is indeed
the key to understanding why Nature has designed the milk
emulsion system as such a complex structure.
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