Food Research International 42 (2009) 659665

Contents lists available at ScienceDirect

Food Research International

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

The use of sedimentation eld-ow fractionation in the size characterization

of bovine milk fat globules as affected by heat treatment
Vassilios Raikos a,*, John Kapolos b, Lambros Farmakis a, Athanasia Koliadima a, George Karaiskakis a
a
b

Department of Chemistry, University of Patras, 26504 Patras, Greece

Department of Agricultural Products Technology, Technological Educational Institute of Kalamata, 24100 Kalamata, Greece

a r t i c l e

i n f o

Article history:
Received 18 September 2008
Accepted 2 February 2009

Keywords:
Milk
Fat globules
Particle size
Denaturation
Temperature

a b s t r a c t
Size distribution of fat globules affects the appearance, taste and stability of milk and milk-based products. Full-fat, semi-fat and chocolate bovine milk were subjected to heat treatment within a temperature
range of 50125 C for 1 h. Sedimentation eld-ow fractionation was employed to determine the
changes in mean particle diameter of milk fat globules as affected by heat treatment. The mean particle
diameter of fat droplets increased with increasing heating temperature for most samples. The particle
size of fat globules increased on average 40 nm (4.65%) for full-fat and 72 nm (8.52%) for semi-fat milk
following the heat treatment (50125 C). Chocolate milk exhibited considerable increase in particle size
(104 nm, 12.53%) within a certain temperature range (50110 C), followed by a decrease in particle size
when heated at 125 C for 1 h. Heat-induced occulation due to attractive interactions between hydrophobic sites on denatured protein molecules on different droplets was assumed to be mainly responsible
for the increases in particle size observed in this study. Extensive heat-induced denaturation of milk proteins was also indicated by Native PAGE. Sedimentation eld-ow fractionation proved to be a useful
technique for adequately monitoring heat-induced changes in particle size distributions in milk.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Milk is a natural, colloidal system whose biological role is to
nourish and protect the mammalian young. It is a relatively complex biological uid and consists of fat globules and casein micelles
dispersed in an aqueous solution containing whey proteins, lactose, minerals and vitamins.
Milk fat is present in milk in the form of dispersed globules, which
in raw milk are stabilized by a native fat globule membrane with the
particle size ranging from 0.59 lm (Jussila, Yohannes, & Riekkola,
1997). Milk fat consists of triacyl glycerols the fatty acid composition
of which is diverse regarding the chain length and the degree of saturation. This composition gives milk fat its specic avor and mouth
feel (Brans, Schron, van der Sman, & Boom, 2004). During processing, milk emulsions are normally homogenized to reduce creaming
during storage. In the homogenization process, the fat droplets are
disrupted into much smaller globules (below 1 lm) by means of
mechanical energy and the extra stabilization of the enlarged surface is taken care of by casein, b-lactoglobulin, a-lactalbumin, phospholipids and glycoproteins (Andersson, Brooker, Cawston, &
Cheeseman, 1977). These emulsiers are basically lm-forming
materials which form an adsorbed layer around the fat globules
developing a fat and avor encapsulating matrix (Jena & Das,
* Corresponding author. Tel.: +30 2610 997109; fax: +30 2610 997144.
E-mail address: vas.raikos@gmail.com (V. Raikos).
0963-9969/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2009.02.001

2006). The protective membrane formed by the milk proteins

prevents droplets from coalescing and as result contributes to the
stability of the milk emulsion (Dickinson, 1992).
One of the important parameters affecting the quality, appearance and taste of the nal food products is the particle size of
the ingredients included. For example, particle size of fat globules
plays predominant role in the stability of the milk emulsion. Large
globules coalesce faster than the small ones and a decrease in the
average globule diameter of a milk emulsion by a factor of two may
decrease the coalescence rate with a factor of 10100 (Bergenstahl
& Claesson, 1990). Furthermore, the shape and size of the particles
affect the closeness of the particle pack, which in turn affects the
milk bulk density. The particle size of protein stabilized emulsions depend largely on the molecular structure and interactions
of the adsorbed protein (Dickinson & McClements, 1995). Moreover, the particle size which determines to a large extent the
appearance, rheology and stability of the food being considered
also depends on the processing and storage conditions that the
food experiences during its lifetime (Demetriades, Coupland, &
McClements, 1997). For instance, the size of the fat globules in
homogenized milk is highly dependent on the temperature, pressure and equipment used in the homogenization process (Anema,
Lowe, & Stockmann, 2005; Dickinson & Parkinson, 2004; Jena &
Das, 2006).
Sedimentation eld-ow fractionation (SdFFF), a sub-technique
of Field-Flow Fractionation (FFF), is an established elution based

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V. Raikos et al. / Food Research International 42 (2009) 659665

separation technique, the theory of which is well documented

(Giddings, 1973). SdFFF has been applied in the past for the characterization of food materials (Farmakis, Koliadima, & Karaiskakis,
2002; Mozersky, Farrell, & Barford, 1991; Saeseaw, Shiowatana, &
Siripinyanond, 2005; Udabage, McKinnon, & Augustin, 2003; Udabage, Sharma, Murphy, McKinnon, & Beckett, 1997). Moreover,
SdFFF has been employed to study the effect of heat treatment
on the particle size of skim milk (de Kruif, 1998). The main objective of the present study was to employ a relatively new, volatile
technique such as SdFFF for size characterization of fat droplets
in thermally processed bovine milk. To the best of our knowledge
this technique has not been applied yet to determine the changes
in particle size of bovine milk fat globules as affected by heat treatment. Results were compared with the ndings of other researchers and interpretations were based on published data obtained
from the literature.

mentally determined retention volume are dened by the geometry of the channel or the experimental set up and are
independently measurable. The major factor limiting the application of SdFFF to the determination of particle size distribution is
the requirement that the density difference between the particles
and the medium be known (Udabage et al., 2003). In the normal
mode of SdFFF, smaller particles travel faster than their larger
counterparts, and the dependence of retention on particle size or
mass is generally quite predictable, provided the force exerted on
the sample particles by the applied eld is a calculable quantity
(Fig. 1). As a consequence, particle size distributions can be obtained from experimental fractograms (detector response vs. time)
on the basis of theoretical relationships. Provided that the exact
geometry of the channel, the eld strength, ow rate and the density difference between particle and carrier liquid are known, then
the diameter, d, of a spherical particle can be calculated from the
following equation (Dondi & Martin, 2000):

2. Theory of SdFFF
Field-ow fractionation is a technique suitable for separation
and characterization of colloidal materials and macromolecules
(Beckett, Nicholson, Hart, Hansen, & Giddings, 1988; Giddings,
1981; Giddings, Karaiskakis, Caldwell, & Myers, 1983). The main
principle is based on a coupling between the parabolic velocity distribution through a ribbon like channel and a perpendicular eld
which compresses suspended particles into layers against one wall
of the channel (Fig. 1).
In SdFFF separation is accomplished by introducing centrifugal
eld forces on the particles suspended in a carrier liquid and colloidal particles are separated by differences in their buoyant mass.
The mean thickness of each particle layer is determined by some
eld-coupled property (e.g. particle size) or a combination of such
properties of the particles. Particles concentrated in slower ow regions closer to the wall migrate through the channel more slowly
than those with greater layer thickness which extend into faster
ow regions nearer the channel center (Farmakis, Sakellaraki, Koliadima, Gavril, & Karaiskakis, 2000). Retention in SdFFF is classied
into two modes; normal (Brownian) and steric. The SdFFF separation in the normal mode of retention can be regarded as a relatively
straightforward method in which particle size can be obtained
without using particle standards. In SdFFF all of the parameters
relating the buoyant mass of the eluting particles to the experi-

36kT

pGW DqV 0

!1=3
Vr1=3

where k is the Boltzmanns constant, T is the absolute temperature,

G is the angular acceleration (G = x2r, where x is the angular velocity around radius r), w is the channel thickness, Dq is the density
difference between solute and solvent, V0 is the column void volume and Vr is the retention time of particle under investigation.
3. Materials and methods
3.1. Materials
Commercially available fresh milk (homogenized, pasteurized)
of bovine origin was purchased from the local supermarket. Fullfat, semi-fat and chocolate milk were utilized for the purposes of
this study. Sodium azide, glycine, bromophenol blue and acetic
acid were purchased from Merck (Germany) and FL-70, a lowfoaming, low-alkalinity, phosphate- , chromate- and silicate-free
mixture of anionic and nonionic surfactants from Fisher Scientic
(UK). Ethanol was purchased from Carlo Erba (Italy), trizma base
and Coomassie blue from Sigma (USA), ammonium persulfate
and glycerol from Promega (USA) and tetramethylethylenediamine
(Temed) and acrylamide/bis from Applichem (Germany). Milk
samples were placed in plastic test tubes and were heated in an
electrically heated oven with a fan mounted in its back wall to provide air circulation (Pye Unicam, Series 104, Cambridge, UK) for a
period of time (1 h) without stirring. The temperature inside the
oven was followed using a thermometer with a temperature probe
located in the oven interior and the timing started when the desired temperature was reached (0.5 C). The sample volume inside
the sealed tube was small (2 mL) to ensure a relatively short thermal equilibration time. After heating the test tubes were cooled
under cold running tap water for approximately 5 min.
3.2. Sedimentation eld-ow fractionation

Fig. 1. Schematic representation of the SdFFF channel. Particles are separated into
zone layers according to their size (A ? C, large ? small).

The SdFFF used in the present study was the S-101 Particle/Colloid fractionator from Postnova Analytik (Germany). The SdFFF
channel was a stainless steel ribbon like channel 89.5 cm long (tip
to tip), 2.0 cm wide and 0.0254 cm thick. The Void volume (Vo)
was 4.45 mL, the injector to channel dead volume was 0.14 mL
and the rotor radius of 15.1 cm. The SdFFF system was equipped
with an HPLC pump (Model PN 1121 Solvent delivery system, Postnova) and the eluted particles were detected using a UV/Vis detector (Model S 3210, Postnova) at the xed wavelength of 254 nm.
The signal was recorded and processed by the computer using inhouse FFF analysis software (SPIN 130, Utah, USA) to generate

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V. Raikos et al. / Food Research International 42 (2009) 659665

Table 1
SdFFF method setup.

Rotor radius (cm)

Carrier liquid
Operating conditions
Analysis type
Initial eld strength (rpm)
Final eld strength (rpm)
Equilibration time (min)
Time held at initial eld strength after
equilibration time (min)
Density difference (delta rho) (g cm3)
Field decay parameter (min)
Channel ow rate(ml/min)
Injection delay (s)
Data rate (points/min)

89.5 long  2.0 wide  0.0254 thick

(cm)
15.1
FL-70 0.02% (v/v), sodium azide
0.02% (w/v) in dH2O

4. Results and discussion

4.1. Fat globules

Normal
1500
50
15
10
0.05
80
1.5
8
7

fractograms. Milk samples (20 lL) were injected into a Rheodyne

model PN 5100 manual injector. The carrier liquid was triply distilled deionized water containing 0.1% (v/v) FL-70 detergent and
0.02% (w/v) sodium azide as a bacteriocide. The channel ow rate
was set at 1.5 mL/min. The SdFFF separation conditions for milk
samples are summarized in Table 1. All samples were diluted 14
times in the carrier liquid before injection into the SdFFF channel
to avoid an overloading effect. Densities of all milk samples were
assumed to be similar to polystyrene latex for calculations, i.e.
1.05 g cm3 (Saeseaw et al., 2005). All measurements were carried
out in triplicate and data are presented as the mean and standard
deviation. The t-test (Gossett, 1908) was used to detect signicance
of differences among means. Condence levels were set at 95%
(P < 0.05). Raw fractograms were converted into size distribution
proles using an Excel spreadsheet (Microsoft Excel 2003, USA).
3.3. Native PAGE
Native polyacrylamide gel electrophoresis (PAGE) was carried
out under non-reducing conditions by following the method of Laemmli (1970) with minor modications using a V10-CDC electrophoresis unit (SCIE-PLAS, UK). The gel system was discontinuous
in which the separating gel contained 10% (v/v) acrylamide (0.15%
v/v ammonium persulfate, 0.08% v/v Temed, 0.375 M Tris, pH 8.8
in dH2O) and the stacking gel contained 5% (v/v) acrylamide (0.2%
v/v ammonium persulfate, 0.2% v/v Temed, 0.19 M Tris, pH 8.8 in
dH2O). The migration buffer (stock solution, 50-fold dilution before
use) was prepared by dissolving 7.5 gr Trizma base and 36 gr glycine in 250 mL of dH2O. Heated milk samples were diluted 4 (v/
v) and unheated (control) samples were diluted 3 (v/v) with
dH2O and were then dispersed in an equal volume of sample buffer.
Sample buffer (stock solution, three-fold dilution before use) was
prepared by dissolving 3 mL glycerol, 0.6 mL migration buffer
(stock solution, 50-fold dilution before use), 6.4 ml dH2O and traces
of bromophenol blue. 15 lL of each sample were loaded in the
wells. Electrophoretic migration was performed at 140 V (constant)
for 1.5 h. The gels were stained with Coomassie Brilliant Blue (0.2%
(w/v) coomassie blue, 7.5% (v/v) acetic acid, 50% (v/v) ethanol), destained for 1 h in dH2O and photographed using a Hewlett Packard
digital camera (HP Photosmart R827, Hewlett Packard, USA).
3.4. Microscopy
A Primo star optical microscope (Carl Zeiss, Germany) was used
at 10 magnication for observation of the milk fat globules. Samples of milk (about 12 drops) were placed on a microscope slide

To determine the contribution of fat globules in milk, fractograms for milk varying in fat content were run under similar running conditions. As illustrated in Fig. 2, the only peak appearing in
the fractogram clearly corresponds to the fat globules dispersed in
milk. Peaks corresponding to whey proteins or caseins could not be
detected under the specied experimental conditions, possibly because they co-elute in the void volume. As expected the fat content
determines the size of the peak with the peak size for full-fat milk
(3.5% fat) being higher than the one corresponding to chocolate
(3.0% fat) and semi-fat milk (1.0% fat). Thus, it was conrmed that
the peak eluting after 15 min. corresponds to the milk fat globules.
Size distributions were monomodal under the experimental conditions of the present study.
Oil droplets are natural systems which are not uniform in size
(ten Grotenhuis, Tuinier, & de Kruif, 2003). As expected for homogenized products, most of the fat globules in the milk samples used
in this study were <1 lm. The mean droplet diameter for the fat
droplets eluted according to the normal mode of operation correlates with the literature values published for commercially available bovine milk (Jussila et al., 1997). Although the values
obtained for milk samples which were not thermally processed
(control) were higher compared to the ones documented by other
researchers using the same methodology (Saeseaw et al., 2005), the
size distributions were within the theoretical limits (<1 lm). Additionally, in agreement with the previous study, chocolate milk
exhibited lower mean particle diameter compared to full-fat milk.
As illustrated in Fig. 3, the size distributions of the non heat-treated milk samples revealed that chocolate milk had the lowest
mean droplet diameter (0.734 0.013) lm, followed by semi-fat
milk (0.805 0.004) lm and full-fat milk (0.823 0.012) lm for
the milk batches analyzed in this study. Furthermore, the unheated
milk samples were analysed by Native PAGE for comparative purposes (Fig. 4). Native PAGE indicated that full-fat and semi-fat milk
are similar in terms of protein content as revealed by the corre-

Full-fat milk
Semi-fat
milk
Chocolate
milk

4000
3500

Response (counts)

Instrument
Column dimensions

and were left uncovered (without cover slip) to avoid destructing

the native fat globules structure. Pictures of the samples were taken using a microscope digital imager at 10 magnication (Celestron, USA).

3000
2500
2000
1500
1000
500
0

10

15

20

25

30

35

40

45

Time (min)
Fig. 2. Representative fractograms of full-fat (3.5%), semi-fat (1.0%) and chocolate
milk (3.0%) diluted by the same factor (14) indicating that the peaks correspond to
the fat globules.

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V. Raikos et al. / Food Research International 42 (2009) 659665

Full-fat milk
Semi-fat milk

Chocolate milk

50 C

95 C

65 C

110 C

80 C

125 C

0.8

Rel. mass

Rel. mass

0.8

0.6

0.6
0.4
0.2

0.4

0
0.2

0.1

0.2

0.3

0.6

0.9

1.0

2.0

Diameter ( m)

0
0.1

0.2

0.3

0.5

0.8

1.0

2.0

Fig. 5. Effect of heating temperature on the particle size distribution of full-fat milk.
Mean particle sizes of fat globules for each temperature have been deducted from
the highest points of each peak.

Diameter ( m)
Fig. 3. Mean particle diameter of non heat-treated full-fat, semi-fat and chocolate
milk (control batches).

sponding band pattern (lanes 1 and 2). However, chocolate milk

showed a relatively different protein band pattern on the gel with
respect to protein concentration. As shown in Fig. 4, the band
intensity of the proteins indicated by the arrows is higher for the
full-fat and semi-fat samples compared to the chocolate milk sample. As a result, we may speculate that the three samples show at
least some quantitative differences with respect to their protein
content.
4.2. Effect of heat treatment on the particle size
The effective particle size of homogenized milk increased when
samples were heated at temperatures above 50 C for 1 h for most

samples. Full-fat and semi-fat milk showed a small but steady increase in mean particle size with increasing temperature. As illustrated in Fig. 5 the mean particle size of fat globules for full-fat milk
shifts from (0.861 0.001) lm (50 C) to (0.901 0.001) lm
(125 C). Semi-fat milk shows a slightly higher increase in particle
size diameter compared to full-fat when heated within the same
temperature levels. At 50 C the mean particle size for semi-fat
milk is (0.845 0.003) lm and reaches the value of (0.917
0.003) lm when the heating temperature increases to 125 C
(Fig. 6). Thus, the particle size of fat globules increases on average
40 nm (4.65%) for full-fat and 72 nm (8.52%) for semi-fat milk following the heat treatment (50 C125 C). For both full-fat and
semi-fat milk samples, temperature has a signicant impact on
the particle size of fat globules. The particle size distribution of
full-fat and semi-fat milk when heated at 50 C for 1 h is statistically different (P < 0.05) to the one obtained when the samples
are heated at 125 C for 1 h. Similar ndings with respect to
changes in the particle size of milk emulsions following heat treatment were documented by other authors (Dickinson & Parkinson,
2004; Millqvist-Fureby, Elofsson, & Bergensthl, 2001).

50C

95 C

65C

110 C

80C

125 C

Rel. mass

0.8
0.6
0.4
0.2
0
0.1

0.2

0.4

0.7

1.0

2.0

Diameter ( m)
Fig. 4. Native PAGE of unheated milk samples; lane 1: full-fat milk, lane 2: semi-fat
milk, lane 3: chocolate milk.

Fig. 6. Effect of heating temperature on the particle size distribution of semi-fat

milk. Mean particle sizes of fat globules for each temperature have been deducted
from the highest points of each peak.

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V. Raikos et al. / Food Research International 42 (2009) 659665

According to the literature, this behavior reects the sensitivity

of whey proteins to heat-induced denaturation and aggregation
(Euston, Finnigan, & Hirst, 2002). Heat treatment of milk is widely
used to modify the properties of milk proteins, which in turn affect
the properties of the nal milk product. The main change that occurs during thermal processing of milk is denaturation of the whey
proteins. Above a certain critical temperature, the whey protein
molecule unfolds and exposes non-polar side, thereby conferring
hydrophobic character on the surface of the emulsion droplets
and enhancing proteinprotein interactions (Demetriades et al.,
1997). The unfolding step is the rst stage of denaturation in solution and it is at least partially reversible, while the subsequent
aggregation stage is irreversible, and is dependent on time and
temperature (Owusu Apenten, 1998). The unfolding temperatures
of the major whey proteins are in the range of 6080 C and are
for a-lactalbumin and bovine serum albumin (BSA) 6065 C and
for b-lactoglobulin 7580 C (Paulsson & Dejmek, 1990). The denaturation temperatures of the major whey proteins documented in
the literature are in agreement with the protein band prole of
the heated milk samples obtained by Native PAGE (Fig. 7).
The protein bands indicated by the arrows, which are clearly
seen when the samples are heated at 50 C for 1 h, disappear when
the temperature of the heat treatment is 95 C or higher. This is an
indication that at least some of the proteins of full-fat and semi-fat
milk unfold due to heat treatment, expose previously hidden
hydrophobic groups and may form aggregates of large molecular
weights. The temperatures at which the highest increase in mean
particle diameter of fat globules was observed were higher compared to the theoretical denaturation temperatures of the whey
proteins. Nevertheless, this may be attributed to the fact that in
this study the protein is mostly in the adsorbed state (homogenized milk), rather than in the normal native state found in aqueous media.
Tangsuphoom and Coupland (2005) suggested that both occulation and possibly a slight degree of coalescence accounted for the
increase in particle size in heated coconut emulsions. Protein stabilized emulsions have been shown to occulate after heating as
the proteinprotein associations formed bind the droplets together
in a network (Siliwinski, Roubos, Zoet, van Boeckel, & Wouters,

2003). Heat-denatured whey proteins form complexes with casein

micelles because of increasing hydrophobicity and at higher temperatures this complex formation is stronger than at lower temperatures (Erdem & Yuksel, 2005). Thus, heat-induced occulation due
to attractive interactions between hydrophobic patches on denatured protein molecules on different droplets may account for
the increases in particle size observed in this study. Coalescence
may then result due to the break down of lamella separating the
occulated droplets. As shown in Figs. 5 and 6 the tailing into
the larger droplet sizes increases with increasing protein denaturation, while the peak remains fairly constant. This provides further
evidence that oil droplet coalescence may occur in the system under investigation.
With respect to chocolate milk a different pattern in particle
size changes relative to heat treatment is observed. Chocolate milk
appears to be more heat-sensitive compared to full-fat and semifat milk. The particle size increases signicantly (P < 0.05) for chocolate milk when heated between 50 and 110 C (Fig. 8). When
heated at 50 C the mean particle size of chocolate milk is
(0.830 0.003) lm, whereas at 110 C the fat globules are (0.934
0.007) lm on average.
This corresponds to a 104 nm (12.53%) increase in average
droplet size. This may be attributed to chocolate milk being more
thermo-labile due to its composition and as a result more sensitive
to thermal processing. Nevertheless, when chocolate milk is subjected to more severe heat treatment (125 C), the mean particle
diameter drops signicantly (0.793 0.008) lm. A similar trend
where a considerable increase in particle size occurred within a
certain temperature range, followed by a decrease in particle size
at higher temperatures, is documented in the literature (Demetriades et al., 1997). Following thermal denaturation, interactions between molecules adsorbed to the same droplet (intradroplet) or
between those adsorbed to different droplets (interdroplet) may
occur. According to Monahan, McClements, and German (1996a),
within the temperature range 50110 C interdroplet interactions
were favored and therefore occulation occurs, whereas at higher
temperatures the intradroplet interactions were favored and therefore the degree of occulation is reduced. A second mechanism
which may account for the particle size reduction above a certain
temperature has also been suggested (Demetriades et al., 1997).
According to this proposed mechanism, the protein molecules are
only partially unfolded below a certain temperature and as a result
a certain degree of hydrophobicity on the droplet surface may lead
to a great tendency for droplet aggregation. At higher temperatures

50C

95C

65C

110C

80C

125C

Rel. mass

0.8
0.6
0.4
0.2
0

0.1

0.2

0.5

0.9

1.0

2.0

Diameter ( m)
Fig. 7. Native PAGE of heat-treated full-fat and semi-fat milk samples; lane 1: fullfat, 50 C, lane 2: full-fat, 95 C, lane 3: full-fat, 125 C, lane 4: semi-fat, 50 C, lane
5: semi-fat, 95 C, lane 6: semi-fat, 125 C.

Fig. 8. Effect of heating temperature on the particle size distribution of chocolate

milk. Mean particle sizes of fat globules for each temperature have been deducted
from the highest points of each peak.

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V. Raikos et al. / Food Research International 42 (2009) 659665

Fig. 10. Micrographs of chocolate milk fat droplets heated at (A) 50 C, (B) 65 C, (C)
80 C, (D) 95 C, (E) 110 C and (F) 125 C for 1 h.
Fig. 9. Native PAGE of heat-treated chocolate milk; lane 1: 50 C, lane 2: 65 C, lane
3: 80 C, lane 4: 95 C, lane 5: 110 C, lane 6: 125 C.

Full-fat milk
Semi-fat milk
Chocolate
milk

0.98

Particle size ( m)

the proteins become fully unfolded (Dalgleish, 1996) and are more
exible. Thus, they can effectively rearrange all non-polar amino
acids towards the oil phase which leads to low surface hydrophobicity and consequently the droplets are less susceptible to aggregation. Fig. 9 shows the effect of temperature on the degree of
denaturation of chocolate milk proteins.
When the samples are heated above 80 C, the protein corresponding to the band indicated by the arrow is not detectable. A
similar temperature-induced effect on the protein structure may
occur to the other milk proteins within the same temperature
range (50125 C) depending on their individual physicochemical
properties, which is not detected on the gel. However, Native PAGE
indicates that the heating conditions (time and temperature) affect
the degree of denaturation of milk proteins and may have an impact on the formation of large molecular weight aggregates. On
the other hand, elevated heating temperatures may lead to extensive denaturation of the whey proteins leading to insolubilization.
As a result, the sharp decrease in mean particle diameter of fat
globules may be related to decreased solubility of the denatured
protein. Furthermore, heat treatment, depending on the degree of
heating, may be responsible for thermomechanical fat deterioration of the milk samples. High heat treatment temperatures produce increased levels of free fat and changes in the globule
membrane. This may account for the fact that heat-treated fat
globules at 125 C were very few when observed under the microscope compared to the prole obtained for all other samples
(Fig. 10).
Fig. 11 shows the particle size change of all different types of
bovine milk used in this study as related to heating temperature.
It is relatively clear that chocolate milk exhibits a steep rise in
mean particle size when heated up to 110 C for 1 h with the fat
globule diameter dropping sharply when heated at 125 C.
On the other hand, full-fat and semi-fat samples show very similar behavior with respect to thermal processing. Fig. 11 provides
indicative information relevant to how processing conditions and
more specically temperature can affect protein functionality

0.93
0.88
0.83
0.78
0.73
30

50

70

90

110

130

150

Temperature ( C)
Fig. 11. Comparative mean particle diameter of full-fat, semi-fat and chocolate milk
as affected by heating temperature. Error bars represent standard deviations based
on triplicate runs.

which in turn determines the physicochemical properties (particle

size distribution) of a food product such as milk. Knowledge of the
behavior of milk samples to various environmental stimuli may be
a useful tool for manipulating their functional properties and nding optimal formulations.
5. Conclusions
The size of fat globules affects physicochemical properties of
milk such as creaming. SdFFF was employed to monitor changes
in size distributions of fat globules as affected by heat treatment.
The mean particle diameter of fat droplets increased signicantly
with increasing heating temperature for most samples. The observed increases in particle size might have been caused by occulation or coalescence of droplets. The extent of heat treatment

V. Raikos et al. / Food Research International 42 (2009) 659665

affects the level of denaturation of milk proteins. It seems likely

that the denaturation and aggregation of surface-bound proteins
accounts for the thermally-induced occulation here. However, it
is difcult to draw any denite conclusions on whether the altered
functionality is due to protein denaturation associated with
changes in protein molecular conformation or the effect of heat
treatment itself.
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