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Food Hydrocolloids 25 (2011) 604e612

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

Emulsifying properties of collagen bers: Effect of pH, protein


concentration and homogenization pressure
R.C. Santana, F.A. Perrechil, A.C.K. Sato, R.L. Cunha*
Department of Food Engineering, Faculty of Food Engineering, University of Campinas (UNICAMP), P.O. Box 6121, 13083-862 Campinas, SP, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 17 March 2010
Accepted 21 July 2010

The emulsifying properties of collagen bers were evaluated in oil-in-water (O/W) emulsions produced
under different conditions of pH, protein content and type of emulsication device (rotorestator and highpressure homogenizer). The stability, microstructure and rheology of the O/W emulsions were measured.
The phase separation and droplet size of the emulsions prepared using the rotorestator device (primary
emulsion) decreased with protein concentration and reduction in pH, allowing the production of electrostatically stable emulsions at pH 3.5. In contrast, emulsions at higher pH values (4.5, 5.5 and 7.5) showed
a microscopic three-dimensional network responsible for their stability at protein contents higher than
1.0% (w/w). The emulsions at pH 3.5 homogenized by high pressure (up to 100 MPa) showed a decrease in
surface mean diameter (d32) with increasing pressure and the number of passes through the homogenizer.
These emulsions showed droplets with lower dispersion and d32 between 1.00 and 4.05 mm, six times
lower than values observed for primary emulsions. The emulsions presented shear-thinning behavior and
lower consistency index and viscosity at higher homogenization pressures. In addition, the emulsions
showed a less structured gel-like behavior with increase in homogenization pressure and number of
passes, since the pressure disrupted the collagen ber structure and the oil droplets. The results of this
work showed that the collagen ber has a good potential for use as an emulsier in the food industry,
mainly in acid products.
2010 Elsevier Ltd. All rights reserved.

Keywords:
Microstructure
Emulsion
Rheology
Stability
High-pressure homogenization

1. Introduction
Emulsions consist of two immiscible liquids (usually oil and
water) where one of the liquids is dispersed in the other as
small (0.1e100.0 mm) spherical droplets. Emulsions are thermodynamically unstable systems, but they can be kinetically stabilized
(without phase separation) for a reasonable period of time by adding
substances known as emulsiers and/or thickening agents prior to
homogenization (McClements, 2005).
Emulsifying processes, such as rotorestator, high pressure,
membrane or ultrasonic homogenization, can also play an important role in emulsion stability by reducing the droplet size. In
rotorestator devices, oil and water are mixed at very high rotation
speed (1000e25,000 rpm) in a narrow gap (50e1000 mm) between
a rotating disk (rotor) and a static disk (stator). This process
promotes the stretch and break-up of the particles of the dispersed
phase by mechanical impingement against the wall, due to high
uid acceleration and shear stress in the turbulent ow in the gap.
Droplet mean with diameters smaller than 1 mm cannot be
* Corresponding author. Tel.: 55 19 35214047; fax: 55 19 35214027.
E-mail address: rosiane@fea.unicamp.br (R.L. Cunha).
0268-005X/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2010.07.018

produced with this system (Jafari, Assadpoor, He, & Bhandari, 2008).
On the other hand, high-pressure homogenizers are used to produce
stable emulsions with droplet diameter smaller than 1 mm and
narrow droplet distribution. In this procedure the pressurized liquid
is forced through a valve that causes a sudden sharp constriction
in the ow. The pressure energy applied at the valve is transferred
into kinetic energy and the particle break-up is initiated by
a combination of turbulence and laminar shear (Dickinson, 1992).
The homogenization pressure is usually between 5 and 50 MPa, but
it may reach 700 MPa (Floury, Belletre, Legrand, & Desrumaux,
2004; Jafari et al., 2008; Marie, Perrier-Cornet, & Gervais, 2002).
During emulsion formation, stabilizers with surface activity
are rapidly adsorbed onto the newly formed oilewater interface,
reducing the surface tension, protecting the oil droplets against
coalescence and thereby providing physical stability to the emulsion
during processing and storage (McClements, 2005). A wide range of
emulsiers is considered as food grade such as small molecular
weight surfactants, phospholipids and biopolymers (Krog & Sparso,
2004). Nevertheless, food industry has presented a growing interest
in the replacement of synthetic emulsiers by natural ones, such as
polysaccharides and proteins (Garti, 1999). The study of emulsifying
properties of proteins, as casein, whey protein, soybean protein and

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612

gelatin, has been the subject of several recently published research


papers (Keerati-u-rai & Corredig, 2009; Lizarraga, Pan, Aon, &
Santiago, 2007; Neirynck, Van lent, Dewettinck, & Van der Meeren,
2007; Surh, Deckes, & McClements, 2006).
Collagen is a protein widely used in food industries to improve the
elasticity, consistency and stability of foods, although its use has only
been carried out in an empirical way (Olivo & Shimokomaki, 2002).
Collagen ber is a new ingredient obtained from collagen in its crude
form, while gelatin is produced from collagen hydrolysis (Nicoleti &
Telis, 2009; Wolf, Sobral, & Telis, 2009). Collagen ber is obtained by
scraping pieces of dermis and subcutaneous tissue. This material
is previously submitted to chemical treatment for defatting, dried at
a mild temperature and ground for the collagen ber production.
During the last step, two fractions are generated according to the
particle size: the ner one is classied as collagen powder, while the
coarser one corresponds to collagen bers. Differences in particle
shape between the fractions can also be expected (Nicoleti & Telis,
2009). More recent studies have shown the gelation properties of
collagen ber (Mximo & Cunha, 2008), and its use as a raw material
for the production of self-biocomposite lms (Wolf et al., 2009).
However, the emulsifying properties of collagen ber have not yet
been extensively explored, in spite of their traditional use as a water
and fat binder in meat products (Asghar & Henrickson, 1982).
In this context, the purpose of this study was to investigate the
potential use of collagen ber as an emulsier in food products,
evaluating the effects of pH, protein content, emulsifying device
(rotorestator and high-pressure homogenizer), homogenization
pressure (20e100 MPa) and the number of homogenization cycles
on the phase separation, droplet size and rheology of the emulsions
stabilized by collagen ber.
2. Material and methods
2.1. Material
Collagen ber extracted from bovine hides was kindly provided
by NovaProm Food Ingredients Ltda (Guaiara, SP, Brazil). The
detailed physico-chemical characterization of collagen ber,
including its amino acid composition, was described by Nicoleti
and Telis (2009), who found a protein content close to 86% (w/w).
Soybean oil (Bunge, Brazil) was purchased from a local market and
all chemicals used were of analytical grade (Sigma Chemical
Company, St. Louis, USA).
2.2. Emulsion preparation
Initially, collagen ber dispersions (aqueous phase) were
prepared using deionized water with gentle magnetic stirring at
room temperature for 1 h. The pH of the samples was constantly
adjusted to the desired value with 1.0 M HCl or 1.0 M NaOH solutions. Sodium azide (0.1% w/w) was added to prevent microbial
growth.
Oil-in-water emulsions (O/W) were prepared using two
different emulsifying processes: (i) primary emulsions were
prepared by mixing the collagen ber dispersions with 10% (w/w)
soybean oil using a rotorestator device (Ultra-Turrax mixer, model
T18 basic, Germany) at 20,000 rpm for 4 min. These emulsions
showed a nal protein content of 0.5, 1.0, 2.0 and 3.0% (w/w) at
different pH values (3.5, 4.5, 5.5 and 7.5); (ii) the primary emulsions
prepared in the rotorestator device and composed by 0.5% protein
(w/w) at pH 3.5, were processed in a two-stage high-pressure
homogenizer (NS1001L2K-PANDA 2K, Niro Soavi S.p.A., Parma,
Italy). The effect of homogenization pressure on emulsion properties was investigated by applying different pressures in the rst
stage of the homogenizer (20e100 MPa). The pressure in the

605

second stage was xed at 5 MPa, since a low pressure homogenizing valve is conventionally used to increase cavitation and
decrease droplet recoalescence phenomenon (Freudig, Tesch, &
Schubert, 2003; Mohan & Narsimhan, 1997). The emulsions were
run through the high-pressure homogenizer once or twice (recycling), and the temperature was measured immediately after.
All systems were evaluated according to its stability and particle
size. Rheological analysis was also done for emulsions homogenized at high pressure.
2.3. Emulsion properties
2.3.1. Stability
Immediately after the emulsifying process, aliquots of the emulsions prepared using the rotorestator device and the high-pressure
homogenizer were placed in 10 mL and 50 mL graduated cylinders,
respectively. The stability of the emulsions to occulation/coalescence was evaluated by visually monitoring the development of
a bottom phase during 7 days of storage at room temperature (25  C).
The creaming index (CI %) of the emulsions was expressed as CI
(%) (Vs/Vi)  100, where Vi represents the initial volume of the
emulsion and Vs the volume of clear serum formed at the bottom of
the tubes (Keowmaneechai & McClements, 2002).
The inuence of the pH value and protein content on the
creaming process was analyzed using a rst order kinetics equation
given as CI (%) CIeq (1  expkt), where CIeq is the equilibrium
creaming index, t is the time and k is the creaming rate in h1.
2.3.2. Optical characterization and size distribution of oil droplets
The microstructure of the emulsions was studied using a Carl
Zeiss light microscope Model mf-AKS 24  36 EXPOMET (Zeiss,
Germany). Emulsions homogenized using the rotorestator device
and high-pressure equipment were analyzed immediately after the
emulsifying process and after 7 days of storage, respectively. The
samples were poured onto microscope slides, covered with glass
cover slips and observed at a magnication of 40 and 100.
At least 20 images were taken for each sample and the best 10 sharp
images were analyzed using the public domain software Image
J v1.36b (http://rsb.info.nih.gov/ij/).
Micrographs of the emulsion prepared using the rotorestator
device were analyzed according to Perrechil and Cunha (2010). The
pictures were transformed into 8-bit grey scale binary images with
640e480 pixels and then segmented by thresholding. The grey
level used to threshold the image was the median of the grey level
histogram of each image. During this process, the pixels were only
deemed as detected if their grey value was lower than the threshold
setting (Pugnaloni, Matia-Merino, & Dickinson, 2005). Droplets
connected to image border were suppressed of image analysis.
Micrographs of the emulsions homogenized by high pressure could
not be analyzed according to Perrechil and Cunha (2010) and Pugnaloni et al. (2005), since their droplet diameters were less than 5 mm, too
small to allow reliable droplet identication. Thus these images were
analyzed by measuring at least 500 droplets (one by one).
After conversion of the pixel-scale into microns using a scaling
factor, surface mean diameter (d32) of the emulsions was calculated
P
P
as d32 (nid3i )/ (nid2i ), where ni was the number of particles
with diameter di.
The droplet size distributions were statistically compared
with a probability density function of a log-normal distribution
(Equation (1)) using the non-parametric KolmogoroveSmirnov test
and the Statistica 5.5 software (Statsoft Inc., Tulsa, USA).

f di

" 
2 #
 ln di  ln dg
1
p exp
ln sg 2p
2 ln2 sg

(1)

606

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612

where dg and sg were the geometric mean and standard deviation


of the geometric mean, respectively, as given by Equations (2) and
(3) (McClements, 2005):

Tukey Procedure and the statistical analyses were applied using the
software Statistica 5.5 (Statsoft Inc., Tulsa, USA).

ln dg

(2)

3. Results and discussion

(3)

3.1. Emulsions homogenized using the rotorestator device


(primary emulsions)

ln sg

ni ln di =N

r
Xh 
2 i
=N
ni ln di  ln dg

where N is the total number of droplets.


2.3.3. Rheological measurements
Rheological measurements were carried out using a Physica
MCR301 rheometer (Anton Paar, Germany). Samples obtained
immediately after the homogenization process were placed in
a parallel plate geometry (with a 0.6 mm gap) made of glass (50 mm
diameter) or stainless steel (75 mm diameter). Steady shear and
oscillatory measurements were carried out in triplicate at 25  C.
The rheological analyses were not applied to the primary emulsions
homogenized in the rotorestator device, since most of them
showed phase separation only a few minutes after preparation.
Flow curves were obtained using an upedowneup step program
with a shear rate range between 0 and 300 s1. The rheological
behavior of the emulsions was analyzed according to the Power-Law
model and expressed as s k  g_ n , where s is the shear stress (Pa), g_
the shear rate (s1), k the consistency index (Pa sn) and n the ow index.
Oscillatory measurements were used to determine the complex
shear (G*), storage (G0 ) and loss (G00 ) moduli over an ascending frequency
ramp of 0.1e10 Hz. All measurements were carried out within the linear
viscoelastic region (deformation less or equal than 1.0%).
2.4. Statistical analysis
The measurements were done in triplicate. Signicant differences (p < 0.05) between treatments were determined by the

The effects of pH (3.5, 4.5, 5.5 and 7.5) and protein content
(0.5, 1.0, 2.0 and 3.0% w/w) were evaluated through their effects on
creaming kinetics and droplet size of the primary emulsions
prepared using the rotorestator device.
3.1.1. Creaming kinetics
Fig. 1 shows the creaming kinetics of the emulsions with storage
time, veried by visual observations. As can be seen from the
proles, phase separation usually occurred after short times (in the
rst 30 min) for very high creaming rates.
In general, emulsion stability improved with increase in protein
concentration and reduction in pH, reducing the equilibrium
creaming index (CIeq) and creaming rate (k) (Table 1). The most
stable emulsions were observed at pH 3.5, making it possible to
produce acid emulsions stabilized by a protein. Emulsions at pH 3.5
with protein concentrations higher than 0.5% (w/w) showed no
signs of serum separation even after 7 days of storage. The opening
of collagen triple helices at pH 3.5 modied the protein surface
hydrophobicity, and could be partially responsible for the better
emulsifying properties of the collagen ber at this low pH.
However, electrostatic interactions between the emulsion droplets
are probably the most important inuence of the pH. In emulsions
stabilized by proteins, the droplet surface is charged if the pH is
far from the protein pI. As the collagen ber pI is between 6.5 and
8.5 (Neklyudov, 2003), the droplets are positively charged at pH
3.5, which led to a strong electrostatic repulsion between them,
80

60

CI (% v/v)

CI (% v/v)

80

40
20

60
40
20

0
0

20

40

60

20

40

80

80

C
60

CI (% v/v)

CI (% v/v)

60

Time (min)

Time (min)

40
20

60
40
20

0
0

20

40

Time (min)

60

20

40

60

Time (min)

Fig. 1. Effect of pH and protein content on the phase separation of emulsions stabilized by collagen bers in the rst 60 min after processing. Protein concentration: 0.5% (eBe),
), 2.0% (e6e) and 3.0% (
). pH: 3.5 (A), 4.5 (B), 5.5 (C) and 7.5 (D).
1.0% (

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612

607

Table 1
Equilibrium creaming index (CIeq % v/v) obtained after 7 days of emulsion storage and creaming kinetics as described by the parameter of the rst order kinetics equation (k1)
tted to the creaming index (R2 > 0.90) of the emulsion homogenized by the rotorestator device during seven days of observation.
% Protein (w/w)

0.5
1.0
2.0
3.0

Equilibrium creaming index (CIeq (% v/v))

Creaming kinetics (k (h1))

pH

pH

3.5

4.5

5.5

7.5

3.5

4.5

5.5

7.5

1.00Aa
0.00Aa
0.00Aa
0.00Aa

63.00Ab
54.33Bb
15.33Cb
0.00Da

56.33Ac
53.67Ab
29.00Bc
11.67Cb

65.67Ab
57.50Bb
29.00Cc
13.33Db

0.02Aa
0.00Aa
0.00Aa
0.00Aa

44.70Ab
19.54Bbc
2.58Cb
0.00Da

34.58Ac
25.69Bb
6.53Cc
1.95Db

46.42Ab
11.63Bd
1.77Cb
0.69Cab

Different superscript letters indicate signicant difference (p > 0.05). Capital letters compare differences within a column and small letters compare differences between
different pH values for the same creaming parameter.

preventing droplets from coming close enough together to aggregate and coalesce (McClements, 2005).
On the other hand, emulsions maintained at pH values close to
the protein pI show uncharged surface droplets and the repulsive
forces may no longer be strong enough to prevent the droplets from
aggregating (McClements, 2005), so that these droplets should
be attracted due to hydrophobic and Van der Walls forces (Chen,
Dickinson, & Edwards, 1999). At this pH condition, the higher
stability observed with increased protein content can be explained
by the viscosity enhancement and steric stabilization of the
collagen ber, decreasing droplets mobility and consequent coalescence and destabilization. In spite of the high pH, emulsions at

pH 7.5 and stabilized by more than 0.5% (w/w) protein, showed an


intermediary creaming rate (k) that could be explained by the
partial solubility of the collagen ber at its pI (Mximo & Cunha,
2008), retarding droplet mobility due to steric hindrance.
3.1.2. Microstructure and droplet size
The microstructures of the emulsions prepared using the
rotorestator device and stabilized by collagen bers are shown
in Fig. 2, revealing polydisperse emulsions with a wide range of oil
droplet sizes. Droplet size distribution is an important parameter for
some emulsion properties such as shelf life and texture, and thus its
control and measurement is important (McClements, 2005).

Fig. 2. Micrographs of O/W emulsions produced using the rotorestator device and stabilized by collagen bers. Scale bar 50 mm.

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612

However, a considerable amount of the process energy added in this


emulsication method is dissipated in the form of heat, so that the
production of monodispersed emulsions with a small droplet size is
difcult (Anton, Benoit, & Saulnier, 2008).
The emulsions showed structural differences depending on the
pH value. True emulsions were observed at pH 3.5 (pH far from that
of the protein pI), with droplets surrounded by a continuous aqueous
phase. On the other hand, emulsions produced at higher pH values
showed a macroscopically three-dimensional network formed due
to the absence of electrostatic repulsion between droplets. In addition, a high content of insoluble protein can be clearly observed in
the emulsions at pH 7.5, 5.5 and 4.5. Thus, collagen ber probably
acts as a stabilizer at these conditions. It was evident that emulsions
formed at higher pH values containing more than 1.0% (w/w) of
protein, were stabilized by a three-dimensional network composed
of an almost intact collagen ber structure that reduced the droplets
movements and decreased the CIeq.
Table 2 shows the average droplet size (d32) of the emulsions
prepared using the rotorestator device. It was only possible to
evaluate the emulsions produced at pH 3.5 and under some
conditions at pH 4.5, due to difculties in visualizing the droplets in
the others conditions, as mentioned before. Surface mean diameter
of the oil droplets varied between 6.71 and 12.76 mm. In agreement
with the stability tests, the smallest droplet sizes were observed at
pH 3.5 with protein content above 0.5% (w/w), conrming emulsifying properties of collagen ber at low pHs.
3.2. Emulsions homogenized by high pressure
The effects of homogenization pressure (20e100 MPa) and the
number of passes through the homogenizer were evaluated using
O/W emulsions composed of 10.0% (w/w) soybean oil and 0.5%
(w/w) collagen ber protein at pH 3.5, which was the best pH
condition to obtain stable emulsions using the rotorestator device.
3.2.1. Temperature rise during high-pressure homogenization
The temperature of the emulsions increased linearly with the
pressure and number of passes through the homogenizer (Fig. 3),
in agreement with Desrumaux and Marcand (2002) and Floury,
Desrumaux, Axelos, and Legrand (2003). The rise in temperature
depends on the uid composition (Corts-Muoz, Chevalier-Lucia,
& Dumay, 2009) and on the conguration and heat capacity of the
homogenizer itself (Sandra & Dalgleish, 2005). The distinct
warming up of the uid was due to viscous stress caused by the
high velocity of the uid ow, which then impinged on the valve
(McClements, 2005).
Heating during homogenization occurs during a short period of
time, and its contribution to modication of the macromolecules
and droplet size is uncertain (Floury et al., 2003). Heat can contribute
to the production of small droplets by decreasing the interfacial
tension between the oil and water, or by reducing the viscosity
of both phases. However, proteins used as emulsiers can lose
their functional properties when heated above the denaturation

Table 2
Surface mean diameters d32 (mm) for emulsions emulsied using the rotorestator
device and stabilized by collagen bers.
Protein concentration
(% w/w)

pH
3.5

4.5

5.5

7.5

0.5
1
2
3

12.76  2.26x
8.60  1.65y
6.71  1.19y
7.76  1.37y

e
e
9.88  1.36xy
11.71  1.56x

e
e
e
e

e
e
e
e

Different superscript letters indicate signicant difference (p > 0.05).

55

Temperature (C)

608

Tpeak

50

Range of
collagen
denaturation

45

Tonset

40
35
30
0

20

40

60

80

100

120

Pressure (MPa)
Fig. 3. Temperature of emulsions as a function of homogenization pressure. Number of
passes through the homogenizer: (C) 1 and (-) 2. Tonset (initial denaturation temperature) 39.5  C and Tpeak (denaturation temperature) 52.5  C (Wolf et al., 2009).

temperature. In these conditions, collagen is disintegrated from


a triple helical structure to random coils, accompanied by changes in
its physical properties (Usha & Ramasami, 2004). At pH 3.0, the
collagen starts to denature at 39.5  C (Tonset), with denaturation
temperature (maximum peak) of 52.5  C (Tpeak) (Wolf et al., 2009).
Thus some structural modications of the collagen bers could
have happened in the emulsions homogenized by pressures above
60 MPa with 1 pass, or 40 MPa with 2 passes, although no emulsion
reached the peak temperature for collagen denaturation (Fig. 3).
3.2.2. Creaming index
All the emulsions homogenized by high pressure showed
good stability, with no signs of phase separation in 10 mL cylinders
(diameter 15.5 mm, height 65 mm). In order to reduce wall
effects, stability tests of these emulsions were also carried out in
50 mL cylinders (internal diameter 25 mm, height 95 mm),
since it is well known that the conning wall can exert an
extra retardation effect on a spherical particle settling in a liquid
(Chhabra, Agarwal, & Chaudhary, 2003). Under these conditions,
the emulsion processed under the mildest conditions (20 MPa with
one pass), which showed a non homogeneous aspect, presented
10% (v/v) of serum phase separation after 7 days of storage. All the
other emulsions showed a homogeneous aspect and no phase
separation even in 50 mL cylinders.
3.2.3. Microstructure and droplet size distribution
The microstructure of the emulsions homogenized by high
pressure is shown in Fig. 4, revealing that the droplet size and its
polydispersity decreased with increase in pressure and number of
passes through the homogenizer. This occurred because the higher
homogenization pressure led to an increase in impact forces that
act on the droplets, causing disruption of the interfacial membranes
(McClements, 2005) with a consequent increase in the interfacial
area and interaction between oil and emulsier (Floury, Desrumaux, & Lardires, 2000). The effect of the number of passes can be
associated with the increase in residence time of the emulsier in
the homogenizer valve, allowing for an improved adsorption onto
the droplet surface before their collision and coalescence (Floury
et al., 2000).
The surface mean diameter (d32) varied between 1.00 and
4.05 mm (Fig. 5), showing a considerable reduction in droplet size
up to 60 MPa, followed by a slight additional decrease up to
100 MPa/1 pass. No further droplet reduction was observed with
treatments above 60 MPa/2 passes, and in such a way that this
point, where the surface mean diameter reached a plateau at
around 1.00 mm, can be identied as the critical limit. The surface
mean diameter can be related to the homogenization pressure (P)

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612

609

Fig. 4. Micrographs of emulsions homogenized by high pressure and stabilized by 0.5% (w/w) of protein. Scale bar 10 mm.

exponent m obtained was around 1. The slight decrease in the


absolute value for m (turbulence increase) when the emulsions
were passed twice through the homogenizer, can be explained from
the reduction in oil droplet packing (Fig. 4), with a consequent
decrease in emulsion viscosity (Fig. 7 and Table 4).
The droplet size distribution of the emulsions was well
described by a log-normal distribution (Fig. 6), as reported by other
authors (Ambrosone & Ceglie, 1997; Coupland & McClements,
2001; McClements, 2005; McClements & Coupland, 1996). Table 3

25

Frequency (%)

using the Power-Law adjustment (d32 f Pm). The exponent m of


the Power Law has been empirically associated with Reynolds
number (Re), which depends on the valve dimension, liquid
viscosity and homogenization pressure. This parameter shows
values between 0.6 and 1.0, depending on the ow regime (Floury
et al., 2003). For a large homogenizer and a low uid viscosity, the
ow regime is predominantly turbulent-inertial and d f P0.6.
For a large homogenizer and a high uid viscosity, the ow regime
is predominantly turbulent-viscous and d32 f P0.75. For small
homogenizers such as those used on a bench-scale, the ow regime
may even be laminar-viscous and d32 f P1.0 (McClements, 2005).
Several authors have discussed the relationship between pressure and droplet size. Walstra and Smulders (1997) obtained
d32 f P0.9 for homogenization at P  15 MPa, while Floury et al.
(2003) obtained d32 f P0.48 for homogenization at P  350 MPa.
For the emulsions stabilized by collagen bers and emulsied at
P  100 MPa, values of d32 f P1.08 (R2 0.95) and d32 f P0.90
(R2 0.90) were found for the samples homogenized using 1 and 2
passes, respectively. It should be noticed that the surface mean
diameter of the emulsions homogenized at 20 MPa with 1 pass was
not included in the Power-Law tting, since this emulsion showed
phase separation. From the obtained results it can be estimated
that the ow in the homogenizer was laminar-viscous, since the

20
15
10
5
0
0.1

1.0

Diameter (m)

5
25

Frequency (%)

d32 (m)

10.0

3
2
1

20
15
10
5

20

40

60

80

100

120

Pressure (MPa)
Fig. 5. Effect of homogenization pressure and number of passes on the surface mean
diameters (d32) of the emulsions homogenized at high pressures. Number of passes:
(C) 1 and (-) 2.

0 .1

1.0

1 0 .0

Diameter (m)
Fig. 6. Effect of homogenization pressure and number of passes on the droplet size
)
distribution in the emulsions homogenized using high pressures. Pressure (MPa): (
) 40, (
) 60, (e e e) 80 and (
) 100. Number of passes: (A) 1 and (B) 2.
20, (

610

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612

Table 3
Parameters obtained from the log-normal distribution of the droplet size.
Homogenization
pressure (MPa)

20
40
60
80
100

Shear stress (Pa)

10

Standard deviation
(sg/mm)

Geometric mean
dg =mm
1 Pass

2 Passes

1 Pass

2 Passes

2.83
2.56
1.36
1.45
0.93

2.66
1.90
0.70
0.85
0.87

1.65
1.71
1.75
1.57
1.46

1.49
1.57
1.54
1.25
1.28

8
6
4
2
0
0

50

100

150

200

250

300

250

300

-1

Shear rate (s )

Shear stress (Pa)

10

8
6
4
2
0
0

50

100

150

200
-1

Shear rate (s )
Fig. 7. Shear stress versus shear rate for the emulsions homogenized using high
pressures and stabilized by collagen bers. Homogenization pressure (MPa): 20 (,),
40 (e), 60 (6), 80 (B) and 100 (>). Number of passes: (A) 1 and (B) 2.

shows the geometric mean and its standard deviation of the


geometric mean as obtained from the log-normal distribution. In
agreement with observations of the micrographs, the droplet
size dispersion (described by a log-normal standard deviation)
decreased with increase in homogenization pressure and number
of passes, specially the emulsions homogenized with pressures of
80 and 100 MPa, which showed a high frequency of droplets on

a submicron scale, reaching values below 1.0 mm. Emulsions


homogenized at 60 MPa showed a high droplet size frequency
at the lowest values observed (between 0.2 and 0.4 mm), while
the emulsions homogenized at 80 and 100 MPa showed lower
polydispersity.
3.2.4. Rheology
The ow curves of all emulsions homogenized at high pressure
and stabilized using collagen bers showed shear-thinning
behavior (Fig. 7). The decrease in apparent viscosity of the emulsions with increasing shear rate could be attributed to the deformation and disruption of clusters or aggregates of droplets, and
their ordering within the ow eld (McClements, 2005).
The Power-Law was tted to the data (Table 4), showing a good
determination coefcient (R2 > 0.999) for all emulsions. Table 4
shows the apparent viscosity at 100 s1 of the emulsions homogenized by high pressure and stabilized by collagen bers.
The emulsion homogenized at 20 MPa with 1 pass showed low
values for the consistency index (k) and apparent viscosity.
The rheological measurements were probably not reliable, since it
showed a weak structure and phase separation. In general, the
emulsions homogenized at high pressure showed higher values for
the ow index and lower values for the consistency index with
increase in pressure and number of passes. Moreover, by comparing
the up and down ow curves for emulsions homogenized at higher
pressures (80 and 100 MPa) (results not shown), less hysteresis was
observed, suggesting that the emulsion structure was broken in the
homogenization process.
The apparent viscosity increased slightly in the emulsions
homogenized at pressures from 20 MPa/2 passes to 60 MPa/2
passes. The increase in viscosity with reduced droplet size could be
attributed to an increase in hydrodynamic interactions between the
droplets, since the mean separation distance between the droplets
decreases when the droplet size is reduced (Pal, 2000). Moreover,
a greater amount of absorbed protein or more tightly packed
proteins at the oil-in-water interface can increase the emulsion
viscosity (Innocente, Biasutti, Venir, Spaziani, & Marchesini, 2009).
On the contrary, emulsions homogenized at 80 and 100 MPa
showed a reduction in the viscosity with decreasing droplet size, as
observed by Desrumaux and Marcand (2002). In this case, smaller
droplet sizes led to lower internal resistance than larger ones
or linear chains of droplets (Schimidt & Smith, 1989). Thus, the
behavior of the viscosity may be well related to mean and droplet
size distribution. As can be observed in Figs. 5 and 6 and Table 4, the
surface mean diameter decreased with decrease in pressure up to
60 MPa, while droplet size polydispersity decreased considerably at
80 and 100 MPa. Floury et al. (2003) and Schulz and Daniel (2000)
showed that the decrease in emulsion viscosity was related to the
reduction in food protein functionality caused by the homogenization pressure, although this was not clear veried in the present
work.
Emulsions homogenized at high pressures and stabilized
using collagen bers showed a gel-like behavior, with the storage
modulus (G0 ) greater than the loss modulus (G00 ) in all cases (data

Table 4
Rheological parameters obtained from the Power-Law model and apparent viscosity at 100 s1 for emulsions homogenized at high pressure and stabilized by collagen bers.
Rheological parameter

n (e)
k (Pa sn)
h100 (mPa s)

Homogenization pressure (MPa) with 1 pass

Homogenization pressure (MPa) with 2 passes

20

40

60

80

100

20

40

60

80

100

0.53Aa
0.25Aa
27.6Aa

0.48Ba
0.56Ba
51.1Ba

0.52Aa
0.48Ca
51.3Ba

0.53Aa
0.38Da
43.4Ca

0.52Aa
0.40CDa
43.2Ca

0.46Ab
0.53Ab
44.3Ab

0.50Bb
0.50Aa
48.5ABa

0.50Bb
0.54Aa
53.3Ba

0.67Cb
0.09Bb
19.1Cb

0.69Cb
0.05Bb
12.3Db

Different superscript letters indicate signicant difference (p > 0.05), capital letters compare differences between homogenization pressure for the same number of passes;
small letters compare differences between number of passes at the same homogenization pressure.

R.C. Santana et al. / Food Hydrocolloids 25 (2011) 604e612

100

611

the stability, structure and rheology of emulsions, showing that the


best conditions to use this ingredient would be at acid pH values.

Acknowledgments

G* (Pa)

10

The authors would like to thank CNPq for their nancial support.
References

0,1
0.1

10

Frequency ( Hz)
100

G* (Pa)

10

0,1
0.1

10

Frequency ( Hz)
Fig. 8. Inuence of homogenization pressure and number of passes on the complex
modulus (G*) of the emulsions homogenized using high pressure and stabilized by
collagen bers. Pressure: 20 (>), 40 (,), 60 (B), 80 (6) and 100 MPa (e). Number of
passes: (A) 1 and (B) 2.

not shown). An increase in homogenization pressure and number


of passes decreased the complex modulus (G*) and increased the
dependence of G0 and G00 on the frequency (Fig. 8), indicating that
this process disrupted the collagen ber structure as well as that of
the oil droplets, resulting in less structured emulsions.
4. Conclusions
The study of the primary emulsions showed that electrostatic
interactions were responsible for the emulsion stability at pH 3.5.
This occurred because the pI of the collagen bers is between 6.5
and 8.5, which is considerably higher than those of conventional
protein emulsiers such as soybean, casein and whey proteins. With
respect to the primary emulsions at higher pH values (4.5, 5.5 and
7.5), the increase in sample stability was only observed as a function
of the protein content. At higher pH values, the collagen bers
showed steric hindrance, forming a lm around the droplets and
reducing their mobility and consequent coalescence. The use of
high-pressure homogenization produced stable and less structured
emulsions, since this process disrupted the collagen ber structure
and the oil droplets. Up to a pressure of 60 MPa, the reduction in
surface mean diameter led to an increase in emulsion viscosity
caused by an increase in hydrodynamic interactions between the
droplets. On the other hand, at higher pressures (80 and 100 MPa),
a considerable decrease in droplet size polydispersity was apparent,
with a consequent reduction in emulsion viscosity. Finally, the
results of this work allowed for the elucidation of some of the
interaction mechanisms of collagen bers, and their inuence on

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