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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
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
Tukey Procedure and the statistical analyses were applied using the
software Statistica 5.5 (Statsoft Inc., Tulsa, USA).
ln dg
(2)
(3)
ln sg
ni ln di =N
r
Xh
2 i
=N
ni ln di ln dg
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% (
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
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
Fig. 2. Micrographs of O/W emulsions produced using the rotorestator device and stabilized by collagen bers. Scale bar 50 mm.
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
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).
609
Fig. 4. Micrographs of emulsions homogenized by high pressure and stabilized by 0.5% (w/w) of protein. Scale bar 10 mm.
25
Frequency (%)
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
Table 3
Parameters obtained from the log-normal distribution of the droplet size.
Homogenization
pressure (MPa)
20
40
60
80
100
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 )
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.
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)
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.
100
611
Acknowledgments
G* (Pa)
10
The authors would like to thank CNPq for their nancial support.
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0,1
0.1
10
Frequency ( Hz)
100
G* (Pa)
10
0,1
0.1
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Frequency ( Hz)
Fig. 8. Inuence of homogenization pressure and number of passes on the complex
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