International Journal of Food Properties

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Nano-Emulsion Production by Sonication and

Microfluidization—A Comparison

Seid Mahdi Jafari , Yinghe He & Bhesh Bhandari

To cite this article: Seid Mahdi Jafari , Yinghe He & Bhesh Bhandari (2006) Nano-Emulsion
Production by Sonication and Microfluidization—A Comparison, International Journal of Food
Properties, 9:3, 475-485, DOI: 10.1080/10942910600596464

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International Journal of Food Properties, 9: 475–485, 2006
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print / 1532-2386 online
DOI: 10.1080/10942910600596464

NANO-EMULSION PRODUCTION BY SONICATION AND

MICROFLUIDIZATION—A COMPARISON

Seid Mahdi Jafari

School of Land and Food Sciences, University of Queensland, Brisbane, Australia

Yinghe He
School of Engineering, James Cook University, Townsville, Australia

Bhesh Bhandari
School of Land and Food Sciences, University of Queensland, Brisbane, Australia

The efficiency of sonication and microfluidization to produce nano-emulsions were evaluated

in this study. The purpose was to produce an oil-in-water nano-emulsion of d-limonene to
apply it in the next step for nano-particle encapsulation. In the entrapment and retention of
volatiles or for the microencapsulation efficiency, emulsion size is one of the critical factors.
In this study, a bench-top sonicator and an air-driven microfluidizer were used to prepare
the emulsions. Results show that, while both methods were capable of producing nano-
emulsions of the size range of 150–700 nm, the microfluidizer produced emulsions with
narrower size distributions and sonication was more convenient in terms of operation and
cleaning. In general, the size of the emulsions decreased with increasing sonication time, or
the microfluidization pressure and duration. However, for both sonication and microfluidi-
zation, optimal conditions were necessary for emulsification beyond which the emulsion
sizes would either increase or have little change with further processing.

Keywords: Microfluidization, Ultrasound, Nano-emulsion, Particle size, Size distribution,

d-limonene, Whey protein concentrate, Hi-Cap.

INTRODUCTION
Nano-emulsions are defined as emulsion systems mostly covering the size range of 50
to 200 nm (transparent) or up to 500 nm that have a milky appearance.[1] The attraction of
nano-emulsions for application in various industrial fields, for example, in personal care and
cosmetics, health care, pharmaceuticals, and agrochemicals, is their very small droplet size
and their high kinetic stability. Nano-emulsions can be prepared by low-energy emulsifica-
tion methods or spontaneous emulsification such as phase inversion temperature (PIT) or
phase inversion composition that are usually used in pharmaceutical and cosmetic indus-
tries.[2] In most cases, however, submicron emulsions can be prepared using a high-energy
input such as ultrasonication or by high-pressure homogenization and microfluidization.

Received 12 September 2005; accepted 25 September 2005.

Address correspondence to Bhesh Bhandari, School of Land and Food Sciences, University of Queen-
sland, Brisbane, Qld 4072, Australia. E-mail: b.bhandari@uq.edu.au

475
476 JAFARI, HE, AND BHANDARI

The Microfluidizer® was originally designed by the Arthur D. Little Co., but was
later taken over by the Microfluidics Corp.[3] This equipment has been traditionally used
in the pharmaceutical industry to make pharmaceutical emulsions and has been used to
produce flavor emulsions or homogenized milk only in the last few years. It works on the
principle of dividing a pressure stream into two parts, passing each part through a fine
orifice, and directing the flows at each other in the heart of the microfluidizer, i.e., the
interaction chamber. The microfluidizer uses a high pressure to guide the flow stream
through microchannels toward the impingement area, which creates a very high shearing
action that provides an exceptionally fine emulsion. Within the interaction chamber,
cavitation, along with shear and impact, reduces emulsion droplet size.[4]
Various studies have shown that emulsification by microfluidization is superior to
the other types of conventional homogenization. The distributions of particle sizes pro-
duced by a microfluidizer appear to be narrower and smaller than the products of tradi-
tional homogenization. Pinnamaneni et al.[5] found the microfluidizer to be more effective
than the homogenizer in producing stable oil in water submicron emulsions at low emulsi-
fier concentrations. Also, submicron emulsions prepared by the microfluidization process
had smaller droplet diameters and exhibited less droplet diameter growth over time com-
pared to high speed homogenization. Some workers have also studied the use of this
device in the homogenization of dairy model emulsions.[6–11] They examined the depen-
dence of the droplet diameter on the emulsification pressure, temperature, and some product
ingredients. Analysis of their data permits prediction of the fat globule size parameters as a
function of process and formulation conditions.
Also, emulsification was one of the first applications of powerful ultrasound, and the
first related patent was taken out more than fifty years ago.[12] Since then, many scientists
and industrialists have used different types of ultrasound devices to make emulsions.
Cavitation is the main phenomenon responsible for ultrasonically induced effects.[13]
Cavitation is the formation and collapse of vapor cavities in a flowing liquid. Such a vapor
cavity forms when the local pressure is reduced to that of the vapor at the temperature of
the flowing liquid because of local velocity changes. The collapse of these cavities causes
powerful shock waves to radiate throughout the solution in proximity to the radiating face
of the tip, thereby breaking the dispersed liquid. A two-step mechanism has been proposed
for ultrasound emulsification:[14] At the first step, a combination of interfacial waves and
instability leads to the eruption of dispersed phase droplets into the continuous phase; and
the second step consists of breaking droplets up through cavitation near the interface. The
intense effect (disruption and mixing) of shock waves explains the very small droplet size,
and the most influential parameters are those affecting cavitation phenomena.
Emulsification is one of the important steps in microencapsulation technology. The
emulsions are usually produced by high-pressure homogenizers and their droplet size is
normally more than one micron. The effect of emulsion sizes larger than 1 micron on the
efficiency and surface oil content of microcapsules have been studied by different work-
ers.[15,16] Even the emulsions produced by two different methods such as ultrasonication
and microfluidization can have different effects on the microencapsulation efficiency as
have been studied by Mongenot et al.[17] The effect of sub-micron emulsions on the
efficiency and surface oil content of microcapsules is unclear and there have been no
studies on the application of nano-emulsions in the microencapsulation of volatiles such
as d-limonene. We decided to define the encapsulation of submicron particles (droplets) in
larger particles as “nano-particle encapsulation.” It is different from nano-encapsulation in
that the final product size of the particles is not in the nano range.
 NANO-EMULSION PRODUCTION BY SONICATION AND MICROFLUIDIZATION 477

The present study investigated the production of submicron emulsions of oil-in-water

type with ultrasonication and microfluidization. During preparation, several parameters
including microfluidization pressure and cycles, ultrasonication time in highest power
input, and emulsion composition were varied to determine their influence on the droplet size
of the emulsions. These sub-micron emulsions would be used in the next step to produce
microcapsules containing nano-particles (droplets) by spray drying (not undertaken in
this study).

MATERIALS AND METHODS

Materials
Whey protein concentrate (WPC) and d-limonene were supplied by Steggall Nutrition
(Brisbane, QLD, Australia) and Quest International (Gold Coast, QLD, Australia), respec-
tively. Modified starch (Hi-Cap) was purchased from National Starch and maltodextrin
(DE 17) from Fieldose Company.

Experimental Parameters
In order to study the effect of dispersed-phase concentration on the emulsion size,
two levels of d-limonene concentration, 20 and 25% of the total solids, which are typical
concentrations in microencapsulation by spray drying, were selected. The continuous
phase of the emulsions consisted of WPC, HI-cap and maltodextrin (aqueous solutions of
40% solids), which are also used extensively in the microencapsulation of volatiles and
non-volatiles.[3] Three different pressures each with 3 cycles and 3 different times were
selected for microfluidization and ultrasonication respectively. The experimental conditions
are summarized in Table 1.

Coarse Emulsion Preparation

All emulsions used were of the oil-in-water type. The aqueous phase was a solution
of maltodextrin with Hi-Cap or WPC, while the oil phase consisted of d-limonene.
Hydrated solution of wall materials were prepared with distilled water one day before
emulsion preparation and kept overnight in water bath to warrant a full saturation of the
polymer molecules. The total concentration of dissolved solid was 40% (w/w) that was com-
posed of 30% maltodextrin, 10% Hi-Cap or WPC, and 60% distilled water. Coarse emulsions
containing different compositions of d-limonene and maltodextrin with Hi-Cap or WPC were
prepared using a high-speed IKA blender (IKA Works Co, Malaysia). The d-limonene content
of the emulsions was in two levels of 20 and 25% of the total existing solids (Table 1).

Table 1 Experimental parameters.

No. Parameters studied Levels

1 Emulsion continuous phase Hi-Cap + MD, WPC + MD

2 Dispersed phase oil(% of total solids w/w) 20, 25
3 Microfluidization pressures (MPa) 35, 70, 105
4 Microfluidization cycles 1, 2, 3
5 Ultrasonication time (s) 20, 40, 60
478 JAFARI, HE, AND BHANDARI

Microfluidization
Previously prepared coarse emulsion was passed through an air-driven microfluidizer
(Model M-110 L, Microfluidics, US) operating from 20 to 124 MPa. This equipment
included a pneumatic pump, a filter, and an interaction chamber. The pump could generate
a pressure up to 124 MPa from the compressed air supply. This system could be operated
continuously or recycled via a closed loop to have different cycles. Emulsions were
homogenized at different pressure and different cycles (Table 1). The interaction chamber
of the microfluidizer was cooled with tap water in order to alleviate the temperature rise.
A thermometer was placed in the reserviour of the samples just after the discharge port to
monitor temperature fluctuations during microfluidization. The temperature rise was
from 27°C for 35 MPa after 3 cycles up to 41°C for 105 MPa after 3 cycles. For each
pressure, 500 mL sample was prepared and passed through a microfluidizer at the set
pressure for one cycle. Then one third of the microfluidized sample was taken away for
size analysis, and the remaining volume was passing through microfluidizer again for
the second cycle and the same procedure was followed for the third cycle. The experiments
were duplicated.

Ultrasonication
Emulsification by sonication was performed using a 24 KHz sonicator (Dr Hielscher
series, Model UP 400S). This system consisted of a generator, a converter, and a horn tip.
The generator converted the conventional alternating electrical current from 60 Hz to
24 KHz. The converter transformed this energy to mechanical vibrations. The heart of the
converter was a lead zirconate titanate piezoelectric quartz crystal that could expand and
contract under an alternating current. The converter, therefore, vibrated and transmitted
this motion to the horn tip, which was 22 mm in diameter and could vibrate at peak-to-peak
amplitude of 100 μm at full power (100%). The horn tip was immersed in the coarse
emulsion after the sonication was turned on to the highest power. All experiments were
performed in a 500 mL glass beaker. A thermometer was placed in the side of beaker for
the determination of temperature rise during sonication. The temperature rise was 32°C
after 20 seconds and 39°C after 60 seconds. Each experiment was duplicated.

Emulsion Droplet Size Analysis

The size distribution of the oil droplets were determined by the laser light scattering
method using Mastersizer 2000 (Malvern Instruments, UK). Two measurements were
done for each sample. The system could detect particle sizes ranging from 0.02 to
2000 μm. The mean diameter of the droplets was expressed as the Sauter diameter
D32 = ∑ ni di3 / ∑ ni di2 , representing a surface average diameter).
In general, volume mean diameter (D 43 = ∑ ni di4 / ∑ ni di3 )represents the existence
of big particles while surface mean diameter (D32) is associated with the smaller particles.
When there is a big difference between D32 and D43, it means that the size distribution is a
bimodal with two peaks and when they are close together, it shows that we have only one
peak. So, by considering volume size distribution of the particles, there can be a misunder-
standing of the average mean diameter especially when there are a lot of small particles
and their contribution to the volume is not as much as big particles such as in our case. But
 NANO-EMULSION PRODUCTION BY SONICATION AND MICROFLUIDIZATION 479

if the frequency size distribution is used, it can represent large number of small particles,
thus the surface mean diameter is more appropriate to consider.[18]
The emulsion size was measured half an hour after emulsification by ultrasound or
microfluidizer to cancel any creaming or coalescence effect. About 1 to 5 mL of the emulsion
was added to 800 mL water whilst being gently stirred and recirculated in the Mastersizer
cell. Particle size distribution was presented as volume percentage vs. droplet diameter.
The volume size distribution was calculated from the intensity of the light diffracted at
each angle using Mie theory. The analysis requires the relative refractive indices of
the dispersed and continuous phases, in our case, d-limonene and water, respectively.
The refractive index of d-limonene, measured using a digital refractometer, was found to
be 1.40 and that for water 1.33. The width or “span” of the droplet or particle size distributions
is calculated from the following formula:[19]

[d(v,90) − d(v,10)]
Span =
d(v,50)

In this formula, d(v,10), d(v,50), and d(v,90) are diameters at 10%, 50%, and 90% cumu-
lative volume, respectively. In other words, [d(v,90) – d(v,10)] is the range of the data and
d(v,50) is the median diameter.

RESULTS AND DISCUSSION

Comparison of Emulsions Produced by Microfluidization vs.
Ultrasonication
In microfluidization, droplet diameter of the emulsions is a function of the emulsifi-
cation pressure, temperature, and product composition. In this study, experiments have
conducted to examine the dependence of emulsion droplet size on system pressure and
number of cycles of processing and also emulsion composition. The results are presented
in Table 2.
Generally, increasing the microfluidization pressure and number of cycles would
result in emulsions of finer sizes. However, this is apparently not the case for results
shown in Table 2 and Fig. 1. In Table 1, the Sauter diameter (D32) before emulsification
was around 10 μm and 400 nm for Hi-cap and WPC emulsions, respectively. Microfluidi-
zation reduced D32 for both emulsions, when treatment pressure was increased from 35 to
70 MPa. Above 70 MPa, D32 increased with increasing pressure.
This phenomenon, where the particle size increases with increasing the input
energy, can be referred to as “over-processing” as stated by Desrumaux et al.[20] This
could be attributed to poorer function of the emulsifiers, and an increase in the Brownian
motion, hence higher probability of collision and coalescence at higher energy input. In
these conditions, the droplet size distribution of the emulsion is a result of the competition
between two opposite processes, drop breakage and drop-drop coalescence. Fresh interface
is created whenever a droplet is formed from breakage of an original droplet. Between its
formation and its subsequent encounter with the other droplets, some surfactant will
adsorb onto this fresh interface. If the timescale of collision is shorter than the timescale of
adsorption, the fresh interface of the newly formed droplets will not be fully covered with
surfactant and leads to coalescence. So, drop coalescence will depend on the relative rates of
surfactant adsorption and drop collision. In our case, at high pressures of microfluidization,
480 JAFARI, HE, AND BHANDARI

Table 2 Effect of microfluidization conditions on the emulsion size of d-Limonene with two different
compositions of continuous phase (40% solids).

Pressure
No (MPa) Cycles D32 (nm) Span D32 (nm) Span D32 (nm) Span D32 (nm) Span

Hi-Cap + MD WPC + MD

Oil = 25% of Oil = 20% of Oil = 25% of Oil = 20% of

solids (w/w) solids (w/w) solids (w/w) solids (w/w)

1* 0 0 9991 1.7 9675 1.5 1056 3.2 387 6.2

2 35 1 497 1.4 445 1.5 5048 1.3 165 4.5
3 35 2 573 1.1 552 1.1 5128 1.1 133 3.6
4 35 3 597 1.1 536 1.1 3185 1.6 125 3.5
5 70 1 449 1.4 488 1.3 7024 1.1 137 3.6
6 70 2 593 1.1 525 1.0 7407 1.2 125 3.6
7 70 3 672 1.0 540 1.0 6875 1.3 135 5.9
8 105 1 716 1.1 477 1.3 7306 1.3 137 3.6
9 105 2 706 1.0 548 1.0 6877 1.4 136 3.7
10 105 3 704 0.9 585 1.0 8501 1.9 138 3.9

*Coarse emulsion (without treatment). Values represent means of duplicate measurements of two separate
samples.

Hi-Cap/MD (20% d-Limonene)

WPC/MD (20% d-Limonene)
700

600

500

400
D32 (nm)

300

200

100

0
35 70 105
Pressure (MPa)

Figure 1 Effect of microfluidization pressure (2 cycles) on the size of emulsions.

the rate of drop collision is greater than drop breakage. Since the newly formed droplets
are not completely covered by the surfactant, a higher rate of collision represents a greater
coalescence rate, leading to an increase in the droplet size (over-processing). This can be
prevented by selecting appropriate surfactant and adjusting its concentration.
 NANO-EMULSION PRODUCTION BY SONICATION AND MICROFLUIDIZATION 481

The results of the emulsions prepared by ultrasonication are summarized in Table 3

and Fig. 2. By looking at these results, it can be explained that emulsification by ultra-
sound is more straight forward and without “over-processing” than with microfluidizer
especially with the emulsions containing Hi-Cap. For Hi-Cap emulsions produced by
ultrasound, the emulsion size is decreasing until 40 seconds of sonication, but after that
the emulsion size is to some extent stable in around 500 nm.
The results presented in Table 2 and 3 indicate that increased pressure and number of
recirculations (cycles) in the case of microfluidization did not change the span (distribution
width) of the emulsions, whereas the emulsion distribution was getting narrower and span

Table 3 Effect of ultrasonication conditions on the emulsion size of d-Limonene with two different
compositions of continuous phase (40% solids).

Frequency
No (KHz) Time (s) D32 (nm) Span D32 (nm) Span D32 (nm) Span D32 (nm) Span

Hi-Cap + MD WPC + MD

Oil = 25% of Oil = 20% of Oil = 25% of Oil = 20% of

solids (w/w) solids (w/w) solids (w/w) solids (w/w)

1* 0 0 9991 1.7 9675 1.5 1056 3.2 387 6.2

2 24 20 701 4.2 657 5.5 854 6.7 243 21.1
3 24 40 523 1.5 484 1.6 867 3.1 285 14.6
4 24 60 522 1.3 460 1.5 754 2.7 298 12.9

*Coarse emulsion (without treatment). Values represent mean of duplicate measurement of two separate samples.

Hi-Cap/MD (25% oil) Hi-Cap/MD (20% oil)

WPC/MD (25% oil) WPC/MD (20% oil)
1000
900
800
700
600
D32 (nm)

500
400
300

200
100
0
20 40 60
Time (s)

Figure 2 Effect of ultrasonication time on the emulsion size.

482 JAFARI, HE, AND BHANDARI

(a)

35 Mpa/1 cycle 70 Mpa/1 cycle 105 Mpa/1 cycle

14 70 MPa
12 105 MPa

10 35 MPa
Volume (%)

8
6
4
2
0
9

3
32

43

56

74

98

77

52

46
12

17

22

29

39

51

67

89
11

15

20
Particle size (nm)

(b)

20 s 40 s 60 s

12
40 S
10
60 S
8
Volume (%)

6
20 S
4

0
32

49

74

3
52

46

97

88

96

2
11

17

25

39

59

89

74

25

60

24
13

20

30

46

70
10

16

24

37

Particle size (nm)

Figure 3 Size distribution of the emulsions produced by (a) microfluidizer for one cycle at 3 different pressures
and (b) Ultrasound at the highest power for 3 different times. Emulsion was containing d-limonene (20% of total
solids) as the dispersed phase and hydrated solution of Hi-Cap/MD (40% solids) as the continuous phase.

was getting smaller with increasing the sonication time (Table 3 and Fig. 3). Nevertheless,
microfluidization produced narrower size distributions (less span) than ultrasound.

Effect of the Dispersed-phase Concentration on the Nano-emulsion

Formation
From Table 2, it can be seen that reducing the amount of dispersed phase or
d-limonene leads to a decrease in the size of microfluidized emulsions especially with
 NANO-EMULSION PRODUCTION BY SONICATION AND MICROFLUIDIZATION 483

WPC emulsions. For example, the emulsions size of WPC composition after microfluidi-
zation at 70 MPa with 25% of d-Limonene for 2 cycles is 7407 nm, while the size for 20%
d-Limonene is 125 nm. Another result that is worth noting is that emulsions of WPC with
higher amounts of d-Limonene have a narrower size distribution than emulsions with
lower amounts of d-limonene, while the distribution of Hi-Cap emulsion seems to be
independent of the oil content.
On the other hand, with WPC emulsions produced by ultrasound, again the concen-
tration of d-Limonene is a critical factor. For higher (> 20%) amount of d-limonene, emul-
sion size is over 800 nm at the beginning and decreased to about 750 nm with increasing
sonication time (Table 3). For WPC emulsions with lower d-limonene (20%), the starting
emulsion size was smaller than 300 nm and increased slightly with increasing sonication
time. All of the WPC emulsions with 25% of d-limonene homogenized by microfluidizer
had an emulsion size of more than 1 μm while the same emulsions homogenized by
ultrasound have an emulsion size lower than 1 μm. Therefore, ultrasonication is more
efficient than microfluidization to produce nano-emulsions of WPC with higher amount of
d-limonene.
The droplet size distribution for ultrasound emulsions was bimodal. It means that
coalescence was happening. Also, D43 of the WPC emulsions with 25% d-limonene by
both emulsification methods, i.e., ultrasonication and microfluidization, was the same and
higher than 1 micron (Data not shown). Lower D32 of the WPC emulsions with ultrasound
was because of their bimodal distribution of these emulsions and skewing minor parts of
the curve toward smaller sizes. In fact, there is not much difference between WPC
emulsions with higher concentration of d-limonene homogenized by microfluidizer or
ultrasound if considering volume-mean diameter (D43) and verifying that WPC concentration
was critical in higher amount of d-limonene. But in case of lower amount of d-limonene
and emulsions with Hi-Cap, there is not much difference in terms of emulsion size
between ultrasonication and microfluidization. In general, sonication was more convenient
regarding operation and cleaning. On the other hand, size distribution of the emulsions
produced by microfluidizer was narrower than emulsions prepared by the ultrasonication
(Fig. 3). Increasing the time of ultrasonication decreases the width of the distribution
(reflected in a decrease in the span as can be seen in Table 3 and Fig. 3).

Emulsifying Properties of WPC vs. Hi-Cap

An additional important result from Fig. 1 is that WPC functioned better than Hi-Cap
as an emulsifier and stabiliser since the size of WPC emulsions with 20% oil is on average
about 350 nm lower than Hi-Cap emulsions. This means that WPC can be adsorbed on the
fresh oil-water interface better than Hi-Cap. However, WPC emulsions were very sensitive
to the concentration of dispersed phase. When the oil concentration was 25%, no nano-
emulsions can be produced with WPC irrespective of the process pressure and cycles.
This can be explained by the works of Tcholakova et al.[21] They found that, with
WPC emulsions, the dependence of D32 on the initial protein concentration consisted of
two distinct regions: a significant decrease of droplet size at low surfactant concentrations
and a plateau region at high surfactant concentrations. Therefore, when the d-limonene
concentration was 25% of the solids, the droplet size was highly dependent on the concen-
tration of WPC. Because it could not cover the fresh interface, droplet size can not be
decreased. On the other hand, when the oil concentration was lower, there is a plateau pattern
in D32, which suggests the concentration of WPC was sufficient and not a critical factor.
484 JAFARI, HE, AND BHANDARI

CONCLUSION
Microfluidization and ultrasonication were used to produce nano-emulsions. The
emulsion droplet size decreased initially with increasing microfluidizer pressure and
number of cycles. However, there was a critical point above which the emulsions become
“over-processed.” The optimum conditions to produce nano-emulsions by microfluidizer
were moderate pressure and number of passes through the equipment. In our particular
case, the optimum pressure and process cycles are about 60–70 MPa with 1 to 2 cycles. A
high-power ultrasound was able to produce nano-emulsions with nearly the same emulsion
size as the microfluidizer, but sonication produced emulsions with wider and bimodal size
distributions. Sonication was better in terms of operation and cleaning. For WPC emul-
sions, the concentration of the dispersed phase (d-limonene) was a critical factor. Using
20% oil, it was possible to produce a nano-emulsion of WPC with 243 nm of emulsion size
after 20 seconds sonication and 125 nm emulsion size after microfluidization in 70 MPa for
2 cycles. For Hi-Cap emulsions, the average droplet size was bigger than WPC emulsions.
For example, the lowest emulsion size for Hi-Cap emulsions with microfluidizer was 445
nm (35 MPa and 1 cycle) and 460 nm with sonication for 60 seconds. Finally, the size dis-
tributions of Hi-Cap emulsions were narrower than those of WPC emulsions.

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