Food Hydrocolloids 39 (2014) 151e162

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

Ultrasonic preparation of stable ax seed oil emulsions in dairy

systems e Physicochemical characterization
Akalya Shanmugam a, Muthupandian Ashokkumar a, b, *
a
b

School of Chemistry, University of Melbourne, Melbourne, Victoria 3010, Australia

Chemistry Department, King Abdulaziz University, Jeddah, Saudi Arabia

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 5 November 2013
Accepted 6 January 2014

This study reports the incorporation of 7e21% of ax seed oil in pasteurized homogenized skim milk
(PHSM) using high intensity ultrasound (US) at 20 kHz between 1 and 8 min and at varying power levels.
A minimum process time of 3 min at an applied acoustic power of 176 W was sufcient to produce
emulsion droplets (7% oil) with an average mean volume diameter of 0.64 mm and they were stable at
least 9 days at 4
2  C. The mechanical, cavitational and cavitation-after-effects of US are responsible for
the production of smaller sized emulsion droplets and process-induced modications of milk proteins. A
very small proportion (less than 20%) of partially denatured whey proteins provided stability to the
emulsion droplets. The emulsion droplets also showed a surface potential of about 30 mV due to the
adsorbed proteins, which provided further stability to the emulsion droplets due to electrostatic
repulsion. In order to see if other high shear techniques can generate stable emulsions, experiments were
carried out using Ultraturrax (UT) at similar energy densities to that of US. UT did not produce stable
emulsions until 20 min of processing suggesting the superiority of US emulsication process.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Ultrasound
Emulsication
Nutraceutical
Flax seed oil
Whey protein
Ultraturrax

1. Introduction
Consuming healthy food has become a major trend in the last
decade leading to the development of novel food processing techniques for the encapsulation and delivery of bioactives/nutraceuticals. Most bioactives and nutraceuticals are hydrophobic
compounds. A poor water solubility of these compounds causes
enormous difculties in delivering them in food. The delivery of
such oil-based bioactives/nutraceuticals as emulsions is wellknown (Garti & Yuli-Amar, 2008; Couedelo et al., 2011). Conventionally, food emulsions (O/W) are obtained using high shear
mixtures such as UT and piston homogenizers with the assistance
of emulsiers and stabilizers to achieve considerable emulsion
stability upon storage (Dapcevic Hadnadev, Dokic, Krstonosic, &
Hadnadev, 2013; Maali & Mosavian, 2013; Santana, Perrechil, &
Cunha, 2013). The use of large quantities of emulsiers and NonGRAS (Generally Recognised As Safe) additives are not permitted
in foods, this makes the food industry to rely only on a few range of
emulsiers and this poses a huge challenge in the area of new
product development.

* Corresponding author. School of Chemistry, University of Melbourne, Melbourne, Victoria 3010, Australia. Tel.: 61 3 83447090; fax: 61 3 93475180.
E-mail address: masho@unimelb.edu.au (M. Ashokkumar).
0268-005X/$ e see front matter 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2014.01.006

US has been used for creating emulsions in foods (Soria &

Villamiel, 2010; Wulff-Perez, Torcello-Gomez, Galvez-Ruiz, &
Martin-Rodriguez, 2009). Much of the existing research work in the
area of ultrasonic emulsication has focussed mainly on simple
matrix such as an emulsion of sunower oil in water. The delivery of
bioactives in a complex/real food matrix (health beverage) using US
remains as a vast area to be explored. Unlike simple matrices, a
complex food matrix is composed of proteins, carbohydrates, fat,
water, vitamins and minerals. Few studies have identied the use of
emulsiers/surfactants in production of smaller oil droplet (Jafari,
He, & Bhandari, 2006; Kentish et al., 2008; Leong, Wooster,
Kentish, & Ashokkumar, 2009), however the stability during the
storage of the product has not been studied in detail. In addition,
formation of emulsions ultrasonically without the use of external
stabilizers and emulsiers (food additives) also remains unexplored. Hence, the purpose of this study is to deliver stable emulsions of a hydrophobic bioactive compound in a complex food
matrix such as milk using ultrasound.
In the past, some studies have reported the preparation of soy
oil emulsions (O/W) using milk fat globular membrane (MFGM) as
an emulsier and by employing high pressure homogenization
(HPH) and microuidizer (Corredig & Dalgleish, 1998; Roesch,
Rincon, & Corredig, 2004). Biasutti, Venir, Marchesini, and
Innocente (2010) have produced 15% O/W model dairy emulsions
using milk cream and emulsiers by HPH. However, the milk cream

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A. Shanmugam, M. Ashokkumar / Food Hydrocolloids 39 (2014) 151e162

by itself is rich in MFGM (Harjinder, 2006) and the storage stability

of these model emulsions was not discussed. In our study, we were
interested in loading a signicant amount of ax seed oil (7e21%) in
pasteurized homogenized skim milk (PHSM) without using MFGM
and other food additives. The incorporation of ax seed oil in PHSM
by employing high intensity US has not been reported in the
literature.
Flax seed oil is a widely popular non polar bioactive among the
functional food category. It is obtained from ax seed (Linum usitatissimum L.) and is a rich source of omega-3-fatty acid, a-linolenic
acid (ALA) (Carter, 1993; Mantzioris, James, Gibson, & Cleland,
1994). ALA is an essential fatty acid and is known to support cell,
nerve & cognitive skills development in children and cardiovascular
functions in humans (Joshi et al., 2006; Mazza, Pomponi, Janiri,
Bria, & Mazza, 2007). In 2002, the Food and Nutrition Board of
the US (Institute of Medicine) has recommended the adequate
intake (AI) levels for ALA in adults (19 years and older): 1.6 g/day for
men and 1.1 g/day for women to avoid any deciency which will
result in symptoms like scaly dermatitis. In addition, Mazza et al.
(2007) have indicated a safe and effective dose of ax seed oil
and are 3e6 g/day to prevent and treat neurodegenerative
disorders.
Broadly, the aim of our study was to emulsify 7e21% of ax seed
oil (59.9% ALA) in PHSM using US at 20 kHz under various experimental conditions. The stability of the emulsions was characterised
by a number of techniques and compared with UT emulsication
process.
2. Materials and methods
2.1. Materials
Fresh PHSM was purchased from a local supermarket and
immediately stored at 4  C until further use. The composition of the
milk was 3.5% protein, 0.1% fat, and 4.9% lactose as labelled by the
manufacturer. The manufacturers specication was cross-checked
in our lab for the proteins. The protein content was 3.46% by
Bradford Assay. We did not observe any creaming-off in the PHSM
sample for about 10 days of storage at 4
2  C indicating that fat
content was very low.
Ultra pure (MilliQ) water was used in all experiments. Unrened
organically grown cold pressed ax seed oil with 59.9% of ALA was a
gift sample from Stoney Creek Oil Products Pty Ltd, Australia.

stable emulsion refers to the system where the oil phase did not
separate until at least 9 days of storage at 4
2  C, respectively.
UT emulsions were prepared in the same jacketed vessel using
Ultraturrax (IKA-Labortechnik) at speed dial value 4; 17,500 RPM
(22.5
0.5  C) and at an energy density equivalent to that of US
(discussed in Results section).
2.3. Particle size and zetapotential measurements
The particle size of the OM emulsion was measured on fresh and
stored samples using a laser diffraction method by Mastersizer
2000 (Malvern Instruments Ltd., Worcestershire, U.K). A few
droplets of the emulsion were suspended directly in recirculating
water (1250 rpm, obscuration (14e16%) and refractive index of ax
seed oil 1.475). The volume size distribution values viz., D(4,3),
Dv90 and Dv50 were recorded. D(4,3) represents volume mean
diameter; Dv90 represents the diameter wherein 90% of the volume distribution is below this value; Dv50 represents the diameter
wherein 50% of the volume distribution is above and below this
value. z-Potential of oil droplets was determined using a Zetasizer
Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK).
The emulsions were diluted 200 fold using MilliQ water prior to
measurements. In this paper, D(4, 3) values are mostly used in the
discussion section.
2.4. Creaming stability
The emulsions were visually checked for phase separation and
oiling-off or creaming. Also, the amount of creaming was measured
by storing them in 6 ml sealed graduated tubes at 4
2  C for 9
days. In this test, Sudan III dye was used to improve the clarity
among separated phases. 0.0025% of Sudan III dye was mixed with
ax seed oil for 2.5 h at room temperature using a magnetic stirrer.
Instead of ax seed oil, oil-colour mixture was used in making the
emulsions. The emulsion stability against creaming was monitored
by measuring the volume of the lipid-rich layer on top (VL) and the
volume of total emulsion (VE) in the tube. Creaming stability in
terms of creaming index (%) was obtained using the equation (1),

Creaming Index % VE  VL =VE  100

(1)

For example, if the creaming index is 100%, there is no phase

separation in the emulsions.
2.5. Hydrophobicity of milk proteins

2.2. Emulsication by US and UT

The emulsion composition was 7% ax seed oil (v/v) in 93%
PHSM, unless mentioned otherwise. Both the water and oil phases
were added sequentially to the water jacketed glass vessel and the
sonicator horn was positioned at a depth of 0.3
0.1 cm. Emulsions
were obtained as 50 ml aliquots using a 20 kHz, 450 W ultrasonic
horn (12 mm diameter, Branson Sonier, Model 102 (CE)) at 88, 132
and 176 W of nominal applied powers (NAP) for different processing times from 1 to 8 min. During sonication, thermostated
water was circulated continuously through a jacket surrounding
the sonication cell and the water temperature was maintained at
22.5
2  C. The emulsied samples were stored in a refrigerator for
about 9 days at 4
2  C. The analysis and storage studies were
performed on both fresh and stored samples. In this paper, ax seed
oil/milk, ax seed oil/water and oil/water emulsions are referred to
as OM, OW and O/W, respectively. The term unstable emulsion
refers to the system where the oil phase separated within 3 h of
standing at room temperature whereas good emulsion refers to
the system where the oil phase did not separate until 2 days and

Changes to the hydrophobicity of the milk proteins were

measured on the aqueous portion of the sonicated emulsions.
Aqueous phase of the emulsions were separated by skimming off
the fat at 14,000 rpm for 15 min using Hermle centrifuge (Z306) at
room temperature (Pearce & Kinsella, 1978). Before uorometric
assay, the aqueous phase was vortexed for 30 s and diluted with
MilliQ water in the ratio of 1:20 in order to prepare the aqueous
milk protein solution. The changes to the protein content of the
aqueous milk protein solution was monitored by the Bradford
Assay according to manufacturers instructions (Sigma Aldrich Pty
Ltd, Sydney, Australia) at 595 nm using UV-VIS Spectrophotometer
(Carey 3E, Varian, Palo Alto, CA, USA). A stock solution of 0.008 M 1anilinonaphthalene-8-sulfonate (ANS) was prepared in 0.1 M pH 7
phosphate buffer. It was wrapped in aluminium foil to prevent light
exposure and stored at room temperature.
In the assay, the required amounts of aqueous milk protein solutions were made up with 10 ml of phosphate buffer and 20 mL of
ANS solution to obtain a set of diluted samples of aqueous milk
protein solution at different dilution factors viz., N, 65, 33, 22, 16

A. Shanmugam, M. Ashokkumar / Food Hydrocolloids 39 (2014) 151e162

2.6. Statistical analysis

When necessary, one-way ANOVA with a 95% condence level
was used. The ANOVA data with p < 0.05 were considered statistically signicant.
All the experiments were at least duplicated. The emulsions
were prepared in fresh PHSM samples of the same batch. All
measurements were performed on the same day of the sonication
and upon storage, wherever necessary.
3. Results
Fig. 1 shows the volume size distribution of 7% OM emulsion
prepared using 20 kHz US at 176 W under different processing time
(Day 1). From the data, it is apparent that an increase in sonication
time from 1 min to 8 min resulted in a decrease in the size of
emulsion droplets, in particular 3 to 8 min of processing resulted in
larger volume of small sized droplets ranging from 0.1 to 1 mm. In
addition, the data also highlights the changes to the size distribution pattern, at longer processing times a bimodal distribution is
noted for 1 and 2 min samples, whereas 3e8 min samples showed
an increasing tendency towards unimodal size distribution.
Fig. 2 compares the volume weighted mean D(4,3) diameter of
ax seed oil droplets in 7% OM emulsions (176 W) at different
processing times and upon storage at 4
2  C for 8 days. The D(4,3)
value shows a 4 fold reduction in size between 1 and 8 min of
processing and these values have not changed until 8 days of
storage.
Fig. 3 shows the creaming stability of 7% OM emulsions (176 W &
with Sudan (III) dye) upon storage at 4
2  C. The creaming index is
a measure of the emulsion stability. Emulsions are stable if the
creaming index is 100%. The emulsions processed from 3 to 8 min
show 100% stability against creaming until 9 days of storage, while
those processed for 1 and 2 min show stability for only 1 day and 2
days, respectively. Fig. 4 supports the above statement and is visual
evidence showing orange coloured cream layer on the top surface
for 1 and 2 min processed samples against the others on the 9th day
of storage.
Until now, the formation of a stable emulsion was considered to
be the effect of an US process and in order to separate the effect/
impact of milk components (in particular, proteins) from the

10.0

Differential Volume Percent (%)

9.0

8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
0.1

10

Particle Diameter (m)

Fig. 1. Volume size distribution of 7% OM emulsions (20 kHz US; 176 W) processed for
different times: 1 min, C 2 min, , 3 min, 4 min, > 5 min, * 6 min, 7 min, and
8 min on Day 1.

formation and stability of OM emulsions, the emulsions of milliQ

water (no milk protein) and ax seed oil were prepared similar to
7% OM emulsions using 176 W 20 kHz US between 1 and 8 min. The
size distribution data of ax seed oil/water (OW) emulsions are
shown in Fig. 5. From the gure, it is clear that the sizes of the OW
droplets and their distribution are not altered by increasing the
processing time until 8 min. The distribution tends to remain
bimodal and the phase separation occurred immediately after 3 h of
standing at room temperature (Fig. 6). This observation indicates
that sonication alone is not sufcient to generate stable emulsions
and milk proteins are important for stabilizing the emulsion
droplets.

1.6
1.4
Volume mean diameter (m)

and 13. By doing this, the protein concentrations of these samples

were varied indirectly. For example, the dilution factor of 65 and 13
refer to 0.00005e0.0002% (w/w) of protein in PHSM and dilution
factor of N refers to absence of aqueous milk protein solution
(Blank). After the addition of ANS, the samples were wrapped in a
foil, vortexed immediately and kept in dark for 15 min. The ANS
probe only binds to the hydrophobic regions of the protein that are
present in sample and uoresce. Thus in every sample set, the increase in the amount of proteins from higher to lower dilution
factor would lead to increased binding of ANS to the hydrophobic
protein and hence increased relative uorescence intensity (RFI).
The RFI was measured using a spectrouorimeter (RF-5301PC,
Shimadzu, Japan). For hydrophobicity determination using ANS, the
excitation and emission slits and wavelengths were set at 5/5 and
390/470 nm, respectively (Chandrapala, Zisu, Palmer, Kentish, &
Ashokkumar, 2011). The RFI of each solution was measured starting from buffer blank and then from the highest to lowest dilution
of the aqueous milk protein solutions. To obtain Net RFI, RFI of each
dilution blank was subtracted from that of the corresponding
protein solution. Plots of RFI values vs dilution factors of each of the
processed sample were used to determine the changes to the hydrophobicity (equal to the slopes) of the milk proteins that were
present in the aqueous phase of the emulsions.

153

1.2
1.0
0.8
0.6
0.4
0.2
0.0
0

Sonication Time (min)

Fig. 2. Comparison between volume weighted mean e D(4,3) diameter of ax seed oil
droplets in 7% OM emulsions (20 kHz US; 176 W) at different processing times and
upon storage at 4
2  C on > Day 1 & C Day 8.

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A. Shanmugam, M. Ashokkumar / Food Hydrocolloids 39 (2014) 151e162

102

9.0

Differential Volume Percent (%)

8.0

Creaming Stability %

100

98

96

94

7.0
6.0
5.0
4.0
3.0
2.0
1.0

92
0.0
0.1

10

Particle Diameter (m)

90
Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Storage Time (Days)

Fig. 3. Creaming stability of 7% OM emulsions (20 kHz US; 176 W) processed at
different times viz., - 1 min,
2 min and
3e8 min at 176 W upon storage at
4
2  C.

To conrm the presence of milk proteins on the surface of OM

emulsion droplets, the absorbance corresponding to the proteins in
aqueous solutions was monitored. The aqueous portion represents
the part of emulsion obtained after centrifugation and dilution with
milliQ water (1:20). Fig. 7 compares the absorbance of proteins (at
595 nm) present in the aqueous solutions of PHSM and 7% OM
emulsions (176 W) processed for 1, 3, 5 and 8 min. The absorbance
due to proteins is lower for all the emulsion samples in comparison
to PHSM.

Fig. 4. Photograph of 7% OM emulsions (20 kHz US; 176 W) with Sudan (III) dye
processed for different times on Day 9. First row (1e3): 0 min US (PHSM), 1 and 2 min
OM emulsion; Second row (4e6): 3e5 min OM emulsion; Third row (7e9): 6e8 min
OM emulsion.

Fig. 5. Volume size distribution of 7% OW emulsions (20 kHz US; 176 W) processed for
different times: 1 min; > 2 min; 3 min; B 4 min; 5 min; 6 min; 7 min;
8 min.

The presence of proteins on the surface of emulsion droplets

may generate charges on the surface of emulsion droplets. The
magnitude of zetapotential gives an indication of potential stability
of a colloidal system. Fig. 8 shows the zetapotential values of OM
emulsions, PHSM and whole milk on the 9th day of storage
(4
2  C). All samples were sonicated under similar conditions, viz.,
20 kHz 176 W for 1, 5 and 8 min. Though creaming was observed in
1 min sample, cream was removed and the sample was withdrawn
after mixing the uncreamed portion. The 0 min samples of PHSM
and whole milk represent the unsonicated (market) milk samples.
Within error limits the zetapotential values are around 30 mV for
all samples irrespective of their composition and constituents. The
average values of zetapotential for whole milk and OM emulsions
are higher than that observed for PHSM.
Fig. 9 shows the changes to uorescence intensity of aqueous
milk protein solutions obtained from 7% OM emulsions (176 W)
processed for 1 min, 3 min and 8 min in comparison to PHSM at
different dilution factors. The slope value indicates the changes to
uorescence intensity of each sample at different dilution levels.
The changes to the slope values at different sonication time are
related to changes in the hydrophobicity of proteins present in the
aqueous phase. A higher slope corresponds to a greater modication to the proteins, i.e., an increase in hydrophobicity. Fig. 9 shows
that the uorescence intensity increases with a decrease in sample
dilution (increase in concentration of protein) at any sonication
time. Thus the hydrophobicity of the samples increases with an
increase in sonication time. The maximum change is noted for
3 min sonicated sample.
Table 1 compares the emulsication capacity of 20 kHz US at
different NAPs, viz., 88, 132 and 176 W and at different processing
times from 1 to 8 min (Day 1). The stability of these emulsions was
checked visually. Also, some of them were tested upon storage at
4
2  C. As already reported, 176 W produced good emulsions from
1 to 8 min of processing; however only those samples obtained
from 3 to 8 min of processing remained stable until 8 days (Figs. 1
and 2). Processing at 22 W did not produce good and stable
emulsions until 8 min of processing (Fig. 10), while processing at
132 W produced good and stable emulsions from 3 to 8 min.
However, the 3 and 4 min samples creamed-off on 2nd day of

A. Shanmugam, M. Ashokkumar / Food Hydrocolloids 39 (2014) 151e162

Fig. 6. Photograph of 7% OW emulsions (20 kHz US; 176 W) at 1 and 8 min processing
time and after 3 h of standing at room temperature.

storage while other samples (5e8 min) remained stable until 8 days
(data not shown). From the volume size distribution (data not
shown), a bimodal distribution pattern was observed between 1
and 7 min at 88 W, 1 and 4 min at 132 W and 1 and 2 min at 176 W;
the tendency towards unimodal distribution increased above these
processing times, i.e., until 8 min. Table 1 provides D(4,3), Dv50 and
Dv90 values of these emulsions. The minimum D(4,3) values
required to produce stable emulsions at 132 and 176 W are 0.53 and
0.64 mm at 5 min and 3 min, respectively. Similarly, the minimum
Dv90 value required to produce stable emulsions at 132 and 176 W
are 1.13 and 1.37 mm at 5 min and 3 min, respectively. Though the
D(4,3) and Dv90 values for 8 min 88 W, viz., 0.59 and 1.29 mm are
considered reasonable and comparable to 5 min at 132 W and 3 min
at 176 W; stable emulsions were not produced in the former.
Fig. 11 represents the volume size distribution of OM emulsions
processed by 20 kHz at 176 W US at different oil percentages, viz., 7,
15 and 21 on Day 1 and Table 2 shows droplet characteristics of 15
and 21% of OM emulsions. This work shows the capability of PHSM
(milk) system to hold larger amounts of oil (15 and 21%) in the form
of emulsion droplets compared to 7% formulation. To make comparison between the three different emulsion formulations, the
process time was kept constant between 3 and 8 min. The stability
of these emulsions was monitored visually on Day 1. The good and

155

stable emulsions were produced between 3 and 8 min in case of

15% loading and between 6 and 8 min in case of 21% loading. The
data from Fig. 11 & Table 2 are compared with Fig. 1 & Table 1 and
the key observations are: 1) an increase in oil % resulted in a
broadening of size distribution, an increase in the volume of larger
sized particles and an increase in processing time from 3 to 6 min in
case of 21% emulsions, 2) D(4,3), Dv50 and Dv90 values of 3 min
15% and 6 min 21% emulsions are similar and are 0.85, 0.66 and
1.7 mm, respectively, 3) minimum D(4,3) and Dv90 values are noted
amongst 8 min emulsion samples and 4) an increase in oil %
resulted in a lowering of the efciency of US to breakup the larger
emulsion droplets, i.e., Dv90. The 15 and 21% loading emulsions
were stable until 5 days of storage without any visual creaming at
4
2  C.
Table 3 shows the process time necessary to prepare 7% OM
emulsions using UT when operated at equivalent energy density of
176 W 20 kHz US at 1, 2, 5, 7 and 8 min. The energy density of 50 ml,
176 W 20 kHz US emulsions at different process times was calculated using Equation (2),

Energy Density J=ml Power Drawn NAP WTimes=

Volume ml
(2)
The process times for UT emulsions were obtained using
Equation (2) and by substituting the values of calculated energy
density (US), volume of solution (50 ml) and power drawn by UT at
a speed dial value of 4 (17,500 RPM; 70 W). The UT processing times
were 2 min 30 s, 5 min, 12 min 30 s, 17 min 30 s and 20 min in
comparison to 1, 2, 5, 7 and 8 min of 176 W 20 kHz US operation.
The UT emulsions were characterized and the data are shown in
Fig. 12 and Table 4. From Fig. 12, it is apparent that all the emulsion
samples made with UT showed bimodal curve with huge amounts
of larger sized particles. Also, D(4,3), Dv90 and Dv50 sizes of the UT
emulsions are larger compared to those off US emulsions. For
example, the Dv90 value ranges between 3.1e7.6 mm for UT

Sonication Time (min)

0

0.5

0.45

-5

0.4

-10

0.3

potential (mV)

Absorbance (a.u.)

0.35

0.25
0.2

0.15

-15

-20

-25

0.1

-30

0.05
0
400

500

600

700

800

-35

Wavelength (nm)
-40
Fig. 7. Changes to the absorbance of protein in aqueous phase (1:20 dilution) obtained
from the PHSM and 7% OM emulsions (20 kHz US; 176 W) processed for
0 min;
1 min,
3 min,
5 min,
8 min at 595 nm
(Measurements are average of two repeats).

Fig. 8. Zetapotential values of storage samples representing

PHSM,
whole milk
and - 7% OM emulsions prepared by sonication (20 kHz; 176 W US) from 0, 1, 5 and

8 min on 9th day at 4
2 C.

156

A. Shanmugam, M. Ashokkumar / Food Hydrocolloids 39 (2014) 151e162

Relative Fluorescence intensity

1200
y = 7E+06x
R = 0.9966

1000

y = 6E+06x
R = 0.9924

800
600

y = 3E+06x
R = 0.999

400

y = 2E+06x
R = 0.9917

200
0 ----------------------------------------------------------------------------------------------
65
33
22
16
13
Dilution Factor of 1:20 Aqueous milk protein solution

Fig. 9. Changes to the uorescence intensity of aqueous milk protein solutions (1:20)
obtained from 7% OM emulsions (20 kHz US; 176 W) processed for 1 min, 3 min
and  8 min in comparison to PHSM at different dilutions are indicated by their slope
values.

samples in comparison to 0.75e3 mm for US samples. In addition,

Fig. 13 shows emulsion instability of the freshly prepared UT samples. It is seen as a creamy layer on the surface of all the emulsion
samples just within few hours of standing at room temperature.

4. Discussion
The results shown in Figs. 1e10 can be summarized as below
(summary points 4.1e4.8):
4.1 The size of OM emulsions decreases with an increase in
sonication time; higher volumes of small sized droplets are
obtained at longer sonication times. A 4-fold reduction of
D(4,3) value is observed at 8 min sonication.
4.2 A bimodal distribution is noted until 2 min of sonication and
a tendency towards unimodal distribution is observed from 3
to 8 min sonication for OM emulsions.
4.3 The stability of OM emulsions is supported by the D(4,3)
values (8 Days; 4
2  C).
4.4 The creaming index of emulsions processed between 3 and
8 min is 100% on the 9th day at 4
2  C, while those processed for 1 and 2 min showed 96 and 99%, respectively.
4.5 The US emulsions prepared using 7% ax seed oil and water
show a bimodal size distribution and are unstable. An increase in process time (1e8 min) does not cause reduction in
the size of emulsion droplets.
4.6 The amount of protein present in the aqueous phase of OM
emulsions is decreased.

Table 1
Emulsication capacity of 20 kHz US to produce 7% OM emulsion at different power
levels, i.e., 88, 132 and 176 W at different times of processing from 1 to 8 min.
US processed sample at
20 kHz and different time and NAP

D(4,3) (mm)a

Dv50 (mm)a

Dv90 (mm)a

5
8
3
4
5
8
3
4
5
8

0.84
0.59
0.75
0.70
0.53
0.39
0.64
0.54
0.48
0.40

0.55
0.38
0.47
0.46
0.36
0.29
0.45
0.38
0.34
0.30

1.90
1.29
1.74
1.56
1.13
0.77
1.37
1.14
0.99
0.79

min;
min;
min;
min;
min;
min;
min;
min;
min;
min;

88 W
88 W
132 W
132 W
132 W
132 W
176 W
176 W
176 W
176 W

a
The results are average of two measurements and the error value varies from
0 to 0.017 mm.

4.7 Within experimental errors, the zetapotential values of 7%

OM emulsion droplets lies around 30 mV on the 9th day of
storage at 4
2  C.
4.8 The slope of the relative uorescence intensity (RFI), i.e.,
hydrophobicity increases with an increase in sonication time
and a maximum change is noted for 3 min OM sample.
Stability of 7% OM emulsions at 176 W 20 kHz US
A 7% loading of the ax seed oil chosen was based on FDAs GRAS
notication number: GRN000256, which recommends the level of
high linolenic acid ax seed oil in milk products. The stability of US
in making 7% OM emulsions at 176 W are discussed based on particle characterization, creaming & storage stability and by understanding changes to the components of the milk which may impart
stability to these emulsions (summary points 4.1e4.4).
The US processing is considered as a high energy emulsication
technique and the emulsions of smaller droplet size are produced
by the physical effects generated in liquids, viz., mechanical vibrations, acoustic streaming, acoustic cavitation, microstreaming,
shear and turbulence (Abbas, Hayat, Karangwa, Bashari, & Zhang,
2013; Ashokkumar et al., 2010). Canselier et al. (2002) have
described US emulsication as a two-step process: in the rst step,
the turbulence caused by the mechanical vibration leads to the
eruption of dispersed phase droplets into the continuous phase and
the second step consists of breaking up of droplets through the
shear forces generated by cavitation at the interface. The emulsication process can also be described as two opposite elementary
steps: droplet breakup leading to formation of several smaller
droplets, and dropletedroplet coalescence leading to formation of a
larger droplet. In general, the droplet-size distribution obtained
during emulsication is governed by the competition between
these two opposite processes (Vankova et al., 2007). The emulsion
stability is closely related to the droplet size distribution, since
larger droplet size distributions may enhance the Oswald ripening,
i.e., increasing the size to larger droplets in turn favours droplet
coalescence and creaming (Gutierrez, Rayner, & Dejmek, 2009).
The amount of US energy incorporated into emulsions not only
breaks the planar interface but also overcomes the Laplace pressure
in order to produce ner droplets (Canselier et al., 2002). Laplace
pressure (PL) is the pressure difference between the convex and the
concave side of a curved interface of an emulsion droplet and is
given by PL g (l/R1 l/R2), where g is the interfacial tension and R1
and R2 are the principle radii of curvature. For a spherical drop of
radius r we thus have PL 2 g/r (Walstra, 1993). Hence, Laplace
pressure is dependent on interfacial tension of the droplet and
therefore larger amounts of shear (physical effects of US) are
required to overcome the interfacial tension in between liquids.
Thus in our system, a) the mechanical forces of US helps in the
mixing of the two immiscible phases and formation of larger
emulsion droplets by breaking the initial planar interface and b) the
shear forces generated by cavitation helps in counteracting the
Laplace pressure to generate ner droplets. However at this stage,
the effect of milk components in lowering the interfacial tension (g)
cannot be neglected because this would also lower the Laplace
pressure and hence lower the amount of shear required to break
the droplets.
From Figs. 1 and 2, a signicant change in size distribution
pattern and reduction in D(4,3) value, i.e., 1.38e0.40 mm is observed
between 1 and 8 min of processing. Here, as the processing time
increases, the amount of input energy increases as well, leading to
disruption of more number of droplets and therefore, a decrease in
average size of emulsion droplets and possibly the changes
observed in the size distribution pattern (summary points 4.1 &
4.2). In a previous study on emulsication by homogenization, the

A. Shanmugam, M. Ashokkumar / Food Hydrocolloids 39 (2014) 151e162

157

Fig. 10. Photograph represents the difference between 7% OM emulsions produced by 20 kHz US at 88 and 132 W (as freshly prepared). First row (Left to right shows good and
stable emulsions): 5 min and 8 min emulsion at 132 W; Second row (Left to right shows unstable emulsions-as yellowness on top surface): 5 min and 8 min emulsion at 88 W.

production of smaller droplets was shown to increase by increasing

the duration of homogenization (McClements, Decker, & Weiss,
2007).
In addition to droplet size characteristics, the physical stability is
an important property of emulsion. An emulsion is physically stable
if its dispersed state does not change, i.e., if its droplet size distribution remains constant for a particular period of time, for
example, 7 days at chill or refrigerated storage (4  C) in case of
market milk products. From Fig. 2 & summary point 4.3, the volume
weighted mean diameter of 7% OM emulsions did not vary until 8
days at 4
2  C. The physical stability is generally achieved by
preventing the droplets from sedimentation (gravitational separation), aggregation or coalescence or Ostwald ripening. It can be
achieved by employing suitable emulsiers or by producing
emulsions of similar droplet sizes (Schubert & Engel, 2004).
In our experiments, the creaming index value is 100% for 3e
8 min emulsions against 96 and 99%, respectively, for 1 and 2 min
emulsions (Figs. 3 and 4 & Summary point 4.4). The creaming index
values indicate the gravitational separation of emulsions in the
form of creaming. Creaming, the upward movement of an emulsion
droplet is hindered in the case of 3e8 min samples. Possible reasons include decrease in particle size, increase in repulsion between the electrical charges on the surface of emulsion droplets,
etc. (Basaran, Demetriades, & McClements, 1998; Chanamai &
McClements, 2000). However, most importantly, the components
of PHSM can also provide stability to these emulsions. PHSM is
majorly composed of proteins, lactose, fat, vitamins and minerals.
Among these, the milk fat globular membrane (MFGM) present on
the surface of milk fat globules and the milk proteins are considered
as the important factors contributing to stability of market milk
against creaming. Here, the stability of milk fat globule is imparted
by different conditions of milk processing, in particular the heating
process and homogenization process (pressure) contributes to

different functionality of MFGM along with their associated proteins (Aiqian, Singh, Taylor, & Anema, 2002; Cano-Ruiz & Richter,
1997). However in PHSM, the stabilized fat globules are present in
minor amounts (<0.1%) along with the modied MFGM and in the
OM emulsions the fat source (ax seed oil) is naturally devoid of
MFGM, which leads to the importance of milk proteins in the stabilization of the emulsion droplets.
The milk proteins are of two major types, viz., caseins and whey
proteins. The isolates of milk caseins and whey proteins obtained
from various processing techniques are used as emulsiers in the
food industry. So these proteins may possibly play a vital role in the
stability of 7% OM emulsions generated by US, as reviewed by
Dickinson, 2001. To conrm this, the emulsion stability of OW
emulsions (devoid of PHSM) was monitored in the current work.
The important observations are indicated in summary point 4.5: in
the absences of PHSM (milk components), the droplet size of
emulsions and their distribution remained unchanged from 1 to
8 min of processing (Fig. 5). Also, none of the emulsions were stable
and the phase separation occurred immediately after 3 h of
standing at room temperature (Fig. 6). This is mainly because of
particleeparticle aggregation (coalescence) and is well-indicated
by its bimodal curve (Kuhn & Cunha, 2012). Hence, the importance of milk components, in particular, the proteins in the making
of stable 7% OM emulsion can be realized as discussed below.
From Figs. 1 and 5, the distribution patterns of 1 and 2 min 7%
OW emulsions are similar to those prepared at 1 and 2 min 7% OM
emulsions (bimodal) but the D(4,3) sizes of 7% OW emulsion are
2.77 and 2.02 mm (Data not shown) in comparison to 1.38 and
0.83 mm of 7% OM emulsion, respectively (Table 4). Thus, the
presence of minimal amount of surfactant, such as a protein, is
required to achieve a reduction in the droplet size of emulsions
(Tcholakova, Denkov, & Danner, 2004). Upon processing from 1 to
8 min, the Dv90 and D(4,3) values of OW emulsions decreased from

158

A. Shanmugam, M. Ashokkumar / Food Hydrocolloids 39 (2014) 151e162

Differential Volume Percent (%)

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0
0.1

10

Particle Diameter (m)

Fig. 11. Volume size distribution of emulsion processed by 20 kHz, 176 W US at

different oil percentages 1) 15% OM emulsions processed for A 3 min, 8 min; and 2)
21% OM emulsions processed for 6 min, : 8 min.

Table 2
Emulsion characterization of OM emulsions produced by 176 W, 20 kHz US at
different oil percentages viz., 15 and 21% between 3 and 8 min.
Amount of ax
seed oil in
emulsion (%) v/v

Process time
at 176 W; 20
kHz (min)

D(4,3) (mm)a

Dv50 (mm)a

Dv90 (mm)a

15
15
15
15
21
21
21

3
4
5
8
6
7
8

0.85
0.75
0.69
0.61
0.85
0.87
0.82

0.67
0.59
0.52
0.46
0.67
0.67
0.63

1.74
1.53
1.41
1.21
1.72
1.75
1.66

a
The results are average of two measurements and the error value varies from
0 to 0.20 mm.

5.54 to 3.72 and 2.77 to 1.87 mm, while the sizes of OM emulsions
reduced from 3.02 to 0.79 and 1.38 to 0.40 mm, respectively. So, a
signicant and larger size reduction is noted in OM emulsions in
comparison OW emulsions at any processing time. This may be due
to adsorption of milk proteins on the surface of oil droplets, which
contribute to the lowering of interfacial tension and the Laplace
pressure as discussed earlier. Though generic, the adsorption of
protein at the surface of the emulsion droplets can be conrmed
from the data shown in Fig. 7. It is indicated by a decrease in the
absorbance of proteins present in the aqueous phase in comparison
to unsonicated PHSM. Therefore, certain amount of protein that

was present in the aqueous phase of emulsions has decreased upon

sonication due to their adsorption on the surface of emulsion
droplets (Summary point. 4.6). However, amongst these OM
emulsions, creaming was observed with 1 and 2 min samples when
they are stored at 4
2  C (Fig. 3). This can be related to incomplete
coverage of proteins on the surface of oil droplets at shorter processing times (Jafari, He, & Bhandari, 2007). The samples processed
between 3 and 8 min showed creaming stability until 9 days at
4
2  C (Fig. 3). Thus in case of 7% OM emulsions, a minimum
processing time of 3 min is recommended for the coverage of sufcient amount of proteins on droplet surface.
The milk protein contribution to the size distribution pattern of
7% OM emulsions can be explained by two-phase adsorption kinetics as discussed by Romero et al. (2011). The rst phase is
characterized by a rapid decrease of interfacial tension and it corresponds to the phase of protein adsorption. The second phase is
attained after a certain amount of time, characterized by a slow
evolution of interfacial tension and it corresponds to the phase of
conformational rearrangements of proteins at the O/W interface.
The OM samples that are obtained between 3 and 8 min of US
process had enough time to pass through all phases of adsorption
kinetics and remained stable upon storage unlike the 1 and 2 min
samples (Fig. 3). In order to conrm this, charge on the surface of
emulsion droplets and protein modications were monitored
(Summary point 4.7 & 4.8).
The emulsion droplets often have an electrical charge, which
plays an important role in their functional performance and stability. From Fig. 8, within experimental errors, the zetapotential
values were 30 mV for all OM emulsions until 9th day of storage.
This net negative charge suggests the presence anionic molecules
on the surface of these emulsion droplets, e.g., caseins, betalactoglobulin, etc. (Matsumiya, Takahashi, Inoue, & Matsumura,
2010). A value of 25 to 30 mV is enough to create high energy
barrier between emulsion droplets which in turn would provide
good colloidal stability (Mirhosseini, 2010; Mohammadzadeh,
Koocheki, Kadkhodaee, & Razavi, 2013; Mora-Huertas, Fessi, &
Elaissari, 2010). Sarkar, Horne, & Singh, 2010, have shown a zetapotential of 30 mV for emulsions obtained using 20% soybean oil
and pure beta-lactoglobulin solution (1%) at pH 7.5. In our emulsion
samples, the pH values were between 6.68 and 6.74 at all processing times (Data not provided). The good electrostatic repulsion
deduced from the high zetapotential at these pH ranges suggests a
better interfacial packing of proteins on the surfaces of the OM
emulsion droplets.
Here, it is also worthwhile to discuss the values of zetapotential
for both unsonicated and sonicated samples of whole milk and
PHSM (1e8 min). They did not change upon sonication of the
sample and remained the same within the experimental errors
(Fig. 8). As mentioned earlier, during conventional processing, the
heat and homogenization conditions have already modied and
created the interfacial layers of the fat globules present in the
market milk and contributed to their stability (Wade & Beattie,
1997). It was mainly due to the association of plasma proteins
with the MFGM of the fat globules and likewise in our work,

Table 3
UT processing of 7% (OM) emulsions at equivalent energy density of 176 We20 kHz US at 1, 2, 5, 7, 8 min of processing.
Sample no

Time of US
processing
(min)

Volume in US
processing
cell (ml)

Power drawn
by 20 kHz
US (W)

Energy density
in the emulsion
(J/ml)

Power
drawn by
speed dial 4 of UT (W)

Time of UT processing at same

energy density of US, by
calculation (min:s)

1
2
3
4
5

1
2
5
7
8

50
50
50
50
50

176
176
176
176
176

211.2
422.4
1056.0
1478.4
1689.6

70
70
70
70
70

02:30
05:00
12:30
17:30
20:00

A. Shanmugam, M. Ashokkumar / Food Hydrocolloids 39 (2014) 151e162

7.0

159

discussions, it is noted that the whey proteins of the milk are highly
susceptible to high intensity sonication process (20 kHz) in comparison to the micellar caseins of the PHSM. Hence, these process
induced modications to the whey proteins are helpful in the formation of stable emulsions of ax seed oil and PHSM. Thus, cavitation bubble liquid interfaces and the shear forces generated by
the high intensity US can contribute to the functionality of whey
proteins in the PHSM.
In summary, the factors responsible for the formation of stable
OM emulsions are: 1) mechanical vibration and acoustic cavitation
of US are responsible for the mixing of two immiscible phases at the
oil and milk interface leading to emulsion formation and 2) the
shear forces generated by the US are responsible for the partial
denaturation, increased hydrophobicity of the whey proteins
(surfactant characteristics) and decreased emulsion droplet size
resulting in stability of the emulsions.

Differential Volume Percent (%)

6.0

5.0

4.0

3.0

2.0

1.0

0.0
0.1

10

Particle Diameter (m)

100

Fig. 12. Volume size distribution of freshly prepared 7% OM emulsions at processing

time A 2.5 min,
5 min, C 12.5 min, 17.5 min, and > 20 min using UT at the
equivalent energy density of US (176 W; 20 kHz at 1, 2, 5, 7 and 8 min).

sonication is considered to be cause for the presence/association of

plasma proteins, viz., casein or whey proteins of the PHSM on the
surface of ax seed oil droplets. In conclusion, it is clear from
summary point 4.7 that the adsorbed protein molecules on the
surfaces of oil droplets prevented the occulation of these oil
droplets possibly via electrostatic repulsion as was indicated by Bos
& van Vliet, 2001; Tcholakova, Denkov, Ivanov, & Campbell, 2002.
Additionally, the above discussion can also be supported by
analysing the changes to hydrophobicity of milk proteins. For
example, the emulsifying capacity of heat denatured proteins is
correlated to surface hydrophobicity index (Nakai, 1983). The increase in hydrophobicity is due to the unfolding of the protein
molecule to expose the hydrophobic residues, thus enhancing their
adsorption at the oilewater interface. During sonication, the increase in the process time from 1 to 8 min resulted in an increase of
the hydrophobicity of the aqueous milk protein solutions (Fig. 9 &
Summary point 4.8). Therefore, emulsication by US has affected
the proteins of the milk that are inherently present in the PHSM.
But these changes are not vigorous enough to make them
completely lipophilic which would rather decrease their emulsication properties. Hence, during protein modication, a balance
between hydrophilic and lipophilic groups is ensured (moderate)
and it contributed to the emulsion stability of our samples. As
mentioned by Nakai, Cheung, and Voutsinas (1983), the electric
charge and hydrophobicity of the adsorbed proteins play an
important role in the emulsication process. Our previous work has
reported similar modications to milk proteins, when PHSM was
sonicated. It has explained the shear induced denaturation & aggregation of whey proteins caused by the phenomenon of acoustic
cavitation in liquids especially at bubble liquid interfaces. Less than
20% of whey proteins were denatured when the PHSM was sonicated until 8 min at 176 W (Shanmugam, Chandrapala, &
Ashokkumar, 2012). Similarly, Chandrapala et al. (2011) have reported the changes to the surface hydrophobicity of whey protein
concentrate (WPC) solutions (4% protein) during sonication. They
have shown an increase in surface hydrophobicity, when the
samples were sonicated for 5 min by 20 kHz US. However
Chandrapala, Martin, Zisu, Kentish, and Ashokkumar (2012) and
Shanmugam et al. (2012) have ascertained the process stability of
caseins micelles upon sonication of milk. Pertaining to the above

Effect of nominal applied power, oil % and processing techniques on the stability of OM emulsions
The results shown in Figs. 11e13 and Tables 1e4 are summarized
below (summary points 4.9e4.16):
4.9 The emulsions processed by 20 kHz US from 1 to 7 min at
88 W; 1 to 4 min at 132 W; 1 to 2 min at 176 W are unstable
with bimodal distribution pattern except 8 min 88 W sample.
4.10 The tendency towards unimodal distribution is observed for
5e8 min samples at 132 W and 3e8 min samples at 176 W
and are stable until 9 days at 4
2  C
4.11 Though the values D(4,3) and Dv90 of 8 min 88 W sample is
comparable with 5 min 132 W and 3 min 176 W samples, the
emulsions are not stable.
4.12 The emulsions at higher oil percentages, viz., 15 and 21% are
stable for 5 days at 4
2  C
4.13 Good and stable emulsions are obtained between 3 and
8 min with 15% emulsions and 6 and 8 min with 21%
emulsions.
4.14 D(4,3), Dv50 and Dv90 values of 3 min 15% and 6 min 21%
emulsions are similar.
4.15 UT process times are calculated at the equivalent energy
density of 50 ml, 20 kHz 176 W (NAP) US.
4.16 UT emulsions are bimodal and unstable. The Dv90 values of
the UT samples are 2.5e4 times larger than the US emulsion
samples.
From summary points 4.9 to 4.11, at constant process times,
increasing the sonication power results in a decrease in emulsion
droplet diameter (Table 1). Also, a notable change in the size distribution can be observed. A similar study by Madadlou, Mousavi,
Emam-Djomeh, Ehsani, and Sheehan (2009) has reported a
higher breakage of re-assembled casein micelles at higher sonication power. In general, at lower frequencies, the bubbles generated in the sound eld are relatively large in size and when the
acoustic power is increased, the size and the number of cavitation
bubbles increase (Ashokkumar & Mason, 2007; Brotchie, Grieser, &
Ashokkumar, 2009; Lee, 2005) followed by the intense collapse of
these bubbles resulting in high shear forces and stronger shockwaves in liquids (Leong, Ashokkumar, & Kentish, 2011) and production of ner emulsion droplets in our system.
The sonication power (NAP) can also be represented as power
density, P (W/ml). The power density, the average energy dissipated
per unit time and unit volume, is a measure of strength of turbulence (shear) in solution and the maximum diameter of the
emulsion droplet in any turbulent ow is given by Equation (3)
(Walstra, 1993):

160

A. Shanmugam, M. Ashokkumar / Food Hydrocolloids 39 (2014) 151e162

Table 4
Comparison of D(4,3), Dv50 and Dv90 value of US and UT process at same energy density.
US process time
20 kHz; 176 W (min:s)

Equivalent UT process
time (min:s)

D(4,3) of US
(mm)a

Dv50 of US
(mm)a

Dv90 of US
(mm)a

D(4,3) of UT
(mm)a

Dv50 of UT
(mm)a

Dv90 of UT
(mm)a

1:00
2:00
5:00
7:00
8:00

2:30
5:00
12:30
17:30
20:00

1.38
0.83
0.48
0.39
0.40

1.12
0.59
0.34
0.30
0.30

3.02
1.81
0.99
0.75
0.79

3.30
2.27
1.54
2.20
1.48

2.44
1.75
1.30
1.30
1.23

7.58
4.93
3.25
3.31
3.15

The results are average of two measurements and the error value varies from 0 to 0.428 mm.
1=5

dmax CP 2=5 g3=5 rc

(3)

where, C is a constant, P is the power density, g is the interfacial

tension and rc is the density of the continuous phase. Hence, the
shearing effect and ultimately the dmax are affected by the power
density. By calculation, the power density is lower for the emulsions obtained at 88 W in comparison to 132 and 176 W. From
Table 1, it is apparent at any particular sonication time that the
droplets produced at lowest power (88 W) are larger compared to
those produced at 132 and 176 W. Therefore, lower the power
density, weaker is the shear generated. In addition to power density, dmax is also dependant on the residence time during the formation of emulsions (Karbstein & Schubert, 1995). The process
carried out at 132 W and at a residence time of 8 min has produced
smaller droplets in comparison to 132 W emulsions prepared at a
residence time of 5 min. This shows the importance of optimum
combination of power density and residence time for the formation
of smaller emulsion droplets. From Table 1, though the D(4,3) and
Dv90 values for 8 min 88 W are comparable to 5 min 132 W and
3 min 176 W, stable emulsions were not obtained for the following
reason: a combination of residence time and power density was
sufcient enough to produce smaller droplet size, but not strong
enough to modify the whey proteins.
The preparation of emulsions at higher power levels can induce
coalescence among emulsion droplets and results in the formation
of larger emulsion droplets. However, coalescence (increase in
D(4,3)), is not observed in our system even at the highest power

level of operation (176 W). These results are similar to the observation recorded by Kentish et al. (2008) who reported an increase
in the droplet sizes of 15% OW emulsions (with emulsiers) only
above 200 W NAP.
Good and stable emulsions of 15 and 21% oil were obtained only
between 3e8 min and 6e8 min of processing, while the rest of the
samples remained unstable. The instability is possibly due to the
presence large emulsion droplets (Fig. 11) in comparison to the data
shown in Fig. 1. It was visually observed in the form of creaming of
samples and is primarily due to the phenomenon of gravity separation. It can also be due to the presence of partially covered protein
surfaces on these larger emulsion droplets. These incompletely
covered surfaces can lead to droplet coalescence. While comparing
the data shown in Figs. 11 and 1, the distribution pattern of all high
oil emulsions overlaps with the 2 min 7% 176 W OM emulsion but
the former samples are stable for 5 days unlike the later stable only
for 2 days. This effect may possibly be due to the optimum combination of residence time and power density in the former
compared to the latter. Here, it is also important to note that the 7%
OM emulsion had higher amounts of milk protein in comparison to
15 and 21% high oil emulsions. The 7% OM emulsions had about 3%
(w/w) of milk protein in the formulation in comparison to about 1%
(w/w) of milk proteins in the 21% OM emulsions.
In the following section we again re-emphasise the importance
of cavitation and shear that are produced by the US system against
a sole shear system (UT). In brief, the emulsication capacity of UT
at equivalent energy densities of 176 We20 kHz US was evaluated
(Summary point 4.15 & 4.16). This work reects the efciency of the
shear forces that are created by UT process. It is deduced by
measuring the particle size and by monitoring the stability of
emulsions. In order to produce UT emulsions at the same energy
densities of 176 W 20 kHz US, it has to be operated at 2.5 times the
process time of US, for example 1 min of US process is equivalent to
2 min:30 s of UT process (Table 3). From Fig. 12 & Table 4, the sizes
of UT emulsions are always higher than US emulsions and the size
distribution remained bimodal for all UT processes. The emulsion
instability is noted in all the fresh UT samples (Fig. 13). As already
discussed, the emulsion stability indirectly refers to the process
induced modications on the whey protein and their efciency in
lowering of the interfacial tension to create ner emulsions. Hence,
it can be suggested from the above discussion that the shear forces
of UT are not effective to produce good and stable emulsions.
Therefore, UT process is not seen as an efcient technique in the
generation of ner and stable OM emulsions.
5. Conclusions

Fig. 13. Photograph represents 7% OM emulsions obtained by UT process after few

hours of standing at room temperature. Second row (Left to right): 2 min 30 s, 5 min
and 12 min 30 s samples, First row (Left to right): 17 min 30 s and 20 min Samples.
Creaming is visible on the surface of all the samples.

Stable emulsions of 7% ax seed oil and PHSM were obtained

using 176 W 20 kHz US. The mechanical, cavitation and cavitation
after-effects of US are responsible for producing stable dairy
emulsions for a minimum of 9 days at 4
2  C. The mechanism
responsible for the stability of the emulsions was evaluated by
measuring the creaming index, zetapotential values and the
changes to the hydrophobicity. The milk proteins, viz., whey

A. Shanmugam, M. Ashokkumar / Food Hydrocolloids 39 (2014) 151e162

proteins have contributed to the stability of these emulsions. A

minimum process time of 3 min is recommended to produce stable
emulsions of 7% ax seed oil and milk. The increase in power
density or NAP resulted in the production of large number of
smaller emulsion droplets until 8 min of processing. The importance of residence time and power density was studied, and an
optimum combination of these two was identied to produce stable emulsions. A combination of 5 min & 132 W and 3 min & 176 W
produced stable emulsions. Also in this work, emulsions with large
quantities of oil, viz., 15 and 21% were obtained using 176 W 20 kHz
US. Minimum process times of 3 min and 6 min are recommended
to manufacture 15 and 21% high oil emulsions at 176 W. In addition,
this work compares two different homogenization techniques, viz.,
US and UT in the preparation of stable 7% OM emulsions. At
equivalent energy densities, US process is efcient in the production of stable emulsions while UT did not produce emulsions at any
processing time. The data discussed suggest that ax seed oil, a
non-polar bioactive, can be effectively incorporated into a complex
food matrix in the form of an emulsion by employing US technique.
In the formulations, the amount of ax seed oil was varied from 7 to
21%, which may provide 4.2 to 12.6% of ALA in the diet. The ALA of
ax seed oil are rich source of u-3 fatty acids and this can substitute
sh oil, used widely in the preparation of u-3 food/infant formulations. Sensorially, the shy avours are not detected in the ax
seed oil/milk US emulsions unlike many of the u-3 food formulations, which were manufactured using the sh oil. As ax seed oil is
an abundant source of poly unsaturated fatty acids (PUFA), the
production of rancid avours are expected upon any processing
technique. However, in all our emulsion samples the rancid avour
is not noted by the human senses (sensorially). The current paper
will remain as the base to a following paper on the functional
characteristics/properties of OM emulsions.
Acknowledgements
The author would like to acknowledge the University of Melbourne for providing Melbourne International Fee Remission and
Melbourne International Research scholarships and also would like
to thank Stoney Creek Oil Products Pty Ltd, Australia for providing
axseed oil as a gift sample for research.
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