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Influence of Disperse Phase Characteristics on Stability, Physical and

Antimicrobial Properties of Emulsions Containing Cinnamaldehyde

Article  in  Journal of the American Oil Chemists' Society · February 2012

DOI: 10.1007/s11746-012-2164-1

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J Am Oil Chem Soc (2013) 90:233–241
DOI 10.1007/s11746-012-2164-1
ORIGINAL PAPER
Influence of Disperse Phase Characteristics on Stability, Physical
and Antimicrobial Properties of Emulsions Containing
Cinnamaldehyde
Cristina Bilbao-Sainz • Bor-Sen Chiou •
Wen-Xian Du • Kay S. Gregorsky • William J. Orts
Received: 20 April 2012 / Revised: 11 October 2012 / Accepted: 17 October 2012 / Published online: 2 November 2012
Ó AOCS (outside the USA) 2012
Abstract Cinnamaldehyde was delivered in emulsion
form using Acetem 90-50K as a carrier and Tween 60 as
emulsifier. Cinnamaldehyde interacted with Acetem
90-50K by forming H-bonds. The effect of disperse phase
characteristics on storage stability, physical and antimi-
crobial properties was investigated. A storage test of
emulsions was carried out for 15 days at two temperatures
(22 and 4 °C). Emulsions and nano-emulsions showed
higher stability at 22 °C than at 4 °C. Nano-emulsions
displayed excellent stability versus creaming and coales-
cence after 15 days storage at 22 °C (z-avg \100 nm).
Physical properties were greatly affected by droplet size
and concentration. Emulsions became less viscous, more
transparent and darker as the droplet size or concentration
decreased. The antimicrobial activity was measured against
Listeria monocytogenes and Escherichia coli. Escherichia
coli was highly resistant to cinnamaldehyde compared to
L. monocytogenes. Incubation with cinnamaldehyde at
2.5 mM caused the complete inactivation of L. monocyt-
ogenes after 1 day and of E. coli after 9 days. There was no
difference in the antimicrobial effect of cinnamaldehyde
due to different droplet sizes (*80 and *5,000 nm).
Keywords Emulsion  Droplet size  Color  Viscosity 
Antimicrobial  Cinnamaldehyde
C. Bilbao-Sainz (&)  B.-S. Chiou  W.-X. Du 
K. S. Gregorsky  W. J. Orts
Western Regional Research Center, ARS, US Department of
Agriculture, 800 Buchanan St, Albany, CA 94710, USA
e-mail: cristina.bilbao@ars.usda.gov
Introduction
Cinnamaldehyde (cinnamic aldehyde or 3-phenyl-2-pro-
penal) represents 60–75 % of the content in cinnamon bark
essential oil. It is generally regarded as safe and is used as a
flavoring agent in the food industry [1]. It is also used as an
ingredient in fine fragrances, shampoos, toilet soaps and
other toiletries as well as in non-cosmetic products such as
household cleaners and detergents [2]. In the last years, there
has been an increased interest in the use of cinnamaldehyde
as a natural antimicrobial agent due to its antibacterial
activity against a number of Gram-positive and Gram-neg-
ative bacteria such as Listeria monocytogenes (L. mono-
cytogenes), Lactobacillus sakei, Clostridium botulinum,
Staphylococcus aureus, Escherichia coli (E. coli), and
Salmonella enterica [3–5]. In addition to exhibiting anti-
bacterial activity, cinnamaldehyde also inhibits mold
growth and mycotoxin production [6]. However, direct
incorporation of cinnamaldehyde to formulations might
encounter technological limitations due to its high volatility
and low water solubility (1.4 mg/g). An alternative
approach to increase the physical stability of this bioactive
compound involves encapsulation technology. Among the
different encapsulation systems, emulsion-based ones allow
highly lipophilic compounds to be incorporated into a suit-
able colloidal delivery system before they can be dispersed
into an aqueous-based system. Emulsion-based delivery
systems have therefore been used increasingly for encap-
sulating poorly water soluble bioactive compounds [7, 8].
Cinnamaldehyde-based antimicrobial emulsions are
suggested for use in foods and beverages. Emulsion-based
formulations are not only characterized by their stability,
but also by their appearance and rheological properties.
Appearance plays a major role in food choice by influ-
encing taste thresholds, sweetness perception, food
123
234
preference, pleasantness, and acceptability [9]. The overall
appearance of the emulsion is determined by its interaction
with radiation in the visible region of the electromagnetic
spectrum, e.g., reflection, transmission, absorption, and
scattering [7]. Scattering is largely responsible for the
turbidity, opacity, or lightness of an emulsion, whereas
absorption is largely responsible for chromaticness (yel-
lowness, redness, greenness, etc.) [10]. The quality attri-
butes of the emulsions might also be described in terms of
creaminess, body and consistency, which can be correlated
to rheological properties. For instance, emulsions can
behave as low viscosity Newtonian liquids, thicker shear-
thinning liquids, or cream-like materials with apparent
yield stresses [11]. The oil phase concentration, the size
and number of oil particles, and their distribution pattern
impart a significant effect on the emulsion appearance and
rheological characteristics [10]. Therefore, the purpose of
this study was to evaluate the effect of the disperse phase
characteristics on the stability, appearance, rheological
behavior and antimicrobial activity of emulsions contain-
ing cinnamaldehyde as an antimicrobial agent.
Materials and Methods
Preparation of Oil in Water Emulsions
Grindsted Acetem 90-50K was kindly provided by Danisco
USA Inc. (New Century, KS, USA). This product is an
acetic acid ester of monoglycerides made from edible,
partially hydrogenated soybean oil. The company provides
the following chemical specifications for this product: The
degree of acetylation is 0.9, the iodine value 45 and the
saponification value 380. Polyoxyethylene sorbitan mon-
ostearate (Tween 60) and cinnamaldehyde were purchased
from Sigma-Aldrich Co. (Milwaukee, WI, USA).
The dispersed phase contained Grindsted Acetem 90-50K
and cinnamaldehyde. The aqueous phase was distilled water
and the surfactant Tween 60 (Table 1). A single formulation
(sample 1) was used to determine the effect of droplet size on
emulsion stability. Samples (1 and 2) with 16 or 25 wt%
droplet concentrations (Acetem ? cinnamaldehyde) were
used to study the effect of droplet size and droplet
Table 1 Composition of the emulsion-based delivery systems
Sample 1 Sample 2 Sample 3
Grindsted Acetem 6 g (10 %) 6 g (15 %) 6 g (10 %)
90-50K
Cinnamaldehyde 4 g (6 %) 4 g (10 %) 2 g (3 %)
Tween 60 3 g (5 %) 3 g (7 %) 3 g (5 %)
Water 50 g (79 %) 28 g (68 %) 50 g (82 %)
123
J Am Oil Chem Soc (2013) 90:233–241
concentration on physical properties. For microbiological
studies, formulations were prepared with 6 and 3 wt% cin-
namaldehyde (samples 1 and 3).
Two techniques were used to prepare the emulsions. The
first method employed a high shear laboratory mixer
(Silverson model L5M-A, East Longmeadow, MA, USA).
A surfactant suspension was initially prepared by dispers-
ing Tween 60 in distilled water with the aid of a magnetic
stirrer. Afterwards, the oil phase was added to the sus-
pension. Emulsions were produced using the high shear
mixer at 5,000 rpm for different lengths of time to produce
emulsions with different mean droplet diameters. Samples
were placed in an ice bath to minimize temperature rise in
the sample. The second method employed a high pressure
homogenizer (M-110Y MicrofluidizerÒ Processor) from
Microfluidics Corp. (Newton, MA, USA). Mixtures con-
taining the aqueous and non-aqueous phase were passed
through the microfluidizer and the number of passes was
varied to produce a range of different droplet diameters.
Homogenization pressure was set at 18,000 psi. The sam-
ples were cooled using a cooling jacket containing ice.
Fourier Transform Infrared Spectroscopy-Attenuated
Total Reflectance (FTIR-ATR) Analysis
FTIR-ATR spectra of cinnamaldehyde, Acetem 90-50K
and cinnamaldehyde–Acetem 90-50K emulsions were
obtained using a Perkin Elmer FTIR spectrometer (Model
System 2000, Perkin Elmer, USA) equipped with a deu-
terated triglycine sulfate (DTGS) detector and the DuraS-
amplIRTM Universal DiCompTM attachment (single
reflection diamond-ZnSe, ASI SensIR Technologies). The
penetration depth of the ZnSe crystal varied from 0.5 to
3.0 lm in the wave number range of 4,000–650 cm-1. All
spectra were collected at 4 cm-1 resolution. A background
spectrum in air was obtained for each sample. A small
sample of the emulsion was then placed onto the diamond
crystal and covered with a small Petri dish. Ten scans were
taken for cinnamaldehyde and cinnamaldehyde–Acetem
90-50K and 15 scans were taken for Acetem 90-50K.
Emulsions Stability
The stability of the emulsions was evaluated by monitoring
the droplet size as a function of storage time under ambient
(22 °C) and cold (4 °C) storage conditions. At least three
measurements were taken for each sample. The emulsions
were first diluted with purified water to 1/100 of their ori-
ginal concentrations. Mean particle diameters (z-average)
and polydispersity index (PdI) were measured right after
preparation using a Malvern Zetasizer dynamic light scat-
tering particle size analyzer (Malvern Instruments Ltd.,
Westborough, MA, USA) at 25 °C. The instrument used the
J Am Oil Chem Soc (2013) 90:233–241
method of photon correlation spectroscopy (PCS) to mea-
sure particle size in constant random thermal, or Brownian,
motion. This motion causes the intensity of light scattered
from the particles to vary with time. Large particles move
slowly than small ones, so that the rate of fluctuation of the
light scattered from them is also slower. PCS uses the rate of
change of these light fluctuations to determine the size
distribution of the particles scattering light.
Optical Properties
Spectral transmittances of emulsions were measured using a
UV–visible spectrophotometer (UV-1700, Shimadzu Corp.,
Kyoto, Japan). The emulsions were diluted to 0.001 wt%
solids with distilled water to avoid multiple scattering. The
samples were placed in quartz cuvettes with a 1-cm path-
length. Spectra were obtained over the wavelength range
400–780 nm using a scanning speed of 700 nm min-1.
Multiple regression analysis was used to develop an
empirical equation to predict transmittance values. The
regression analysis was performed using Minitab 15 sta-
tistical software, where the stepwise regression selection
procedure was used to determine the best fitting regression
model. A P value \0.15 was used for the inclusion and
exclusion of independent variables during model fitting.
The color of the emulsions was measured using a col-
orimeter (Minolta Chroma Meter CR-200, Minolta Co.,
Ltd., USA). For each sample, 20 mg of red dye solution
was added to 30 g sample. For dye-free emulsions, the
a and b values are approximately equal to zero therefore a
dye was used to evaluate the effect of the droplet size and
concentration on the color parameters a* and b*. After
mixing, 10 g of the colored emulsion sample was poured
into the measurement cup surrounded with a black paper
strip, and covered with a white tile. The instrument pro-
vides the color of the samples in terms of the L*, a*, b*
color space system. In this color space, L* represents the
lightness, which is a measure of the total amount of light
that is reflected from the emulsion surface, and a* and b*
are color coordinates with ?a* being the red direction,
-a* the green direction, ?b* the yellow direction, and
-b* the blue direction. Three samples of each emulsion
were prepared to evaluate the optical properties.
Rheological Properties
The emulsion viscosity was measured at 23 °C using a
rheometer (TA Instruments, model AR2000, New Castle,
DE, USA) with a concentric cylinder geometry. The
diameter of the inner cylinder was 14 mm and the diameter
of the outer cylinder was 15 mm. Samples were placed in
the rheometer and allowed to equilibrate to the required
temperature (23 °C) for 10 min before the start of each
235
experiment. The viscosity was measured for shear rates
ranging from 0.01 to 1,000 s-1. Samples were analyzed in
triplicate.
Bacterial Strain and Growth Conditions
The Food and Drug Administration (FDA) provided the
E. coli O157:H7 bacteria (our strain designation of
RM1484; original designation of SEA13B88) isolated from
tomato juice associated with an outbreak. L. monocytoge-
nes was obtained from the University of California,
Berkeley (our strain designation of RM2199; original
designation strain of F2379) isolated from cheese associ-
ated with an outbreak. Frozen cultures of E. coli O157:H7
and L. monocytogenes were streaked on Trypticase Soy
Agar (TSA) and then incubated at 37 °C for 24 h. One
isolated colony was re-streaked on TSA and then incubated
at 37 °C for 24 h. This was followed by inoculating one
isolated colony into a tube with 5 ml Trypticase Soy Broth
(TSB) and incubating at 37 °C overnight with agitation.
Kinetics of Killing
Overnight bacterial cultures were added at 1 % v/v (0.1 ml
bacterial culture ? 9.9 ml solution) to undiluted and to 10-
and 100-fold dilutions of the emulsions. Aliquots of sam-
ples were taken from those inoculated emulsions after
1–9 days of storage at room temperature and immediately
diluted in 0.1 % peptone water at room temperature. For
viable counts, samples were either spread plated 0.1 ml
onto duplicate TSA plates or spot plated in duplicate
(20 ll) onto TSA plates, if CFU decreased to 1 log CFU/g
or less, plated 0.25 ml on each of four plates. Plates were
counted by hand after 24 h of incubation at 37 °C.
Results and Discussion
Delivery of Cinnamaldehyde in Emulsion Form
In a series of initial experiments, the optimal disperse
phase composition was evaluated using Acetem 90-50K as
a carrier for cinnamaldehyde. Emulsions prepared with
neat Acetem 90-50K showed higher stability compared
with neat cinnamaldehyde. Also, when Acetem 90-50K
was used as a carrier for cinnamaldehyde at different ratios,
the sample became more stable than that with neat cinna-
maldehyde (Fig. 1).
FTIR-ATR spectroscopy was used to identify chemical
interactions between Acetem 90-50K and cinnamaldehyde.
Figure 2 shows the spectra of cinnamaldehyde, Acetem
90-50K, and their mixture. The carbonyl (C=O) stretch of
the ester in Acetem 90-50K (1,743 cm-1) showed a shift to
123
236
Fig. 1 Evolution of droplet size
during storage at 22 °C of
emulsions prepared with
different disperse phase
compositions
Fig. 2 FTIR spectra of cinnamaldehyde, Acetem, and their mixture.
The Acetem and cinnamaldehyde spectra have been shifted 0.5
absorbance units above the spectrum below it
lower frequency, whereas the carbonyl (C=O) stretch of the
aldehyde in cinnamaldehyde (1,668 cm-1) showed a shift
to higher frequency in the mixture. These changes suggest
that the cinnamaldehyde and Acetem 90-50K formed
hydrogen bonds between the aldehyde H and the ester
carbonyl O. This hydrogen bonding weakened the ester
C=O bond and thus decreased its stretch frequency. It also
decreased the resonance effect of the alkene group in cin-
namaldehyde on the aldehyde C=O bond, resulting in
higher stretch frequency. The hydrogen bonding also
affected the motions of the phenyl (1,625 cm-1) and
the alkene groups (1,450 cm-1) of cinnamaldehyde and the
C–C–O of the ester (1,218 cm-1) in Acetem 90-50K as
shown in the changes of their frequencies.
Effect of Droplet Size and Temperature on Emulsion
Stability During Storage
Storage stability of emulsions and nano-emulsions con-
taining Acetem 90-50K and cinnamaldehyde as the disperse
123
J Am Oil Chem Soc (2013) 90:233–241
phase was evaluated over 2 weeks. For this study, a diameter
of 100 nm was used as the boundary between nano-emulsion
and emulsion because the samples’ appearance and rheo-
logical properties changed appreciably below this value.
Emulsions and nano-emulsions showed higher stability at
22 °C than at 4 °C (Fig. 3). Storage of nano-emulsions at
22 °C resulted in the oil droplet size remaining below
100 nm for at least 15 days. From the z-avg data, emulsions
exhibited lower stability than nano-emulsions, the smaller
the initial droplet size the higher the stability. After 15 days
of storage, emulsion droplet diameter increased 16 % at
22 °C and 54 % at 4 °C when the initial droplet size was
84 nm; however, the droplet size increased 24 % at 22 °C
and 137 % at 4 °C when the initial droplet size was 229 nm.
Like conventional emulsions, nano-emulsions are non-
equilibrium systems; nevertheless, nano-emulsions have
been reported to have better stability to particle aggregation
and gravitational separation [12, 13]. Therefore, the higher
stability of nano-emulsions in comparison with emulsions
was attributed to the enhanced shelf stability of nano-
emulsions against gravitationally driven creaming since the
creaming rate is proportional to the square of the particle
radius as it is given by Stokes’ law equation.
Effect of Droplet Size and Droplet Concentration
on the Spectral Transmittance of Emulsions
The transmittance of oil-in-water emulsions containing a
range of different mean droplet diameters was measured
for emulsions with 16 and 25 wt% droplet concentrations,
respectively (Fig. 4a, b). These results clearly showed that
the transmittance spectra were dramatically influenced by
droplet concentration and droplet size. The emulsions
containing small droplet sizes (d \ 100 nm) appeared
either transparent or only slightly turbid; this was due to the
droplet size being relatively small compared with the
wavelength of light. Consequently, the droplets only scatter
light weakly. However, the spectral transmittance
decreased with increasing droplet size and/or droplet
J Am Oil Chem Soc (2013) 90:233–241 237

Fig. 3 Mean droplet size and 700

PdI over time of emulsions with
different initial droplet sizes 600
stored at 4 or 22 °C
500 Zavg=84nm, T=22°C
Zavg=84nm, T=4°C

Zavg (nm)
400
Zavg=156nm, T=22°C

300 Zavg=156nm, T=4°C

Zavg=229nm, T=22°C
200 Zavg=229nm, T=4°C

100

0
0 2 4 6 8 10 12 14 16

Storage time (days)

0.9

0.8

0.7 Zavg=84nm, T=22°C

Zavg=84nm, T=4°C
0.6
Zavg=156nm, T=22°C
PdI

0.5
Zavg=156nm, T=4°C
0.4
Zavg=229nm, T=22°C
0.3
Zavg=229nm, T=4°C
0.2

0.1

0
0 2 4 6 8 10 12 14 16
Storage time (days)

100 100
a b
90 90
80 80
Transmittance (%)

Transmittance (%)

70 70
71 nm
60 60
83 nm 66 nm
50 50
145 nm 90 nm
40 40
159 nm 112 nm
30 30
247 nm 267 nm
20 20
474 nm 549 nm
10 10
3340 nm 3356 nm
0 0
400 500 600 700 800 400 500 600 700 800
Wavelength (nm) Wavelength (nm)

Fig. 4 Transmittance spectra of a series of oil in water emulsions with a range of droplet diameters. a 16 wt% droplet concentration. b 25 wt%
droplet concentration

concentration. At high droplet size or droplet concentra-
tion, the scattering became so intense that the light wave
was unable to travel through the solution without being
reflected or absorbed. For small droplet size (\145 nm),
the following regression equation T (%) = 133 - 2.48
droplet conc. (%) ? 0.12k (nm) - 0.971 droplet size (nm)
(R2 = 82.4) was obtained using multiple linear regression
analysis for the transmittance value (T) when droplet
concentration, droplet size and wavelength (k) were chosen
as independent regression variables.

123
238 J Am Oil Chem Soc (2013) 90:233–241

The main factor controlling the transmittance of the emul- 25% 16%
sion with droplet size\145 nm was the droplet concentration. 90
88

Effect of Droplet Size and Droplet Concentration 86

on Emulsion Color 84
82
A series of oil-in-water emulsions were prepared with two L* 80
different droplet concentrations (16 and 25 wt%) and a 78
range of mean droplet diameters. The color of the emul- 76
sions containing a red dye (0.07 wt%) was measured using 74
a colorimeter and presented in the form of the L*, a*, b* 72
tristimulus system (Fig. 5). 70
The emulsion lightness (L*) increased with increasing 0 500 1000 1500 2000 2500 3000 3500
droplet diameter. For emulsions containing 16 wt% droplet droplet size (nm)
concentration, the lightness increased steeply as the droplet
size increased up to 473 nm and then leveled off at 25% 16%
approximately 86 % at larger diameters. Similar behavior 0 500 1000 1500 2000 2500 3000 3500
-2.00
was observed for the emulsions containing 25 wt% droplet
concentration. These results indicated that the emulsions -3.00

became lighter as the droplet size increased for the range -4.00
studied. The maximum in the scattering efficiency of the -5.00
droplets occurs when their diameter is approximately equal -6.00
to the wavelength of light. The influence of droplet size and a*
-7.00
concentration on the color of oil-in-water emulsions con-
-8.00
taining a red food dye was also studied by Chantrapornchai
et al. [14]. They also found that the lightness of the -9.00

emulsions increased with increasing droplet concentration -10.00

and droplet size up to 500 nm. After this point, the light- -11.00
ness decreased with increasing droplet size up to 26 lm. droplet size (nm)
The difference between their results and our results could
be due to the higher PdI observed in our emulsions, which 25% 16%
could have resulted in the absence of a clear maximum in 27

the emulsion lightness for a specific droplet size. 26

A decrease in droplet concentration or an increase in
25
droplet size results in the a* values becoming less negative
and the b* values increasingly positive, indicating the 24
emulsions became more red and more yellow (Fig. 5b, c). b*
23
This is because the scattering efficiency of the droplets
decreased as the droplet concentration decreased or the size 22
increased [14]. In this case the light beam penetrates further
21
into the emulsion before being reflected back to the detector,
and therefore be subject to more absorption by the dyes. The 20
emulsion color coordinates are strongly affected by droplet 0 500 1000 1500 2000 2500 3000 3500
size up to 159 nm. For larger droplet sizes, the a* value droplet size (nm)
remains constant and the b* value steadily increased. The a*
Fig. 5 Dependence of L*, a*, b* on droplet size and droplet
and b* values also increased with an increase in droplet size or concentration for oil in water emulsions containing cinnamaldehyde
a decrease in droplet concentration from 24 to 1 wt% oil.

Effect of Droplet Size and Droplet Concentration
on Rheological Behavior
The flow curves for 25 wt% droplet concentration (Fig. 6b)
exhibited three distinct regions: the low shear region where
viscosity is constant, the shear-thinning region in the
intermediate shear rate range where viscosity decreases
with an increase in shear rate, and the high shear region
where the viscosity again becomes constant. It is assumed
that the position of the shoulder on each curve between the

123
J Am Oil Chem Soc (2013) 90:233–241
1
viscosity (Pa.s)
0.1
0.01
0.001
0.0001
0.01 0.1 1 10
shear rate (1/s)
Fig. 6 Apparent viscosity of O/W emulsions prepared with different droplet diameters. a 16 wt% droplet concentration. b 25 wt% droplet
concentration
constant viscosity at low shear region and the shear-thin-
ning region is indicative of the breakdown stress of the
putative flocculated network structure. The flow curves for
16 wt% droplet concentration (Fig. 6a) exhibited the shear-
thinning region followed by the high shear Newtonian
region. In this figure no constant viscosity at low shear rate
was observed since the flocculation of droplets did not take
place because of little of droplets. The shear-thinning
behavior had been widely reported for emulsions [15, 16].
During shearing, the oil droplets are deformed and even-
tually disrupted, leading to a decrease in overall resistance
to flow and therefore a reduction in viscosity.
From Fig. 6, emulsion viscosity increased with an
increase in droplet concentration from 16 to 25 wt%. This
increase in viscosity for higher dispersed phase concen-
tration had been found in previous studies [15, 16]. One
important factor that influences emulsion viscosity is the
disperse phase volume fraction (/), which is equal to the
volume of emulsion droplets divided by the total volume of
the emulsion [17]. In dilute emulsions (/ \ 0.05), the
dependence of viscosity (g) on oil phase volume fraction
can be described by the Einstein equation:
g ¼ gl ð1 þ 2:5/Þ
where gl is the viscosity of the aqueous phase. As the
concentration of oil phase increases, colloidal interactions
(electrostatic and steric) between droplets causes the
effective volume fraction of the dispersed phase to become
significantly greater than its actual volume fraction, leading
to higher emulsion viscosity [17, 18].
The viscosity of the emulsions also greatly depended on
the droplet size. The shear-thinning effect was more
apparent for emulsions with larger droplet diameters. For
emulsions with smaller droplet diameters (z-avg\100 nm),
the apparent viscosity did not depend as much on shear
rate. This is because large droplets are easily deformed but
239
a 1
b
3340 nm
viscosity (Pa.s)
1547 nm 1060 nm
83 nm 0.1 549 nm
267 nm
90 nm
0.01
0.001
100 1000 0.01 0.1 1 10 100 1000
shear rate (1/s)
small droplets are assumed to behave more like hard
spheres due to their large Laplace pressure. Also,
the emulsions exhibited a much lower viscosity when the
droplet size decreased. The effects of droplet size on the
rheology of oil-in-water emulsions had been shown to
depend on dispersed phase concentration [16, 18]. Pal
observed that for highly concentrated oil-in-water emul-
sions ([60 %), a reduction in droplet size results in a
dramatic increase in viscosity. Also, the shear-thinning
effect becomes stronger when the droplet size is reduced
[16]. The authors explained the observed increase in vis-
cosity and shear-thinning effect with the decrease in
droplet size at high concentrations of dispersed phase in
terms of flocculation of fine droplets. Fine emulsions
exhibit a greater tendency to flocculate. This increased
tendency of flocculation could be due to at least two dif-
ferent mechanisms; first, the Brownian motion becomes
important in the case of fine emulsions, as it tends to
increase the collision, and hence flocculation, between
droplets; and, second, the droplets are subject to van der
Waals attraction force.
Antimicrobial Activity Against L. monocytogenes
and E. coli
Emulsions with different cinnamaldehyde concentrations
(2.5 and 4.8 mM) were tested for their antimicrobial
activity against L. monocytogenes and E. coli. Friedman
et al. [19] reported that cinnamaldehyde 1.4 and 4.3 mM
decreased 50 % the number of CFU of L. monocytogenes
and E. coli, respectively. The Gram-positive bacterium
L. monocytogenes was more sensitive to cinnamaldehyde
than the Gram-negative bacterium E. coli. Twenty-four
hours of incubation at the lowest cinnamaldehyde con-
centration of 2.5 mM caused the complete inactivation of
L. monocytogenes (data not shown), but had no effect on
123
240
8 antimicrobial effect of nano-emulsions derived from the
7 active ingredients incorporated into the oil and not from
6 their nano dimensions. Therefore based on our findings and
Log CFU/ml
5 NE, 4.8 mM
EM, 4.8 mM
4
NE, 2.5 mM
3 EM, 2.5 mM
2
1
0
0 1 2 3 4 5 6 7 8 9 10
time (days)
Fig. 7 Effect of different concentrations of cinnamaldehyde deliv-
ered in emulsion (EM) or nano-emulsion (NE) form on E. coli
viability
E. coli. Du et al. [20] also found that the inhibitory zone
induced by the vapor phase of cinnamon oil was greater for
L. monocytogenes than for E. coli. However, Gill and
Holley [5] reported greater susceptibility of E. coli to
cinnamaldehyde compared to L. monocytogenes. They
found that cinnamaldehyde progressively inhibited the
membrane bound ATPase activity of E. coli as cinnamal-
dehyde concentration increased from 0.1 to 10 mM,
whereas significant ATPase inhibition of L. monocytogenes
only occurred above 5 mM. The difference between our
results and those from Gill and Holley [5] might be due to
the complex formation between Acetem 90-50K and cin-
namaldehyde (see Fig. 2). This complex might interact
with the bacteria cells in a different manner than the neat
cinnamaldehyde.
To compare the effect of droplet size on antimicrobial
activity, emulsions and nano-emulsions were prepared with
droplet sizes of 4,964 ± 392 and 79 ± 2 nm, respectively.
Figure 7 shows the kinetics of E. coli inactivation for
different droplet sizes and cinnamaldehyde concentrations.
Cinnamaldehyde concentration had a greater effect on
E. coli viability than mean droplet size. For a cinnamal-
dehyde concentration of 4.8 mM, E. coli was reduced
about 2 log cycles after 2 days of incubation and was
completely inactivated after 3 days. In comparison, for a
cinnamaldehyde concentration of 2.5 mM, E. coli was
reduced about 2 log cycles after 7 days and was com-
pletely inactivated after 9 days. Figure 7 also shows that
emulsions and nano-emulsions have the same level of
antimicrobial effect against E. coli. The effect of emulsion
droplet size on antimicrobial properties against bacteria
had been previously investigated by Buranasuksombat
et al. [21]. These authors found that the antimicrobial
properties of lemon myrtle oil emulsions against E. coli,
L. monocytogenes, S. Typhimurium, P. aeruginosa, and
B. cereus did not depend on droplet sizes, which ranged
from 100 nm to 100 lm. They concluded that the
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J Am Oil Chem Soc (2013) 90:233–241
those reported in the literature it seems that the interfacial
area does not play a significant role in determining the
antimicrobial effect of cinnamaldehyde against E. coli.
Conclusions
The antimicrobial agent cinnamaldehyde was delivered in
emulsion and nano-emulsion forms using Acetem 90-50K
as a carrier. Cinnamaldehyde interacted with Acetem
90-50K by forming H-bonds. The complex formation
increased the stability of the cinnamaldehyde without los-
ing its antimicrobial activity against L. monocytogenes and
E. coli. All formulations had antibacterial activity with
cinnamaldehyde concentrations ranging from 2.5 to
4.8 mM.
Storage stability and physical properties were strongly
influenced by droplet size. The most dramatic changes
occurred at 100 nm. Below this size, no changes in droplet
size or PdI took place during 15 days storage at 22 °C.
Nano-emulsions appeared more transparent, were darker
and had lower viscosity than emulsions with larger droplet
size. However, the antimicrobial properties of cinnamal-
dehyde did not depend on droplet sizes.
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