Journal Pre-proof

Effect of sodium alginate and different types of oil on the physical

properties of ultrasound-assisted nanoemulsions

Ivanise Guilherme Branco, Kacoli Sen, Carlos Rinaldi

PII: S0255-2701(20)30399-8
DOI: https://doi.org/10.1016/j.cep.2020.107942
Reference: CEP 107942

To appear in: Chemical Engineering and Processing - Process Intensification

Received Date: 19 December 2019

Revised Date: 25 March 2020
Accepted Date: 11 April 2020

Please cite this article as: Branco IG, Sen K, Rinaldi C, Effect of sodium alginate and different
types of oil on the physical properties of ultrasound-assisted nanoemulsions, Chemical
Engineering and Processing - Process Intensification (2020),
doi: https://doi.org/10.1016/j.cep.2020.107942

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Effect of sodium alginate and different types of oil on the physical

properties of ultrasound-assisted nanoemulsions

Ivanise Guilherme Brancoa*, Kacoli Senb and Carlos Rinaldib,c

a
Department of Biological Sciences, Universidade Estadual Paulista (UNESP), Assis, SP
19806-900, Brazil.
b
Department of Chemical Engineering, University of Florida (UF), Gainesville, FL

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32611, United States.
c
J. Crayton Pruitt Family Department of Biomedical Engineering University of Florida

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(UF), Biomedical Sciences Building, Gainesville, FL 32611-6131, United States.
*Corresponding author. E-mail: ivanise.branco@unesp.br
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Graphical abstract

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Highlights
 Nanoemulsions with different sodium alginate concentrations and oil types
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were formulated.
 Nanoemulsions were produced by ultrasound and physicochemical properties
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were evaluated.
 Corn oil and oleic acid nanoemulsions with 0.5% sodium alginate showed the
best quality parameters.
 Oleic acid and corn oil nanoemulsions were stable over 28 days of storage.
 - Corn oil and oleic acid nanoemulsions showed spherical and asymmetric
shape, respectively.
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Abstract

The aim was to develop stable nanoemulsions as potential delivery systems for bioactive

ingredients, pharmaceutical products, and nanoparticles dispersed in organic solvents.

The effect of sodium alginate (SA) concentration (0.5, 1.0 and 1.5% w/w) and the type of

oil (corn oil (CO), oleic acid (OA), olive oil, and unrefined and refined coconut oil), in

combination with tween 80, on the quality of nanoemulsions produced by ultrasound was

evaluated. CO and OA nanoemulsions with 0.5% SA showed the lowest droplet size,

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polydispersity index, and zeta potential. After ultrasonication, CO and OA nanoemulsions

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exhibited a reduction in droplet size, polydispersity index, and viscosity. Coarse

emulsions showed near-Newtonian behavior while the nanoemulsions exhibited

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Newtonian behavior. Fourier-transform infrared spectroscopy did not reveal differences

among SA solutions with OA and CO nanoemulsions. CO and OA nanoemulsions

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possessed a spherical and asymmetric branched structure, respectively. Stability studies
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revealed that nanoemulsions may be stable upon storage for 28 days at room temperature.
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Keywords: nanoemulsion; ultrasonication; sodium alginate; oleic acid; corn oil; stability.

1. Introduction
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Nanoemulsions, also referred to as mini-emulsions, are described as colloidal

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dispersions produced from water, oil, and surfactants [1]. They have been widely utilized

in biological, food, and medical applications [1] since they can act as carriers or delivery

systems for bioactive ingredients [2], lipophilic compounds in functional foods [3],

cosmetics, pharmaceutical products [4], and nanoparticles dispersed in organic solvents

[5].
 4

Generally, nanoemulsions can be fabricated using mechanical (high energy) or

chemical (low energy) treatments. The high energy method is widely used in the

production of nanoemulsions and it depends on intense disruptive forces to break the

water/oil phase into small droplets to obtain a thermodynamically stable nanoemulsion

[6]. An ultrasonic homogenizer is often used to improve the emulsification process [7, 8]

since it produces acoustic waves of higher intensity (low frequency), resulting in droplet

collapse due to cavitation effects, yielding droplets with smaller size [6]. Usually, before

application of ultrasonic waves, a coarse emulsion may be obtained using high speed

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mechanical agitators, which can also create cavitation conditions [9]. However,

ultrasound is more efficient in terms of energy consumption for obtaining emulsions with

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reduced drop size compared to the mechanical process [10]. In addition, studies have
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observed that ultrasound-assisted emulsification can be used for the preparation of stable

nanoemulsions with lower droplet size and polydispersity [7, 8, 9, 10].

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Nanoemulsions can be obtained in oil-in-water (O/W) or water-in-oil (W/O)
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systems. However, different oils can bring considerable changes in the emulsification

process. Several oils have been utilized to formulate nanoemulsions which vary in fatty

acid composition. Corn oil (CO) is composed of long chain triglycerides including
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unsaturated fatty acids, mainly ω-6 linoleic acid and ω-9 oleic acid, and saturated fatty

acids, such as palmitic acid, stearic acid, and minor quantities of other fatty acids [11].
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Unrefined (UCO) and refined (RCO) coconut oil are composed of medium chain
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triglyceride (lauric and myristic) saturated fatty acids [11]. Oleic acid (OA) is a long-

chain monounsaturated triglyceride (ω-9 oleic acid) [11] approved as a penetration

enhancer by the United States Food and Drug Administration, and utilized in formulating

nanoemulsions [12], similar to olive oil (OO) which consists of unsaturated fats, mainly

OA [11].
 5

To obtain nanoemulsions it is necessary to use a surfactant capable of adsorbing

at the oil-water interface of droplets, providing a flexible film around the droplets and

reducing surface tension [13]. Polysorbates, such as tween 80, are among the most widely

used surfactants for emulsification [14, 15, 16]. Tween 80 is a high molecular weight

emulsifier [4], and has a hydrophilic head group consisting of polyethoxylated sorbitan

and a lipophilic tail group consisting of OA [17]. The balance of hydrophilic and

lipophilic groups may be described as hydrophilic-lipophilic balance (HLB). High HLB

values (8-18) are preferred for the formation of O/W emulsions since the surfactant is

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more hydrophilic in nature [4]. The HLB value of tween 80 is relatively high at 15 [18],

making it suitable for O/W emulsions. It has been used due to its ability to produce steric

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stability, since it might keep droplets apart by means of steric hindrance [4].
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Polysaccharides such as sodium alginate (SA), an anionic polysaccharide found

in the cell wall of brown algae, may be added in the aqueous phase of nanoemulsions
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[19]. The addition of SA to the aqueous phase may improve the stability of nanoemulsions
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against aggregation since these molecules can cause steric and/or electrostatic repulsion

between droplet interfaces [13], preventing the destabilization caused by droplet

coalescence or gravitational separation [13, 15, 20]. The functional groups in SA, such as
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carboxylates, readily dissociate in the aqueous phase and impart negative charge to

emulsions [13]. Additionally, the interaction of tween 80 and SA influences the surface
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charge of lipid droplets [15]. However, the addition of SA in the aqueous phase must be
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carefully controlled since it may have either a positive or negative effect on the physical

stability of the nanoemulsions [3].

Some important parameters of interest in preparing nanoemulsions include droplet

size, polydispersity index (PDI), zeta potential, and rheological behavior. The size and

polydispersity of nanoemulsions strongly influences stability, rheology, and shelf life of

 6

the formulation [14]. The zeta potential and viscosity of the nanoemulsion is strongly

affected by the presence of SA in the continuous phase due to its anionic nature and

texturization properties [15]. The characteristics of the dispersed phase have significant

impact on the rheological properties of nanoemulsions [16]. Rheological characterization

of emulsions facilitates understanding of structural organization and interactions between

components [21], which can be obtained using conventional macro scale instruments,

such as controlled shear or controlled strain rate rheometers [22].

Considering that the composition of the dispersed and continuous phases and the

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type of mechanical treatment applied influences the physical properties of

nanoemulsions, the objective of this work was to study the effect of SA concentration in

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the aqueous phase and the use of different oils on the physical properties of
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nanoemulsions obtained by ultrasonication. Nanoemulsions with superior physical

qualities were pursued for further physiochemical analysis, which included stability
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studies, droplet morphology, and Fourier-transform infrared spectroscopy (FTIR)
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spectroscopy.

2. Materials and methods

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2.1 Materials
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Corn, extra virgin olive, unrefined and refined coconut oils were purchased at
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local stores. Oleic acid was purchased from Sigma-Aldrich Co. Ltd. (USA) and tween 80

was obtained from Fisher Chemical (USA). Ultrapure water obtained from a Milli-Q

filtration system was used for the preparation of all samples and solutions.
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2.2 Methods

2.2.1 Nanoemulsion preparation

The components of the nanoemulsions prepared here include the oil phase, the

aqueous phase and the non-ionic surfactant. Aqueous phases were prepared by dissolving

varying concentrations of SA (0.5, 1.0 and 1.5% w/w) in ultrapure water at 25°C. Briefly,

the oil phase (1% w/w of different oils) and tween 80 (1% w/w) were added to the aqueous

phase containing SA (98% w/w) using a high speed homogenizer (PowerGen 125, Fisher

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Scientific, USA) with a rotor diameter of 7 mm, at 15000 rpm for 5 min to produce a

coarse emulsion. The nanoemulsions were obtained using a Q750 ultrasound sonicator

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(750 W full power, 20 kHz frequency, Qsonica, USA) for 5 min with a power amplitude
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of 40%. This instrument displays the power (W) and the total amount of energy delivered

to the system during the process. An ultrasound probe (6 mm diameter) was used to treat
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10 mL of coarse emulsion contained in a glass tube. The microtip was placed at the same
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distance from the base of the liquid level for all ultrasound treatments.

2.2.2 Characterization of nanoemulsions

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2.2.2.1 Droplet size, PDI, and zeta potential

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The hydrodynamic size, PDI, and zeta potential were determined by photon
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correlation spectroscopy using a Brookhaven Instruments Zeta PALS/BI-MAS dynamic

light scattering (DLS) system. In all analyses, the samples were diluted with ultrapure

water at a dilution ratio of 1:99 sample-to-solvent to minimize multiple scattering effects.

The PDI is an important parameter to determine the size homogeneity of nanoemulsion

globules. A PDI value close to zero is desirable since it represents a homogenously

 8

dispersed sample, whereas higher PDI values, tending towards 1, indicate non-uniform

globule size distribution [23]. The zeta potential (mV) is a measure of the surface charge

of the oil droplets dispersed in the aqueous phase [15, 16, 17, 24]. The zeta potential of

coarse and nano- emulsions was determined using a capillary cell that measures

electrophoretic mobility. The surface charge of the droplets was measured over 10 runs,

in duplicate, and the data are reported as the mean of three samples.

2.2.2.2 pH and electrical conductivity

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The conductivities of the nanoemulsions were measured using a conductivity

meter (CON 11/110 Conductivity/TDS, Thermo Fisher Scientific, USA). The pH of the

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nanoemulsions was recorded using a pH meter (Orion Star A111, Thermo Fisher

Scientific, USA) at 25ºC.

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2.2.2.3 Flow behavior
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Rheological measurements of coarse emulsions, nanoemulsions, SA solutions and

different oils were performed at 25ºC using an Anton Paar MCR 302 rheometer equipped

with a DG26.7 double-gap concentric cylinder geometry. For each measurement,

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approximately 10 mL of the sample was carefully placed inside an external cylinder. All

samples were allowed to rest for 3 min after loading to allow temperature equilibration.
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Flow curve measurements were carried out in the range of 1 to 100 s-1 for 5 min.
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Rheological data (shear rate, shear stress, and viscosity) of samples were obtained from

the Rheoplus/32 software package and fitted to the Ostwald-de Waele function, to obtain

rheological parameters, or to a linear function, to calculate the shear viscosity [24].

 9

2.2.2.4 Stability studies

The nanoemulsions were stored at room temperature ( 25ºC) and their stability

was evaluated by monitoring the droplet size and PDI (item 2.2.2.1) for a storage period

of 28 days.

2.2.2.5 FTIR spectroscopy

To assess the presence of functional groups (tween 80, CO, OA and SA solution

0.5%) in coarse emulsions and nanoemulsions, samples were characterized by using an

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infrared spectrometer (Frontier FTIR spectrometer, PerkinElmer, USA) in the range of

4000 to 400 cm-1 at a resolution of 0.5 cm−1.

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2.2.2.6 Transmission electron microscopy (TEM)
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The morphology of CO and OA nanoemulsions was determined by using TEM
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(Hitachi, model H-7600, Japan) at an accelerating voltage of 80 kV. Samples were diluted
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appropriately, carefully instilled on a carbon coated copper grid and stained with 2%

(w/v) ammonium molybdate (pH 7.4) as previously described by Banerjee et al. [25]. The

sample was dried before capturing the TEM images.

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2.3. Statistical analysis

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Data were analyzed statistically by one-way analysis of variance, followed by the

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Tukey’s test. Differences between means were considered significant at a p-value ˂ 0.05

(95% confidence level). Results were reported as mean ± standard deviation.

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3. Results and discussion

3.1 Effect of SA concentration and type of oil on the droplet size and PDI of

nanoemulsions

The concentration of SA has a positive correlation with droplet size (Fig. 1).

Increasing the SA concentration from 0.5 to 1.5% resulted in an increase in globule size

for nanoemulsions, except for those made with UCO. Previous studies have reported

apparently conflicting results on the influence of concentration of SA added to the

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emulsion on the droplet size. Hosseini et al. [26] verified higher droplets with

polysaccharide concentration increase while Salvia Trujillo et al. [3] observed that

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increasing the SA concentration from 0.5 to 1.0% resulted in a reduction in the droplet
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size. The effect was attributed to depletion, flocculation, and coalescence of droplets at a

lower concentration, while at a higher concentration of alginate, the polysaccharide

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protects the droplets from coalescing. These results are contrary to those obtained in the
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present study, and this may be due to differences in the method of synthesis of the

nanoemulsion, as well as the type of oil base used, which have specific differences in

molecular weight, polarity, conformation, and adsorptive properties of the surfactant [16].
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Nanoemulsions prepared at higher SA concentrations had the smallest droplet sizes when

using CO. Smaller and close droplet size values were obtained using 0.5% SA solution in
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the aqueous phase with CO and OA. The differences in the droplet size between various
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oils may be attributed to their molecular structure and interfacial tension or surfactant

affinity [16]. The smallest size droplets can be produced when the oil phase has low

viscosity and/or interfacial tension as this facilitates droplet disruption by mechanical

processes [1]. OA has similar surface tension values [27] and lower viscosity (Table 1)

than the others oils studied, with the exception of RCO. With regard to interfacial tension,
 11

it has been shown that OA and linoleic acid, a major component in CO [11], could

concentrate at the oil-water interface, competing with other surfactants and further

decreasing this property [28]. In addition, the geometric structure of triglyceride chains

have an effect on droplet size. Short chain triacylglycerols form the biggest droplets as a

consequence of Ostwald ripening effects, which is favored because of their high water

solubility. In contrast, oils with a higher molecular mass have reduced water solubility,

inhibiting Ostwald ripening and providing emulsion stability [29]. The bigger droplet size

was verified with RCO and UCO, since they are rich in saturated medium chain fatty

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acids. In contrast, in the case of OO, CO and OA, which have long chain triglycerides,

OA and CO nanoemulsions yielded reduced droplet sizes, which could be due to

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differences in OA content and saturated fatty acids content [11]. These results indicate a
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clear influence of SA concentration and the type of oil used on the nanoemulsion globule

size. The PDI value for the nanoemulsions varied with the nature of the oil used and with
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the concentration of SA, in the range of 0.17 (obtained for OA; 0.5%) to 0.29 (obtained
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for OO; 1.5%). In general, OA nanoemulsions had lower PDI values compared to the

other oils used. These observations are in line with previous studies which reported the

influence of the type of oil on droplet size, while little effect was observed on the
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polydispersity of the emulsions [8, 30, 31]. Formulations with larger droplet sizes and

PDI values greater than <0.2 were obtained with 1.5% SA, regardless of the oil studied,
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and hence this concentration was excluded from subsequent studies.

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3.2 Effect of the type of oil on the zeta potential of nanoemulsions

Fig. 2 shows the surface charge of oil droplets in the nanoemulsions. The zeta

potential value is a measure of the surface charge of the droplet, and in an electrostatically

stabilized nanoemulsion the zeta potential should have a value lower than -30 mV or

greater than +30 mv [15, 16]. All nanoemulsions exhibited zeta potential values below
 12

−30 mV, except RCO (0.5% SA), which suggests electrostatic stability. These results

indicate that the presence of SA, with carboxylate (−CO2) and hydroxyl (−OH) groups,

played a role in the electrostatic stabilization of nanoemulsions, as suggested previously

[13]. The zeta potential values obtained for the nanoemulsion with 0.5% alginate is

appreciably higher than reported by Salvia-Trujillo et al. [3] for a nanoemulsion with the

same alginate concentration. In general, an increase in SA concentration led to a more

negative zeta potential, indicating that a higher SA concentration in the continuous phase

imparted a higher negative surface charge to the lipid droplets. The adsorbtion of SA

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around the surface of the droplet may have increased the thickness of the electric double

layer and, consequently, may have increased the negative charge and size of the droplet.

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Differences observed in the zeta potential with different oils might be due to differences
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between adsorption of the surface-active compounds at the oil-water interface [16]. In

addition, the degree of unsaturation and fatty acid composition varies with oil type, which
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may have an effect on zeta potential. OA had a greater contribution than linoleic in
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decreasing the droplet surface charge, possibly due to differences in geometric shape

which is more linear for OA, allowing easier access to the droplet surface [28]. OA is

composed of pure oleic acid whereas CO contains, mainly, linoleic acid and oleic acid.
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This explains the higher zeta potential of OA with respect to CO nanoemulsions.

However, a previous study showed little effect of oil type on zeta potential [8]. Adequate
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zeta potential values were obtained with OA or CO nanoemulsions containing 0.5% SA

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in the aqueous phase, precisely the samples that had the lowest droplet sizes and PDI

values. Since the OA and CO nanoemulsions had the best quality parameters in this study

(low droplet size, PDI, and zeta potential), they were selected for stability testing and for

estimating the effects of ultrasound.

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3.3 Effect of ultrasound on CO and OA emulsions

3.3.1 Droplet size, PDI, zeta potential, pH and conductivity

The effect of ultrasonication on coarse emulsions was evaluated by analysis of

droplet size, PDI, zeta potential, pH, and conductivity, post ultrasound. Sonication

significantly reduced the droplet size of nanoemulsions and promoted the formation of

smaller and uniform droplets. OA showed a higher reduction of droplet size, with

significant difference compared to CO nanoemulsions. Regarding PDI, previous studies

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have shown that the ultrasound process forms monodispersed emulsions [8, 9, 10].

However, OA showed a significant reduction of PDI after ultrasonication, whereas no

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significant effect was observed with CO. These results suggest that ultrasonication
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contributed more to reduction of droplet size and PDI with OA emulsions than with CO.

This may be attributed to the power applied under the OA and CO emulsions, which
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resulted in average values of 23 W and 21 W, respectively, as given by the ultrasound
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equipment, corresponding to the power density of 2.3 W/mL and 2.1 W/mL, respectively.

The use of the high-speed homogenizer in the formation of the coarse emulsion of OA

resulted in less breakdown of the globules compared to CO, which may be due to the
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slightly higher viscosity of the OA system. In the synthesis of coarse emulsions, high

dissipation rates are generated with a rotor stator mixer, which combines the high speed
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of rotation of the rotor and the small gap between the rotor and the surrounding stator
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[32]. Drop breakup mechanisms in this type of rotor-stator mixer may be due to the

"guillotine" effect which droplets suffer when passing through the rotor-stator [33] or due

to cavitation generated by rotation of the rotor in a fluid and its impact against the stator

[9]. However, the breakdown forces applied by the high-speed homogenizer in this study

was not sufficient to reduce to nano scale, as verified by the larger globule size. In
 14

addition, higher rotation speeds may lead to the formation of super cavitation, resulting

in a collapse of cavities with reduced intensities, which affects the overall cavitation

intensity [9].

Coarse emulsions exhibited strong surface charge, which was more negative than

in corresponding nanoemulsions. The zeta potential values of nanoemulsions were

significantly different, whereas there were no significant differences between coarse

emulsions. Similar behavior was reported by Hosseini et al. [26], who observed that the

zeta potential values of SA (0.1% w/w) with -lactoglobulin increased from -59.89 to -

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49.08 mV after ultrasonication. The authors attributed this phenomenon to the reduction

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of alginate reactivity and structural changes after the sonication process. SA pretreated

with ultrasound could probably undergo sono-chemical reactions including conjugation,

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oxidation, C-D, C-heteroatom, and C-C bond formations [34] which may lead to the

elimination of some of its negatively charged groups [26]. However, some previous
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studies [13, 14] have reported a decrease in zeta potential for nanoemulsions when
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compared to coarse emulsions. These results may be due to differences in the

methodological aspects of the coarse emulsion and nanoemulsion syntheses. The

nanoemulsions of this study showed zeta potential values below -30 mV, indicating that
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all have good stability. The OA nanoemulsion had a zeta potential value more negative

than the CO nanoemulsion, which may be attributed to slight triglyceride hydrolysis

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during sonication, resulting in an increased negative charge of the droplets [5].

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The system pH is an important parameter as it influences the physical stability of

multilayer emulsions [35]. OA and CO nanoemulsions were found to be in a neutral pH

range between 5.95 to 6.98. The pH of the CO nanoemulsion was not significantly

different from its coarse emulsion or the 0.5% SA solution. The OA nanoemulsion had

the lowest pH with no significant difference (p ˂ 0.05) from the respective coarse
 15

emulsion; however, it was statistically different to the SA solution. The lower pH values

found for OA nanoemulsions can probably be attributed to the ionization potential of the

carboxyl group of OA in pure water [36].

Conductivity is the ability of a material to conduct an electric current and, for

emulsions, it depends on the water and oil contents [37]. Higher conductivity values have

been observed when the water content is greater than the oil content in emulsions, as in

O/W emulsions, since water is a much better electrical conductor than oil [38]. So, the

type of emulsion can be identified or confirmed by measuring the conductivity [38].

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Regardless of the oil studied here, there was no significant difference in conductivity

between coarse emulsion and the nanoemulsion. The only significant difference was

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observed with respect to the starting solution of 0.5% SA, which suggests that the
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polysaccharide had the greatest contribution to conductivity. Watthanaphanit and Saito

[39] reported that increasing the SA concentration leads to increased conductivity of the
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SA solution.
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3.3.2 Rheological properties

Viscosity and concentration of the dispersed phase are important factors which
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influence the formation of nano-sized emulsions. Flow curves for SA solutions followed

the Ostwald-de Waele model (Fig. 3, Table 1). SA solutions showed shear thinning
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behavior, mainly in the presence of high levels of polysaccharide (1.5%), as evidenced

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by the flow behavior index values. The apparent viscosity increased with a high

polysaccharide concentration [3, 15, 39] and decreased with increasing shear rate [3, 24].

All oils studied exhibited Newtonian behavior (Table 1).

Fig. 3 presents flow curves for OA and CO emulsions, and their corresponding

nanoemulsions. OA (K = 60.15 mPa.sn; n = 0.92) and CO (K = 54.63 mPa.sn; n = 0.93)

 16

coarse emulsions behaved like a near-Newtonian fluid, without significant difference

between the oils studied, and they were accurately described by the Power Law model.

Nanoemulsions showed Newtonian behavior with no statistically significant differences

between them. Low viscosity values were exhibited by other nanoemulsion rheological

studies [8, 40]. The apparent viscosity of the coarse emulsions at a shear rate of 75 s-1 was

considerably higher than for the corresponding nanoemulsions (Table 2).

The ultrasonication process can cause chemical and physical degradation of the

alginate, due to the shear stress, micro-streaming and turbulence force generated by

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cavitation microbubbles, resulting in a decrease in molecular weight of the polysaccharide

chains [26, 34, 41] as well as a decrease in thickening properties and viscosity of the

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corresponding nanoemulsions [14]. The untreated SA solution exhibited pseudoplastic
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behavior while alginate molecules sonicated at 50 kHz had near Newtonian behavior [41],

indicating that ultrasonication can affect the rheological properties of alginate. The
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reduction of emulsion viscosity, as a consequence of the ultrasonic power applied, may
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enhance the deformation rate of droplets due to cavitational intensity and improve droplet

size and the PDI of the nanoemulsions [40].

Although the viscosity of CO is higher than pure OA (Table 1), the viscosity of
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the OA coarse emulsion and nanoemulsion were slightly higher than those formulated

with CO, which may be in relation to the pH of the systems, since reports indicate that
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SA solution viscosity increases with decreasing pH [42].

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3.3.3 Stability Studies

Nanoemulsions are thermodynamically unstable systems and their kinetic stability

during storage is affected by collisions, aggregation, or growth of the droplets [1]. Thus,
 17

the storage stability of OA and CO nanoemulsions were monitored for 28 days by analysis

of droplet size and PDI.

OA nanoemulsion results showed lower PDI and droplet size values than CO

nanoemulsions. PDI values were below 0.25, indicating that OA and CO based

nanoemulsions are uniform and show good stability, since PDI values lower than 0.25

indicate a close size distribution which provides good physical stability of nanoemulsions

due to reduced Ostwald ripening [23]. The low values of PDI may be due to the viscosity

of the continuous phase, which reduced the mobility of the oil droplets, delaying system

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instability phenomena, and as a consequence, reducing the heterogeneity in particle size

[43].

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The underlying phenomena causing nanoemulsion destabilization include
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aggregation, coalescence, and Ostwald ripening, which happens in polydispersed

emulsions, whereby larger droplets grow at the expense of smaller droplets due to
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molecular diffusion of the oil through the continuous phase [1]. Ostwald ripening may be
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delayed if the oil phase is insoluble in the aqueous phase. As OA and CO nanoemulsions

showed low PDI and both oils are long-chain fatty acids that are virtually insoluble in

water, the main destabilization mechanisms of nanoemulsions may have been

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flocculation and coalescence [5].

Droplet size is one of the most important factors affecting the kinetic stability of
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emulsions [37]. The droplet sizes of the OA nanoemulsions were significantly lower than
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for the CO nanoemulsions, at the different storage times (Table 3). The stability of

nanoemulsions as well as the droplet size is directly related to the emulsifier's HLB value

with respect to that of oil (rHLB) [18]. OA has a rHLB value of 11 [44], that is closer to

the HLB value of tween 80 surfactant [15] in comparison to CO (rHLB 9) [45]. Hence

OA has a higher probability of forming smaller globules but a stable emulsion. Regardless
 18

of the type of oil studied, there was a significant increase in droplet size in the first three

days of storage and, after this period, no significant variation in droplet size was observed

over a period of 19 days.

There was a significant decrease in CO droplet size after 19 days of storage, whilst

no variation was observed in OA droplet size. Studies have shown that OA may have

considerable stabilization effects against droplet coalescence because of accumulation of

negatively charged carboxylic groups at the interface, favoring the use of OA as a co-

surfactant in lipid nanoemulsion systems [40]. Another study reported that OA and

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derivatives have higher stability in conjunction with a non-ionic emulsifier because OA

is highly unsaturated and would require a more hydrophilic non-ionic emulsifier [46].

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Non-ionic surfactants, such as tween 80, may produce charged droplets due to the
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presence of H3O+ and OH− ions, depending on the pH of the system, since a negative

charge has been observed for nanoemulsions in acidic pH (pH 3–6) whereas a positive
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charge has been observed for nanoemulsions in basic pH (pH more than 7) [4]. According

to results obtained in this study, OA nanoemulsions (pH  6) were more acidic, with
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negative charge on the droplets, compared with CO nanoemulsions (pH  7) which

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exhibited a more basic nature, with this behavior verified for 0.5% SA solution. However,

nanoemulsions with pH in the range of 6-9 have been reported to be stable [23], which

was also verified in this study (Table 2). In addition, the microbiological stability of the
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nanoemulsion improves at lower pH [47], since bacterial growth is considerably inhibited

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in acidic conditions (visual observation), suggesting that OA nanoemulsions are less

prone to degradation than CO nanoemulsions. Another visible change in the OA

nanoemulsions was the formation of a thin opaque layer at the top of the glass tubes after

28 days of storage, indicating that the nanoemulsion stability depends on storage time, as

reported by Rao and McClements [17].

 19

3.3.4 FTIR

FTIR spectroscopy was used for characterization of interactions between

nanoemulsion components at the molecular level. FTIR spectra and typical peaks of SA

0.5%, CO, OA, tween 80, coarse emulsions and nanoemulsions are shown in Fig. 4. FTIR

spectral similarities were observed between SA solutions and coarse and nano- emulsions.

CO and, more prominently, OA nanoemulsions showed a slight decrease of the -OH

(3303 cm-1, asymmetric stretch, H-bonded) when compared with SA 0.5% indicating the

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presence of water in the emulsions. Another major peak was verified at 1642 cm-1 which

corresponds to that of asymmetric vibrations of the carboxylate groups of the

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mannuronate and guluronate moieties in alginate present in the form of the sodium salt
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[19]. Coarse and nano- emulsions spectra showed the appearance of small peaks between

2939 and 2860 cm-1, which are close and correspond to tween 80 (2923 - 2860 cm-1) [2]
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and CO (2925 - 2854 cm-1) [48], and OA (2932 – 2859 cm-1) [49]. These results indicate
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that both oils, as well as the tween 80, remained within the biopolymer structure after

encapsulation. The remaining peaks observed with OA and CO coarse emulsions and

their respective nanoemulsions are the same, indicating that no changes occurred during
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emulsification.
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3.3.5. Morphology of OA and CO nanoemulsions

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TEM is a useful technique to complement information obtained by DLS

measurements. It provides resolution to visualize nanosized structures [20] and has been

applied to study the morphology of nanoemulsions [5, 20]. TEM images of CO and OA

nanoemulsions (Fig. 5) revealed differences in the shape and disposition of SA around

the droplets. The CO nanoemulsion droplets exhibited a spherical shape, as also reported
 20

previously in the case of microfluidized lemongrass oil-alginate nanoemulsions [20].

These nanoemulsions presented a spherical shape with two concentric layers, which may

be the surfactant being overlaid by a layer SA (Fig. 5A). The presence of the two layers

adsorbed on the surface of the oil droplets could be clearly visualized, which may have

contributed to the increase in CO droplet size, since the droplet hydrodynamic radius is

the sum of the radius of the oil core and the thickness of the interface [1]. The OA

nanoemulsions exhibited an asymmetric shape (Fig. 5B), showing a grafted brush OA

layer. This behavior was also verified by Jung et al. [50], who reported dendritic structures

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for OA emulsions. The influence of pH on the OA nanoemulsion (Table 2) is an important

factor to take into account, as reported by Ferreira et al. [51] who verified that sonicated

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dispersions of OA were predominantly spherical at pH 8.0, elongated or tubular at pH
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7.4, and aggregated and difficult, or impossible, to visualize, at pH values between 5.0

and 6.5, demonstrating that the protonation state of the fatty acid governs the type of
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microstructures formed. In another study, it was verified that the disposition of alginate
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around the oil droplets is due to the breaking of biopolymer chains after microfluidization,

which was dependent on the procedure used for their incorporation during the preparation

of the nanoemulsion [13]. However, as the same process (sonication) was used in both
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studied oils, the results indicate that the type of oil used is a major determinant of the

droplet morphology. Besides, it has been verified that the sonication process had no
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structural effect on lipids of turmeric oil [10] or rapeseed nanoemulsions [52]. The
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presence of SA on oil droplets is also confirmed by the negative charge (zeta potential)

of nanoemulsions (Table 2).

4. Conclusions
 21

Ultrasound-assisted nanoemulsions containing OA and CO were successfully

formulated using 0.5% SA in the aqueous phase. The nanoemulsions obtained showed

uniform size, low droplet size, low and negative value of zeta potential, and Newtonian

rheological behavior, with adequate stability over a period of 28 days at 25°C. CO and

OA nanoemulsions showed a spherical and asymmetric shape, respectively. This study

showed that the application of sonication to an oil-in-water and polysaccharide system

can be a feasible method to obtain a nanoemulsion with satisfactory physiochemical

properties, thus providing a platform for designing nanoemulsions with potential

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applications as a delivery system for nutraceuticals, drug and/or nanoparticles synthesized

in organic solvents.

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Ivanise Guilherme Branco: Conception and design of study, acquisition and analysis of
data, drafting of article and critical revision, final approval of manuscript.
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Kacoli Sen: Acquisition and analysis of data, drafting of article and critical revision, final
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approval of manuscript.

Carlos Rinaldi: Conception and design of study, drafting of article and critical revision,
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final approval of manuscript.

Conflict of interest
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There are no conflicts of interest regarding this paper.

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Acknowledgements

Ivanise G. Branco is grateful to all members of Prof Carlos Rinaldi's laboratory for being

supportive in all endeavors and to Department of Chemical Engineering and J. Crayton

Pruitt Family Department of Biomedical Engineering at the University of Florida

Gainesville for infrastructural and instrumental support to conduct this study.

 22

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FIGURES

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(a) (b)
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Fig. 1. Comparison of droplets size (nm) (a) and polydispersity (PDI) (b) of
nanoemulsions obtained with different types of oil and sodium alginate concentrations.
UCO: Unrefined coconut oil; RCO: Refined coconut oil; CO: Corn oil; OA: Oleic acid;
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OO: Olive oil.

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Fig. 2. Zeta potential of nanoemulsions formulated with different types of oil and sodium
alginate concentrations. UCO: Unrefined coconut oil; RCO: Refined coconut oil; CO:
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Corn oil; OA: Oleic acid and OO: Olive oil.
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OA coarse emulsion
40 Sodium alginate 0.5% 4 CO coarse emulsion
Sodium alginate 1.0% OA nanoemulsion
Sodium alginate 1.5% CO nanoemulsion
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30 3
Shear stress (Pa)
Shear stress (Pa)

20 2
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1
10
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0
0
0 20 40 60 80 100
0 20 40 60 80 100
Shear rate (1/s)
Shear rate (1/s)

(a) (b)

Fig. 3. Flow curves of (a) SA, and (b) OA and CO coarse emulsions and its respective
nanoemulsions.
 31

0.4
0.3

IR Abs
(a) 0.2 CO Coarse emulsion
0.1 CO Nanoemulsion
Sodium alginate
0.0 Water

4000 3500 3000 2500 2000 1500 1000 500

0.4
0.3
IR Abs

(b) 0.2 OA Coarse emulsion

0.1 OA Nanoemulsion
Sodium alginate
0.0 Water

0.5 4000 3500 3000 2500 2000 1500 1000 500

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0.4
(c) 0.3
IR Abs

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0.2 Corn oil
Oleic acid
0.1
Tween 80
0.0 Sodium alginate

4000 3500 3000 2500 -p2000

Wavelength (cm )
-1
1500 1000 500
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Fig. 4. FTIR spectra of CO (a) and OA (b) coarse emulsions, and its respective
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nanoemulsions, 0.5% SA, and water; (c) CO and OA pure oils, 0.5% SA, and tween 80
at 4000 - 400 cm−1 region.
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(a) (b)
Fig. 5. Transmission electron microscopy (TEM) images of CO (a) and OA (b)
nanoemulsions. -p
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 33

TABLES

Table 1
Flow properties of oils and sodium alginate solution. Viscosity (), consistency
coefficient (K), flow behavior index (n) and R2 correlation coefficient.
Type of oils  (m Pa.s) R2 K (m Pa.sn) n R2
Olive oil (OO) 64.7 1.0
Corn oil (CO) 54.2 1.0
Unrefined Coconut oil (UCO) 46.4 0.99
Oleic acid (OA) 31.1 0.99
Refined coconut oil (RCO) 24.7 0.99
0.5% Sodium alginate 55.6 0.93 0.99
1.0% Sodium alginate 319.1 0.85 0.99

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1.5% Sodium alginate 1106.0 0.78 0.99

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Table 2
Droplet size (nm), polydispersity index (PDI), viscosity (m Pa.s), apparent viscosity at 75 s-1,

emulsions and their respective nanoemulsions.

Droplet size PDI
-p
zeta potential (mV), pH, and conductivity (S) of OA (oleic acid) and CO (corn oil) coarse

Viscosity Zeta pH Conductivity

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(nm) (mPa.s) potential (S)
(mV)
OA Coarse emulsion 8065.9 ± 396.7a 0.404 ± 0.10a 42.65 ± 2.6a -59.83 ±4.1a 5.95 ± 978.0 ± 24a
0.11b
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CO Coarse emulsion 2813.3 ± 735.1b 0.275 ± 41.62 ± 1.9a -49.10 ±5.1a 6.74 ± 981.0 ± 27a
0.03b 0.12a
OA nanoemulsion 155.7 ± 0.3c 0.172 ± 0.01c 8.47 ± 0.5b -40.29 ±2.6b 5.98 ± 986.7 ± 28a
0.01b
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CO nanoemulsion 179.4 ± 7.9d 0.248 ± 7.66 ± 0.2b -32.99 ±4.8c 6.98 ± 944.3 ± 15a
0.01b 0.12a
0.5% Sodium 6.98 ± 1054.0 ± 24b
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alginate 0.11a

Values are averages of two experiments, with each individual sample analyzed in triplicate. Means in
same column with different letters are significantly different at p ˂ 0.05.
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Table 3
Droplet size (nm) and polydispersity index (PDI) during 19 days for oleic acid (OA) and corn
oil (CO) nanoemulsions.
Droplets size (nm) PDI
Days OA CO OA CO
aA aA aA
0 147.0 ± 6.6 176.0 ± 7.8 0.18 ± 0.004 0.25 ± 0.011aB
3 169.4 ± 10.4bA 235.3 ± 17.3bcB 0.17 ± 0.01abA 0.22 ± 0.015bB
7 165.9 ± 7.5bA 236.2 ± 12.7bcB 0.16 ± 0.01bA 0.21 ± 0.014bB
 34

12 166.6 ± 21.8bA 240.0 ± 13.6bcB 0.18 ± 0.05aA 0.23 ± 0.030abB

19 165.0 ± 4.3bA 289.4 ± 11.6bC 0.18 ± 0.01aA 0.24 ± 0.011acB
28 174.9 ± 11.0bA 223.5 ± 19.8cB 0.19 ± 0.01aA 0.24 ± 0.016acB
Values are averages of two experiments, with each individual sample analyzed in triplicate.
a,b
Means in same column with different letters are significantly different at p ˂ 0.05.
A,B
Means in same line with different letters are significantly different at p ˂ 0.05.

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