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Received: 17 August 2020    Revised: 14 December 2020    Accepted: 16 December 2020

DOI: 10.1111/jfpp.15181

ORIGINAL ARTICLE

Effect of the use of ethanol and chia mucilage on the

obtainment and techno-functional properties of chia oil
nanoemulsions

Sibele Santos Fernandes1  | Juan Carlos Cuevas Bernardino2  |

Patricia Quintana Owen3  | Carlos Prentice1  |
Myriam de las Mercedes Salas-Mellado1  | Maira Rubi Segura-Campos4

1
Laboratory of Food Technology, School of
Chemistry and Food, Federal University of
Rio Grande, Rio Grande, Brazil
2
CONACYT−Centro de Investigación y
Asistencia en Tecnología y Diseño del
Estado de Jalisco, Mérida, Mexico
3
Laboratory of National Nano and
Biomaterials, CINVESTAV Unidad Mérida,
Mérida, Mexico
4
Laboratory of Food Science, Faculty
of Chemical Engineering, Autonomous
University of Yucatán, Mérida, Mexico
Correspondence
Sibele Santos Fernandes, Laboratory of
Food Technology, School of Chemistry and
Food, Federal University of Rio Grande,
Av Italy km 8, Carreiros, 96203-900 Rio
Grande, Brazil.
Funding information
Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior, Grant/Award
Number: Finance 001
Abstract
Chia oil contains a high content of linoleic and linolenic acid, which are essential for
human metabolism. Chia nanoemulsion is a technique that can aid in preserving the
components of this oil. This study aimed to develop and characterize chia oil nanoe-
mulsions using chia mucilage as wall material. The nanoemulsions were developed
varying the use of ethanol to check for any influence in the stability and the content
of chia oil (0.043, 0.100, and 0.233 ml) and chia mucilage (0.125, 0.250, and 0.375 g)
through high-speed stirrer. The smallest particle size, polydispersity index, zeta po-
tential suitable, high encapsulation efficiency, and stability of agitation were the pa-
rameters for choosing treatments with (T4E) and without (T4) ethanol with 0.375 g
of chia mucilage and 0.233 ml of chia oil. The chosen nanoemulsions were character-
ized, being verified that the encapsulation was efficient. Also, during the stability
evaluation, it was seen that the T4 presented smaller particle size and lower polydis-
persity index. The rheological parameters indicated that all nanoemulsions presented
shear-thinning and semi-liquid viscoelastic behaviors. The nanoemulsions produced
can be used in foods, because they facilitate the incorporation and preservation of
the chia oil in food formulations and making them more nutritious.
Novelty impact statement: The development of chia oil nanoemulsion was per-
formed using different concentrations of chia mucilage as an encapsulating material
and varying the use of ethanol as an aid to the formation of drops. Physicochemical,
morphological, stability, and technological characteristics were evaluated. Chia oil
nanoemulsions with the addition or not of ethanol can be applied to food preserving
chia oil from oxidation, in addition to facilitating the incorporation of oil into the food.

1 |  I NTRO D U C TI O N from cardiovascular disease (Ateya et al., 2017), neurodegenera-

Omega-3 fatty acids are an essential part of cell membrane
structure and are responsible for several physiological functions
(Bilinski et al., 2019). Therefore, they are associated with a vari-
ety of health benefits such as a significant reduction in mortality
tive disorders (Thomas et al., 2015), cancer (Aredes et al., 2019;
Fabian et al., 2015), and among other pathologies. Since essential
fatty acids (omega-3 and omega-6) are not synthesized by human
metabolism, there is a need for them to be ingested in the diet
(Mudgil, 2019). These polyunsaturated fatty acids are mainly

J Food Process Preserv. 2021;45:e15181. wileyonlinelibrary.com/journal/jfpp |

© 2020 Wiley Periodicals LLC.     1 of 16
https://doi.org/10.1111/jfpp.15181
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2 of 16       FERNANDES et al.
consumed from oils derived from fatty fish. However, a source
that has gained potential prominence is chia seeds (Fernandes
et al., 2019).
Chia (Salvia hispanica L.) is an herbaceous plant, cultivated an-
nually, and native to southern Mexico and northern Guatemala
(Capitani et al., 2012). Chia seed has about 28 to 32% of oil, which
is rich in polyunsaturated fatty acids, especially omega-3 (linolenic
acid, ~68%), greater than any known plant source, and omega-6 (lin-
oleic acid, ~19%) (Fernandes et al., 2019). Moreover, this oil presents
other valuable components such as tocopherols, polyphenols, phy-
tosterols, carotenoids, and phospholipids (Fernandes et al., 2019;
Julio et al., 2019). Therefore, chia oil is an important raw material for
obtaining enriched omega-3 food products (Castejón et al., 2019).
Although the chia oil fatty acid profile is nutritionally favorable, the
high degree of unsaturation makes them very susceptible to oxida-
tion (Ixtaina et al., 2015).
Oxidation leads to the formation of unpleasant tastes and
odors, reducing the shelf life of the product and promoting the
generation of free radicals, which can have negative physiologi-
cal effects on the body. Thus, the use of alternative technologies
such as encapsulation is necessary to slow down the oxidation
process while maintaining the stability and nutritional properties
of chia seed oil and making it possible to be incorporated into food
(Alcântara et al., 2019). Among the encapsulation techniques, the
nanoemulsion-based system stands out for its prospective applica-
tion in oil encapsulation to avoid oxidative degradation, in addition
to the ease of addition to food formulation (Meneses et al., 2019;
Sharma et al., 2019). Nanoemulsions have several advantages
compared to other encapsulation techniques such as good water
dispersibility, better (more translucent) optical properties, better
physical stability (against gravitational separation and particle ag-
gregation), and increased bioavailability of the lipophilic bioactive
compounds incorporated into them (Firoozi et al., 2020; Karthik
et al., 2017). The use of ethanol in nanoemulsions is a strategy
because it reduces the dielectric constant of the medium resulting
in a uniform formation of particles (Joshi et al., 2017). Besides,
ethanol is a GRAS solvent (generally recognized as safe), making
it a green process.
Many compounds can be used as wall material in encapsu-
lation; however, most use whey protein in nanoemulsion (Farshi
et al., 2019). Chia mucilage is a high viscosity anionic heteropoly-
saccharide, even at low concentration, with dietary fibers (72%)
being its major component (Akcicek & Karasu, 2018; Fernandes &
Salas-Mellado, 2017). The protein content of chia mucilage (10%) fa-
vors emulsifying properties, while the high carbohydrate (80%) and
fiber content favor encapsulation properties as it tends to form gels
(Campo et al., 2017; Fernandes & Salas-Mellado, 2017; Fernandes
et al., 2020). In addition, it has an adequate oil retention capacity,
which is advantageous for the retention of oil-based active com-
pounds (Segura-Campos et al., 2014). Because of this, chia muci-
lage is a promising polymer to be incorporated as a wall material. It
has already been used in various encapsulation techniques, either
individually (Campo et al., 2017; Stefani et al., 2019) or together with
chia protein isolate (Timilsena, Adhikari, Kasapis, et al., 2016) and
sodium caseinate, lactose, maltodextrin, and chia protein-rich frac-
tion (Us-Medina et al., 2018). Recently, Cuomo et al. (2020) obtained
promising results for protection action using chia mucilage in lemon-
grass essential oil emulsions.
Thus, combining the challenge of maintaining the properties of
chia seed oil and polyunsaturated fatty acids with the need for their
presence in the human diet, this study aimed to encapsulate chia oil
through nanoemulsions, evaluating the use of ethanol and chia mu-
cilage as a wall material, and to characterize the nanoemulsions with
the best techno-functional properties.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Material
Commercial chia seeds used for chia mucilage extraction were pur-
chased from a local market in Mérida, México. Chia oil was obtained
from pressing (Fernandes et al., 2019). All reagents used in the for-
mulation of nanoemulsions and chemical determinations were ana-
lytical grade.
2.2 | Obtaining chia mucilage
Chia mucilage was obtained according to Fernandes and Salas-
Mellado (2017) with modifications. The chia seeds and water were
placed, in the proportion of 1:40 (w:v), with agitation of 6,500 rpm
(IKA, T25 digital, México) for 1 hr and 30 min at room temperature.
After that, the mucilage formed was separated from the seed by
filtration. The filtrate was centrifuged (Beckman, GS-15R, USA) at
5,985 x g for 15 min. The supernatant (mucilage) was dried at 50°C
for 10 hr.
2.3 | Nanoemulsions preparation
Chia oil nanoemulsions were developed using chia mucilage as
wall material and Tween 20 (0.1  ml) as an emulsifying agent. In
addition, the use of ethanol was tested. The oil-in-water (O/W)
emulsions were made according to Campo et al. (2017), with
modifications. Firstly, the organic phase, formed by Tween 20,
chia oil and ethanol (if in treatment with ethanol), and the aque-
ous phase, formed by chia mucilage and water, were prepared.
For the development of nanoemulsions, the organic phase was
slowly added to the aqueous phase and was mixed for 15 min at
10,000 rpm, using the Ultraturrax homogenizer (IKA, T25 digital,
Mexico).
The experimental conditions of operation were determined
in factorial design 22 with three central points, varying the chia
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TA B L E 1   Formulation of encapsulation of chia oil by nanoemulsion

Ethanol (ml)
Chia mucilage Tween 20
Treatments X1 X2 (g) Chia oil (ml) (ml) Water (ml) Without With

1 −1 (0.5) −1 (30) 0.125 0.043 0.1 25 0 4

2 +1 (1.5) −1 (30) 0.375 0.043 0.1 25 0 4
3 −1 (0.5) +1 (70) 0.125 0.233 0.1 25 0 4
4 +1 (1.5) +1 (70) 0.375 0.233 0.1 25 0 4
5 0 (1.0) 0 (50) 0.250 0.100 0.1 25 0 4
6 0 (1.0) 0 (50) 0.250 0.100 0.1 25 0 4
7 0 (1.0) 0 (50) 0.250 0.100 0.1 25 0 4

Note: X1: coded value for chia mucilage variable; X 2: coded value for the chia oil variable.

mucilage concentration, from 0.5% to 1.5% (Antigo et al., 2020;
Cuomo et al., 2020), and the ratio of chia oil to Tween 20 surfactant
between 30% and 70% (vol/vol). Table 1 shows the experiments per-
formed following the planning as well as the quantities used of each
component in each treatment.
The nanoemulsions were stored under refrigeration in sealed
glasses and protected against light and gas permeation until the
evaluation. The responses analyzed were particle size, polydisper-
sity index, zeta potential, encapsulation efficiency, and stability.
filtrate (solvent) was first evaporated at room temperature and then
at 60°C to constant mass. The oil that was not encapsulated (free
oil) was determined by difference. Equation 1 presents the deter-
mination of the encapsulation efficiency, where Oiltotal is the total
amount of oil added to the nanoemulsion formulation and Oilsurface is
the amount of free oil in the nanoemulsion.
Oiltotal − OilSurface
Encapsulation efficiency ( % ) = (1)
Oiltotal

2.4 | Particle size, polydispersity index, and
zeta potential
The average particle size, particle size distribution, polydispersity
index (PDI), and zeta potential of nanoemulsions were determined
using Dynamic Light Scattering - DLS (Zetasizer Nano ZS, Malvern
Instruments, Worcestershire, UK), with refractive indexes for chia
mucilage and water of 1.335 and 1.330, respectively. The nanoe-
mulsion samples were diluted in ultrapure water at ambient tem-
perature. Each sample was analyzed in a disposable polystyrene
cell (DTS0012, Malvern Instruments). The zeta potential of the na-
noemulsions was determined with a universal dip cell (ZEN1012,
Malvern Instruments). The results were analyzed by the Zetasizer
7.12 software program.
2.5 | Encapsulation efficiency
The encapsulation efficiency was verified by measuring the total
nanoencapsulated oil and free oil on the surface of the nanoparti-
cles. The free oil was determined according to Timilsena, Adhikari,
Barrow, et al. (2016) with some modifications. First, to 20 ml of na-
noemulsion was added 20 ml of hexane, and the suspension was
stirred for 1 min at room temperature. The suspension was filtered
and the retentate was washed twice more with 20 ml of hexane. The
2.6 | Centrifugation test
For the preliminary stability, the centrifugation test was performed.
The nanoemulsion samples were subjected to cycles of 677, 2,415,
and 3,381 ×g (Beckman, GS-15R, USA) for 15 min, for the evaluation
of the separation of phases (Maruno & Rocha-Filho, 2010). Equation
2 presents the calculation of stability (%).
Height of separation of the nanoemulsion
Stability = × 100 (2)
Total height of the nanoemulsion
2.7 | Evaluation of selected nanoemulsions
The nanoemulsions that presented the nanoparticles produced in
the best conditions (treatment 4 with and without ethanol) were
characterized in relation to the chemical (pH), thermal (stress test
and differential scanning calorimetry‒DSC), morphological (trans-
mission electron microscopy‒TEM), and storage stability (syneresis,
rheology, and color) properties.
2.7.1 | Chemical property: pH
The pH of the nanoemulsions was determined in pH meter (Quimis,
Q400AS, Brasil) at room temperature, without dilution, right after
the nanoemulsion preparation (Campo et al., 2017).
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2.7.2 | Thermal properties
Thermal stress test
Thermal stress was evaluated according to Bernardi et al. (2011) with
modifications. The nanoemulsions were heated in a bath at tempera-
tures ranging from 40 to 100°C. The temperature was increased by
5°C every 30 min. At the end of each cycle (30 min), the macroscopic
appearances of the nanoemulsions were evaluated.
Differential scanning calorimetry (DSC) analysis
The thermal characteristics of unencapsulated chia oil and na-
noemulsions were determined by DSC (Shimadzu, model DSC-60,
Japan). DSC analysis was realized with the heating rate of 10°C/min,
at the temperature range from −25 to 300°C and nitrogen atmos-
phere flow rate of 50 ml/min.
2.7.3 | Morphological property
The internal morphology of the chia oil nanoemulsions was analyzed
by TEM using a Tecnai G2-12 SpiritBiotwin FEI microscope (Oregon,
USA), with an acceleration voltage of 120  kV. The nanoemulsions
were diluted in ultrapure water (1:10 m/v) and a drop of the dilution
was added on the grid for 5 min. Subsequently, the excess sample
was withdrawn and one drop of uranyl acetate (5% m/v) was depos-
ited. Then, the grid was left for rest for 24 hr (Campo et al., 2017).
The images were observed at a magnification of 200 k.
2.7.4 | Storage stability
Storage stability for chia seed oil nanoemulsions was tested by stor-
ing the samples at −18°C, 4°C, and room temperature (~25°C) for
28 days monitoring particle size, polydispersity index, and zeta po-
tential according to item 2.3, and as well as syneresis, rheology, and
color.
Syneresis
Syneresis was evaluated in the nanoemulsions stored at refrigeration
temperature (4°C) and freezing temperature (−18°C). The exuded
water height was measured and the syneresis was determined by
Equation 3. Samples were measured at time 0, 7, 14, 21, and 28 days.
Height of exuded water
Syneresis ( % ) = × 100
Initial height of nanoemulsion
Rheological measurements
Rheological measurements were carried out using a controlled
stress rheometer (Discovery HR-2, TA Instruments, New Castle,
DE, USA) using a parallel plate geometry (diameter  = 40 mm and
gap = 1,000 µm) (Erçelebi & Ibanoğlu, 2009). The apparent viscosity
as a function of shear rate was determined in the shear rate range
from 0.1 to 1,000 s−1at 25ºC. The rheological behavior was fitted to
the power-law model using TRIOS software (TA Instruments, New
Castle, DE, USA) by the following Equation 4:
𝜏 = K 𝛾̇ n (4)
where η = apparent viscosity (Pa s), K = consistency coefficient
(Pa sn), 𝛾̇ = shear rate (s−1), and n = flow behavior index.
The dynamic oscillatory tests of nanoemulsions were carried out
in the linear viscoelastic region (LVR). Frequency sweeps were de-
termined by applying a constant strain of 1% and a frequency range
from 0.1 to 10 rad/s at 25ºC. The mechanical spectra were plotted in
terms of the elastic or storage modulus (G′) and viscous or loss mod-
ulus (G″) as a function of frequency. All measurements were done in
duplicate at time 0, 14, and 28 days.
Color
The color of nanoemulsions was determined using a Colorimeter
(BYK Gardner, USA). The analysis was based on the CIE L*a*b*
system that was defined by the CIE (International Commission on
Illumination) in 1976 for determining the L* (lightness), a*, and b*
(chromaticity coordinates) values. From these values, the Chroma
parameter (intensity or purity of the color‒C*), the Hue angle (true
color‒h°), and the total color difference (TCD) according to Equations
5‒7, respectively, were calculated. Samples were taken at time 0, 7,
14, 21, and 28 days.
√
C∗ = a∗2 + b∗
2
(4)
b∗ (5)
h ◦ = tan − 1
a∗
√
TCD =
(
L ∗ − L0∗
)2 )2 (
+ a ∗ − a0∗ + b ∗ − b0∗
( )2
(6)
where a0∗, b0∗, and L0∗ parameters were obtained on day zero and a*,
b , and L* parameters were obtained on the day of sampling.
*
2.8 | Statistical analysis
All determinations were performed at least in triplicate and data
were compared using variance (ANOVA). The average values ob-
tained were compared using Tukey's test through Statistical 5.0
(3)
software (Statsoft, USA), with statistical significance (α) at p < .05.
Experimental design responses and properties evaluated during
storage stability were also analyzed using multifactorial analysis
(ANOVA). Exploratory factor analysis with significance (α) at p < .05,
using Statgraphics, was used to determine the treatment with the
best techno-functional (particle size, PDI, zeta potential, encapsula-
tion efficiency, and centrifuge test responses) properties, using as
 FERNANDES et al.

TA B L E 2   Responses of the experimental design

Stability (%)
Encapsulation
Treatment Particle size (nm) PDI Zeta potential (mV) efficiency (%) 677 ×g 2,415 ×g 3,381 ×g
a a c b a b
Without ethanol 1 637.33 ± 16.34 0.637 ± 0.040 −30.84 ± 1.45 93.33 ± 1.65 100.0 ± 0.0 87.6 ± 1.9 87.5 ± 1.2b
2 153.68 ± 3.64d 0.414 ± 0.009d −36.66 ± 1.86d 93.49 ± 1.45b 100.0 ± 0.0a 96.4 ± 2.0a 94.8 ± 0.3a
b cd a a a a
3 291.25 ± 19.16 0.455 ± 0.045 −23.04 ± 0.39 97.01 ± 0.99 100.0 ± 0.0 95.8 ± 1.5 93.9 ± 1.4a
4 160.48 ± 2.12d 0.547 ± 0.027abc −27.18 ± 0.89b 96.61 ± 0.36a 100.0 ± 0.0a 95.4 ± 0.6a 92.7 ± 2.1a
c b a ab a a
5 247.65 ± 8.84 0.554 ± 0.007 −24.38 ± 1.70 94.55 ± 0.92 100.0 ± 0.0 97.3 ± 0.0 92.2 ± 0.4a
6 245.50 ± 4.53c 0.570 ± 0.063ab −25.53 ± 1.02ab 95.30 ± 0.14ab 100.0 ± 0.0a 98.4 ± 1.4a 93.1 ± 2.3a
c ab ab ab a a
7 236.75 ± 8.55 0.573 ± 0.010 −25.06 ± 1.20 94.80 ± 1.49 100.0 ± 0.0 97.4 ± 0.1 94.8 ± 0.3ª
A A BC A A A
With ethanol 1 363.23 ± 21.80 0.572 ± 0.007 −30.06 ± 1.75 94.07 ± 0.16 100.0 ± 0.0 99.2 ± 1.4 98.3 ± 1.5A
E A A B A A
2 170.28 ± 4.69 0.491 ± 0.076 −25.80 ± 1.19 88.72 ± 1.48 100.0 ± 0.0 100.0 ± 0.0 75.6 ± 0.8C
3 181.03 ± 8.88DE 0.501 ± 0.042 A −28.50 ± 0.99B 97.25 ± 0.67A 100.0 ± 0.0A 98.4 ± 1.4A 97.5 ± 2.5A
C A D A A A
4 213.25 ± 9.81 0.600 ± 0.043 −39.83 ± 1.24 96.12 ± 2.58 100.0 ± 0.0 98.3 ± 1.5 84.1 ± 1.8B
5 293.10 ± 17.42B 0.518 ± 0.121A −32.15 ± 0.64C 96.45 ± 1.06A 100.0 ± 0.0A 100.0 ± 0.0A 98.8 ± 1.4A
C A C A A A
6 214.15 ± 7.07 0.515 ± 0.014 −31.60 ± 0.64 94.70 ± 2.12 100.0 ± 0.0 100.0 ± 0.0 99.2 ± 1.3A
7 208.78 ± 7.75CD 0.504 ± 0.040A −46.13 ± 1.07E 95.33 ± 1.76A 100.0 ± 0.0A 100.0 ± 0.0A 100.0 ± 0.0A

Note: Treatment 1:0.125 g of mucilage and 0.043 ml of chia oil; Treatment 2:0.375 g of mucilage and 0.043 ml of chia oil; Treatment 3:0.125 g of mucilage and 0.233 ml of chia oil; Treatment 4:0.375 g of
mucilage and 0.233 ml of chia oil; Treatment 5, 6, and 7:0.250 g of mucilage and 0.1 ml of chia oil; PDI = polydispersity index. The average with standard deviation, the same letter in the column indicates
that there is no significant difference between the means according to the Tukey test (p > .05).
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factors the use of ethanol and treatments performed, and the study
of storage nanoemulsions using the use of ethanol, temperature, and
sampling times as factors.
3 |  R E S U LT S A N D D I S CU S S I O N
3.1 | Preparation and selection of the nanoemulsion
formulation
3.1.1 | Particle size, polydispersity index, and
zeta potential
Table 2 presents the parameters evaluated during the experimental
design. The results referring to the factorial analysis are available
in the supplementary material (Figure 1 of supplementary material)
and the ANOVA of the treatments is represented by the sub-indices
(uppercase or lowercase) in the same column.
Except for treatment 1 without ethanol, all other treatments
showed particle size within the characteristic range (50–500 nm) of
nanoemulsions used for industrial preparations such as food, cos-
metics, and pharmaceuticals (Chaari et al., 2018). Treatment 1 was
formulated with 0.125 g of chia mucilage and 0.043 ml of chia oil, in-
dicating that when ethanol was not used, low amounts of main com-
ponents contributed to larger particle sizes because the amounts
added were small and, thus, the particles tend to aggregate.
Regarding factor analysis (supplementary material), all inter-
actions between factors (use of ethanol and treatments) showed
homogeneity (p > .05). Treatments 2, 6, and 7 did not differ signifi-
cantly (p  > .05) in particle size when considering the use of etha-
nol. The other treatments presented a significant difference. In the
treatments without ethanol (treatment 2 and 4), it was found that
the more chia mucilage added in nanoemulsions, the smaller the par-
ticle size, regardless of the amount of chia oil added. Thus, this could
corroborate that the size of the encapsulated chia oil particles may
be directly related to the added amount of chia mucilage. Treatment
2 promoted the smallest particle size, whether or not ethanol was
used.
Although there are some studies using chia mucilage as an encap-
sulating agent in nanoemulsions, they have not studied the adequate
amounts of each component. Campo et al. (2017) also encapsulated
chia oil with chia mucilage and found values for the mean diame-
ter of 205 ± 4.24 nm. Stefani et al. (2019) used chia seed mucilage
for nanoencapsulation of flaxseed oil and found higher values for
the average diameter (356 ±  2.83  nm). Variations in the diameter
of nanoparticles using gum/mucilage as wall material may be influ-
enced by factors such as type of wall material, gum concentration,
agitation, and emulsifier used (Ghayempour et al., 2015).
Polydispersity Index (PDI) is dimensionless and indicates the het-
erogeneity (monodisperse or polydisperse) of sizes of particles in a
mixture. Regarding the PDI in factor analysis, the values were be-
tween 0.4 and 0.6, which is characteristic of a very wide distribution
(Figure 2 of supplementary material). All treatments using ethanol
showed no significant difference between them, unlike those that
did not use ethanol. The varying sizes of the particles are directly
correlated with the dielectric constant of the medium, so the use of
solvents in the formulation of nanoemulsions, and the use of ethanol
compared to other solvents, such as methanol, is more homogeneous
due to uniform particle formation, as it promotes a greater reduc-
tion in the dielectric constant of the medium (Joshi et al., 2017).
Similarly, Yang et al. (2009) examined the effect of adding ethanol to
nanoemulsions and found that ethanol keeps the initial droplet size
of nanoemulsions constant.
Although the use of ethanol alone (ANOVA) did not cause a
significant difference, when the interaction between the factors
ethanol use and different treatments were tested (Figure 1 of sup-
plementary material), it was found that in the PDI, the effect of etha-
nol promotes significant difference for treatments 2 and 6.
Zeta potential reflects the indirect measurement of the particle
surface, which is influenced by changes in the interface with the
dispersing medium due to the dissociation of functional groups on
the drop surface or the adsorption of ionic species present in the
dispersing aqueous medium. Particles with a zeta potential greater
than 30  mV and less than −30  mV are considered to be physically
stable. However, depending on the wall material used the drops may
be stable even with zeta potential values of around 20 mV, or even
lower (Mishra et al., 2009).
The obtained drops had negative zeta potential due to the pres-
ence of chia mucilage, which conferred a negative potential to the
drop interface due to the presence of carboxylic chains in its compo-
sition (Lin et al., 1994). As the surfactant used (Tween 20) is nonionic
in character, it does not interfere with the potential presented by
the particles.
Regarding the factor analysis (Figure 1 of supplementary mate-
rial) of zeta potential, only treatment 1 did not show a significant
difference with the interaction of the factors. For the other treat-
ments that showed a significant difference, and except for treatment
2, nanoemulsions without ethanol had a higher zeta potential than
nanoemulsions with ethanol. This change in zeta potential is related
to ethanol hydroxyl ions, formed at the oil-surfactant-water inter-
face (Liu et al., 2020).
Analyzing individually the effect of ethanol, the zeta potential
of nanoemulsions without ethanol indicated that they were stable
when less chia oil was added to the formulations (treatment 1 and 2),
with the lowest potential reaching −36.66 mV for treatment 2 and
−30.84 mV for treatment 1. While for ethanol nanoemulsions (also
stable) the opposite happened, the addition of greater amounts of
chia oil resulted in higher zeta potential than nanoemulsions with low
ethanol, with the highest value obtained (−25.80 mV) for treatment
2 and the lowest value (−46.13 mV) for treatment 7. The zeta poten-
tial results indicated that the nanoemulsions produced are physically
stable, showing no tendency to create aggregates between particles.
The zeta potential values found in this study were better
than those found by Campo et al. (2018), where they obtained
−14.8  ±  0.42  mV for nanoemulsion of Zeaxanthin with Opuntia
FERNANDES et al.
monacantha mucilage, and by Stefani et al. (2019), where they ob-
tained −22.75 ± 3.89 mV for nanoemulsion of linseed oil with chia
mucilage.
According to Liu et al. (2020), the correct amount of ethanol
tends to increase the stability of the nanoemulsion reducing particle
size, suitable zeta potential, and stable PDI. The hydrogen bond be-
tween the OH groups (hydroxyl ions) of ethanol with the surfactant
(Tween 20) decreases the fluidity of the interfacial film and the ap-
parent viscosity of the system.
3.1.2 | Encapsulation efficiency
Encapsulation efficiency is an important parameter to evaluate na-
noparticle formation and corresponds to the quantification of the
encapsulated bioactive compound (Campo et al., 2018). According
to Table 2, all treatments, except treatment 2 with ethanol, had en-
capsulation efficiency greater than 93%, proving the efficacy of the
encapsulation technique used and the potential of chia mucilage
as a structuring material for nanoemulsion bioactive compounds.
The high encapsulation efficiency may have been caused by the
high emulsifying effect of chia mucilage, about 63.7% (Fernandes
& Salas-Mellado, 2018), which reduces losses to the aqueous phase
and contributes to the encapsulation efficiency and stability of these
compounds. Besides, another factor that may have contributed to
the encapsulation efficiency, trapping the chia oil in the oil-mucilage
matrix, is the oil retention capacity of the chia mucilage, which cor-
responds to about 9 g of retained oil/g of sample (Fernandes & Salas-
Mellado, 2018).
Through factor analysis (Figure 1 of supplementary material),
except for treatment 2 which showed higher efficiency when eth-
anol was not used, the other treatments showed no significant
difference in the interaction of the factors. This shows that the
use of ethanol does not change the efficiency of encapsulation.
Analyzing individually (ANOVA), it was found that the treatments
using the lowest chia oil contents resulted in significantly lower
encapsulation efficiencies, possibly by the form of determination
that was gravimetric.
3.1.3 | Centrifugation test
Under unfavorable environmental conditions, nanoemulsions can
be easily destabilized due to their thermodynamic instability. The
presence of centrifugal force can accelerate the Brownian motion,
so particles can get closer, and thus, increase the chances of destabi-
lization processes occurring (Carpenter & Saharan, 2017; Zhai et al.,
2018). Centrifugation test is important for checking how nanoemul-
sions behave when added to foods that require agitation during their
production.
Stability was 100% in all treatments when centrifugation at
677 ×g was used. For the factor analysis of the centrifugation
test at 2,415 ×g, it was found that treatments 1 and 2 showed a
|
      7 of 16
significant difference when considering the use of ethanol, with
greater stability for those with ethanol. For the factorial analy-
sis of the centrifugal test (supplementary material) at 3,381 ×g,
treatment 2 presented the largest significant difference in factor
interaction. Moreover, it was found that at the central points of
the planning (treatments 5, 6, and 7) the use of ethanol promoted
greater stability of nanoemulsion.
3.1.4 | Determination of nanoemulsions
for evaluation
Both, ethanol and the different treatment conditions were major
factors affecting particle size. The particles encapsulated with
treatment 4 had small sizes, 160.48 nm for nanoemulsion without
ethanol, and 213.25 nm for nanoemulsion with ethanol. The particle
distribution was considerable, showing that in these treatments a
good particle distribution was obtained. The high zeta potential of
the chosen treatments confirmed that chia mucilage was deposited
on the surface of chia oil particles by steric stabilization as well as the
high encapsulation efficiency. Besides, centrifugation tests showed
that nanoemulsions were stable at low agitation and that there was
not a significant difference with increased agitation in these same
treatments.
Considering all the individual and factorial effects, treatment 4
without (T4) and with ethanol (T4E) were chosen to perform a more
specific characterization for future application.
3.2 | Evaluation of nanoemulsions
3.2.1 | pH
pH is a parameter that monitors the stability of nanoemulsions
since possible changes in its value indicate the occurrence of
chemical reactions or bacterial growth, which may compromise
the quality of the final product. The pH of the T4 nanoemulsion
was 5.90 ± 0.01 and the T4E nanoemulsion was 5.79 ± 0.17, show-
ing no significant difference between them. The acidic pH is due
to the carboxylic chains in the composition of the chia mucilage
(Campo et al., 2017). Besides, for the T4E nanoemulsion, ethanol
through hydroxyls also favors acidic pH. Similar results were ob-
tained by Pengon et al. (2018), who evaluated the chemical prop-
erties of coconut oil nanoemulsions and reported pH values from
5.0 to 6.0.
3.2.2 | Thermal properties
The thermal stability of nanoemulsions should be carefully evalu-
ated to ensure that there is no adverse influence of environmental
conditions on the course of processing, transport, and application in
food (Shu et al., 2018). During food processing, for example, there is
 |
8 of 16       FERNANDES et al.
thermal destruction of pathogens and inactivation of enzymes, for
their preservation, through thermal treatments such as pasteuriza-
tion and sterilization (Micali et al., 2016).
Thermal stress test
Thermal stress tests are based on the fact that an increase in kinetic
energy occurs when heating a particle system, which may lead to de-
hydration of hydrophilic surfactants, in the case of Tween 20, chang-
ing their solubility in water. Sudden changes in temperature are good
ways of assessing the stability of nanoemulsion, predicting its shelf
life, and what food to use (Seibert et al., 2019).
T4 nanoemulsion had a higher resistance to macroscopic appear-
ance modification than T4E nanoemulsion (Table 1 of supplemen-
tary material). T4 nanoemulsion showed slight appearance change,
due to color change, starting from 65°C, while T4E nanoemulsion
already had a different appearance starting from 55°C. Up to 85°C,
for both nanoemulsions, there was not creaming or phase separa-
tion, maintaining the initial appearance, and from this temperature,
the nanoemulsions began to exude water and from 90°C the pres-
ence of air bubbles began. T4E nanoemulsion at 90°C was modified
with phase separation, whereas T4 nanoemulsion only at 100°C. The
appearances occasioned by the temperature increase in nanoemul-
sions can be visually observed through Figure 2 of the supplemen-
tary material.
Thus, it was verified that the nanoemulsion without ethanol (T4)
in its formulation was more stable in the face of the temperature
increase and can be used at temperatures below 100 ºC without al-
tering its structure.
Differential scanning calorimeter (DSC)
Figure 1 shows the thermal properties obtained by DSC. The nanoe-
mulsions obtained in treatments T4 and T4E showed a well-defined
endothermic peak, which coincides with the endothermic peaks of
the chia oil and the chia mucilage suspension used. The DSC curve
for chia mucilage has an endothermic peak, but it is not possible to
visualize it on the scale presented.
Endothermic peaks observed at 0°C represent ice melting. It is
possible to verify that at negative temperatures the nanoemulsions
remained stable, suggesting that they can be applied in frozen foods.
The endothermic peaks referring to the melting temperature was
51.68°C (∆Hfusion = 5.45 J/g) for chia oil, 50.94°C (∆Hfusion = 15.11 J/g)
for chia mucilage, 69.66°C (∆Hfusion  = 1,090.00 J/g) for the na-
noemulsion without ethanol (T4), and 57.62°C (∆Hfusion = 537.04 J/g)
for the nanoemulsion with ethanol (T4E). These results indicate that
nanoencapsulation promoted a solid-liquid transition at high tem-
peratures. Also, nanoemulsions showed high fusion enthalpy value.
This suggests a high organization in the internal structure of the
encapsulated particles because the fusion of a highly organized
material requires more energy to break the cohesive forces of the
structure than the fusion of an amorphous or slightly ordered mate-
rial (Zavareze et al., 2014).
3.2.3 | Morphology
Figure 2 presents the TEM microphotographs of nanoemulsions.
TEM analysis confirmed that nonaggregated were droplets were
well-defined, similar to the spherical form and that the size was in
the nanometric range (which had previously been determined), this
indicates that the shear, turbulence, and cavitation effects produced
in the agitation process promoted small micelles. The particle size
presented from the TEM image was smaller than the size determined
by DLS. Fundamentally, different techniques may result in differ-
ent size particle values due to the different measurement principles
F I G U R E 1   Differential scanning
calorimetry (DSC) of chia oil, chia
mucilage, T4 nanoemulsion (without
ethanol), and T4E nanoemulsion (with
ethanol)
FERNANDES et al. |
      9 of 16

F I G U R E 2   Transmission electron
microscopy (TEM) micrographs of a) T4
nanoemulsion (without ethanol), and b)
T4E nanoemulsion (with ethanol)

TA B L E 3   Syneresis at 4°C and particle size, polydispersity index, and zeta potential at different temperatures the samples nanoemulsion
on different days of storage

Days

0 7 14 21 28
a,A a,A a,A
Syneresis (%) T4 4 ºC ‒ 2.68 ± 0.04 2.68 ± 0.04 2.68 ± 0.04 2.68 ± 0.04a,A
T4E ‒ ND ND ND ND
Particle size (nm) T4 −18 ºC 160.48 ± 2.12c 183.13 ± 5.52c,C 356.95 ± 26.43a,A 313.00 ± 13.72b,A 297.13 ± 13.72b,A
4 ºC 160.48 ± 2.12b 814.50 ± 4.57ª,A 161.27 ± 3.55b,C 161.25 ± 5.10 b,C 159.85 ± 8.20 b,C
e b,B c,B d,B
Room 160.48 ± 2.12 243.38 ± 6.54 232.30 ± 2.60 198.28 ± 1.56 264.20 ± 7.62a,B
T4E −18 ºC 213.25 ± 9.81d 436.83 ± 9.40 c,B 547.90 ± 14.87b,A 530.13 ± 5.09b,B 729.60 ± 11.96a,B
c d,C b,B a,C
4 ºC 213.25 ± 9.81 128.36 ± 3.94 367.73 ± 5.94 384.90 ± 2.35 374.70 ± 6.29ab,C
Room 213.25 ± 9.81d 847.07 ± 12.15a,A 242.58 ± 9.99c,C 606.80 ± 3.05b,A 871.87 ± 16.03a,A
a a,A a,B b,B
Polydispersity index T4 −18 ºC 0.547 ± 0.027 0.486 ± 0.068 0.533 ± 0.035 0.363 ± 0.025 0.363 ± 0.037b,B
4 ºC 0.547 ± 0.027ab 0.531 ± 0.030 b,A 0.657 ± 0.026a,A 0.560 ± 0.057ab,A 0.550 ± 0.066ab,A
Room 0.547 ± 0.027a 0.459 ± 0.036b,A 0.390 ± 0.012c,C 0.390 ± 0.017c,B 0.410 ± 0.017c,B
ab ab,A ab,A b,A
T4E −18 ºC 0.600 ± 0.043 0.626 ± 0.031 0.604 ± 0.074 0.538 ± 0.022 0.683 ± 0.020a,A
4 ºC 0.600 ± 0.043b 0.387 ± 0.017c,C 0.568 ± 0.042b,A 0.362 ± 0.051c,B 0.759 ± 0.025a,A
a b,B ab,A ab,A
Room 0.600 ± 0.043 0.467 ± 0.040 0.542 ± 0.088 0.506 ± 0.069 0.517 ± 0.099ab,B
Zeta potential (mV) T4 −18 ºC −27.18 ± 0.89b −37.02 ± 1.81c,B −50.30 ± 1.60 d,C −23.37 ± 0.12a,B −36.40 ± 1.64c,C
c d,C b,A a,A
4 ºC −27.18 ± 0.89 −42.78 ± 0.85 −22.75 ± 1.54 −20.07 ± 0.23 −21.25 ± 1.46ab,A
Room −27.18 ± 0.89a −29.87 ± 1.00 b,A −31.22 ± 0.92b,B −26.43 ± 0.46a,C −31.03 ± 0.61b,B
c d,B d,B a,A
T4E −18 ºC −39.83 ± 1.24 −44.23 ± 0.83 −44.00 ± 1.45 −29.20 ± 0.95 −36.03 ± 1.53b,A
4 ºC −39.83 ± 1.24c −26.00 ± 0.66a,A −40.20 ± 0.89c,A −40.60 ± 0.78c,B −35.00 ± 0.56b,A
b c,B b,A b,B
Room −39.83 ± 1.24 −43.80 ± 1.53 −39.77 ± 1.19 −39.68 ± 1.26 −35.95 ± 0.87a,A

Note: T4 = Treatment 4 without ethanol: 0.375 g of mucilage and 0.233 ml of chia oil; T4E = Treatment 4 with ethanol: 0.375 g of mucilage, 0.233 ml
of chia oil and 4 ml of ethanol. Room = ~25 ºC. ND = Non-detectable. The same lowercase letter in the line indicates that there were no significant
differences between each treatment, and the same uppercase letter in the column indicates that there were no significant differences in the same
nanoemulsion in the same day and different temperature according to Tukey's test (p > .05).

used. Therefore, the discrepancy of results obtained with different
techniques sizing is expected (Chuesiang et  al.,  2018). Droplet ag-
gregates can be triggered by the droplet flocculation during the na-
noemulsion formulation (Shanmugapriya et al., 2019).
Sarheed et al. (2019) also found the presence of spherical micelles
in loratadine nanoemulsions with coconut oil, Tween 80, and oleic acid.
The authors attributed this form to the higher added concentration of
loratadine, which causes a more concave curvature for oil. In our study,
chia mucilage concentration was also higher than in the oil phase.
3.2.4 | Storage stability
Particle size, PDI, and zeta potential
Particle size, PDI, and zeta potential control are important parame-
ters for confirming the physical stability of nanoparticle formulations
during storage (Chaari et al., 2018). Table 3 presents the syneresis at
4°C and particle size, PDI, and zeta potential at different tempera-
tures and storage times.
10 of 16       | FERNANDES et al.

(a) 102
(b) 102 F I G U R E 3   Apparent viscosity versus
0 d T4
shear rate of T4 nanoemulsion and T4E
0 d T4
14 d T4 14 d T4
28 d T4
nanoemulsion at different storage times.
28 d T4
0 d T4E 0 d T4E
101 101
Apparent viscosity (Pa s)

Apparent viscosity (Pa s)

14 d T4E
Storage temperatures: (a) −18°C, (b) 4°C,
14 d T4E
28 d T4E 28 d T4E

and (c) room (~25°C). T4, nanoemulsion

100 100 without ethanol; T4E, nanoemulsions with
ethanol
10-1 10-1

10-2 10-2
10-1 100 101 102 103 10-1 100 101 102 103
-1 -1
Shear rate (s ) Shear rate (s )

(c) 102
0 d T4
14 d T4
28 d T4
0 d T4E
Apparent viscosity (Pa s)

101 14 d T4E
28 d T4E

100

10-1

10-2
10-1 100 101 102 103
-1
Shear rate (s )

Regarding factor analysis, for particle size and PDI, it was found 21 days there was a decrease in the values of the parameter. The
that there was no statistically significant effect of double and triple decrease obtained is related to the increase in particle size.
interactions between factors (days, temperatures, and treatments, At the end of 28 days of storage, most nanoemulsions had a
p > .05). Besides, there was no significant statistical effect of indi- zeta potential modulus greater than 30 mV, indicating that it was
vidual factors days and temperature (p > .05). However, a significant high enough to stabilize nanoemulsions by electrostatic repulsion
statistical effect regarding the treatment factor was reported for and steric interactions during storage, as repulsive forces predom-
particle size and PDI (p = .0406 < .05). Therefore, when studied the inated over van der Waals forces of attraction, the system soon
relationship between the use of ethanol (treatment), day and tem- dispersed. The zeta potential at the end of the storage time was
peratures of storage, it was found that only the effect of ethanol satisfactory for both nanoemulsions, but these values were not
in the constitution of nanoemulsion presented a difference during sufficient to promote flocculation since there was an increase in
storage. particle size. That is, the stabilization mechanism is not only due to
When an analysis of treatments was performed in an individually electrostatic repulsion, but is also mainly due to steric hindrance.
form, it could be seen that, except for T4 nanoemulsion stored at The increase in size was favored because the rate of adsorption
4°C, all others treatments showed an increased particle size with the of mucilage at the oil–water interface is relatively slow due to the
storage time. According to Henry et al. (2010), the increase in par- high molecular weight of the polysaccharides. Further, areas that
ticle size over time is due to the movement of the dispersed phase were not covered by emulsifying agents were susceptible to the
to the continuous phase, thus, increasing the chance of collisions flocculation of the drops.
between droplets and favoring the coalescence process. The tem- In summary, there was a considerable increase in the average
perature has a direct influence on the particle motion, therefore, T4E particle diameter during storage. It is known that the frequency of
nanoemulsion storage at 25°C (room) resulted in larger droplet sizes droplet collisions decreases with increasing droplet size, while the
than storage at −18 and 4°C. Nejadmansouri et al. (2016) evaluated efficiency of coalescence increases. Furthermore, Ostwald's ripen-
the stability of fish oil nanoemulsions for 28 days and found that par- ing rate increases with decreasing particle size, increasing polydis-
ticle size also remained relatively constant at 4°C and a considerable persity, and increasing the concentration of free surfactant (Walker
increase at 25°C from day 14. et al., 2015). The increase in particle size was smaller in the T4 na-
In the factor analysis, no significant statistical effect of double noemulsion, which can be attributed to the absence of ethanol in its
and triple interactions between factors and individual factors (days, structure. The zeta potential was more negative at the end of storage
temperatures, and treatments, p  > .05) on zeta potential was ob- in the T4E nanoemulsions, which indicates that the nanoemulsion
served. Except for the T4E treatment at 4 ºC, the others treatments would be more stable since the charged particles repel each other
initially presented an increase in zeta potential modulus and after and this force exceeds the van der Waals forces, which promote
FERNANDES et al. |
      11 of 16

(a) (A)
10

10

G'' (Pa)
G' (Pa)
1

0 d 4T
0 d 4T
14 d 4T
14 d 4T
28 d 4T
28 d 4T
0 d 4TE
0 d 4TE
14 d 4TE
14 d 4TE
28 d 4TE
28 d 4TE
0.1 1
0.1 1 10 0.1 1 10

Frequency (rad/s) Frequency (rad/s)

(b) (B)
10

10

G'' (Pa)
G' (Pa)

0 d 4T 0 d 4T
14 d 4T 14 d 4T
28 d 4T 28 d 4T
0 d 4TE 0 d 4TE
14 d 4TE 14 d 4TE
28 d 4TE 28 d 4TE
0.1 1
0.1 1 10 0.1 1 10

Frequency (rad/s) Frequency (rad/s)

(c) (C)
10

10
G'' (Pa)
G' (Pa)

0 d 4T 0 d 4T
14 d 4T 14 d 4T
28 d 4T 28 d 4T
0 d 4TE 0 d 4TE
14 d 4TE 14 d 4TE
28 d 4TE 28 d 4TE
0.1 1
0.1 1 10 0.1 1 10

Frequency (rad/s) Frequency (rad/s)

F I G U R E 4   The dynamic oscillatory behavior of the T4 nanoemulsion and T4E nanoemulsion at different storage times. (a) Storage
modulus (G′) and (A) loss modulus (G″) at −18°C; (b) storage modulus (G′) and (B) loss modulus (G″) at 4°C; (c) storage modulus (G′) and (C)
loss modulus (G″) at room (~25°C). T4, nanoemulsion without ethanol; T4E, nanoemulsions with ethanol

aggregation. However, as it was seen that there was an increase in
size, what may have occurred was the generation of some free fatty
acids caused by the hydrolysis of the triglyceride molecules during
storage (Walker et al., 2015).
Syneresis
The two nanoemulsions tested did not present syneresis when
stored at −18°C. This could be advantageous for possible appli-
cation in frozen foods such as ice cream. In addition, T4E nanoe-
mulsion did not show syneresis when stored for 28 days at 4°C.
When nanoemulsions were subjected to elevated temperatures
(up to 95°C), it was previously seen that T4 nanoemulsion showed
greater stability compared to the constant temperature increase.
However, when stored at refrigeration temperature (4ºC), T4E
nanoemulsion was more stable than T4 nanoemulsion. These ef-
fects indicated that nanoemulsion with ethanol could be capable
of promoting greater stability at low temperatures than at high
temperatures.
Although T4 nanoemulsion promoted syneresis, this value was
very low (2.7%) throughout the storage time. What favored the
high stability of nanoemulsions against syneresis was the use of
chia mucilage as a wall material. Chia mucilage is mainly composed
of soluble dietary fibers, which can be portrayed through wa-
ter-holding capacity (WHC), which increases the viscosity of the
system, decreasing the movement among the drops, which delays
the possible coalescence among them. According to Fernandes &
Salas-Mellado (2018), chia mucilage presents an excellent value of
WHC (161.00 g of retained water/g of sample). This is not only
due to the increased hydrodynamic volume of the mucilage, but
also, more important, to its degree of hydrophobicity, which may
also be contributed by the protein fraction and uronic acid content
(Chiang & Lai, 2019).
 |
12 of 16       FERNANDES et al.

TA B L E 4   The color at different temperatures the samples nanoemulsion on different days of storage

Days

0 7 14 21 28
b a,A a,A a,A
L* T4 −18 ºC 48.63 ± 0.15 56.50 ± 0.27 56.42 ± 2.36 58.09 ± 0.59 58.55 ± 1.00a,A
4 ºC 48.63 ± 0.15b 46.73 ± 0.23c,B 49.75 ± 0.22a,B 50.70 ± 0.43a,C 50.75 ± 0.69a,C
Room 48.63 ± 0.15c 57.25 ± 0.44a,A 56.78 ± 0.62a,A 54.08 ± 1.46b,B 56.71 ± 0.36a,B
c b,A b,B b,A
T4E −18 ºC 55.73 ± 0.35 58.05 ± 0.16 58.35 ± 0.45 57.82 ± 0.21 59.55 ± 0.20a,A
4 ºC 55.73 ± 0.35b 54.73 ± 0.27c,C 55.89 ± 0.39b,C 56.17 ± 0.22b,B 57.47 ± 0.46a,B
c b,B a,A b,A
Room 55.73 ± 0.35 56.71 ± 0.51 59.35 ± 0.23 57.51 ± 0.17 56.84 ± 0.17b,B
a* T4 −18 ºC 0.82 ± 0.10a 1.21 ± 0.49a,A 1.53 ± 0.48a,A 1.28 ± 0.38a,B 1.46 ± 0.09a,B
d cd,A bc,A ab,AB
4 ºC 0.82 ± 0.10 1.00 ± 0.11 1.20 ± 0.21 1.40 ± 0.06 1.57 ± 0.07a,B
Room 0.82 ± 0.10 b 1.10 ± 0.28b,A 1.14 ± 0.33b,A 2.02 ± 0.35a,A 2.56 ± 0.12a,A
a a,A a,B a,A
T4E −18 ºC 1.47 ± 0.16 1.01 ± 0.25 1.25 ± 0.04 1.42 ± 0.38 1.56 ± 0.06a,A
4 ºC 1.47 ± 0.16ab 1.15 ± 0.28b,A 1.47 ± 0.08ab,B 1.55 ± 0.06ab,A 1.64 ± 0.05a,A
ab b,A a,A ab,A
Room 1.47 ± 0.16 1.15 ± 0.36 2.16 ± 0.38 1.68 ± 0.16 1.64 ± 0.06ab,A
b* T4 −18 ºC 5.10 ± 0.26b 9.60 ± 0.10a,A 8.89 ± 0.78a,A 9.63 ± 0.88a,A 9.94 ± 0.10a,B
e d,B c,B b,A
4 ºC 5.10 ± 0.26 6.67 ± 0.22 7.35 ± 0.13 8.28 ± 0.21 8.84 ± 0.16a,C
Room 5.10 ± 0.26d 6.89 ± 0.35c,B 9.07 ± 0.58b,A 9.99 ± 0.79ab,A 10.98 ± 0.27a,A
a b,B b,B b,B
T4E −18 ºC 10.07 ± 0.44 9.02 ± 0.47 8.70 ± 0.13 8.28 ± 0.35 8.14 ± 0.13b,C
4 ºC 10.07 ± 0.44b 10.41 ± 0.08b,A 10.44 ± 0.28b,A 10.76 ± 0.12ab,A 11.29 ± 0.26a,A
ab b,AB a,A ab,A
Room 10.07 ± 0.44 9.83 ± 0.32 11.39 ± 0.95 10.47 ± 0.25 10.53 ± 0.13ab,B
C* T4 −18 ºC 5.17 ± 0.25b 9.68 ± 0.13a,A 9.03 ± 0.68a,A 9.70 ± 0.81a,AB 10.05 ± 0.10a,B
e d,B c,B b,B
4 ºC 5.17 ± 0.25 6.74 ± 0.21 7.45 ± 0.10 8.39 ± 0.21 8.98 ± 0.15a,C
Room 5.17 ± 0.25d 6.98 ± 0.35c,B 9.13 ± 0.61b,A 10.13 ± 0.67ab,A 11.28 ± 0.24a,A
a b,B b,B b,B
T4E −18 ºC 10.17 ± 0.41 9.08 ± 0.46 8.79 ± 0.16 8.41 ± 0.29 8.29 ± 0.13b,C
4 ºC 10.17 ± 0.41c 10.48 ± 0.08bc,A 10.54 ± 0.28bc,A 10.87 ± 0.11ab,A 11.41 ± 0.26a,A
b b,A a,A ab,A
Room 10.17 ± 0.41 9.90 ± 0.28 11.60 ± 0.86 10.61 ± 0.23 10.65 ± 0.13ab,B
hº T4 −18 ºC 80.87 ± 1.44b 82.85 ± 1.65ab,A 80.06 ± 3.99b,A 87.08 ± 2.66a,A 81.68 ± 0.30ab,A
a a,A a,A a,B
4 ºC 80.87 ± 1.44 81.46 ± 1.01 80.69 ± 1.76 80.42 ± 0.26 79.95 ± 0.59a,B
Room 80.87 ± 1.44ab 80.89 ± 2.30ab,A 84.02 ± 2.09a,A 78.15 ± 1.01b,B 76.85 ± 0.92b,C
abc a,A ab,A c,B
T4E −18 ºC 81.66 ± 1.21 83.59 ± 1.72 81.84 ± 1.49 78.53 ± 0.45 79.16 ± 0.40 bc,B
4 ºC 81.66 ± 1.21a 83.71 ± 1.52a,A 81.98 ± 0.52a,A 81.82 ± 0.35a,A 81.74 ± 0.05a,A
a a,A a,A a,A
Room 81.66 ± 1.21 83.30 ± 2.28 80.69 ± 1.03 80.88 ± 1.01 81.14 ± 0.31a,A
TCD T4 −18 ºC ‒ 9.08 ± 0.38c,A 10.07 ± 1.06bc,A 10.55 ± 0.37ab,A 11.46 ± 0.25a,A
b,B b,B a,C
4 ºC ‒ 2.50 ± 0.10 2.55 ± 0.07 3.83 ± 0.58 4.39 ± 0.40a,C
Room ‒ 8.82 ± 0.36ab,A 9.10 ± 0.35ab,A 7.42 ± 1.73b,B 10.14 ± 0.46a,B
b,A b,B b,A
T4E −18 ºC ‒ 2.60 ± 0.45 3.02 ± 0.44 2.82 ± 0.57 4.30 ± 0.06a,A
4 ºC ‒ 1.18 ± 0.19b,B 0.61 ± 0.37b,C 1.09 ± 0.17b,B 2.15 ± 0.39a,B
b,B a,A b,B
Room ‒ 1.07 ± 0.58 4.09 ± 0.38 1.86 ± 0.21 1.00 ± 0.06b,C

Note: TCD: Total color difference. T4 = Treatment 4 without ethanol: 0.375 g of mucilage and 0.233 ml of chia oil; T4E = Treatment 4 with ethanol:
0.375 g of mucilage, 0.233 ml of chia oil and 4 ml of ethanol. Room = ~25 ºC. The same lowercase letter in the line indicates that there were no
significant differences between each treatment, and the same uppercase letter in the column indicates that there were no significant differences in
the same nanoemulsion in the same day and different temperature according to Tukey's test (p > .05).

Rheological properties
Viscosity is an important parameter for the physicochemical charac-
terization of nanoemulsions since it is related to stability and appli-
cability in foods (Kaplan et al., 2019). The apparent viscosity curves
as a function of the shear rate of nanoemulsions with and without
ethanol at different temperatures and storage times are shown in
Figure 3. As observed, the flow behavior decreased with an increas-
ing shear rate (0.1 − 1,000 s−1) corresponding to a non-Newtonian
fluid with a shear-thinning behavior.
As the shear stress increases, the hydrodynamic forces dom-
inate the Brownian motion effects causing the droplets to be-
come ordered along the fluid-flow lines, which leads to less
FERNANDES et al.
energy dissipation and a lower nanoemulsion viscosity (Zhang &
Mcclements, 2018). The apparent viscosity values were obtained
within the range of 0.171 and 0.728 Pa.s at 50 s−1 (Table 2 of sup-
plementary material). The flow behavior showed high correlation
values (R 2  =  0.986  −  0.998) with a power-law model. The flow
index values of the nanoemulsions varied from 0.170 to 0.415,
which demonstrates the pseudoplastic behavior (shear-thinning
behavior; n  < 1) of the nanoemulsions, while that the K values
varied from 3.824 to 10.832 Pa.sn , which represents the na-
−1
noemulsion consistency at 1.0 s . These results suggest that the
effects of ethanol alter the nanoemulsion structures formed under
these conditions; however, the storage temperatures dramatically
change the rheological parameters. The nanoemulsions made with
ethanol presented higher viscosities compared to the nanoemul-
sions prepared without ethanol at 4°C (Figure 3b). However, the
nanoemulsions without ethanol showed the highest viscosities at
−18°C and 25°C, which indicates that each nanoemulsion may find
application in food industries (Figure 3a,c, respectively).
G′ and G″ modulus of the nanoemulsions with and without eth-
anol carried out in an angular frequency range from 0.1 to 10 rad.s−1
are shown in Figure 4. The G″ values were higher than the G′ values
over the entire frequency range for all the nanoemulsions tested,
indicating typical viscoelastic behavior predominantly viscous (G″
> G′). This liquid behavior is typical for nanoemulsions containing
only negative droplets because of the strong electrostatic repulsion
between them (Zhang & Mcclements,  2018). The nanoemulsions
without ethanol at −18°C had much better viscoelastic behavior at
10 rad/s than at 0.1 rad/s (Figure 4a). The nanoemulsions made with
ethanol at 4°C had the best viscoelastic behavior in comparison to
all nanoemulsions prepared (Figure 4b). These results suggest that
the ethanol applied in the formulation could affect the viscoelastic-
ity of the nanoemulsion at 4°C during their self-life, which could in-
crease their applications within functional food products as delivery
systems. However, the nanoemulsions were highly unstable during
storage, especially at 25°C (Figure 4c), which could be attributed
to coalescence and Ostwald ripening. Saberi, Fang, & Mcclements
(Saberi et al., 2013) reported that ethanol alters the interactions
with the oil, water, surfactant molecules, and biopolymers in the
nanoemulsions.
Color
The evaluation of color during storage at different temperatures is
important to evaluate how it behaves during storage and how a pos-
sible application to some food may influence the color. Table 4 pre-
sents the color values at different temperatures of the nanoemulsion
samples on different storage periods.
In relation to factorial statistics, it was observed that there
was no significant statistical effect of the triple interactions be-
tween the factors (days, temperatures and treatments, p  > .05)
on all color parameters. In relation to double interactions, a sig-
nificant statistical effect of the interaction between temperature
and treatment for the L* (p  = .0127 < .05), b* (p  = .0041 < .05),
C* (p = .0047 < .05), and TCD (p = .0020 < .05) parameters was
|
      13 of 16
observed. In addition, the parameters b* (p  = .0028 < .05) and
C* (p = .0031 < .05) presented a significant statistical effect be-
tween the days and treatment. All individual factors had a signif-
icant statistical effect (days: p = .0039; temperatures: p = .0004;
treatment: p  = .0001 < 0.05) on L*. For chroma a*, only the in-
dividual factor days (p  = .0378 < .05) reports a significant sta-
tistical effect. The treatment and days individuals factors had a
significant statistical effect on chroma b* (days: p = .0037; treat-
ment: p = .0001 < .05) and chroma C* (days: p = .0035; treatment:
p  = .0001 < .05). And the treatment and temperature individual
factors had a significant statistical effect (temperature: p = .0001;
treatment: p = .0000 < .05) on the TCD parameter.
Nanoemulsions presented an opaque-whitish appearance during
initial storage. The decrease in the luminosity (L*) of nanoemulsions
has been caused by the chia mucilage which has a darker coloration
when rehydrated (Fernandes & Salas-Mellado, 2018). After 28 days
of storage, the nanoemulsions showed a lighter color, indicated by
the increase of luminosity (L*). Changes in the color of nanoemul-
sions can be promoted by lipid oxidation. The evaluated nanoemul-
sions showed positive values for parameter b*, which indicated a
yellow contribution to color, and positive values for parameter a*,
which indicated a red contribution to color, also coinciding with their
appearance. Color instrumental measurements suggested that there
was some change in the chemical stability of nanoemulsions during
storage, with a progressive increase in b* (yellowing) over time for
T4 nanoemulsion and at 4°C and 25°C storage temperatures for T4E
nanoemulsion. TCD values varied according to storage time, with
the highest values after 28 days of storage. The temperature of −18
ºC promoted in the two nanoemulsion formulations tested a bigger
color difference.
4 | CO N C LU S I O N
Chia oil was encapsulated by nanoemulsion, and the use of ethanol
as auxiliary and chia mucilage as wall material was evaluated. The
treatments with (T4E) and without (T4) ethanol, with 0.375 g of mu-
cilage and 0.233 ml of chia oil, presented suitable particle size, poly-
dispersity index, and zeta potential, high encapsulation efficiency,
and stability of agitation. These treatments presented techno-func-
tional properties, with the stability of phase separation up to 90°C
with better stability at low temperatures in nanoemulsion with etha-
nol (T4E) and high temperatures in nanoemulsion without ethanol
(T4). Besides, these treatments presented similar spherical unglued
organization inside the particles, and low syneresis values at 4 ºC
during storage.
The results obtained in this study indicate that chia oil na-
noemulsions with chia mucilage and with or without ethanol can
be used in food production to maintain the properties of chia oil
and provide more nutritious foods. Considering all the character-
istics evaluated, the T4E nanoemulsion would be more suitable for
lower temperature processing foods, such as ice cream, and the
T4 nanoemulsion would be more suitable for application in dairy
 |
14 of 16       FERNANDES et al.
products, such as yogurt, in order to maintain the functional prop-
erties of chia oil.
AC K N OW L E D G M E N T S
This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) -
Finance Code 001. The authors would like to thanks Universidad
Autonoma de Yucatán; to LANNBIO Cinvestav Mérida, projects
FOMIX-Yucatán 2008-108160 CONACYT LAB-2009-01-123913,
188345, 204822, 292692, 294643, 299083; to “Centro de
Microscopia Eletrônica do Sul” (CEME-SUL) FURG/ Brazil for the
TEM analysis and to the project CYTED 119RT0567.
C O N FL I C T O F I N T E R E S T
The authors have declared no conflicts of interest for this article.
DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the
supplementary material of this article.
ORCID
Sibele Santos Fernandes  https://orcid.org/0000-0002-4698-8865
Juan Carlos Cuevas Bernardino  https://orcid.
org/0000-0002-2830-8280
Patricia Quintana Owen  https://orcid.org/0000-0002-8727-7605
Carlos Prentice  https://orcid.org/0000-0003-4679-3870
Myriam de las Mercedes Salas-Mellado  https://orcid.
org/0000-0002-8153-2011
Maira Rubi Segura-Campos  https://orcid.
org/0000-0002-7664-6647
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S U P P O R T I N G I N FO R M AT I O N
Additional Supporting Information may be found online in the
Supporting Information section.
How to cite this article: Fernandes SS, Bernardino JCC, Owen
PQ, Prentice C, Salas-Mellado MDLM, Segura-Campos MR.
Effect of the use of ethanol and chia mucilage on the
obtainment and techno-functional properties of chia oil
nanoemulsions. J Food Process Preserv. 2021;45:e15181.
https://doi.org/10.1111/jfpp.15181