Food Hydrocolloids xxx (2018) xxx-xxx

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Food Hydrocolloids

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journal homepage: www.elsevier.com

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Pickering emulsions stabilized with native and lauroylated amaranth starch
Everth J. Leal-Castañedaa, Yunia García-Tejedaa, Humberto Hernández-Sáncheza, Liliana Alamilla-Beltrána,
Darío I. Tellez-Medinaa, Georgina Calderón-Domíngueza, Hugo S. García-Galindob, Gustavo F. Gutiérrez-Lópeza, ∗
a
Graduados e Investigación en Alimentos, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación del Carpio y Plan de Ayala s/n, Col. Santo

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Tomás, 11340 Ciudad de México, Mexico
b
Unidad de Investigación y Desarrollo de Alimentos, Instituto Tecnológico de Veracruz, M.A. de Quevedo 2779, Col. Formando Hogar, Veracruz, Ver. 91897, Mexico

ARTICLE INFO ABSTRACT

Article history: Pickering emulsions were prepared by using native and modified (esterification with lauroyl chloride) ama-
Received 29 August 2017 ranth starches as stabilizing particles of an oil phase formed with a mixture of canola oil and α-tocopherol

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Received in revised form 16 December 2017 (1% v/v). The effect of different concentrations of native (NS) and modified starch (MS) ranging from 2 to
Accepted 30 January 2018
30 %wt was evaluated in the emulsions regarding average droplet size, emulsion stability, zeta potential (ζ)
Available online xxx
and emulsification index. Optical microscopy showed that both NS and MS were adsorbed at the oil-water
interface forming a barrier that delayed phase separation. The MS gave place to a smaller emulsion droplet
Keywords: size than NS, and no phase separation was detected for the emulsions prepared by using 20 and 30 % wt of
Pickering emulsions NS or MS, producing stable emulsions.
Amaranth starch © 2017.
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Lauroylated amaranth starch
Emulsion stability
Lauroylation
Modified starches

1. Introduction bility of the particles which allows their accumulation at the oil-water
interface (Marto, Ascenso, Simoes, Almeida, & Ribeiro, 2016). Edible
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Solid-stabilized emulsions, also known as Pickering emulsions
(Pickering, 1907), have been recently applied in food related fields
given their potential applications (Rayner et al., 2014). Many food
products are formed of immiscible phases, so that an emulsifier makes
it possible to form a homogeneous mixture, i.e., an emulsion which
may be a suitable option for the delivering of a number of com-
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particles replacing synthetic surfactants include: colloidal celluloses
and various cellulose derivatives (Duffus, Norton, Smith, Norton, &
Spyropoulos, 2016), proteins (Gao, Wang, Yue, Xiong, Wu, Qiao, &
Liao, 2017), solid lipid nanoparticles and fat crystals (Weiss et al.,
2008), chitin nanoparticles (Tzoumaki, Moschakis, Kiosseoglou, &
Biliaderis, 2011), starch granules from different sources (Li, Sun, &

pounds, including vitamins. In particular, α -tocopherol is the most Yang, 2013; Marefati, Bertrand, Sjöö, Dejmek, & Rayner, 2017), and
active form of vitamin E which has a potent antioxidant capacity starch derivatives (Haaj, Thielemans, Magnin, & Boufi, 2014).
(Wysota, Michael, Hiew, Dawson, & Rajabally, 2017) and the prepa- As a potential biopolymer for Pickering emulsions, starch has re-
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ration of emulsions containing this vitamin has been reported using
different polymers as wall materials (Granillo et al., 2017). On the
other hand, a recommended dose of α-tocopherol of 22.4 IU/day has
been reported (Wysota et al., 2017) equivalent to 15 mg/day for adults.
This amount may be the basis to prepare Pickering emulsions con-
taining this vitamin, using a suitable vehicle such as vegetable oil in
which it is soluble. Pickering emulsions are stabilized by edible solid
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ceived increased attention and different starch sources have been eval-
uated for their capacity to prepare Pickering emulsions, including:
waxy maize, wheat, potato and rice. Li, Li, Sun, and Yang (2013) re-
ported that starches having small granules (i.e., rice starch) better sta-
bilize Pickering emulsions. Moreover, native starches enhance their
performance in Pickering emulsions by means of esterification such as
the hydrophobization with octenyl succinic anhydride (n-OSA) which

particles instead of the commonly used surfactants. Stabilization of
emulsion droplets by solid particles is attributed to the dual wetta
Abbreviations: NS, native starch; MS, modified starch; NSE, native starch emul-
sions; MSE, modified starch emulsions; PDI, polydispersity index; ζ, zeta-poten-
tial; Global TSI, global Turbiscan stability index; EI, stability index; DS, degree of
substitution
∗
Corresponding author.
Email address: gusfgl@gmail.com (G.F. Gutiérrez-López)
confers the starch molecule with amphiphilic properties (Bormann,
Pierucci, Leite, & Miguez da Rocha, 2013).
The amphiphilic nature of starches obtained by means of hy-
drophobic modifications makes them attractive in a wide and in-
teresting spectrum of applications. For instance, as rheology modi-
fiers, emulsion stabilizers, surface modifiers and drug delivery ve-
hicles among others (Namazi & Dadkhah, 2010). Timgren, Rayner,
Dejmek, Marku, and Sjöö (2012) studied n-OSA-starches from dif-
ferent sources as potential stabilizers of Pickering emulsions; they

https://doi.org/10.1016/j.foodhyd.2018.01.043
0268-005/© 2017.
2 Food Hydrocolloids xxx (2018) xxx-xxx

observed that the size of the granule had a big influence on the sta-
bilizing capacity, and in particular, the smallest starch granules from
quinoa had the best emulsifying properties. The chemical modifica-
tion of starch by lauroyl chloride produces amphiphilic macromole-
cules consisting mainly of a hydrophilic backbone and hydrophobic
side chains (Namazi, Fathi, & Dadkhah, 2011). These hydrophobic
2.2. Isolation of starch
Starch was isolated from amaranth seeds, based on the method
reported by Rayner, Timgren, Sjöö, and Dejmek (2012), for quinoa
starch isolation. Amaranth seeds were immersed in distilled water dur-
ing 24 h at 4 °C, then the seeds were blended with distilled water (wa-

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side chains have a greater affinity for the dispersed phase than the ter:seeds ratio 1:1). The blended seeds pulp was sieved through a
native starch, giving place to high-energy interactions at the oil-par- cloth, and the obtained paste was then washed with distilled water for

ticle interface that might be a good alternative for stabilizing emul-
sions (Rayner et al., 2014). Recently, Marefati et al. (2017) reported
the preparation of Pickering emulsions with native and modified ama-
ranth starch with different degrees of substitution (DS) of OSA. These
authors demonstrated that DS and the type of granule influenced the
size of the droplet. A number of works suggest that it is possible to
create a Pickering emulsion with the n-OSA-starch (Agama & Bello,
2017; Marefati et al., 2017; Saari, Heravifar, Rayner, Wahlgren, &
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at least three times until the washing water was clean. The filtrate,
which contained the starch suspension, was centrifuged at 3000 × g
during 10 min and the supernatant was removed. Proteins were elim-
inated by washing the starch twice with an alkaline solution (NaOH,
0.3%). Finally, the starch was dried at 40 °C for 24 h in a laboratory
oven (SMO1E, Shel-Lab, USA).

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2.3. Lauroylation reaction
Sjöö, 2016), but the potential use of starches esterified with fatty acid
chlorides has not yet been reported. The use of fatty acid chlorides to
Starch esterification was carried out by following the method of
modify different types of starch has a number of advantages in com-
Namazi et al. (2011). A 50 g sample of starch was dispersed in 500 mL
parison with other classic methods of modification, such as the use of
of NaOH 0.1 M at 25 °C during 10 min; then, lauroyl chloride (1.5 mL/
water instead of organic solvents as the media for the reaction. Re-
g of starch) was added drop by drop to the starch dispersion under stir-
action time is relatively short and the hydrophobic modified products
ring conditions by using a magnetic stirrer at 300 rpm at room tempera-
can precipitate in water and may be separated without the addition of
ture. After the esterification process, the slurry was centrifuged during

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solvents (Namazi et al., 2011); besides, the byproducts of the reaction
10 min at 3000 × g and the lauroylated starch was recovered as the sed-
are non-toxic (water and NaCl). Lauroyl chloride, is a medium-size
iment from the tubes. After centrifugation, the lauroylated starch was
chain fatty acid that has proven to be a good candidate for chemical
washed three times with methanol and once with distilled water, then,
modifications of starches, and that allows achieving high degrees of
it was centrifuged and dried in a convection oven (SMO1E, Shel-Lab,
substitution with high yields as compared to the rest of the short and
USA) at 40 °C during 24 h. The dried starch was milled in a Hamil-
large chain fatty acid chlorides (Namazi et al., 2011; Vanmarcke et
ton Beach mill model 80,350 and sieved thorough a mesh having a
al., 2017). Moreover, the Food and Drug Administration (FDA) does
0.149 mm of opening size.
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not have published restrictions for lauric acid chloride regarding the
maximum amount that can be used in different food applications. Lau-
royl chloride has been used for the modification of alginate to which 2.4. Qualitative determination of the modification of starch
antimicrobial characteristics have been attributed. The product is cur-
rently patented as Na-lauroyl arginate ethylester, LAE®, which is suit- Qualitative determination of the modification of starch was car-
able for human consumption and was regarded as GRAS by the Food ried out by means of Fourier transformation infra-red (FTIR) spec-
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and Drug Administration (FDA) and by the European Food Safety
Authority (EFSA) in 2007 (Coronel-León et al., 2016). In this study,
amaranth starch was selected as stabilizing material of the emulsions
because it forms one of the smallest starch granules found in nature,
typically 1–3 μm in diameter (Middlewood & Carson, 2012; Xia et al.,
2015), which suggests that native and modified amaranth starch (es-
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troscopy aiming to demonstrate that the modification of the starch
had been carried out. The equipment used was a FTIR module IR2
equipped with an Indium Gallium Arsenide (InGaAs) detector, cou-
pled to a Jobin-Yvon LabRam HR800 spectrometer (Horiba, Kioto,
Japan). The starch samples were placed in a holder and analyzed over
a wave number range between 4000 and 450 m−1 with a spectral res-
olution of 4 cm−1 and performing 36 scans per measurement, using an

terified with lauroyl chloride) may be good options for the preparation
of Pickering emulsions. ATR contact objective.
The aim of this work was to prepare and characterize oil in water
(O/W) Pickering emulsions using native and modified (lauroylation)
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2.5. Degree of substitution (DS)

amaranth starches to stabilize an oil phase formed by α-tocopherol and
canola oil as the vehicle to supply this vitamin.
The degree of substitution (DS) was determined according to the
method of Jeon, City, Viswanathan, Lowell, and Gross (1999). A 1 g
2. Materials and methods
sample from each starch (NS and MS) was dissolved in 10 mL of di-
methyl sulfoxide during 10 min at 70 °C, left to cool at ambient tem-
2.1. Materials
perature (25 °C) and five drops of phenolphthalein were added. Then,
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the obtained solution was titrated with a standard solution of 0.05 M of

Seeds of amaranth (Amaranthus hypochondriacus) were obtained
sodium hydroxide until a pale pink color was obtained. DS was calcu-
in the Xochimilco, Mexico City market, α-tocopherol, lauroyl chlo-
lated with the equation reported by Cao, Song, Deng, and Ragauskas
ride, 1, 2-propanediol (99%), Nile Blue and Nile Red, were acquired
(2011):
from Sigma-Aldrich (Mexico City). Sodium hydroxide 98% and
methanol were acquired from Luzeren Laboratories (Mexico City).
The canola oil was acquired from Mazola (México City). Ultra-pure
water with a resistivity of 18.25 MΩ⋅cm was used throughout the ex-
periment.
(1)
 Food Hydrocolloids xxx (2018) xxx-xxx 3

Where A is the titrated volume of the standard solution of NaOH in
mL; M is the molarity of the NaOH solution; W is the dry weight of
the esterified starch.
2.6. Particle size measurements of starch granules
used to determine the absorbance at 595 nm in a Thermo Scientific
spectrophotometer Nanodrop 2000, (Wilmington, USA).
2.9. Preparation of the pickering emulsions

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A high amount of water (70%) was used in respect to the added oil
Particle size distributions (PS) and polydispersity index (PDI) of phase (30 %v) which was prepared as a mixture of canola oil and α-to-
native and modified starch granules were measured by placing 1 mL copherol (99:1). Emulsification of canola oil and α-tocopherol (99:1)

of a water suspension of particles at a concentration of 0.01 %wt, in a
Malvern Zetasizer Nano ZS (Malvern, USA).
2.7. Scanning electron microscopy (SEM)
The morphology of starch granules was observed in a Field Emis-
sion Scanning Electron Microscope JSM-7800F (JEOL, JAPAN), us-
ing an acceleration voltage of 5 kV. The starch samples were fixed in
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in water with the aid of starch granules were formulated as shown in
Table 1. Starch dispersions were prepared in glass test tubes by using
native or lauroylated amaranth starches at concentrations of 2, 6, 10,
20 and 30 % wt, and each one containing 7 mL of ultra-pure water and
3 mL of oil. The mixtures were mixed at 11,000 rpm for 1 min by using
a homogenizer IKA T18 Basic Ultra-Turrax at 25 °C.

stubs containing a double-faced adhesive metallic tape and they were
cover with gold before fixing them to the microscope stage.
2.8. Chemical characterization of starch
Starch purity was measured as the total starch content based on the
heat-stable α-amylase and amyloglucosidase method, for which the to-
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2.10. Droplet size by optical microscopy
Optical microscopy of the Pickering emulsions was performed in
an Eclipse H550S (Nikon, Chiyoda-ku, Japan) optical microscope
equipped with a Kodak DC 120 digital camera (Sevier County, Ten-
nessee, USA). The size of the emulsion droplets was obtained by plac-
ing a drop of the emulsion between a channeled and a cover slides and
capturing images of the emulsions as reported by Li et al. (2013), con-

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tal starch measurement kit from Megazyme International Ireland Ltd sidering 200 droplets for each emulsion. Image processing was car-
(Bray Business Park, Ireland) was employed. Procedure A was fol- ried out by using the ImageJ v 1.49 software (Rasband, 1997–2015).
lowed for samples not containing resistant starch. The surface to volume diameter (d32d) and the volume mean diameter
The amylose content (% dry basis) of the starch was determined by (d43d) of the droplets were calculated by applying Eqs. (2) and (3) (Li
the method reported by Williams, Kuzina, and Hlynka (1970), which et al., 2013). Also, the polydispersity index (PDI) was obtained by us-
is based on the reaction of iodine with amylopectin in which a char- ing Eq. (4).
acteristic color is obtained. The concentration of amylose was ob-
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tained by interpolating in a standard curve generated by using different
concentrations of amylose standard (Sigma-Aldrich). 5 mL of a 0.5 N
KOH solution were added to 20 mg of starch (20 mg). Subsequently, (2)
this solution was diluted with 100 mL of distilled water and 10 mL of
this solution were transferred to a 50 mL volumetric flask to which
5 mL of 0.5 N HCl and 0.5 mL of iodine solution were added and total
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volume completed by adding distilled water. The absorbance was read

at 625 nm after 5 min. In parallel, a blank was run without the starch (3)
and used as a reference. Amylopectin content was calculated by sub-
tracting amylose from the total starch content.
Total protein content of native and modified starch was deter-
mined by Bradford (Thermo Scientific). Starch Solutions were pre-
(4)
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pared at a concentration of 0.2% (w/v), then a 10 μL aliquot was

added with 10 μL of the Bradford reagent. The mixture was cen-
trifuged at 3000 rpm for 5 min, and after 1 min, a 2 μL sample was In which di is the diameter of a droplet, N is the total number of
droplets.
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Table 1
Size parameters of droplets (d32, d43), Polydispersity Index, ζ, Global Turbiscan Stability Index and Emulsification index of emulsions at different concentrations of starch.

Concentration of starch in the emulsion Area (μm2) Perimeter (μm) d32 (μm) d43 (μm) PDI ζ (mV) Global TSI EI
a a a a
NS-2%wt 1041.0 ± 213.4 124.9 ± 13.6 40.0 40.5 1.04 −19.2 ± 0.7 16.79 ± 1.4 0.8 ± 0.2a
NS-6%wt 560.3 ± 105.6b 101.3 ± 11.2b 28.7 28.9 1.02 −20.7 ± 0.5a 11.02 ± 2.8b 0.9 ± 0.2a
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NS-10%wt 147.5 ± 29.5c 49.6 ± 5.1b 15.3 15.4 1.02 −21.2 ± 0.7a 3.99 ± 0.64c 1.0 ± 0.1a
NS-20%wt 110.8 ± 22.7c 40.6 ± 9.2b 10.1 10.2 1.05 −22.3 ± 0.9a 1.39 ± 0.13d 1.0 ± 0.1a
NS-30%wt 57.0 ± 14.4d 33.6 ± 5.0b 9.90 10.0 1.04 −24.5 ± 0.1a 2.40 ± 0.35c 1.0 ± 0.0a
MS-2%wt 53.3 ± 13.8d 28.9 ± 4.7b 8.90 9.10 1.05 −12.2 ± 0.5b 11.26 ± 0.8b 0.7±.1a
MS-6%wt 45.4 ± 12.3d 26.2 ± 3.9b 8.30 8.50 1.08 −11.0 ± 0.7b 10.360 ± 1.0b 0.8 ± 0.1a
MS-10%wt 39.3 ± 10.3d 24.0 ± 3.2b 7.70 7.80 1.05 −10.1 ± 0.3b 4.63 ± 0.7c 0.9 ± 0.1a
MS-20%wt 37.8 ± 7.7d 24.1 ± 2.7b 7.50 7.70 1.03 −9.1 ± 0.2b 3.14 ± 0.7c 1.0 ± 0.1a
MS-30%wt 27.5 ± 5.3e 24.4 ± 4.1b 6.80 6.90 1.06 −4.9 ± 0.3c 3.73 ± 0.5c 1.0 ± 0.0a
Each value in the last three columns represents the mean ± standard deviation of three replicates. Different lowercase letters within the same row denote significant differences (≤0.05)
different starch concentration and different type to starch.
4 Food Hydrocolloids xxx (2018) xxx-xxx

2.11. Confocal laser scanning microscopy (CLSM)

(6)
A Confocal Laser Scanning Microscope (CLSM) (LSM 710 NL0
Carl Zeiss, Germany) was used to visualize the structure of the Picker-
Where Vemuls is the volume occupied by the emulsion, and Vtotal is the
ing emulsion droplets. Nile Blue dye was used to stain the native and
total volume of all phases (oil phase, added starch, and water).
lauroylated amaranth starches (Yusoff & Murray, 2011) in an aque-

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ous solution and Nile Red dye was used to stain the oil phase (Yusoff
2.14. Statistical analysis
& Murray, 2011). Pickering emulsions were then prepared by mixing

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stained Nile Blue and Nile Red amaranth and oil samples respectively,
Data were reported as mean values of three replicates for each ana-
as described in Section 2.9. Then, the emulsions were immediately
lytical determination. One-way ANOVA and Tukey's significant mul-
placed between two cover glasses for observation in the CLSM.
tiple comparison test were used to determine statistical differences be-
The CLSM was operated in fluorescence mode with an Apochro-
tween treatments (p ≤ 0.05).
mat- Plan objective (63×/1.4 Oil DIC M27) at 60×/1.4. The fluores-
cent dyes were excited by using an argon laser at 488 nm for Nile
3. Results and discussion
Red or a helium neon (He-Ne) laser at 633 nm for Nile Blue. The pro-
jected area of the droplets was evaluated using the software ImageJ

v 1.49 software (Rasband, 1997–2015), applying the method reported
by Garcia-Armenta et al. (2016).
2.12. ζ of starch particles
To determine ζ to the NS and MS starch particles, they were dis-
persed (0.01 wt%) in water and measured by using a Zetasizer ZS
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3.1. Characterization of starches
3.1.1. Chemical characterization of starch
The obtained amylose contents of NS and MS were 22.3 ± 0.3 and
21.2 ± 0.1 as %dry basis, respectively. Recently, Marefati et al. (2017)
reported 20.95% of amylose content for amaranth hypocondriacus
starch, native from Mexico. Esterification did not generate significant
differences in the amylose content. Similar results were reported by

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(Malvern, USA) at 25 °C.
2.13. Stability kinetics of the emulsion and emulsifying capacities of
the starches
Stability of the emulsions with time was analyzed by light scat-
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tering in a Turbiscan Lab Expert (Formulation, Toulouse, France).
The detection head is composed of a pulsed near-infrared light source
(λ = 880 nm) and two synchronous detectors. Stability of the emulsions
with time was analyzed by light scattering in a Turbiscan Lab Expert
(Formulation, Toulouse, France) by evaluating the Turbiscan Stabil-
ity Index (TSI). The detection head of the equipment is composed of
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He, Song, Ruan, and Chen (2006) for different varieties of rice starch
modified with n-OSA. The authors obtained protein contents of 4.9
and 3.2 as % dry basis for NS and MS, respectively. Similar results
were also reported by Marefati et al. (2017) for amaranth starch mod-
ified with n-OSA at different DS for which the protein content con-
comitantly decreased with esterification: 0.112 to 0.032 for native and
modified starch, respectively. Reported esterification with fatty acid
chlorides and n-OSA were carried out in alkaline media which gener-
ated a release of the protein during chemical modification (Rayner et
al., 2012). On the other hand, total starch contents were 86% ± 0.9 and
90% ± 1.1 for NS and MS, respectively.
3.1.2. Analysis of lauroylated starch by FTIR

a pulsed near-infrared light source (λ = 880 nm) and two synchronous

detectors. The calculation of this index was carried out with equation The FTIR spectra of amaranth starch and lauroylated starch are
(5): shown in Fig. 1. The native amaranth starch presented its characteris
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(5)
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Where xi is the average backscatter for each minute of the measure-

ment, xbs is the mean value of xi and n is the number of scans. TSI
considers all the variations detected in the samples in terms of size
and/or concentration with time so that it allows to infer on the stability
of the sample and compare among different systems. The higher the
TSI value, the less stable the emulsion is (Kang et al., 2011). A 25 mL
sample of the emulsion was poured into a glass cell and placed in the
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equipment to obtain the stability kinetics during 6 h and the Global

Turbiscan Stability Index (Global TSI) of the emulsion at the end of
the experiment is then evaluated as the average value of the individu-
als TSI: at the bottom, in the middle and in the top sections of the cell.
The emulsifying capacities of the starches were determined one
day after their preparation as the emulsion index (EI) (Saari et al.,
2016) as follows:
Fig. 1. The FT-IR spectra of native (a) and lauroylated (b) amaranth starches. Numbers
in the spectra are the wavenumbers in the corresponding axis.
 Food Hydrocolloids xxx (2018) xxx-xxx
tic fingerprint region (Fang, Fowler, Sayers, & Williams, 2004), ad-
ditionally, the absorption band for NS at 1544 cm−1 was associated
to a residual protein amide II. These absorption band has been re-
ported for amaranth protein (Aceituno-Medina, Mendoza, Lagaron, &
López-Rubio, 2015). After esterification, lauroylated amaranth starch
showed a characteristic band of the carbonyl group at 1624 cm−1 of
the fatty ester. In addition, the C-H stretching vibrations of the alkyl
groups of the fatty ester chain were clearly present at 2920 and
2833 cm−1. The decrease of hydroxyl vibrations in the range of
3000–3600 cm−1 and at 1624 cm−1 suggested the esterification of hy-
droxyl groups of starch (Namazi et al., 2011). In this work, FTIR was
used as a first step for determining if chemical modification of starch
had occurred. The analysis of FTIR has not been regarded as a reli-
able technique to quantitatively evaluate DS (Freire, Silvestre, Neto,
& Rocha, 2005).
3.1.3. Determination of the degree of substitution of lauroylated
starch
Lauroylated amaranth starches have not been employed as Pick-
ering emulsion stabilizers before. It has been reported that the incor-
poration of hydrophobic groups to the amylose, results in surface ac-
tive properties, it is believed that functional groups are located mainly
at the surface of the granules (Whitney, Reuhs, Ovando, & Simsek,
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2016.), which is useful for stabilizing emulsions. The properties of es-
terified amaranth starch depend on the degree of substitution (DS). In
this work, the DS obtained for lauroylated amaranth starch was 0.01.
Variables such as the source of the starch, length of the fatty acid chain
and type of chemical modification carried out have an influence on
DS. Fang, Fowler, Tomkinson, and Hill (2002) chemically modified
four starches (corn, Hylon VII, Hylon, Amioca) and reported that the
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esterification reaction was successful only with acid chlorides contain-
ing 6–10 carbon chains. Longer and shorter fatty acids chains were hy-
drolyzed due to the conditions of reaction. Namazi et al. (2013) carried
out the esterification reaction of potato and corn starch with longer
carbon chain fatty acid chlorides with some modifications with respect
to the work by Fang et al. (2002) such as a lower NaOH concentration,
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temperature and reaction time. These authors reported a lower DS for
the modification with lauryl chloride than for octenyl chloride (0.35
and 0.48, respectively) and even lower for palmitoyl chloride (0.11)
and corn starch. Gao et al. (2014) esterified corn starch with lauric acid
using lipase as a catalyst and reported similar DS (0.0109 and 0.0151)
than those obtained in our work for two different concentrations of
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lauric acid with an amylose content of 47.31 and 48.69%, respectively
and using the same technique reported in our work. Morphology and
size of native and modified starches.
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3.1.4. Morphology and size of native and modified starches
Granule size plays an important role in the formation of an emul-
sion, it has been reported that small granules with smooth surfaces,
have a better contact in the oil-water interface (Saari et al., 2016).
The shapes and size of starch granules are dependent on their source.
Scanning electron micrographs of native starch are shown in Fig. 2,
in which it is possible to observe granules with a polyhedral morphol-
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ogy; the size of the granules ranged from 0.6 to 1.7 μm and the av-
erage particle size was 1.13 μm. After modification, the size of ob-
tained granules was 0.6–1.2 μm with an average size of 1.09 μm and
PDI found were larger for NS than MS (0.87 and 0.49, respectively.
Jackson, Choto-Owen, Waniska, and Rooney (1988) reported that
sodium hydroxide could depolymerize starch, generating a smaller di-
ameter of the starch granule. Rayner et al. (2012) and Wang et al.
(2014) found that NaOH gave place to proteins removal from the sur
5
face of the granule which could reduce its size (as reported by us).
In our work, protein content decreased by 35% (4.9 and 3.2 for NS
and MS, respectively). The damage generated by the chemical modi-
fication depends on many factors, such as the source of the starch, the
type of modification made, the media of reaction, the extent of agita-
tion, among others (Namazi et al., 2010, 2011). Chemical conditions
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at modification may affect the amorphous region of starch granules by
exposing the -OH groups of the amylose which in turn as are exposed
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to the alkaline media that damage the granule surface (Wang et al.,
2014). Extreme erosion of the amorphous region can practically dis-
appear such region so giving place to crystals as reported by Li et al.
(2013) who reported that emulsions can be stabilized by starch crys-
tals. The surface of the modified granule, unlike the native, showed a
smoother and damaged surface, while the native starch had a polygo-
nal shape and a rougher surface. These morphometric changes might
be attributed to the chemical modification although, to the best of our
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knowledge, there is no evidence of the morphometry of starch gran-
ules modified with fatty acid chlorides. Song, He, Ruan, and Chen
(2006) reported surface damage of rice starch granules after treatment
with OSA. In this case, native rice starch granules were polygonal
with well-defined edges, much like the lauroylated starch granules
of amaranth. Starch OSA granules exhibited rough surfaces and their
edges lost definition. Pores and cavities were generated on the granule
surface. Gao et al. (2014) reported no obvious differences in the shape
of waxy corn starch granules after lipase esterification and only slight
morphometric changes could be appreciated on the surface of esteri-
fied starch granules as compared to that of the native starch. Esterified
starch granules exhibited slightly rough surfaces with appearance of
apophysis and pores. Marefati et al. (2017), on the other hand, did not
report significant changes in the diameter of amaranth starch granules
modified with different OSA concentrations.
3.2. Characterization of emulsions
3.2.1. Droplet size of pickering emulsions
Two key factors influencing the emulsion droplet size during Pick-
ering emulsion preparation process are: (i) the concentration of starch
particles initially dispersed in the aqueous phase and (ii) the amount
of oil phase (Frelichowska, Bolzinger, & Chevalier, 2010). The influ-
ence of starch concentration in the water phase was assessed by mea-
surements of droplet size distributions over a wide range of starch con-
tent. Pickering stabilization does not require the complete coverage
of closely packed particles in the interface (Dickinson, 2013). Emul-
sions prepared at starch concentrations lower than 10% wt (Table 1)
were unsuccessful. In this case, the surface coating by solid particles
was not large enough to form a compact layer in the oil-water inter-
face and coalescence occurred, which could be observed by optical
microscopy. On the other hand, efficient stabilization against coales-
cence was achieved with 20 and 30% wt of starch for both NS and
MS. Pickering emulsion droplets prepared at the following starch con-
centrations: NS-2%wt, NS-2% wt, NS-30% wt and WS-30% wt are
shown in Fig. 3. The droplets are conformed by an oil core coated
by granules of starch. In the micrographs, it is difficult to observe the
granules of starch because of their small size. This observation has
also been reported by Rayner et al. (2012). At NS-2% wt, only a few
starch granules on the surface of droplets can be seen (Fig. 3a). As
the solids concentration increased from 2% wt to 30 %wt, larger parti-
cles were observed in Fig. 3c, the droplet interface was more densely
coated with particles at 30% wt than at 2%wt for both NSE and MSE.
Average size of individual droplets was related to the concentra-
tion of the solid particles forming the emulsions shown in Fig. 3. As
6 Food Hydrocolloids xxx (2018) xxx-xxx
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Fig. 2. Particle size distribution of native (NS) and lauroylated (MS) starches granules (top) and scanning electron microscopy images of a) native and b) modified amaranth starch
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granules (bottom). Scale bar = 1 μm.
the amaranth starch concentration increased, the droplet size of Pick-
ering emulsions decreased from 40.0 to 9.8 μm and from 8.9 to 6.8 μm
for NS and MS respectively. Authors have shown that the average size
of the droplet increases at short times after preparation of the emulsion
and quickly reaches a limit value that depends on the amount of solid
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particles. It is likely that the studied emulsions showed limited coa-
lescence (Arditty, Whitby, Binks, & Schmitt, 2003). When concentra-
tion of particles was low they did not adsorb on the oil surface in suf-
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ficient amount to stabilize the emulsion, larger droplets were formed
which had sufficient amount of particles to stabilize them as compared
with droplets stabilized with larger amount of particles in which lim-
ited coalescence gave place to smaller droplets (see Table 1) (Binks
& Olusanya, 2017; Leal-Calderon & Schmitt, 2008; Tzoumaki et al.,
2011). When comparing the droplet size of the NS and MS, it was ob-
served that MS emulsions had a smaller droplet size than NS. Marefati
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et al. (2017) reported an increment in the emulsifying capacity of mod-
ified amaranth starch with OSA, which generated an increase in the
thickness of the emulsion starch layer as the modification level in-
creased. Also, Saari et al. (2016) reported droplet diameters of 13.4 μm
and 4.7 μm, when using 200 and 1200 mg of n-OSA quinoa starch/mL
of oil, respectively.
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3.2.2. Laser scanning confocal microscopy
In Fig. 4a and d, the CLSM images of NS and MS stabilized emul-
sions are shown; blue regions correspond to amaranth starch and red
areas are those of the oil phase. Upon separation of the blue and red
channels, it was possible to observe that droplets of emulsion prepared
with NS (Fig. 4c) had a lower coverage of the oil phase in respect to
those prepared with MS (Fig. 4f). Oil phases in both cases are shown
in Fig. 4b and e for NS and MS emulsions respectively. All droplets
in Fig. 4 have an average thickness which is dependent on the diam-
eter of the adsorbed particles. Additionally, according to Dickinson
(2013) due to the increased hydrophobicity of the modified particles,
they might tend to aggregate with each other in the aqueous phase so
that sometimes, aggregates adsorb on the oil surface rather than indi-
vidual particles giving place to local larger thicknesses in the sites of
adsorption.
3.2.3. Stability of emulsions determined by backscattering
Pickering emulsions usually present phase and reverse sedimen-
tation (Rayner et al., 2014). For a conventional emulsion, this type
of phenomena is considered as destabilizing, which significantly af-
fects the physical properties of the emulsion. However, for Pickering
emulsions, sedimentation, flocculation and Ostwald maturation are
not considered important factors that modify physical properties since
they are commonly found in these types of emulsions and in which
 Food Hydrocolloids xxx (2018) xxx-xxx 7

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Fig. 3. Amaranth starch granule stabilized Pickering emulsions (optic microscopy image 40× magnification) at different concentrations a) 2 % wt of NS, b) 2 % wt of MS, c) 30 %
wt of NS and d) 30 % wt of MS. Scale bar = 10 μm.
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Fig. 4. CLSM micrographs of NSE prepared with 20%wt of starch (a, b and c) and of MSE prepared with 20 % wt of starch (d, e and f).
8 Food Hydrocolloids xxx (2018) xxx-xxx
coalescence has to be avoided (Rayner et al., 2014). The backscatter-
ing profile of emulsions stabilized by NS and MS are shown in Fig. 5.
The X-Axis represents the height of the tube, while Y-axis represents
the percent of change of BS relative to the initial stage. As shown in
Fig. 5, there are few changes in NSE-30% wt and NSE-10% wt, indi-
cating that there is no particle migration in the emulsion. The MS-2%
wt and NS-2% wt emulsion samples, which contain a low concentra-
tion of starch, showed the decrease of the size of the emulsified region
with time. Phase separation was larger for MSE and NSE at 2% wt
starch concentration.
The Global TSI after 6 h of storage under different starch concen-
tration is shown in Table 1. The Global TSI of NSE decreased from
16.8 to 2.4 as starch concentration increased from 2 to 30 % wt indi-
cating increased stability and the Global TSI of MSE decreased from
11.26 to 3.73, as starch concentration increased from 2 to 30 % wt;
which indicated improved stability. The increase of solids content pro-
duced a lower Global TSI after storage, which was attributed to small
sizes of particles that were associated to more stable emulsions. Global
TSI values of 1.39 and 2.4, obtained for NS-20% wt and NS-30% wt
emulsions respectively, indicated the emulsions with the highest sta-
bility with time.
Finally, to compare the effect of the lauroylation of amaranth
and starch concentration in the emulsions, the emulsification index
ED
(EI) evaluated at 6 h after preparation is depicted in Table 1. Ele-
vated starch concentration favored the formation of a higher EI6h in
both types of starch. However, NS produced a greater stability for
the emulsified region (Fig. 5) as from 10% of starch concentration
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Fig. 5. Turbiscan Lab Expert backscattering light graphs at different concentrations of starch: 2, 10 and 30 %wt of NS; and 2, 10 and 30 %wt of MS; and images of the corresponding
emulsions.
(EI6h = 1.0) upwards in comparison with MSE at the same concentra-
tion (EI6h = 0.9). Particle size and its density influenced the EI (Rayner
et al., 2012). Successful Pickering emulsions with different sizes and
thickness of their starch cover were found. The smaller the size of the
particles, the larger the thickness of their starch covers which caused
an eventual droplets flocculation. In this case, high concentrations of
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starch generated droplets with different densities (Rayner et al., 2012),
thus generating a stable emulsion without separation of visible phases
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and with an IE6h = 1 for the concentrations of 20 and 30%wt of native
and modified starches.
4. Conclusions
Food grade Pickering emulsions stabilized by native and lauroy-
lated amaranth starches were prepared. It was found that both, na-
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tive and modified amaranth starches acted as good stabilizers of Pick-
ering emulsions. The starch concentration had an influence on the
droplet size of the emulsion, modified starch generated smaller droplet
size and a greater emulsion stability attributed to the affinity of the
particle with the oil phase. NSE-10% wt, NSE-20% wt, NSE-30%
wt, MSE-20% wt, MSE-30% wt were stable during 6 h. The stabil-
ity studies presented in this work showed that the lauroylated ama-
ranth starch improved emulsifying capacity as compared with the na-
tive amaranth starch because it produced smaller droplet sizes of the
emulsion, slower creaming and a better stabilization of the Pickering
emulsion as compared with emulsions prepared with native starch.
 Food Hydrocolloids xxx (2018) xxx-xxx
Acknowledgments
Author Leal-Castañeda acknowledges the support from CONA-
CYT-México. Thanks go to Center of Nanosciences and micro and
nanotechnologies from IPN and to Ms. Ilse Monroy for help with
formatting references. Authors acknowledge the financial support of
CONACYT-Mexico through the grants (CB-2014-01-242371) and
SIP-20171502.
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://
doi.org/10.1016/j.foodhyd.2018.01.043.
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