Synthesis and characterization of rice starch laurate ...

Carbohydrate Polymers 194 (2018) 177–183

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Synthesis and characterization of rice starch laurate as food-grade emulsifier for canola oil-in-water emulsions☆

T

⁎

Y.V. García-Tejedaa, , E.J. Leal-Castañedaa, V. Espinosa-Solisb, V. Barrera-Figueroac a

Instituto Politécnico Nacional, Av. Wilfrido Massieu Esq. Cda. Miguel Stampa s/n, Gustavo A. Madero, Ciudad de México, C.P. 07738, Mexico Universidad Autónoma de San Luis Potosí, Coordinación Académica Región Huasteca Sur de la UASLP, km 5, Carretera Tamazuchale-San Martín, 79960, Tamazuchale, S.L.P., Mexico c Instituto Politécnico Nacional, Sección de Estudios de Posgrado e Investigación, UPIITA, Avenida Instituto Politécnico Nacional No. 2580, Col Barrio la Laguna Ticomán, Gustavo A. Madero, Ciudad de México, C.P. 07340, Mexico b

A R T I C LE I N FO

A B S T R A C T

Keywords: Hydrolyzed rice starch Starch laurate Oil-in-water emulsion Canola oil

The effect of esterification on hydrolyzed rice starch was analyzed, for this aim rice starch was hydrolyzed and subsequently esterified with lauroyl chloride at three modification levels. Starch derivatives were characterized regarding their degree of substitution (DS), water solubility index, z-potential, gelatinization, and digestibility properties. DS of derivatives of rice starch laurate ranged from 0.042 to 1.86. It was determined that after esterification the water solubility index increased from 3.44 to 53.61%, the z-potential decreased from −3.18 to −11.27, and the content of slowly digestible starch (SDS) decreased from 26.22 to 5.13%. Different emulsions with starch concentrations ranging from 6 to 30 wt% were evaluated. The most stable emulsions were those having 20 and 30 wt% of rice starch laurate.

1. Introduction Canola oil is rich in unsaturated fatty acids, having the best ratio of omega-6 to omega-3 (5.6/1) with respect to other vegetable oils, and provides organoleptic properties to foods as well as health benefits to humans (Monteiro, Souza, Costa, Faria, & Vicente, 2017). There exist potential applications of canola oil in sauces and dressings, since it may improve the lipid profile with an increase in the linolenic fatty acid content. The presence of an emulsifier into an inmiscible o/w mixture makes it possible the incorporation of lipophilic products such as polyunsaturated fatty acids, essential oils and vitamins. Nowadays, there exists a trend in the development of emulsions based on the usage of natural particle stabilizers such as vegetable proteins and polysaccharides (Burgos-Díaz, Wandersleben, Marqués, & Rubilar, 2016). Edible particles from different starch sources have been evaluated for their emulsifying properties, including: waxy maize, wheat, potato, amaranth, and rice starches. The reports show that rice is a good stabilizer of emulsions because its granules have small size and regular shape (Simsek, Ovando-Martinez, Marefati, Sjӧӧ, & Rayner, 2015). Moreover, native starches enhance their emulsifying properties by means of esterification with reactants, such as acid anhydrides, octenyl succinic anhydride (n-OSA), dodecenyl succinic anhydride, fatty

acids, and fatty acid chlorides (Namazi, Fathi, & Dadkhah, 2011). In (Marefati, Wiege, Haase, Matos, & Rayner, 2017) it was reported that the smallest starch granules from quinoa esterified with n-OSA showed better emulsifying properties than other granules of larger sizes. Among the reactants used for starch esterification, the most widely used is the n-OSA. However, the Food and Drug Administration (FDA) restricts its use at the maximum level of 3% for starch modification (21 CFR-172.892). At this level, it is obtained a low degree of substitution (0.1 DS) in the presence of an alkaline catalyst in an aqueous medium (Simsek et al., 2015). On the other hand, the FDA does not restrict the use of fatty acid chlorides. The lauroyl chloride has been employed for the manufacture of Na-lauroyl arginate ehylester (LAE®), a substance which is suitable for human consumption and was regarded as GRAS by the FDA and by the European Food Safety Authority (EFSA) in 2007 (Coronel-León et al., 2016). A method for the esterification of starch with long chain fatty acid chlorides in the absence of organic solvents was reported (Namazi et al., 2011), it consists of two steps: first, native starch is dispersed into an alkaline medium by using sodium hydroxide; and then, the selected amount of fatty acid chlorides is added dropwisely to the reaction until the modified products precipitate, and sodium chloride is eliminated by washing. To the best of our knowledge, papers dedicated to the preparation

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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Corresponding author. E-mail addresses: [email protected] (Y.V. García-Tejeda), [email protected] (E.J. Leal-Castañeda), [email protected] (V. Espinosa-Solis), [email protected] (V. Barrera-Figueroa). ⁎

https://doi.org/10.1016/j.carbpol.2018.04.029 Received 11 December 2017; Received in revised form 24 March 2018; Accepted 6 April 2018 Available online 11 April 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.


Y.V. García-Tejeda et al.

equipment.

and characterization of the esterification of rice starch with fatty acid chlorides for the stabilization of oil-in-water emulsions have not been reported in the literature up to this moment. For starch particles used as emulsifying agents it is important to partially confer them hydrophobic properties, and a good option for the esterification of rice starch could be using a medium-chain fatty acid such as lauric acid (C12). A suitable substitute of wheat starch is rice starch since it is hypoallergenic, and is available worldwide (Bouyer, Mekhloufi, Rosilio, Grossiord, & Agnely, 2012); for this reason, it is important to analyze the properties of oil-in-water emulsions for their possible application in edible products. In this work, rice starch was hydrolyzed with hydrochloric acid and subsequently esterified with lauroyl chloride at three concentrations. The influence of the kind of starch in the stabilization of emulsions of canola oil-in-water is also investigated. In addition, the effects of the modification on the granule morphology, solubility, zpotential, crystallinity, gelatization, and digestibility properties of starch derivatives are discussed.

2.2.2. Fourier transformation infra-red (FTIR) spectra The esterification of rice starch was studied with a FTIR module IR2 equipped with an Indium Gallium Arsenide (InGaAs) detector, coupled to a Jobin-Yvon LabRam HR800 spectrometer (Horiba, Kioto, Japan). For the measurements, the starch samples were placed in an object holder and analyzed over the wave-number range of 450–4000 m−1 with a spectral resolution of 4 cm−1 and 36 scans per measurement, using an ATR contact objective. 2.2.3. Degree of substitution The degree of substitution (DS) was determined by 1H Nuclear magnetic resonance spectroscopy (NMR), using a Bruker Avance 750 spectrometer (Bruker Biospin, Karlsruhe, Germany) operating at 750.1 MHz and equipped with a 5 mm TXI probe at 298 K. The starch samples were dissolved in deuterated DMSO and placed in NMR tubes; spectra were obtained using the pulse program 1D NOESY-presat. DS was obtained according to the method reported by (Vanmarcke et al., 2017), namely, DS was calculated from the ratio of the area of the proton peak at 0.86 ppm to that of the proton peak between 4.40 and 5.10.

2. Materials and methods 2.1. Plant material and chemicals Canola oil was acquired from Capullo® (México City, México). Normal rice starch with an amylose content of 20%; lauroyl chloride; amyloglucosidase (A7095-50 mL) from Aspergillius niger (300 U/mL); pepsin (P7000-25G) from porcine stomach mucosa (1:10 000 U/mg); pancreatin (P7545-500G) from porcine pancreas (8 x U.S.P); invertase (I4504-1G) grade VII from bakers yeast (401 U/mg); and guar gum were purchased from Sigma-Aldrich Chemical Co. (Toluca de Lerdo, México). The Megazyme D-Glucose (glucose oxidase/peroxidase; GOPOD) assay kit and total starch kit (KT-STA) were acquired from Megazyme International Ireland Ltd. (Wicklow, Ireland). Hydrochloric acid, sodium hydroxide and dimethyl sulfoxide (DMSO, 99.7%) were purchased from J. T. Baker (USA).

2.2.4. Water solubility index (WSI) The sample (2.5 g) was vigorously mixed in 30 mL of water in a 50 mL centrifuge tube. After that, the mixture was incubated in a water bath at 30 °C for 30 min, and then it was centrifuged at 3000 × g during 15 min. The supernatant was then collected in a pre-weighed Petri dish, and the residue was weighed after oven drying overnight at 105 °C. The amount of solids in the dried supernatant as a percentage of the total dried solids in the original 2.5 g sample is an indicator of WSI (Anderson, Conway, & Peplinski, 1970). 2.2.5. Measurement of z-potential The z-potential of starches and emulsions prepared in Section 2.3 was measured using a commercial Zetasizer (Malvern Nano, USA). Measurements of z-potential were carried out at 25 °C using a maintenance-free capillary cell, and ultra-pure water at pH 7 with a resistivity of 18.25 MW×cm for diluting the sample. Samples of starches and emulsions (0.01% w/v) were diluted and injected directly into the capillary cell of the equipment; the filling was done by using a 1 mL syringe in order to avoid the presence of air bubbles. The measurements were carried out in triplicate for the interval of time of 30 s for each injection; the Zetasizer software converted the electrophoretic mobility measurements into values of z-potential by using the Smoluchowsky mathematical model.

2.2. Preparation of starch derivatives Prior esterification, rice starch was hydrolyzed with hydrochloridric acid. Starch slurry was prepared by dispersing 400 g of starch (dry basis) in 1 L of HCl 0.384 N. The reaction was performed during 6 h in a water bath at 50 °C. Starch esterification was carried out by following the method of Namazi et al. (2011): 100 g of starch were dispersed into 1000 mL of NaOH 0.1 M at 25 °C during 10 min; then 4, 50 and 100 g of lauroyl chloride was added to the starch dispersion drop-wisely during 10 min, and the reaction was completed after 10 min. After esterification, the slurry was centrifuged during 10 min at 1600 × g, and the starch laurate was recovered. After centrifugation, the starch laurate was washed three times with ethanol and one time with distilled water, then centrifuged and dried in a convection oven at 40 °C during 24 h. The dried starch was milled in a coffee grinder (Hamilton Beach® model 80350) and sieved in a mesh 100 of 0.149 mm opening size. The obtained samples were: hydrolyzed rice starch (HS for short); hydrolyzed rice starch laurate modified at the level of 4% (w/w) (HSL4 for short); hydrolyzed rice starch laurate modified at the level of 50% (w/w) (HSL50 for short); and hydrolyzed rice starch laurate modified at the level of 100% (w/w) (HSL100 for short).

2.2.6. Thermal properties of starches Thermal properties of starches were determined in a DSC (Discovery, TA Instruments, USA) equipped with a mechanical refrigeration system (RCS – refrigerated cooling accessory). The equipment was calibrated with indium which has a melting point of 156.4 °C and an enthalpy H = 6.8 cal/g. The onset (To), and peak (Tp) temperatures of gelatinization and the enthalpy (ΔHgel) in J/g of native and derivative starches were determined. For each sample, approximately 3 mg (dry basis) were directly weighed into aluminum trays, and deionized water was added in a relation of 2:1 of starch weight. The heating program used had a heating rate of 10 °C/min in a temperature range from −20 to 160 °C.

2.2.1. Morphology and particle size of starch granules The morphology and size of starches were evaluated by SEM (XL30 ESEM, EDAX, Philips Electronics N.V., Amsterdam, the Netherlands), by using an acceleration voltage of 20 kV. The samples were fixed on stubs with double-faced adhesive metallic tape. The mean particle size (Zaverage) and polydispersity index (PDI) of starch granules were measured in water suspensions by using a Malvern Zetasizer Nano ZS (Malvern Nano, USA). A sample of approximately 1 mL was placed in a cell and the determination of the particle size was made on the

2.2.7. XRD analysis The X-ray diffraction patterns (XRD) of native and derivative starches were obtained on a Rigaku Miniflex 600 (Rigaku Denki Co. Ltd, Japan) diffraction instrument operating at 40 kV and 15 mA with CuK radiation wavelenght of l = 1.54 Å. The sweeping angle ranged from 2° to 60° on a 2Ɵ scale with a step size of 0.01 and 0.03°/min. The relative 178


Y.V. García-Tejeda et al. i=1

crystallinity (Rc) of starch granules was calculated as the ratio of the crystalline area to the total area under the major diffraction peaks (Farooq, Dhital, Li, Zhang, & Huang, 2018). The software used to analyze the spectrum was OriginPro 8 (OriginLab Corporation, MA, USA).

D3,2 =

∑

i=1

ni di3/ ∑ ni di2,

N

D4,3 =

i=1

i=1

∑

ni di4 / ∑ ni di3, N

(2)

PDI = di / ∑ di43,

(3)

N

2.2.8. Starch digestibility In vitro starch digestibility of hydrolyzed rice starch and laurate starches were analyzed using the method described in a previous work (Espinosa-Solis, Sanchez-Ambriz, Hamaker, & Bello-Pérez, 2011). An enzyme solution containing pancreatin, amyloglucosidase, and invertase was prepared immediately before usage. Starch samples (200 mg) were mixed with 2 mL of deionized water in glass tubes, the mixtures were heated to 37 °C, and 4 mL of a solution of pepsin/guar gum/hydrochloric acid were added to the tubes; the reactions were carried out during 30 min. Then 2 mL of 0.25 M sodium acetate and one glass bead were added to each tube, which then were put in a slanted position with shaking at 160 rpm during 20 min. After that, 2 mL of the enzyme cocktail were added to each sample in intervals of 1 min; after 20 min, 50 mL of the samples were removed from tubes. Finally, the tubes were centrifuged at 14 857 × g for 5 min, and 50 mL of the hydrolyzed samples were analyzed for their glucose content using the GOPOD method. Each sample was analyzed in duplicate. The rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) were determined as percentage (%). Total starch content was measured by using the total starch kit developed by Megazyme International (2017).

where di is the droplet diameter, N is the total number of droplets and ni is the number of droplets having a diameter di (Li, Li, Sun, & Yang, 2013). 2.3.2. Stability of the kinetic of the emulsion The stability of emulsions was investigated on samples of HRS and HRS100 by light scattering in a Turbiscan Lab Expert (Formulation, Tolouse, France). The measurements were taken every 5 min during the first hour, and every 20 min during the last 5 h at 25 °C. The analysis of stability was performed as a variation of backscattering (BS) profiles as a function of time at the middle and top layer of the samples and then exported as%BS and peak thickness respectively by Turbisoft Lab 2.2 software. The curves obtained by subtracting the BS profile at t = 0 from the profile at t (DBS = BSt − BS0), showed a typical shape that allows a better quantification of creaming, flocculation, and other destabilization processes (Raikos, 2017). The global Turbiscan Stability Index (TSI) was calculated to compare the stability of the different formulations under analysis using the following formulae:

2.3. Preparation of canola oil-in-water emulsions

BS = 1/ λ* ,

(4)

λ* (φ , d ) = 2d/3ϕ (1 − g ) Qs,

(5)

i=1

6 wt% 10 wt% 20 wt% 30 wt%

0.2 0.33 0.67 1

7:3 7:3 7:3 7:3

(6)

2.4. Statistical analysis Experiments were performed in triplicate. The software Sigma-Stat Version 4 (Systat Software Inc., San José Calif., USA) was used to conduct an analysis of variance (ANOVA) in order to determine differences between means of treatments. Treatment means were considered significantly different at P ≤ 0.01 using pair-wise multiple comparison procedures (Tukey Test). 3. Results and discussion 3.1. Analysis of starches 3.1.1. Degree of substitution of starches The FTIR spectra of HS, HSL4, HSL50 and HSL100 are shown in Fig. 1. The FTIR spectra of HSL4, HSL50 and HSL100 were similar to their corresponding counterpart of HSL, but the spectra of both HSL50 and HSL100 showed two new peaks of low intensity at 1735 cm−1 and 2845 cm−1, which confirm the reaction of esterification. The peak at 1735 cm−1 confirms the formation of ester carbonyl groups (Namazi et al., 2011) in the starches, and the peak at 2845 cm−1 is attributed to the CeH stretching vibrations of the alkyl groups of the fatty ester. No new peaks were detected for LHS4 in its FTIR spectrum owing to its low level of modification. 1H NMR spectroscopy was used to quantitatively estimate the esterification of the rice starch. The presence of lauric acid was confirmed by integration of the characteristic signals of fatty acid protons from 0.89 to 2.34 ppm, and of the starch backbone from 5.50 to

Table 1 Formulation and composition of canola oil-in-water emulsions. Water:oil proportion (ml)

,

where λ* is the photon transport mean free path in the analyzed dispersion; φ is the volume fraction of particles; d is the mean diameter of particles; g and Qs are the optical parameters given by Mie theory; xiis the average backscattering for each minute of measurement; xBS is the average xi; and n is the number of scans (Raikos, 2017).

2.3.1. Optical microscopy for the measurement of the droplet size Optical microscopy of the prepared emulsions was performed in an Eclipse H550S (Nikon, Chiyoda-ku, Japan) microscope equipped with a Kodak DC 120 digital camera (Sevier Country, Tennessee, USA). In order to determine the droplet sizes in each emulsion, several pictures of the emulsion droplets were taken. The processing of the images was performed in the ImageJ software (Schneider, Rasband, & Eliceiri, 2012). Populations of 200 droplets were counted in the distribution. The surface-to-volume ratio (D3,2), the volume mean diameter (D4,3), and the polydispersity index (PDI) of droplet sizes were calculated by using formulae (1)–(3), respectively:

g of starch/ml of oil

1/2

⎞ ⎛ TSI = ⎜∑ (x i − xBS )2 /(n − 1) ⎟ ⎠ ⎝ n

Canola oil-in-water emulsions were prepared from ultra-pure water and canola oil in a volume proportion of 7:3. Starches were added to the resulting mix of canola oil and water in several concentrations, as is shown in Table 1; the formulations were selected on considering that salad dressings have high viscosity and fat content of 20–70%. Dispersions of starches were prepared in glass test tubes by using HS and HSL100 at concentrations of 6, 10, 20 and 30% (w/w), each one in a volume of 10 mL of the mix of oil-water. The emulsions were mixed at 11,000 rpm during 1 min by using a homogenizer (IKA T18 Basic UlTRA-TURRAX®) at 25 °C. The resulting emulsions are named as follows: 6%-HS, 10%-HS, 20%-HS, 30%-HS, 6%-HSL100, 10%-HSL100, 20%-HSL100, and 30%-HSL100, depending on the concentrations of starches and the level of modification.

Starch content

(1)

N

179



Fig. 1. FTIR spectra of hydrolyzed rice starch (HS) and esterified starches (HSL4, HSL50, HSL100).

3.00 ppm (Vanmarcke et al., 2017). The DS increased as the level of modification increased, the obtained values were 0.042, 0.06, 1.86 for HRS4, HRS50, and HRS100, respectively. The esterification of rice starch at the level of 100% was favored because of the increase of lauroyl chloride in the reaction medium, and a moderately substituted rice starch was obtained (1.86 DS). The DS obtained in this work for HSL50 is 0.06, a lower value was reported for laurylated maize starch (0.015) that was enzyme-catalyzed at the same ratio of weight starch:lauric acid (Gao et al., 2014). Hydrolyzed starches have higher degree of substitution after esterification or etherification than native starches because the hydrolysis gives to the substituent groups more access to the subsurface of granules (Fouladi and Mohammadi Nafchi, 2014; Karim, Sufha, & Zaidul, 2008; Whistler, Madson, Zhao, & Daniel, 1998).

Fig. 2. Scanning electron images of native rice starch and rice starch laurate.

OSA (DS of 0.0186). 3.1.3. Morphology and granule size of starches Granule size plays an important role in the formation of emulsions; small granules with smoother surfaces have a better contact at the o/w interface (Saari, Heravifar, Rayner, Wahlgren, & Sjӧӧ, 2016). Noticeable changes in the surface of granules were observed only in the HSL100 sample (see Fig. 2b), since the surfaces show a little damage that may be attributed to the acid hydrolysis and alkaline reaction conditions. The results obtained from dynamic light scattering analyses are shown in Table 2. The mean diameter (Z-average) of rice starch includes individual and clustered granules. After acid hydrolysis it was observed an increase in the mean diameter of starch, which may be attributed to the agglomeration of starch granules. After esterification with lauroyl chloride, a considerable reduction in particle size

3.1.2. Water solubility index and zeta potential Table 2 shows the values of water solubility index (WSI) for native, hydrolyzed and esterified rice starches. The value of WSI increased after acid hydrolysis because hydroxonium ions hydrolyze the glycosidic bonds, leading to an increase of hydroxyl groups that retain more water molecules by means of hydrogen bonds. Moreover, lauroylation increased WSI as DS did. The inverse tendency was observed in the behaviour of z-potential, i.e., the z-potential decreased (from −2.93 to −11.27 mV) as DS increased. The more negative values of z-potential may originate a larger repulsion between starch molecules, thus facilitating WSI. Similarly, Miao et al. (2014) reported a decrease from −1.8 mV to −7.8 mV after esterification of waxy maize starch with n-

Table 2 Physicochemial characterization, gelatinization and digestibility properties of native and rice starch laurate derivatives. Parametersa

RS

Z-Average (mm) PDI WSI (%) z (mV) Thermal properties To(◦C) Tp(◦C) ΔH (J/g) Digestibility properties RSD SDS RS TS

8.11 0.72 1.39 0.28

HS

HSL4

HSL50

HSL100

13.19 ± 0.76c 0.32 ± 0.06a 3.44 ± 0.14b −2.93 ± 0.58c

4.38 ± 0.27a 0.24 ± 0.02a 24.38 ± 0.32c −6.30 ± 0.39b

8.63 ± 2.32b 0.81 ± 0.22c 29.83 ± 0.25d −7.33 ± 1.12b

4.11 ± 1.16a 0.89 ± 0.19c 53.61 ± 0.26e −11.27 ± 1.53a

52.77 ± 0.02a 54.06 ± 0.05a 11.74 ± 0.06a

62.85 ± 0.06b 68.32 ± 0.48b 4.35 ± 0.05b

63.19 ± 0.02c 67.76 ± 0.09b 4.38 ± 0.04b

63.68 ± 0.06d 69.38 ± 0.32c 4.77 ± 0.01c

63.49 ± 0.05e 72.68 ± 0.50c 5.11 ± 0.04d

N.D. N.D. N.D. N.D.

58.62 ± 0.61a 26.22 ± 0.79a 9.36 ± 1.04a 94.20 ± 1.6a

68.15 ± 0.94c 21.74 ± 0.98c 2.91 ± 0.06c 92.80 ± 1.9ac

73.11 ± 1.84d 19.95 ± 1.20c 0.81 ± 0.69cd 93.90 ± 0.5ac

84.03 ± 0.19b 5.13 ± 0.72b 1.23 ± 0.09bd 90.40 ± 1.30bc

± ± ± ±

0.04b 0.02b 0.22a 0.06d

a Z-Average: the mean particle size; PDI: polydispersity index; WSI: water solubility index; z: zeta potential; To: onset temperature; Tp: gelatinization temperature; ΔH: gelatinization enthalpy; RDS: rapidly digestible starch; SDS: slowly digestible starch; RS: resistant starch; TS: total starch; N.D: not determined. Results show the mean value ± standard error from.

180



Table 3 Effect of starch concentration on the mean droplet size, TSI, and z potential on emulsions produced with HS and HSL100. Sample

D32

D43

PDI

TSI

z (mV)a

6%-HS 10%-HS 20%-HS 30%-HS 6%-HSL100 10%-HSL100 20%-HSL100 30%-HSL100

39.1 29.0 21.1 18.2 38.0 31.8 17.6 13.2

39.7 29.3 22.7 19.6 39.2 32.2 18.0 13.4

1.3 2.4 1.6 1.3 1.1 1.0 1.1 1.1

20.95 ± 5.3 17.63 ± 3.1 5.12 ± 0.9 2.20 ± 0.3 26.55 ± 4.9 18.79 ± 3.8 2.66 ± 0.5 2.02 ± 0.3

−32.0 ± 2.2 −26.3 ± 1.6 −20.1 ± 1.2 −18.3 ± 0.9 −10.6 ± 0.38 −0.26 ± 0.09 −0.11 ± 0.09 0.37 ± 0.37

a D32: surface-weighted mean (mm); D43: volume-weighted mean (mm); PDI: polydispersity index; TSI: turbiscan stability index.

Fig. 3. Diffraction patterns and relative crystallinity of native (RS) and modified rice starch derivatives (HS, HSL50, HSL100).

3.2. Characterization of emulsions 3.2.1. Droplet size and stability of emulsions As the concentration of starch increased in the aqueous phase, the droplet size of emulsions decreased (see Table 3). A higher concentration of starch lead to a higher viscosity, which improves the intra- and inter-molecular interactions in the emulsion system (Hong et al., 2018). According to Hong et al. (2018), the ability of biopolymers in increasing viscosity of continuous phase could reduce the rate of particle sedimentation. A comparison of the droplet size of the emulsions obtained with HS and HSL100 is shown in Table 3, at the levels of 20 and 30% of concentration, HSL100 shows lower droplet size values than HS. This behavior is attributed to the esterification reaction, which confers an active surface on HSL100 and improves the solubility of starch due to a greater repulsion of charges between particles as the DS increased (see Table 2). The effect of esterification on the stability of emulsions prepared with HS and HSL100 was analyzed by light scattering. The backscattering (BS) profiles of emulsions are shown in Fig. 4. In each BS profile, the horizontal axis represents the tube’s height, while the vertical axis represents the percent of change of BS with respect to the initial stage. The coloring of curves represents the various testing times. As is shown in Fig. 4, few changes are noticeable in the BS profile of emulsions prepared at 20 wt% and 30 wt%. This behavior could be related to the high content of solids and the smaller droplet size, which avoid the coalescence of canola oil droplets. In this sense, emulsions stabilized at 20 wt% and 30 wt% exhibited the lower values of the global TSI because few changes in BS profiles were observed during six hours of evaluation. As can be seen in Table 3, the increase of starch concentration improved the TSI, and HSL100 presented the best performance compared to HS.

accompanied by an increase in PDI was observed. According to Wu, Zhang, and Watanabe (2011), values of PDI greater than 0.5 correspond to a broad distribution of particle sizes. On the other hand, a lower zpotential at the highest level of modification leads to a major repulsion between starch granules, thus a low amount of starch agglomerates was detected by the Zetasizer equipment, as is shown in Table 2.

3.1.4. Thermal and crystalline analyses Table 2 shows thermal parameters of native, hydrolyzed and rice starch laurate derivatives. Differential scanning calorimetric (DSC) analysis showed that the gelatinization properties (T0 and Tp) of the hydrolyzed rice starch were higher than native starch. Moreover, DSC showed that ΔH in hydrolyzed rice starch was lower than in native starch. This can be explained on considering that during acid hydrolysis, the amorphous region of the granule is easily accessible to the hydrochloric acid, then amylose and amylopectin chains get hydrolyzed. The results of XRD (Fig. 3) showed a reduction of the crystalline region from 31.34 to 30.93%. This reduction in crystallinity is related to a reduction in ΔH, for this reason the hydrolyzed starch is gelatinized at a higher temperature (Gunaratne & Corke, 2007). The gelatinization properties Tp and ΔH of esterified starches were slightly higher than those of hydrolyzed starch. This behavior is attributed to the addition of covalent bonds (Gao et al., 2014), due to the introduction of fatty acid groups into starch molecules. In this sense, the energy required for disrupting the starch structure is higher than for the native starch (Zhang, Mei, Chen, & Chen, 2017). The rice starch displayed A-type diffraction pattern with strong reflections at 14.98°, 17.85°, 19.7° and 22.87° 2Θ, these values are in agreement with previous results reported by Farooq, Dhital, Li, Zhang, and Huang (2018) for rice starch. The esterification did not change the crystalline pattern of starches.

3.2.2. Optical microscopy of emulsions Emulsion droplets are conformed by a center of canola oil that is coated by starch. As can be seen in Fig. 5, the 6%-HS sample possesses few starch granules at the oil-water interface. But as the starch concentration increases from 6% to 30%, a more densely packed oil-water interface is observed. Emulsions with starch concentrations lower than 6% for all the treatments were unsuccessfully prepared since the surface coating of solid particles was not enough to form a compact monolayer in the oil–water interface so that coalescence occurred between oil droplets. Emulsion stabilization requires a complete monolayer coverage of closely packed particles (Dickinson, 2013). Unlike emulsion prepared with 6%-HSL, a higher surface coverage of particles is clearly observed in droplets of emulsions prepared with 6%-HSL100, which is due to a greater affinity between rice starch laurate and canola oil. As the concentration of HS increased from 6 to 20%, the droplet size increased as well. This effect is attributed to the coalescence between oil droplets; in contrast, for HSL100 the droplet size decreased as the starch

3.1.5. Digestibility properties The properties of digestibility in vitro of hydrolyzed starch and rice starch derivatives are shown in Table 2. There we can see that RDS increases as DS does, consequently, a lower content of SDS accompanied by a decrease of RS is observed after esterification of hydrolyzed rice starch by lauroyl chloride. The decrease of both the crystalline level and TS could be responsible of the higher digestibility properties of starch derivatives. Digestibility properties depend on the botanical sources of starches. Simsek et al. (2015) reported a higher content of SDS in starches from maize, tapioca, potato and wheat after esterification with n-OSA; however a decrease from 81.33 to 76.83% in SDS was observed after the esterification of rice starch with a DS=0.026.

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Fig. 4. Backscattering profiles and the corresponding photographs of canola oil emulsions stabilized by HS and HSL100 at different concentrations.

the most stable emulsion is near to zero (0.3 mV) since the oil droplets are almost covered by starch granules. A similar tendency is shown in the work Ricaurte, Perea-Flores, Martinez and Quintanilla-Carvajal (2016), namely the lower droplet size (163 nm) is associated to a less negative z-potential (−29.7 mV), and the larger droplet size (2268 nm) is associated to a more negative z-potential (−47.2 mV). Observe that the standard theory of stability establishes that conventional emulsion are stable if z-potential satisfies ζ > 30mV (Barfod & Sparsø, 2007), and if z-potential is about zero the emulsion shall precipitate. To the difference with the standard theory, in the analyzed emulsions the precipitation did not take place when z-potential is near to zero, on the

concentration increased. An efficient stabilization against coalescence was achieved by using 20 and 30 wt% of starch. Canola oil emulsions prepared with HSL100 presented a higher stability than those prepared with HS at the same starch concentration (see Table 3). 3.2.3. Zeta potential The effect of starch concentration on z-potential was evaluated in emulsions prepared with HSL and HSL100. The increase of starch concentration leads to less negative values of z-potential, which may be attributed to the electrostatic shielding of the oil droplets by starch granules at the oil-starch interface. For this reason, the z-potential of

Fig. 5. Light microscopy images of emulsions stabilized with HS and HSL100 at different concentrations. 182



contrary, these emulsions were the more stable.

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4. Conclusions Esterification of hydrolyzed rice starch with lauroyl chloride was confirmed by FTIR and 1H NMR; a medium degree of substitution (DS) of 1.86 was obtained. A decrease in both SDS and RS was observed as DS increased, then a potential application of rice starch laurate consists in high energy supplements for special medical purposes. The emulsifying capacity of rice starch laurate was improved in comparison with hydrolyzed rice starch. Laurate starches presented more desirable characteristics than hydrolyzed rice starch including greater solubility and lower zeta potential, which are associated with better emulsion-stabilizing properties, due to a larger electrostatic repulsion between starch granules. Smaller emulsion droplet size and better stabilization were obtained by using HSL100 at 20 and 30 wt% of starch. According to these results, rice starch laurate has a potential application of food-grade emulsifier for canola oil-in-water. Aknowledgments YVGT acknowledges to CONACyT for the postdoctoral founding. VBF acknowledges to SIP project 20180438. The authors recognize the experimental support of the CNMN-IPN in the realization of the presented work. References Anderson, R. A., Conway, H. F., & Peplinski, A. J. (1970). Gelatinization of corn grits by roll cooking, extrusion cooking and steaming. Starch—Stärke, 22(4), 130–135. Barfod, N. M., & Sparsø, F. V. (2007). 5—Structure and function of emulsifiers and their role in microstructure formation. In D. J. McClements (Ed.). Understanding and controlling the microstructure of complex foods (pp. 113–152). Woodhead Publishing. Bouyer, E., Mekhloufi, G., Rosilio, V., Grossiord, J., & Agnely, F. (2012). Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: Alternatives to synthetic surfactants in the pharmaceutical field? International Journal of Pharmaceutics, 436(1–2), 359–378. Burgos-Díaz, C., Wandersleben, T., Marqués, A. M., & Rubilar, M. (2016). Multilayer emulsions stabilized by vegetable proteins and polysaccharides. Current Opinion in Colloid & Interface Science, 25, 51–57. Coronel-León, J., López, A., Espuny, M. J., Beltran, M. T., Molinos-Gómez, A., Rocabayera, X., et al. (2016). Assessment of antimicrobial activity of Nα −lauroyl arginate ethylester (LAE®) against Yersinia enterocolitica and Lactobacillus plantarum by flow cytometry and transmission electron microscopy. Food Control, 63, 1–10. Dickinson, E. (2013). Stabilising emulsion-based colloidal structures with mixed food ingredients. Journal of the Science of Food and Agriculture, 93(4), 710–721. Espinosa-Solis, V., Sanchez-Ambriz, S. L., Hamaker, B. R., & Bello-Pérez, L. A. (2011). Fine structural characteristics related to digestion properties of acid-treated fruit starches. Starch—Stärke, 63(11), 717–727. Farooq, A. M., Dhital, S., Li, C., Zhang, B., & Huang, Q. (2018). Effects of palm oil on structural and in vitro digestion properties of cooked rice starches. International Journal of Biological Macromolecules, 107, 1080–1085.

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