carboxymethyl starch complex nanogels as novel Pickering stabilizers: Physical stability and rheological properties

Food Hydrocolloids 93 (2019) 215–225

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Chitosan hydrochloride/carboxymethyl starch complex nanogels as novel Pickering stabilizers: Physical stability and rheological properties

T

Xiao-Min Lia,d, Qiu-Tao Xieb, Jie Zhuc, Yi Pana,d, Ran Menga,d, Bao Zhanga,d,∗, Han-Qing Chena,d, Zheng-Yu Jine a

Engineering Research Center of Bio-process, Ministry of Education, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui, 230009, PR China Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha, PR China c School of Chemical Engineering and Energy Technology, Dongguan University of Technology, Dongguan, 523808, PR China d School of Food and Biological Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui, 230009, PR China e The State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Chitosan hydrochloride/carboxymethyl starch complex nanogels Pickering emulsions Stability Microstructure Rheological property

The Pickering emulsions stabilized by chitosan hydrochloride - carboxymethyl starch (CHC-CMS) nanogels prepared through a facile covalent cross-linking method using 1-ethyl-3-(3-dimethyl-aminopropyl-1-carbodiimide) were investigated. The smallest mean size of CHC-CMS nanogels (378.2 nm) was obtained when the volume ratio of CHC: CMS was 2:1. The three-phase contract angle of CHC-CMS nanogels was 89.3°, which exhibited that the CHC-CMS nanogels could be used as effective Pickering emulsifiers. The effects of CHC-CMS nanogels concentration, oil phase fraction and environment factors viz, pH, ionic strength on the stability of emulsions were evaluated. An increase in CHC-CMS nanogels concentration led to a formation of smaller droplets, and the droplet size of Pickering emulsions was increased with the rise of oil phase fraction. The Pickering emulsions were highly stable at pH 6 and above. Increasing NaCl concentration was found to generate the aggregation of droplets. Confocal laser scanning microscopy revealed that the CHC-CMS nanogels could be adsorbed on the oil-water interface and form a densely packed layer at the surface of spherical oil droplets, which exhibited long-term stability for 3 months storage. Rheological results illustrated that all emulsions showed the typical pseudoplastic fluid characteristics and satisfied the Herschel-Bulkley model. The fact that the elastic modulus (G') was higher than the loss modulus (G″) in all the samples was indicative of the formation of an elastic gel-like structure. These results provided a potential way for Pickering emulsions preparations, which could be used as an effective delivery carrier of bioactives.

1. Introduction Emulsion was a mixture of two immiscible liquids with one dispersed in the other, which was extensively used in food, pharmaceuticals or personal care products (Lu, Zhang, Li, & Huang, 2018). The preparation and stabilization of emulsion were usually developed by adding small molecular weight surfactants and biopolymers (polysaccharides and proteins) (Dickinson, 2012; Xiao, Li, & Huang, 2016). However, the application of emulsions was limited by their thermodynamic instability, which tended to breakdown during storage due to droplet aggregation, gravitation separation, or Ostwald ripening mechanisms (McClements, 2012). Forming and manipulating interfacial structures via solid particles provided a promising method to fabricate stable emulsions. Pickering emulsions were known that solid particles

∗

accumulated at the surfaces of oil droplets and formed a steric barrier to prevent droplet aggregation (Pickering, 1907). Recently, considerable focus has been attracted on Pickering emulsions because of their high stability against aggregation, coalescence, and Ostwald ripening (Yang et al., 2017). Numerous types of inorganic particles were shown as effective Pickering stabilizers for O/W emulsions, such as silica (Ashby, Binks, & Paunov, 2004; Wu, He, Cui, & Jin, 2019), latex (Binks & Whitby, 2004), and clay (Nonomura & Kobayashi, 2009; Wu et al., 2018). However, the lack of consumer satisfaction resulted in a limited application of inorganic particles in food industry. Therefore, there was a potential requirement for Pickering emulsions with natural, environmental-friendly, biodegradable and food-grade colloidal characteristics (Song et al., 2015). A large number of food-grade particles, such as cellulose nanocrystals (Yan et al., 2017), chitin nanocrystals

Corresponding author. School of Food and Biological Engineering, Hefei University of Technology, PR China. E-mail address: [email protected] (B. Zhang).

https://doi.org/10.1016/j.foodhyd.2019.02.021 Received 18 December 2018; Received in revised form 9 February 2019; Accepted 11 February 2019 Available online 13 February 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.

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reactants were transferred into 30 °C water bath for 60 min. Subsequently, 10 g of chloroacetic acid (CA) was added, and the system was reacted at 40 °C for 240 min. At the end of reaction, the solution was filtered and the filtrate was removed. The residues were dispersed in 80% ethanol and 5 M HCl was added to neutralize the un-reacted sodium hydroxide. The slurry was filtered and washed 3 times with 80% ethanol and 2 times with absolute ethanol till no chloride ions was detected with 0.2 M AgNO3. The sediment was collected and placed in an oven at 45 °C for 24 h. The finally product was broken up into fine power for further analysis. The degree of substitution (DS) was measured by titration method (Volkert, Loth, Lazik, & Engelhardt, 2004). A blank titration of the unmodified corn starch was performed as a control. The DS of carboxymethyl starch was 0.612 (DS = 0.612).

(Barkhordari & Fathi, 2018), starch nanoparticles (Ge et al., 2017; Ye et al., 2017), and protein colloid particles (Liu & Tang, 2013; Xiao, Wang, Gonzalez, & Huang, 2016), had already been applied to make emulsions stabilized through the Pickering mechanism (Lam, Velikov, & Velev, 2014). Chitosan was a natural-based polysaccharide, which derived from crustacean shells and was widely used in food industry because of its unique characteristics such as biocompatibility, biodegradability and non-toxicity (Xia, Liu, Zhang, & Chen, 2011). It had cationic character in solution allowing for electrostatic interaction with negatively charged molecules to form nano-complexes by ionic gelation (Younes & Rinaudo, 2015). However, the charge of chitosan molecules was absent near their pKa of 6.5 under neutral and basic environments, leading to precipitation and limited application of chitosan (Liang et al., 2015). Therefore, chitosan hydrochloride (CHC), one of the most important water-soluble chitosan derivatives, was chose to prepare nanoparticles (Ge, Yue, Chi, Liang, & Gao, 2018). Native starches had been widely used in food industry due to their low cost, biocompatibility, biodegradability and non-toxicity (Chen et al., 2019; Tao et al., 2018; Tao, Xiao, Wu, & Xu, 2018). However, native starches got more and more limited for various applications due to poor solubility, bad resistance to mechanical shear, instability at high temperature and other reasons (Ma et al., 2019; Zhang et al., 2018). Therefore, physical or chemical methods were used to improve their functional characteristics or advantages (Chi et al., 2018; Lv et al., 2018; Xie, Li, Chen, & Zhang, 2019). Among these starch derivatives, carboxymethyl starch (CMS) had attracted extensive interests in both research and industry. The presence of function group (CH2COOe) yielded starch with polyanionic property, which was beneficial to prepare nanoparticles with polycationic polymer by complex coacervation. 1-ethyl-3-(3-dimethyl-aminopropyl-1-carbodiimide) (EDC) was used to activate carbohydrates for reaction with amino groups of proteins to form amide linkage. However, little information was available on nanogels of CHC-CMS through EDC cross-linking. What's more, there was no a systematic study on CHC and CMS complex colloidal particles as Pickering emulsion stabilizers. In this research, CHC and CMS complex nanogels were prepared by covalent cross-linking method as novel renewable natural particle-stabilizers of Pickering emulsions, which were characterized by using droplet size, images, optical microscopy, confocal laser scanning microscopy (CLSM), and rheological measurements. Moreover, the stability of Pickering emulsions against environment factors (pH and ionic strength) were also discussed.

2.3. Preparation of CHC-CMS nanogels CHC-CMS nanogels were prepared by the formation of amide linkage between CHC and CMS through an EDC-mediated reaction (Chen, Lee, & Park, 2003; Khalili et al., 2015). Briefly, the CHC (0.25 wt %) was dissolved in distilled water with high speed stirring until completely dissolved, followed by adjusting to pH 4. CMS (1 wt%) was also dissolved in distilled water using magnetic stirring at 1200 rpm for 30 min, adjusting the same pH as CHC solution, then the EDC (EDC/ CMS = 0.1 wt%) was added dropwise to the solution at 800 rpm for 30 min to activate carboxyl group. Afterwards, CMS solution was added dropwise to CHC solution with different mixing ratios and the mixtures were stirred at 800 rpm for 30 min to form amide bonds. The total volume of mixture solution was fixed at 80 mL, then the volume ratios of mixtures were adjusted to 3:1, 2:1, 1:1, 1:2 and 1:3 (CHC/CMS, v/v). At the end of reaction, the pH of the mixtures was adjusted to 8.5–9 using NaOH to precipitate the CHC-CMS nanogels. Subsequently, CHCCMS nanogels were washed using ethanol and distilled water to remove un-reacted substance and no desired product. The nanogels were freezedried for further analysis. 2.4. Characterization of CHC-CMS nanogels 2.4.1. Particle size measurements The CHC-CMS nanogels were dispersed in distilled water, then the particle size was measured by Nano-ZS Zetasizer analyzer (Malvern Instruments, U.K.). The samples were carried out at 25 °C and 1.33 of refractive index in triplicate (Xi, Luo, Lu, & Peng, 2018). 2.4.2. Wettability measurement of CHC-CMS nanogels The three-phase contact angle of particles (CHC, CMS, and CHCCMS nanogels) was measured using an OCA 15 EC (Dataphysics Instruments GmbH, Germany) following the method described by Wang et al. (2016) with some modifications. The sample powders were compressed to pellets with diameter of 13 mm and thickness of 2 mm. A drop of deionized water (5 μL) was gently placed on the surface of the pellets using a high-precision injector. After 30 s for equilibration, the droplet was photographed using a high-speed video camera, and the contour of imaged drop was simulated with Laplace-Young equation to obtain the three-phase contact angle. Measurements were performed over at least three times.

2. Material and methods 2.1. Materials Corn starch was purchased from Hebei Gusong Agricultural Products Co., Ltd. (China). Chitosan hydrochloride (CHC) (deacetylation degree 85.47%, viscosity 30 mpa.s) was obtained from Jinan Haidebei Marine Biological Engineering Co., Ltd. (China). Chloroacetic acid (CA) (purity > 99.5%) was obtained from Tianjin Fuchen Chemical Reagent Factory (China). 1-ethyl-3-(3-dimethyl-aminopropyl1-carbodiimide) (EDC) (purity > 98%), Nile Red and Nile Blue A were provided by Bomei Biotechnology company (Hefei, Anhui province China). Corn oil was purchased from the local supermarket (Hefei, China). All other chemicals were of analytical grade.

2.5. Preparation of Pickering emulsions The Pickering emulsions were prepared using various CHC-CMS nanogels concentrations (0.1%, 0.3%, 0.5%, 0.7%, 1%, 1.5% and 2%, w/v) and different oil phase fractions (φ = 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7, v/v). The total volume of Pickering emulsions was fixed at 20 mL. In short, the corn oil was added into the CHC-CMS nanogels suspensions, and the resultant mixtures were sheared using a high speed homogenizer IKA T18 (Germany) at 12000 rpm for 3 min at room temperature to prepare Pickering emulsions. The emulsification

2.2. Preparation of carboxymethyl starch (CMS) Sodium carboxymethyl starch (CMS) was prepared under alkaline conditions as described in a previous study by Zhang et al. (2017). Briefly, corn starch (10 g, dry basis) and sodium hydroxide (7 g) were added into three-necked flask, then 90% ethanol (120 mL) was added under continuous stirring until the solution dispersed evenly. The 216

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1500 PDI

C e

Size (d. nm)

1200

2.6. Emulsion type The type of emulsion (either O/W or W/O) was inferred by observing what happened when a drop of emulsion was added to either corn oil or water. For O/W emulsions, when a drop of emulsion was added to water, it was dispersed. On the other hand, the emulsion droplets remained agglomerated when placed in water, which exhibited that it belonged to W/O emulsions (Hu et al., 2016).

0.8

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2.7. Particle size and zeta potential measurements of emulsions

2:1

0.4

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CHC:CMS (v/v)

Fig. 1. Mean size and PDI of CHC-CMS nanogels at different CHC to CMS volume ratios. Samples designated with different lower case letters (a, b, c, d, e) were significantly different (P < 0.05) when compared size between different mixing ratio. Samples designated with different capital letters (A, B, C) were significantly different (P < 0.05) when compared polydispersity index (PDI) between different mixing ratio.

The droplet size distribution of O/W emulsions was determined using a Laser particle size analyzer BT-9300HT (Dandong Instrument, China). Droplet size measurement was reported as the volume-average droplet size (D[4,3]) using the following equations:

D [4,3] =

1.0 Size

PDI

efficiency was examined by droplet size and visual inspection of their appearance. In general, the droplet size of emulsions markedly reduced, suggesting that more nanogels were participated to form and stabilize the newly created interface during the emulsification. This result indicated that the nanogels had better emulsification efficiency.

∑ ni Di4 ∑ ni Di3

The zeta potential of the droplets in the emulsions was measured using Nano-ZS Zetasizer analyzer (Malvern Instruments,U.K.). All the samples were diluted 10 times with distilled water before analysis to avoid multiple scattering effects. All measurements were performed in triplicate (Zhang, Zhang, & McClements, 2016). Fig. 2. Water contact angles of CHC, CMS and CHC-CMS nanogels.

2.8. Microscopy analysis of emulsions Light microscope (AE 2000, China) with a digital camera was used to character the morphology of Pickering emulsion-stabilized by CHCCMS nanogels. One drop emulsion was placed on a glass microscopic slide and no cover glass was used in order to avoid deformation of droplets (Li et al., 2019). In addition, the interface structure of Pickering emulsions stabilized by CHC-CMS nanogels was observed by confocal laser scanning microscopy (CLSM) (LSM 710, Germany). Nile Red dye was used to stain oil phase and Nile Blue A was applied to stain CHC-CMS nanogels. The sample was dyed with a mixed fluorescent dye solution consisting of 1 mg mL−1 Nile Red and 1 mg mL−1 Nile Blue A. The stained sample was placed on concave confocal microscope slides. The fluorescent dyes were excited by either an argon laser at 488 nm for Nile Red or a helium neon (HeeHe) laser at 633 nm for Nile Blue A (Ge et al., 2017).

2000 g for 10 min. Centrifugation accelerated phase separation and generated an oil phase at the top, an emulsion phase in the middle and an aqueous phase on the bottom. The initial volume of the emulsion (VInt) and the volume of remaining emulsion after centrifugation (VCen) were evaluated. Emulsion stability (ES) was measured using the following equations:

ES (%) =

VCen × 100 VInt

2.10. Emulsion index (EI) Pickering emulsions were transferred to a glass tube for 1 day and 7 days of storage in order to evaluate the environmental factors (pH and ionic strength) on the stabilization capacity of emulsions. The EI was determined by measuring the volume of the observed emulsion and the volume of all the phases in the tube. The equation was used to calculate the EI as following:

2.9. Evaluation of emulsions stability to different influencing factors a) Effects of pH: In order to study the effects of pH on the properties of the emulsions, a series of emulsion samples were prepared by 1.5% CHC-CMS nanogels with 0.5 oil phase fraction and the pH was adjusted to 2, 3, 4, 5, 6, 7 or 8 using either HCl or NaOH (Shah et al., 2016). b) Effects of ionic strength: To evaluate the effects of ionic strength on the emulsions stability, an appropriate amount of NaCl powder was directly added to 10 mL of freshly prepared emulsions (1.5% CHCCMS nanogels with 0.5 oil phase fraction) to a concentration of 0–0.6 M. c) Effects of storage time: In order to investigate long-term stability of the CHC-CMS nanogels stabilized Pickering emulsions, the emulsions were stored at room temperature for 3 months. d) Physical stability of emulsions: Centrifugation method was used to evaluate the physical stability of emulsions (Barkhordari & Fathi, 2018). Briefly, emulsions in graduated tubes were centrifuged at

EI (%) = Hs / He Where Hs was the serum layer height and He was the total emulsion height. 2.11. Rheological measurement of Pickering emulsions The rheological properties of Pickering emulsions were measured at 25 °C using a DHR-3 rheometer (TA company, USA) with a plate clamp (40 mm diameter, gap height 1 mm) (Chen et al., 2018; Wang et al., 2019; Wang, Zou, Gu, & Yang, 2018). For each measurement, the emulsions were placed in the plate and waited for 5 min to allow temperature equilibrium before measurements. The sample was continuously sheared from 0.1 to 100 s−1 for the sweep testing. All the dynamic tests were performed within the linear viscoelastic region 217

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Fig. 3. Effects of particles concentrations (0.1%, 0.3%, 0.5%, 0.7%, 1%, 1.5% and 2%, w/v) on the diameter (A) and appearance photograph (B) of Pickering emulsions stabilized by CHC-CMS nanogels. Fig. 4. Effects of oil phase fractions (φ = 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7, v/ v) on the diameter (A) and appearance photograph (B) of Pickering emulsions stabilized by CHC-CMS nanogels.

(Wang, Yuan, Liu, Xu, & Cui, 2019; Yuan, Xu, Cui, & Wang, 2019). The frequency was oscillated from 0.1 to 100 rad/s and the strain was made at 1%. The storage modulus (G′) and loss modulus (G″) were recorded versus frequency. All measurements were performed for three times.

Pickering emulsions. The suitable wettability could promote particles to adsorb on the oil/water interface and form a steric hindrance preventing the droplet coalescence (Tzoumaki, Moschakis, Kiosseoglou, & Biliaderis, 2011). Contact angle measurements of CHC, CMS and CHCCMS nanogels (2:1) were shown in Fig. 2. The contact angle of CHC was 116.20°, which suggested that it was predominantly hydrophobic. The contact angel of CMS (around 37.45°) indicated that it was preferentially wetted by the aqueous phase. Therefore, CHC and CMS were hardly usable for stabilizing oil-in-water Pickering emulsions. As expected, the contact angle of CHC-CMS nanogels was closed to 90°, suggesting a potentially suitable for acquiring stable Pickering emulsions. Dai et al. (2018) previously reported that zein-propylene glycol alginate composite particles with wettability values near 90° were able to yield stable Pickering emulsions.

2.12. Statistical analysis All measurements were performed in triplicate and the data reported above were exhibited as the mean values ± standard deviations. Data were analyzed using an analysis of variance (ANOVA) procedure of the SPSS 13.0 statistic analysis program, and the difference between means of the trials were detected by a least significant difference (LSD) test (P < 0.05). 3. Result and discussion 3.1. Characterizations of CHC-CMS nanogels

3.2. Effect of CHC-CMS nanogels concentration and oil phase fraction on Pickering emulsions

The particle size and polydispersity index (PDI) of CHC-CMS nanogels prepared with different volume mixing ratios of CHC-to-CMS were displayed in Fig. 1. The smallest particle size of nanogels (378.2 nm) was obtained when the mixing ratio was 2:1. Under the optimum conditions, the PDI of the dispersion solution was 0.215, exhibiting a better dispersibility and stability. A similar result was reported by Dai et al. (2018), who prepared a smaller zein - propylene glycol alginate particles at a zein-to-PGA mass ratio of 10:1. The smaller size contributed to the adsorption of nanogels on the oil/water interface, which was beneficial for the formation of food-grade Pickering emulsions (Xi et al., 2018). Therefore, the mixing ratio of 2:1 was selected for further analysis. The interfacial wettability of the nanogels was key to prepare stable

Initially, it was verified that the CHC-CMS nanogels stabilized Pickering emulsions were O/W type because they dispersed in distilled water, rather than in corn oil. 3.2.1. Effect of CHC-CMS nanogels concentration on Pickering emulsions The emulsification efficiency of Pickering emulsions stabilized by solid particles was related to the ability to cover the oil-water interface. Fig. 3 showed the droplet size and visual appearance of different CHCCMS nanogels concentrations stabilized Pickering emulsions at the fixed oil phase fraction (φ 0.5). The droplet size of emulsions stabilized 218

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Fig. 5. Effect of pH on the diameter of the droplets and emulsion index (EI) upon storage for 1 day and 7 days (a); Zeta potential of Pickering emulsions at different pH conditions (b).

Fig. 6. Optical microscopic images of Pickering emulsions stabilized by CHCCMS nanogels with different pH after 7-day storage. The blank bars: 20 μm in scale.

by 0.1% CHC-CMS nanogels was 42.03 ± 0.86 μm. It decreased to 7.35 ± 0.16 μm when the CHC-CMS nanogels concentration was increased to 1.5%. This phenomenon could be attributed to the increasing in the number of solid particles adsorbed and anchored onto oil-water interface to cover a larger interfacial area (Mwangi, Ho, Tey, & Chan, 2016). When the CHC-CMS nanogels concentration was beyond to 1.5%, the droplet size of the emulsions remained unchanged. Therefore, the 1.5% CHC-CMS nanogels were selected for further analysis.

surface charge and surface wettability of CHC-CMS nanogels. In principle, the suitable wettability was the origin of the strong anchoring of solid particles at the oil-water interface (Yuan et al., 2017). The droplet size, EI and zeta potential of Pickering emulsions with different pH were presented in Fig. 5. The zeta potential of emulsions decreased from +35.7 ± 0.9 mV to −64.26 ± 5.35 mV when the pH changed from 2 to 8. Under the condition of the low pH (pH < 4.0), the stability of emulsions decreased with the decrease of zeta potential from +35.7 mV to + 1.01 mV. At pH 4.0 and 5.0, the droplets became flocculated (Fig. 6), and the droplet size of emulsions significantly increased. The decrease in electrostatic repulsion of droplets might lead to instability of emulsions, thus the EI decreased. When pH was above 6.0, the emulsions were highly stable against droplet coalescence and creaming due to the high electrostatic repulsions between the droplets. Meanwhile, the droplet size and EI were almost constant. The stability of CHC-CMS nanogels stabilized emulsions appeared to response to pH.

3.2.2. Effect of oil phase fraction on Pickering emulsions The effect of oil phase fraction (φ = 0.1–0.7) on the visual appearance and droplet size of the Pickering emulsions determined at a fixed nanogels concentration (1.5% CHC-CMS nanogels) were shown in Fig. 4. The droplet size of emulsions increased from 4.43 ± 0.31 μm to 17.19 ± 0.87 μm as the oil phase fraction increased from 0.1 to 0.7. This result was consistent with the previous study reported by Xiao et al., 2016a,b, which illustrated that the increasing oil phase fraction resulted in higher emulsified phase volume fraction and larger droplet size of the Pickering emulsions. At the low oil contents, the solid particles were sufficient to maintain the oil-water interface, which could form the uniform droplets. However, at high oil contents (φ = 0.6, 0.7), the oil off phenomenon could be observed at the top of the emulsion because the nanogels particles were insufficient to cover the oil-water interface. Meanwhile, the droplets were packed closely together and some of the interfacial layers were destroyed.

3.4. Effect of ionic strength on emulsion stability The ionic strength was found to effect the packing of charged particles at the interface as the electrostatic repulsion between particles being screened (Tzoumaki et al., 2011). The droplet size, EI and zeta potential under different concentration of NaCl (0–0.6 M) were shown in Fig. 7. The droplet size of emulsions increased from 7.74 μm to 23.89 μm when the NaCl concentration was raised from 0 M to 0.6 M, suggesting droplets aggregation (Zhu, Chen, Mcclements, Zou, & Liu, 2017). After 7 days of storage, the droplet size of Pickering emulsions increased markedly (from 7.74 μm to 93.58 μm) with increasing NaCl

3.3. Effect of pH on emulsion stability The pH of the aqueous phase triggered changes in the overall 219

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D[4,3] m

EI Day 1 EI Day 7

D[4,3] m Day 1 D[4,3] m Day 7

100

100

80

80

60

60

40

40

20

20

0

0.0

0.1

0.2

0.3

0.4

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Emulsion index, EI (%)

a

0

NaCl concentration (mol/L) NaCl concentration (mol/L)

b

-10

0.0

0.1

0.2

0.4 d

-20

Zeta-potential (mV)

0.3

0.5

0.6

d

d

0.7

c b

-30

b

-40 -50

-60

a

-70 Fig. 7. Effect of NaCl concentration on the diameter of the droplets and emulsion index (EI) upon storage for 1 day and 7days (a); Zeta potential of Pickering emulsions at different NaCl concentrations (b).

widely. The droplet size and visual appearance of CHC-CMS nanogels emulsions under 3 months storage were displayed in Fig. 9. The droplet sizes of Pickering emulsions with 0.1 and 0.2 oil phase fraction increased significantly while those of other samples were nearly unchanged after 1 month of storage. However, it should be noted that the droplet size and visual appearance of emulsions prepared by high oil phase fraction (φ 0.5) was almost kept constant after 3 months of storage. This phenomenon was attributed to the fact that the oil droplets in the emulsions were tightly packed together with increase of oil phase fractions, which was beneficial to form network architecture and enhance its stability against coalescence. The network architecture could trap emulsions droplets and restrict flowing around of liquids, thus inhibiting phase separation of Pickering emulsions. Besides, emulsions prepared by high oil phase fraction had high viscosity, which might slow down the destabilization phenomena. This result further revealed that the CHC-CMS nanogels stabilized Pickering emulsions highly resisted unstable phenomenon, such as creaming, flocculation, coalescence and Ostwald ripening.

concentration up to 0.6 M. Microscopic images confirmed that the droplets aggregation happened (Fig. 8). This result could be attributed to the fact that the zeta potential decreased from −63.06 mV to −17.83 mV as the NaCl concentration increased from 0 M to 0.6 M. The electrostatic repulsion between the droplets was reduced due to the electrostatic shielding effect, resulting in the instability of emulsions. EI was also applied to evaluate the stability of the Pickering emulsions. It was observed that the emulsion stability was dependent on the ionic strength. The EI decreased with increasing ionic strength. After 7 days of storage, the EI decreased slightly, which was attributed to the occurrence of the aggregation. A similar result was reported in hydrophobic starch particles stabilized Pickering emulsions (Song et al., 2015). 3.5. Storage stability of nanogels-stabilized Pickering emulsions Storage stability was an important factor of food product because it determined to some degree whether or not the product could be used 220

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Fig. 8. Optical microscopic images of Pickering emulsions stabilized by CHCCMS nanogels with different NaCl concentrations after 7-day storage. The blank bars: 20 μm in scale.

The microstructure of emulsions stabilized by different oil phase fractions were shown in Fig. 10. After 24 h of storage, emulsions prepared by CHC-CMS nanogels with different oil phase fractions had similar droplet sizes. Interestingly, emulsions prepared by high oil phase fraction (φ 0.5) remained stable at room temperature for 3 months. However, at low oil phase fraction, the droplets with large droplet size appeared. These consequences were in accordance with the particle size measurement.

Fig. 9. The droplet size of Pickering emulsions stabilized by 1.5% CHC-CMS nanogels at various oil phase fractions of 0.1–0.5 for 0, 1, and 3 months storage (a); Visual observation of Pickering emulsions stabilized by CHC-CMS nanogels at various oil phase fractions of 0.1–0.5 for 3 months storage (b). Samples designated with different lower case letter (a, b, c, d, e, f) indicated significant difference (p < 0.05) when compared between different samples (same storage time). Samples designated with different capital letter (A, B, C) indicated significant difference (p < 0.05) when compared between different storage time (same sample).

3.6. Physical stability

nanogels stained (C). The green oil phase was in the interior of the droplets while the red complex nanogels particles were presented as a shell around the droplets, which confirmed that the oil-in-water Pickering emulsions were successfully prepared. It was found that the composite particles had a tendency to adsorb on the oil-water interface, forming a densely packed layer on the surface of spherical oil droplets. This interfacial structure generated a physical barrier for Pickering emulsions against flocculation, coalescence and Ostwald ripening. This phenomenon was in agreement with previous studies (Dai, Sun, Wei, Mao, & Gao, 2018; Ge et al., 2017).

Centrifugation accelerates the creaming process of emulsions to force the droplets concentrate (Hu et al., 2016). After centrifugation, the excess water was excluded from emulsions, resulting in close packing conditions. Meanwhile, a layer of free oil released at the top of emulsions. The ES of emulsions after the centrifugation was used to characterize emulsion stability (Fig. 11). The ES was decreased from 90.22% to 6.72% when the oil phase fractions decreased from 0.5 to 0.1. This result was consistent with the storage stability, further indicating that the stability of Pickering emulsions was influenced by oil phase fractions.

3.8. Flow behavior of O/W emulsions 3.7. CLSM The flow plots of CHC-CMS nanogels stabilized O/W Pickering emulsions were displayed in Fig. 13a. The Pickering emulsions showed the typical non-Newtonian pseudoplastic behavior (shear thinning) at the shear rates of 1–100 1/s. The viscosity of emulsion was high at low shear rate and then decreased with increasing shear rate. The

The microstructure, especially interfacial framework and network structure of the emulsions, was determined using CLSM (Fig. 12). Three pictures of the stained Pickering emulsions were shown (i) CHC-CMS nanogels stained red (A); (ii) Oil stained green (B); (iii) Both oil and 221

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100% a 80%

ES%

60% b

40%

c 20% d 0%

0.5

0.4

0.3 0.2 Oil phase fractions (v/v)

d 0.1

Fig. 11. Emulsion stability against physical stresses with different oil phase fractions. Samples designated with different lower case letters (a, b, c, d) were significantly different (P < 0.05).

relationship between shear stress-shear rate of O/W emulsions by CHCCMS nanogels was non-linear, indicating the shear-thinning behavior of emulsions as a non-Newtonian fluid. This behavior could be explained by breaking the entangled polymer network during shearing, in which the intermolecular entanglement rate of disruption was greater than that of reformation, resulting in less intermolecular resistance to flow and lower apparent viscosity as described by Park, Chung, and Yoo (2004). As shown in Table 1, the higher value of correlation coefficient confirmed the reliability of the obtained parameters for Pickering emulsions. Midmore (1999) reported that σ0 was an indication of how well the emulsion droplet and network could resist the sedimentation or creaming. A low σ0 of W/O emulsions with low oil phase might indicate a tendency to separation as reported by Torres, Iturbe, Snowden, Chowdhry, and Leharne (2007). The K value increased with increasing oil phase fractions, which exhibited that apparent viscosity of the examined emulsions increased. The value of n was related to the degree of non-Newtonian behavior and all values shown in Table 1 were less than 1, which verified the pseudoplastic fluid characteristics of O/W emulsions (Torres et al., 2007).

Fig. 10. Optical micrograph of Pickering emulsions stabilized by 1.5% CHCCMS nanogels with different oil phase fractions (φ = 0.1–0.5). Micrographs a-e and a1-e1 were taken after 24 h and 3 months of quiescent storage, respectively. The blank bars: 20 μm in scale.

3.9. Viscoelastic properties of O/W emulsions The rheological properties, one of the most important factors of Pickering emulsions, indicated their stability and functionality. For all Pickering emulsions, the value of storage modulus (G') was obviously higher than loss modulus (G″) in the whole frequency range (Fig. 14). It indicated an elastic gel-like structures of Pickering emulsions prepared, which was in agreement with previous studies (Chen et al., 2016). Moreover, higher oil phase fractions resulted in a gradual increase in G' and G″, contributing to a tightly packed structure. Xiao et al., (2016a,b) and Dai et al. (2018) reported a similar result that the G' and G″ of Pickering emulsions were positively related with the oil content. Additionally, both G' and G″ showed a slight dependence on the whole frequency sweep range for all Pickering emulsions, indicating that the non-covalent physical interactions were mainly responsible for the formation of gel-like emulsion network (Zou, Guo, Yin, Wang, & Yang, 2015).

pronounced shear thinning behavior of emulsions was a typical of weak associative interactions, indicating the formation of a weak droplet network structure in Pickering emulsions stabilized by CHC-CMS nanogels. The similar pseudoplastic flow property was observed in the case of O/W Pickering emulsions stabilized by zein/gum arabic complex colloidal nanoparticles or hydropholic phytoglycogen nanoparticles (Dai et al., 2018; Song et al., 2015; Ye et al., 2018). Moreover, the viscosities of Pickering emulsions was progressively improved with the increase of oil phase fraction. These results were consistent with flow parameters of emulsions stabilized by soy glycinin or kafirin nanoparticles (Liu & Tang, 2016; Xiao et al., 2016a,b). Plots of shear stress versus shear rate data for emulsions stabilized by CHC-CMS nanogels with different oil phase fractions were shown in Fig. 13b. The Power law (σ = Kγ n ) and Herschel-Bulkley (σ = σ0 + Kγ n ) models were used to describe the flow behavior (Table 1), where σ was the shear stress (Pa), σ0 was the apparent yield stress (Pa), K was the consistency index (Pa.sn), γ was the shear rate (s−1), n was the flow behavior index. The higher value of correlation coefficient confirmed that the experimental data were fitted Herschel-Bulkley model better. The parameters of Herschel-Bulkley model exhibited that the

4. Conclusions In conclusion, the stable Pickering emulsions were prepared with CHC-CMS nanogels. A near-netural wettability of CHC-CMS nanogels was observed with smallest particle size (378.2 nm) at the optimal ratio (CHC:CMS = 2:1). The emulsion droplet size was influenced by CHC222

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Fig. 12. CLSM images of 1.5% CHC-CMS nanogels stabilized Pickering emulsions at 0.5 oil phase fraction: CHC-CMS nanogels was stained by Nile Blue (red) excited at 633 nm (A); Corn oil was stained with Nile Red (green) excited at 488 nm (B); C was combined image of A and B. The blank bars: 50 μm in scale.

100

a

0.5 0.3 0.1

0.4 0.2

Viscosity (Pa.s)

10

1

0.1

20

100

Shear stress (Pa)

b

0.5 0.3 0.1

40

60 Shear rate (1/s)

80

100

80

100

0.4 0.2

10

1

0.1

0

20

40

60 Shear rate (1/s)

Fig. 13. Shear-rate dependence of viscosity for O/W emulsions stabilized by CHC-CMS nanogels (a), and shear-rate dependence of shear-stress for O/W emulsions stabilized by CHC-CMS nanogels (b).

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Table 1 Effect of oil phase fraction on fit Power law model and Herschel-Bulkley model of CHC-CMS nanogels stabilized Pickering emulsions. Power law

Herschel-Bulkley n

2

oil fraction

K (Pa.s )

n

R

0.1 0.2 0.3 0.4 0.5

0.9683 1.6931 3.2221 5.9830 9.7230

0.55665 0.48621 0.38736 0.38152 0.31120

0.99029 0.97970 0.94681 0.95541 0.98684

σo(Pa)

K (Pa.sn)

n

R2

0.0339 0.3239 1.6100 1.8710 3.210

1.302 1.493 1.800 4.323 6.048

0.4724 0.4966 0.5861 0.4375 0.4139

0.9988 0.9879 0.9788 0.9809 0.9888

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Fig. 14. Dynamic frequency sweep curves for O/W emulsions stabilized by CHC-CMS nanogels with different oil phase fractions. G', solid shapes. G″, hollow shapes.

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