Engineering functional alginate beads for encapsulation of Pickering emulsions stabilized by colloidal particles

Abstract

Pickering emulsions are widely used as delivery systems in food, cosmetics, and pharmaceutical industries for the encapsulation and sustained release of hydrophilic compounds. The solid colloidal particles stabilized in oil–water interface can prevent the oil droplets from undesired coalescence and endow additional functionality. In order to impact further protection and pH-responsive deliver of the encapsulated component, we used two kinds of mechanisms. For internal interaction, the excess Ca²⁺ on the surface of zein–tannic acid (TA)/Ca²⁺ nanoparticles (NPs) interacted with alginate added to form a second layer. In the case of external gelation, a cross-linking agent (Ca²⁺) was dissolved in the aqueous gelling bath outside. When added the mixture of the emulsions to Ca²⁺ solution, alginate–Ca beads were formed to encapsulate the emulsions and further impact the pH-responsive deliver of VD3 in stomach and rapid release of it in small intestine.

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1. Introduction

Oil-in-water (O/W) emulsions play an important role in a wide range of industrial processes and commercial products.¹ Among them, Pickering emulsions, wherein solid colloidal particles instead of molecular surfactants or amphiphilic polymers adsorb at the oil/water interface, are known to display long-term stability against coalescence.² Thereby, this kind of emulsions can provide variety of promising applications for texture modification, calorie and fat reduction, as well as bioactive compound delivery.^3–5 Compared with conventional emulsions, solid colloidal particles stabilized in oil–water interface can endow additional functionality including the response to a trigger, forming bilayer shell and the like.^2,6,7

Different types of solid particles,^8–11 such as silica,^12–14 calcium carbonate (CaCO₃) particles,¹⁵ titanium dioxide (TiO₂) particles,¹⁶ polystyrene lattices¹⁷ and gold nanoparticles¹⁰ have been used as emulsifiers to stabilize Pickering emulsions. However, in regard to their suitability for food applications and environmental problems, the direct application of such particulate emulsifiers is still very limited.¹⁸ Although promising emulsifiers based on protein particles,¹⁹ polysaccharide particles^20,21 and protein–polysaccharide complex²² have emerged, studies on food-grade Pickering emulsions for food applications are still in a limited number. Thus, the development of effective colloidal particles based on food-grade ingredients to serve as Pickering emulsion stabilizers is still a significant challenge in the food emulsion area.²³

As an alcohol-soluble protein obtained from corn, zein contains sharply defined hydrophobic and hydrophilic domains and is capable of forming self-assembled NPs to stabilize Pickering emulsions.^24–26 Therefore, zein-based particles possess great potential to be used as food-grade Pickering emulsion stabilizers. de Folter and co-workers have first used zein colloidal particles as effective particle-stabilizers of oil-in-water Pickering emulsions.²⁷ Then they demonstrated the behavior of zein particles in the formation of stable Pickering emulsions as a function of particle concentration, pH and ionic strength. The followers devoted to further increasing the stabilization of oil–water interfaces with zein complex colloidal particles. Gao et al. prepared the complexation of zein particles and surfactant sodium stearate which increased the adsorption and accumulation of zein particles at oil–water interface.⁴ The resultant Pickering emulsions stabilized by the complexation exhibited superior stability against both coalescence and creaming. Then in order to reduce the lipid oxidation of emulsified oil, Wang et al. fabricated zein/chitosan complex particles loaded with antioxidant curcumin, and further prepared antioxidant Pickering emulsions, giving rise to the emulsions with favorable oxidative stability.²² Food-grade colloidal complexes based on zein and tannic acid were prepared and successfully used to stable Pickering emulsion gels.²⁵

As a kind of natural polyphenol and a food-grade material, tannic acid (TA), which contains numerous terminal hydroxyl groups, has unique structural properties.^28,29 The dominant constituents facilitate interactions with a variety of materials including proteins,³⁰ polysaccharides³¹ and synthetic polymers^32,33 via multiple reaction pathways, such as electrostatic, hydrogen bonding, and hydrophobic interactions.³⁴ Furthermore, the strong metal chelation ability and materials surface binding affinity make it a perfect material to act as a polydentate ligand for metal ion coordination.^35,36 In our previous work, TA has been well recognized as a promising material to self-assembly of coordination bonding architecture with metal ions on zein NPs.^31,37 In addition, the excess metal ions on the surface of NPs could further form connections with other materials. The newly formed layer may achieve one or more desirable effects including protection from evaporation or oxidation of the loaded compound, and controlled-release applications.

Alginate is a characteristic example of natural polyelectrolytes undergoing chain–chain association and forming hydrogels upon addition of divalent cations (e.g., Ca²⁺).^38,39 Gelation is controlled by chelation between the carboxyl groups of a-L-guluronic acid in chains of alginate with calcium.^40–42 Alginate hydrogels have been extensively investigated due to their biocompatibility and pH-responsive characteristics which can provide a controlled release of encapsulated component.^43–45

In this study, we first formed zein–TA/Ca²⁺ NPs, and consequently use this kind of particles to prepare stable Pickering emulsions for encapsulation of an unstable nutrient, VD3. In order to induce the gelation of the droplets, we mainly employed two kinds of mechanisms, namely internal interaction and external gelation.⁴⁶ For internal interaction, the excess Ca²⁺ on the surface of NPs will interact with alginate added to form a second layer. In the case of external gelation, cross-linking agent was dissolved in the aqueous gelling bath outside. When added the mixture of the emulsions to Ca²⁺ solution, alginate–Ca beads were formed to encapsulate the emulsions and further impacted pH-responsive release of VD3 in stomach and rapid release of it in small intestine.

2. Materials and methods

2.1. Materials

Zein (Z0001) was purchased from Tokyo Chemistry Industry, Co., Ltd. (Tokyo, Japan). VD3 (99%) and tannin acid was purchased from Aladdin Chemistry Co., Ltd. Alginate and CaCl₂·2H₂O was obtained from the Sinopharm Chemical Reagents Co., Ltd. (Shanghai, China). Medium chain triglyceride (MCT) was purchased from Boxing Chemical Reagent Co. Ltd. (Wuhan, China). Other chemicals used were of analytical grade. All the solutions used in the experiments were prepared using ultrapure water through a Millipore (Millipore, Milford, MA, USA) Milli-Q water purification system.

2.2. Preparation of zein–TA/Ca²⁺ NPs

All solutions were freshly prepared for immediate use. The standard preparation process was described as follows: zein (10 mg mL⁻¹, 20 mg mL⁻¹ and 30 mg mL⁻¹) was dissolved in 75% alcohol-aqueous solution. Then 1 mL of the above mentioned solution was rapidly added into 9 mL of ultrapure water. Next, 100 μL of TA solution (24 mM) was added and the dispersion was briefly vortexed. The solution was heated in a water bath at 45 °C for 1 h with slow stirring. Following this, 100 μL of fresh metal solution (25 mM, 50 mM, 75 mM, 100 mM or 125 mM of CaCl₂ solution) was added and the dispersion was vortexed. The product was then purified by successive dialysis (MWCO 3500) against deionized water for 36 h. The final product was freeze-dried and kept in a desiccator until use.

2.3. Characterizations of zein–TA/Ca²⁺ NPs

Dynamic laser scattering (DLS) and zeta potential. Dynamic Light Scattering (DLS) and zeta potential measurement were performed on a commercial laser light scattering instrument (Nano-ZS90, Malvern, UK). For the DLS experiment, disposable cuvettes were used and each sample was measured with 20 numbers of runs. All measurements were carried out at 25 °C, and the results reported were the average of three readings.

Transmission electron microscope (TEM). Images were taken on a JEM-2100F microscope (JEOL, Japan). The samples were prepared by dropping solution onto copper grids coated with carbon following by drying under ambient conditions. The photographs were taken at various magnifications and the accelerating voltage was 100 kV.

Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) analysis. FT-IR spectra were obtained with a Jasco 4100 series with an attenuated total reflection cell (Jasco Inc., Easton, MO). All samples were prepared as KBr pellets and were scanned against a blank KBr pellet background. X-ray photoelectron spectroscopy (XPS) observations were conducted on an axis ultra DLD apparatus (Kratos, U.K.).

2.4. Preparation of Pickering emulsions

The prepared zein–TA/Ca²⁺ NPs were utilized to prepared Pickering emulsions. The emulsions were formulated at oil/water ratio of 0.2. In brief, 20 mL of the zein–TA/Ca²⁺ NPs suspensions (with zein final concentration of 0.1%, 0.2% or 0.3%) in a glass vial, and 4 mL of MCT (with or without VD3) was added to the dispersion. Then the resultant mixtures were sheared using an IKA Ultra-Turrax T25 homongenizer at 6000 rpm for 5 min at room temperature. The obtained emulsions were directly subject to analysis or storage stability experiments.

2.5. Morphology observation

The microstructures of the freshly formed emulsions were visualized by using static micro particle image analyzers (Winner 99D, China) with a 10 or 20× objective. The samples were diluted with deionized water.

2.6. Particle size distribution of Pickering emulsions

Droplet size distribution of various freshly prepared emulsions was determined at room temperature, using a Malvern Mastersizer 2000 (Malvern Instruments Ltd., Malvern, Worcestershire, U.K.). Volume mean diameter (d_4,3) was calculated from particle size profiles of the Pickering emulsions according to previous study.⁴⁷

2.7. Preparation and characterizations of alginate–Ca beads

Ten milliliters of zein–TA/Ca²⁺ NPs stabilized Pickering emulsions were mixed with ten milliliters alginate solution with different concentrations (1%, 2%, 3%, 4% or 4.5%). Then the above mixed solution was extruded through a 0.55 mm needle and was dripped into Ca²⁺ gelling solution to form oil-loaded alginate–Ca beads. The gelling solution was gently stirred with a magnetic stirrer to prevent the beads sticking together.⁴⁸ The beads that formed then hardened for 30 min in the gelling bath. A digital camera was used to capture the images of the wet beads. Also the microstructure of the freshly formed beads was observed by optical microscopy.

2.8. Release profile of VD3 from alginate–Ca beads

The prepared alginate–Ca beads were used for in vitro kinetic release test in different conditions. For a kinetic release test at pH 7.4, a certain amount of sample was re-suspended in phosphate buffer containing 0.2% Tween 20 to provide sink condition. The experiment was carried out under the water bath at 37 °C with shaken speed of 100 rpm. At each predetermined time interval, 1 mL of the supernatant containing the released VD3 was collected and freeze-dried. The VD3 percentage released was calculated as a function of time (up to 8 h).

The accumulated release profiles of the beads in the SGI with digestive enzymes were obtained using the method as previously reported.⁴⁹ The samples were first incubated in 50 mL of simulated gastric fluid (SGF) with 0.1% pepsin (w/v) for 0.5 h and the supernatant containing the released VD3 was collected and freeze-dried. Digestion was stopped by raising the pH to 7.4 using NaOH, and the sample was then centrifuged to separate aggregates from supernatant. While the precipitate was re-dispersed using 50 mL of simulated intestinal fluid (SIF) with 1.0% pancreatin (w/v) at 37 °C and digested for 6 h under mild stirring. After digestion, the supernatant was collected and used for VD3 measurement. All measurements were performed in three replicates.

3. Results and discussion

3.1. Preparation of zein–TA/Ca²⁺ NPs

In this work, we fabricated a novel kind of Pickering emulsions stabilized by colloidal particles based on Ca²⁺–TA coated zein NPs. The excess Ca²⁺ on the surface of NPs was used to initiate alginate interaction to form a second layer. In the case of external gelation, the mixture of the emulsions was added to Ca²⁺ solution and alginate–Ca beads were formed to encapsulate the emulsions and further impacted pH-responsive deliver of VD3 in stomach and rapid release of it in small intestine.

For the preparation of zein–TA/Ca²⁺ NPs, we first formed zein NPs and then added TA into zein NPs solution. After that we heated the formed NPs to enhance their interaction. During incubation, it can be predicted that hydrogen bonds of intramolecular may be broken which may provide complexation sites for TA to interact.²⁵ In addition, TA contains numerous terminal hydroxyl groups, which endow it stronger surface binding affinity to proteins than other polyphenols.^34,35 As shown in Scheme 1, Ca²⁺ was used to coordinate bonded with –OH units of TA. Also Ca²⁺ might interact with –COOH groups of zein to harden the NPs. Moreover, the Ca²⁺ on the surface of the NPs would combine with alginate, which would provide a second protection of the loadings in emulsions (Scheme 1).

Scheme 1 Illustration of the synthesis and structures of metal–TA coated zein NPs and the preparation for alginate–Ca gel.

The influence of zein and Ca²⁺ concentrations on particle size, PDI and zeta potential in different formulations were summarized in Table 1. The particle size of zein–TA NPs without mental ion added was 75.4 ± 0.6, 105.2 ± 1.2, 129.4 ± 0.7 at zein concentration of 0.1%, 0.2%, 0.3% respectively (data was not shown). After connected by mental ions, the particle size varied with calcium concentration added. For each zein concentration, increase the amount of Ca²⁺ added led to a decrease trend of the particle size. Then further increase the Ca²⁺ concentration, the particle size increased until aggregation occurred. In addition, with the increase of zein concentration, the aggregation would occur at high Ca²⁺ concentration. The PDI in all formulations was less than 0.2, indicating that particles in these formulations may have uniform particle sizes. The addition of Ca²⁺ caused the decrease in zeta potential. As more Ca²⁺ was added and cross-linked with TA and zein, the zeta potential became less negative. In order to have more complexation sites for alginate to interact, we chose Z₁–TA/Ca₅₀, Z₂–TA/Ca₇₅ and Z₃–TA/Ca₁₀₀ for further study.

Table 1 Characterization of zein–TA/Ca²⁺ NPs, the results were displayed as the mean ± standard deviation (n = 3)^a

Samples	Size (nm)	PDI	Zeta potential (mV)
a Z1–TA/Ca25, Z1–TA/Ca50 and Z1–TA/Ca75 represented zein–TA/Ca^2+ NPs at zein concentration of 0.1% with the initial used metal ion concentration of 25 mM, 50 mM, and 75 mM respectively; Z2–TA/Ca25, Z2–TA/Ca50, Z2–TA/Ca75 and Z2–TA/Ca100 represented zein–TA/Ca^2+ NPs at zein concentration of 0.2% with the initial used metal ion concentration of 25 mM, 50 mM, 75 mM and 100 mM respectively; Z3–TA/Ca25, Z3–TA/Ca50, Z3–TA/Ca75, Z3–TA/Ca100 and Z3–TA/Ca125 represented zein–TA/Ca^2+ NPs at zein concentration of 0.3% with the initial used metal ion concentration of 25 mM, 50 mM, 75 mM, 100 mM and 125 mM respectively. The number labeled after Z or Ca means the different concentration of zein or Ca^2+ used.
Z1–TA/Ca25	81.2 ± 1.6	0.15 ± 0.04	−37.1 ± 0.9
Z1–TA/Ca50	78.2 ± 2.3	0.19 ± 0.01	−29.5 ± 1.3
Z1–TA/Ca75	Large aggregates
Z2–TA/Ca25	131.1 ± 1.1	0.11 ± 0.01	−32.5 ± 0.8
Z2–TA/Ca50	103.5 ± 0.9	0.18 ± 0.03	−28.9 ± 1.0
Z2–TA/Ca75	123.5 ± 0.9	0.14 ± 0.04	−25.1 ± 0.6
Z2–TA/Ca100	Large aggregates
Z3–TA/Ca25	164.4 ± 1.0	0.12 ± 0.03	−30.7 ± 0.8
Z3–TA/Ca50	132.4 ± 2.1	0.14 ± 0.03	−27.9 ± 0.6
Z3–TA–Ca75	131.7 ± 1.1	0.10 ± 0.02	−25.5 ± 0.5
Z3–TA–Ca100	151.7 ± 4.0	0.13 ± 0.02	−23.3 ± 0.7
Z3–TA–Ca125	Large aggregates

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3.2. Characterization of zein–TA/Ca²⁺ NPs

Fig. 1 showed the TEM images and typical size distribution profiles of zein–TA NPs and zein–TA/Ca²⁺ NPs. As shown in the TEM images (Fig. 1a–c), zein–TA NPs without Ca²⁺ shared features of a spherical shape, but most of the particles were clumped and connected to each other. After zein–TA NPs were connected by metal ions, it was possible to see individual NPs clearly with well-defined spherical shape and homogeneous distribution (Fig. 1d–i). In addition, the particle size increased with zein and Ca²⁺ concentration increased.

Fig. 1 TEM image of Z₁–TA NPs (a), Z₂–TA NPs (b), Z₃–TA NPs (c), Z₁–TA/Ca₅₀ NPs (d), Z₂–TA/Ca₇₅ NPs (e) and Z₃–TA/Ca₁₀₀ NPs (f); size distribution of Z₁–TA/Ca₅₀ NPs (g), Z₂–TA/Ca₇₅ NPs (h) and Z₃–TA/Ca₁₀₀ NPs (i). Z₁–TA/Ca₅₀ NPs represented zein–TA/Ca²⁺ NPs at zein concentration of 0.1% with the initial used metal ion concentration of 50 mM, Z₂–TA/Ca₇₅ NPs represented zein–TA/Ca²⁺ NPs at zein concentration of 0.2% with the initial used metal ion concentration of 75 mM, Z₃–TA/Ca₁₀₀ NPs represented zein–TA/Ca²⁺ NPs at zein concentration of 0.3% with the initial used metal ion concentration of 100 mM. The number labeled after Z or Ca means the different concentration of zein or Ca²⁺ used.

Then, FT-IR method was used to verify the existence of the specific intermolecular interactions between Ca²⁺, TA and zein components in the metal/TA coated NPs. The representative spectra of zein, TA, zein–TA NPs and zein–TA/Ca²⁺ NPs were shown in Fig. 2. In the infrared spectra, a characterization peak was in the range of 3200–3400 cm⁻¹, indicating the hydrogen bonding.⁴⁹ The O–H stretching band of the hydroxyl groups in zein and TA was at 3420 cm⁻¹ and 3398 cm⁻¹, respectively, and shifted to 3347 cm⁻¹ and 3317 cm⁻¹ in zein–TA NPs and zein–TA/Ca²⁺ NPs respectively, suggesting the hydrogen bonding was formed between TA and zein. The amide I band of zein at 1648 cm⁻¹ demonstrated a prominent C=O stretching, while the amide II band at 1546 cm⁻¹ demonstrated C–N stretching.⁵⁰ Comparing the spectra of zein with zein–TA NPs or zein–TA/Ca²⁺ NPs, the band of amide I and amide II group shifted to 1658 cm⁻¹ and 1533 in zein/TA NPs or zein–TA/Ca²⁺ NPs, indicating that the electrostatic interaction was an intermolecular force between zein and TA. Seen from the FTIR spectrum of all of zein–TA/Ca²⁺ NPs, the reduced intensity of the HO–C stretching peak at 1202 cm⁻¹ also indicated that the phenolic groups coordinated with metal ions peak when compared with the spectrum of the zein–TA sample and TA.⁵¹

Fig. 2 Fourier transform infrared spectroscopy (FTIR) spectra of different samples. Z, zein powder; TA, tannic acid powder; Z₁–TA, zein–TA NPs at zein concentration of 0.1%; Z₁–TA/Ca, zein–TA/Ca²⁺ NPs at zein concentration of 0.1% with the initial used metal ion concentration of 50 mM; Z₂–TA/Ca, zein–TA/Ca²⁺ NPs at zein concentration of 0.2% with the initial used metal ion concentration of 75 mM; Z₃–TA/Ca, zein–TA/Ca²⁺ NPs at zein concentration of 0.3% with the initial used metal ion concentration of 100 mM. The number labeled after Z or Ca means the different concentration of zein or Ca²⁺ used.

To further confirm the surface chemical information of the NPs, XPS analysis was performed. Fig. 3 displayed the survey scan spectrum of zein–TA/Ca²⁺ NPs, in which C 1s, O 1s and N 1s core-levels existed obviously. Three peak components of C 1s core-level photoelectron spectrum located at 284.2 eV, 285.5 eV, and 287.3 eV (Fig. 3c), which are coordinated to C–C, C–O, and C=O or O–C=O group. From Fig. 3b, Ca 2p signal was observed in the survey scan spectrum of zein–TA/Ca²⁺. In addition, the C/O ratio of zein, zein–TA/Ca²⁺ were obtained to be 4.18 and 3.23, respectively (Table 2). As is well-known, TA was rich in oxygen, about 43.27%, much higher than in zein which also identified the metal–phenolic films coating on zein NP.⁵²

Fig. 3 XPS survey spectra of Z₁–TA/Ca₅₀. Z₁–TA/Ca₅₀ represented zein–TA/Ca²⁺ NPs at zein concentration of 0.1% with the initial used metal ion concentration of 50 mM. The number labeled after Z or Ca means the different concentration of zein or Ca²⁺ used.

Table 2 Element composition and content on the surface of zein and zein–TA/Ca²⁺ NPs^a

Samples	C	O	N	S	Ca
a Z1 represented zein NPs at zein concentration of 0.1%; Z1–TA/Ca50 represented zein–TA/Ca^2+ NPs at zein concentration of 0.1% with the initial used metal ion concentration of 50 mM. The number labeled after Z or Ca means the different concentration of zein or Ca^2+ used.
Z1	68.65	16.44	14.34	0.57	—
Z1–TA/Ca50	67.30	20.83	11.25	0.52	0.12

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3.3. Characterization of zein–TA/Ca²⁺ NPs based Pickering emulsions

VD3 is a kind of liposoluble vitamin which is very sensitive to various environmental factors i.e., light, heat, and oxygen and could rapidly induce isomerization or oxidation and then decrease its physiological benefits.^49,53 Additionally, the oxidation of lipids adversely affects the property of VD3. So in this study, we used TA, a kind of polyphenols, to surface coated zein NPs to impact the antioxidant activity of zein NPs. In addition, according to our precious work, TA in the NPs adsorbed at the oil/water interface was able to provide great protection again UV light-induced degradation of VD3.²⁹

In this study, Z₁–TA/Ca₅₀, Z₂–TA/Ca₇₅ and Z₃–TA/Ca₁₀₀ NPs were used to support the formation of stable Pickering emulsions. First, we evaluated the influence of zein and Ca²⁺ concentration in the aqueous phase on the microstructure and the droplet size distribution of the fresh emulsions. Table S1† showed the d_4,3 value of droplets in different sets of fresh zein–TA/Ca²⁺ NPs stabilized emulsions, determined using water as the dispersing solvent. For the fresh prepared emulsions, we can see that increasing concentration of zein and Ca²⁺ resulted in a reduction in d_4,3 first and then followed by an increase trend (Table S1†), with the droplet size widely ranging from 34.64 to 101.91 μm.

The flocculated state of oil droplets in the fresh Pickering emulsions were evaluated using microscope, as displayed in Fig. 4. As expected, the droplet size of these emulsions stabilized by zein–TA/Ca²⁺ NPs considerably varied with zein and Ca²⁺ concentration. For all the formulations, it can be observed that most of the droplets were present in the separated and unflocculated form (Fig. 4a–c).

Fig. 4 Microscopy images of Pickering emulsions stabilized by Z₁–TA/Ca₅₀ NPs (a), Z₂–TA/Ca₇₅ NPs (b), Z₃–TA/Ca₁₀₀ NPs (c). Typical particle size distribution of Pickering emulsions stabilized by Z₁–TA/Ca₅₀ NPs (d), Z₂–TA/Ca₇₅ NPs (e) and Z₃–TA/Ca₁₀₀ NPs (f). The scale bar is 20 μm. Z₁–TA/Ca₅₀ NPs represented zein–TA/Ca²⁺ NPs at zein concentration of 0.1% with the initial used metal ion concentration of 50 mM, Z₂–TA/Ca₇₅ NPs represented zein–TA/Ca²⁺ NPs at zein concentration of 0.2% with the initial used metal ion concentration of 75 mM, Z₃–TA/Ca₁₀₀ NPs represented zein–TA/Ca²⁺ NPs at zein concentration of 0.3% with the initial used metal ion concentration of 100 mM. The number labeled after Z or Ca means the different concentration of zein or Ca²⁺ used.

Fig. 4d–f showed the typical droplet size distribution profile of the various fresh emulsions as a function of zein and Ca²⁺ concentration. All the emulsions fresh prepared at varying concentration exhibited a similarly monomodal size distribution profile. This indicated that all the emulsions were present in the unflocculated form which was corroborated by the microstructural observation of the emulsions using optical microscopy.

3.4. Preparation and characterizations of alginate–Ca beads

In order to impact a second protection and pH responsible release of VD3, we further used alginate–Ca gels to cover the emulsions. First, alginate was mixed with the fresh prepared emulsions, forming a second layer between Ca²⁺ on the surface of zein–TA/Ca²⁺ NPs. Then the mixture was slowly added into Ca²⁺ solution to form alginate–Ca beads.

It was expected that the concentration of alginate solutions would affect the stability of the emulsions as well as the formation of the gel beads. Thus, this work was undertaken to determine the optimal concentrations of alginate for making the emulsions with the highest stability. In Fig. 5c–g, it can be observed that most of the droplets were present in the separated and unflocculated form, and the droplet size progressively decreased with increasing the alginate concentration. Moreover, the increase in the concentration of alginate from 1% to 4.5% led to an increase in stability of the emulsions (Fig. 5a–b). After storage for 3 days, the emulsions became unstable, containing water phase in the bottom except the emulsions with 4.5% alginate (Fig. 5b).

Fig. 5 (a) Digital photograph of Pickering emulsions encapsulated by different concentration of alginate. (b) Digital photograph of Pickering emulsions encapsulated by different concentration of alginate after storage 3 day. (c)–(g) Microscopy images of Pickering emulsions encapsulated by alginate with the concentration of 1%, 2%, 3% 4% and 4.5% respectively. The emulsions stabilized by Z₁–TA/Ca₅₀ NPs. The scale bar is 20 μm.

Then we used emulsions with 4.5% alginate to prepare alginate–Ca beads. The surface morphology of microparticles was observed by optical light microscopy. In Fig. 6a–e, we could see the droplets were homogenously entrapped within the gel network. With the increase of alginate concentration, the amount of oil droplet on the surface decreased and almost all particles were encapsulated by the gel.

Fig. 6 (a)–(e) Microscopy images of Pickering emulsions stabilized Z₁–TA/Ca₅₀ NPs encapsulated by alginate with the concentration of 4.5% in alginate–Ca gel. The scale bar is 20 μm.

3.5. Release of VD3 from alginate–Ca beads

The intestinal-targeted characteristics of alginate–Ca beads are investigated by studying the dissolution of beads in SGF and SIF. Fig. 7 showed the release rate of alginate–Ca beads after immersed in pH 1.2 SGF and pH 7.4 SIF. As can be seen from the picture, the alginate–Ca beads did not show significant changes in shape in SGF, while exist dramatic increase in beads size in SIF. If prolonged the incubation time, the beads trended to dissolve.

Fig. 7 Photographs of alginate–Ca beads encapsulating Pickering emulsions stabilized by Z₁–TA/Ca₅₀ NPs (a) or Z₂–TA/Ca₇₅ NPs (b) in gastric fluid (up) or intestinal fluid (down).

Then we monitored the kinetic release profile of beads in PBS and the cumulative release profile in SGI with digestive enzymes. In PBS medium, all formulations showed a first-order release profile, with biphasic kinetic releasing trend, that is, a burst effect within 2 h followed by a sustained release for up to 6 h with nearly 90% and 80% VD3 released for emulsions stabilized by Z₁–TA/Ca₅₀ and Z₂–TA/Ca₇₅. Under the SGI condition with digestive enzymes, after incubation at 37 °C in SGF for 30 min, only about 18% of the VD3 was released from emulsions stabilized by Z₁–TA/Ca₅₀ or Z₂–TA/Ca₇₅. When transferred to the SIF (pH 7.4), at most 95% of VD3 was released from the polymeric matrix within 6 h for emulsions stabilized by Z₁–TA/Ca₅₀. In contrast, 85% of the VD3 was detected in the releasing medium for emulsions stabilized by Z₂–TA/Ca₇₅. For emulsions stabilized Z₂–TA/Ca₇₅ NPs, more Ca²⁺ could interact with more alginate forming robuster coating which provide a relative slow release rate.

It has been reported that alginate gel is pH sensitive, which shrinks at lower pH and swells at higher pH. As known, VD3 will decrease the stability in acidic conditions. This property of alginate beads was contributed to prominent protection on VD3 against digestion in the gastric fluid, together with an increased amount of VD3 being delivered to the small intestine (Fig. 8).

Fig. 8 Kinetic release of VD3 from alginate–Ca beads in pH 7.4 media (a). Release of VD3 in SGF and SIF from alginate–Ca beads (b).

4. Conclusion

In this work, we fabricated a novel kind of Pickering emulsions stabilized by zein–TA/Ca²⁺ NPs. This kind of particles was irreversibly anchored at the oil–water interface to form particle-based network architecture therein, producing ultrastable O/W Pickering emulsions. The excess Ca²⁺ on the surface of NPs was used to initiate alginate interaction to form a second layer. In the case of external gelation, cross-linking agent (Ca²⁺) was dissolved in the aqueous gelling bath outside. When added the mixture of the emulsions to Ca²⁺ solution, alginate–Ca beads were formed. Compared with previous VD3 delivery system about NPs^49,54 or nanoemulsions,⁵⁵ this alginate beads demonstrated the pH-responsive deliver of VD3 in stomach and rapid release of it in small intestine to provide a comprehensive protection.

Acknowledgements

This work was financially supported by the Fundamental Research Funds for the Central Universities (Program No. 2662016PY092). The authors would like to express their sincere gratitude to many conveniences offered by colleagues of Key Laboratory of Environment Correlative Dietology of Huazhong Agricultural University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra21755a

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