W Emulsion Gels Based on Self-Assembled Saponin

KEYWORDS: multiple W/O/W emulsion gels, glycyrrhizic acid, saponin nanofibrils, fibrillar. 36 network, photo-sensitive cargo protection, water diffusi...
2 downloads 0 Views 9MB Size
Download PDF
Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX-XXX

pubs.acs.org/JAFC

Multiple Water-in-Oil-in-Water Emulsion Gels Based on SelfAssembled Saponin Fibrillar Network for Photosensitive Cargo Protection Lulu Ma,† Zhili Wan,*,† and Xiaoquan Yang*,†,‡ †

Research and Development Center of Food Proteins, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China ‡ Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou 510640, China S Supporting Information *

ABSTRACT: A gelled multiple water-in-oil-in-water (W1/O/W2) emulsion was successfully developed by the unique combination of emulsifying and gelation properties of natural glycyrrhizic acid (GA) nanofibrils, assembling into a fibrillar hydrogel network in the continuous phase. The multiple emulsion gels had relatively homogeneous size distribution, high yield (85.6−92.5%), and superior storage stability. The multilayer interfacial fibril shell and the GA fibrillar hydrogel in bulk can effectively protect the double emulsion droplets against flocculation, creaming, and coalescence, thus contributing to the multiple emulsion stability. Particularly, the highly viscoelastic bulk hydrogel had a high storage modulus, which was found to be able to strongly prevent the osmotic-driven water diffusion from the internal water droplets to the external water phase. We show that these multicompartmentalized emulsion gels can be used to encapsulate and protect photosensitive water-soluble cargos by loading them into the internal water droplets. These stable multiple emulsion gels based on natural, sustainable saponin nanofibrils have potential applications in the food, pharmaceutical, and personal care industries. KEYWORDS: multiple W/O/W emulsion gels, glycyrrhizic acid, saponin nanofibrils, fibrillar network, photosensitive cargo protection, water diffusion

■

INTRODUCTION In recent years, complex emulsions with hierarchical microstructures, such as multiple emulsions, have been the focus of research interest due to their great potential in many applications, including foods, pharmaceuticals, cosmetics, and agriculture.1−4 Due to their compartmentalized internal structure, multiple emulsions (or double emulsions) such as water-in-oil-in-water (W/O/W) emulsions are an ideal delivery platform for the encapsulation of various hydrophilic and hydrophobic actives that need to be protected from external stresses.3−8 In food processing, it has been demonstrated that the multiple emulsions have a high potential to encapsulate and protect sensitive and functional components such as antioxidants, flavors, vitamins, and minerals.4,9,10 The active agents, especially the hydrophilic cargos, can be loaded within the inner droplets, providing a high loading and protection efficiency of the encapsulated agents. The use of multiple emulsions also can offer a potential strategy for producing foods with reduced fat and sodium contents.4,10−12 With consumers increasingly looking for food products with added health benefit, the multiple emulsions offer a promising technological strategy to incorporate nutritional and bioactive compounds for the development of new functional foods. Moreover, multiple emulsions are widely used as a template for generating advanced materials such as microcapsules to deliver cargos with different polarities or as microreactors for chemical synthesis.13,14 Although multiple emulsions show great potential in a wide variety of applications, the production of stable multiple © XXXX American Chemical Society

emulsions remains a big challenge for large-scale utilization due to their inherent thermodynamic instability.2,9,15,16 In most practical cases, a combination of hydrophilic and hydrophobic emulsifiers as well as a two-step emulsification process is typically required to produce multiple W/O/W emulsions.2,9 The high interfacial area due to the presence of two interfaces in these complex multiphase systems can induce many destabilization pathways and thus lead to emulsions with limited stability. For instance, the inner water droplets of double W/O/W emulsions may diffuse or coalesce with the outer water phase because of osmotic and chemical potential differences, disrupting the multicompartmented structure and finally leading to the formation of conventional O/W simple emulsions after processing and storage.17−19 Many strategies have been attempted to improve the physical and structural stability as well as to ensure high encapsulation efficiency of the bioactive cargos entrapped in multiple emulsions.4,9,15,20−29 Among these strategies, the incorporation of natural macromolecules such as polysaccharides into the outer water phase as texture modifiers, perhaps to gel the phase and form structured emulsions, has been demonstrated to be an effective approach to increase the yield and stability of multiple emulsions.24−29 Weiss et al. prepared a gelled multiple emulsion Received: Revised: Accepted: Published: A

August 29, 2017 October 18, 2017 October 23, 2017 October 23, 2017 DOI: 10.1021/acs.jafc.7b04042 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

emulsions at room temperature (25 °C). The oil phase was first prepared by mixing PGPR with sunflower oil and stirring for 30 min at 25 °C. The primary W1/O emulsions were prepared by adding 30 wt % of internal water phase (W1, only water) to 70 wt % of oil phase, and then the resulting mixtures were strongly sheared using an Ultra-Turrax T10 (IKA-Werke GmbH & Co., Germany) at 30 000 rpm for 2 min. In the second step, the freshly prepared W1/O emulsions were incorporated into an external water phase (W2) in a 50:50 (w/w) ratio. The W2 phase was prepared by dissolving appropriate amounts of GA powder in water in a sealed vial and heating it at 80 °C under mild agitation until a clear solution was obtained. The W1/O/W2 emulsions were produced by first dispersing the primary W1/O emulsions in hot GA fibril solutions (80 °C) under mild agitation for 2 min, and then the resulting dispersions containing 4 wt % GA fibrils were homogenized with the Ultra-Turrax T10 at 11 500 rpm for 2 min. The resultant samples were immediately cooled in an ice bath to induce the formation of gelled multiple emulsions. The prepared emulsion gels were stored at room temperature (25 °C) before further measurements. Droplet Size Measurements. The droplet size and size distribution of multiple emulsions were measured using a Mastersizer 3000 (Malvern Instruments Co. Ltd., Worcestershire, UK) at 25 °C. The gelled samples were first diluted 1:10 using water and slightly stirred for 5 min to obtain a homogeneous double emulsion. No significant differences were observed in the values of mean diameter, ranging from 1:10 to 1:100 dilution ratios. The refractive indices of the oil droplets and water continuous phase were taken as 1.47 and 1.33, respectively. Absorption index was set at 0.01. The droplet size was reported as the surface area mean diameter d32 = ∑nidi3/∑nidi2 and volume mean diameter d43 = ∑nidi4/∑nidi3, where ni is the number of droplets with diameter di. Confocal Laser Scanning Microscopy (CLSM). The gelled multiple emulsions and diluted multiple emulsion droplets were visualized by using confocal laser scanning microscope (CLSM, Leica Microsystems Inc., Heidelberg, Germany) under confocal and brightfield modes. To obtain homogeneous emulsion droplets, the gelled samples were diluted 1:10 using water and slightly stirred for 5 min. For confocal visualization, we added Nile Red (0.1 wt %) into the oil phase prior to the multiple emulsion preparation. The samples were examined using an argon krypton laser (ArKr, 488 nm), and the emission fluorescence was observed between 510 and 580 nm. ThT was used to label the GA fibrous aggregates and fibrillar network in the multiple emulsions.30,31 ThT (0.001 wt %) was first dissolved in GA fibril solutions prior to the sample preparation. The ThT concentration used was very low to avoid dye self-assembly. The 458 nm line of an argon laser was used to excite the samples, and the emission fluorescence was observed between 470 and 560 nm. The oil phase dyed with Nile Red is green, whereas the GA fibrous aggregates and fibrillar network dyed with ThT are blue. Differential Scanning Calorimetry (DSC). The inner water amount within the oil droplets (defined as yield) of multiple emulsions was determined using a differential scanning calorimeter (TA Q200 instrument, New Castle, DE, USA). This measurement is based on the fact that the inner and outer water phases of multiple W1/O/W2 emulsions can be distinguished due to their different freezing temperatures.33,34 Since the heat quantity released during freezing of the droplets is directly correlated to the water mass, the latent heat of freezing can be used to calculate the amount of water in the inner droplets.33,34 The samples (8−12 mg) were hermetically sealed in aluminum pans and introduced into the calorimeter. After equilibrating at 10 °C for 1 min, the samples were steadily cooled to −60 °C at a cooling rate of 5 °C/min. All the measurements were carried out under nitrogen atmosphere at a flow rate of 25 mL/min. To determine the energy needed for water freeze, the area under the relevant peak in the thermogram was integrated by using TA Universal Analysis. The yield of multiple emulsions can be calculated by analyzing the freezing energy of the inner aqueous dispersed phase in the primary W1/O emulsion and the corresponding W1/O/W2 multiple emulsion. Details of this method are given elsewhere.33,34 Rheological Measurements. The rheological measurements were carried out on a Haake RS600 rheometer (HAAKE Co., Germany)

by introducing a combination of alginate and Ca2+ into the outer water phase and found that compared to polysaccharide gels the gelled double emulsions showed a slower release of encapsulated hydrophilic components.28 Benna-Zayani et al. reported that the addition of different polysaccharides such as scleroglucan, xanthan, and locust bean gum into the external water phase, forming a weak gel, could improve the stability of W/O/W double emulsions.29 However, none of these texture modifiers mentioned above are able to act as an emulsifier simultaneously, and thus additional emulsifying agents are required to be combined in order to produce stable multiple emulsions. Recently, we reported that the naturally occurring saponin glycyrrhizic acid (GA, see Figure S1, Supporting Information, SI) can be used as a structuring agent to fabricate soft-solid emulsion gels.30,31 GA, the main ingredient of licorice root extract, is a monodesmosidic saponin comprised of a hydrophobic triterpenoid aglycon moiety (18β-glycyrrhetinic acid) attached to a hydrophilic diglucuronic unit. Owing to the amphiphilic structure, GA molecules have a complex self-assembly behavior in water, forming long nanofibrils, which with increasing concentrations can further assemble and entangle to create a supramolecular hydrogel with fibrillar network. 30−32 As amphiphilic assemblies, GA nanofibrils were found to have a multilayer adsorption at the oil−water interface and can be used as natural emulsifiers for fabricating stable oil-in-water (O/W) emulsions.30,31 The unique combination of gelation and emulsifying properties endows these saponin nanofibrils with the ability as excellent building blocks to construct stable emulsion gels. Based on these previous works,30,31 it is reasonable to suggest that the GA fibrils have great potential as a bifunctional building block (texture modifiers and emulsifiers) in the outer water phase of W/O/W emulsions, to stabilize the outer O/W interface and structure the water phase simultaneously, for creating a gelled multiple emulsion system with superior stability. Therefore, in this work, we aimed to incorporate selfassembled GA nanofibrils into the outer water phase, creating a gelled W1/O/W2 multiple emulsion with improved yield and stability, through utilization of the synergistic combination of gelation and emulsifying behaviors of GA fibrils. To achieve our aim, we first used polyglycerol polyricinoleate (PGPR) as the lipophilic emulsifier to prepare a stable primary emulsion (W1/O emulsion). We investigated the effects of GA fibril and PGPR concentrations on the yield and stability of gelled multiple emulsions. We also characterized their microstructure and mechanical properties by using confocal laser scanning microscopy and a small-deformation rheological test, respectively. Riboflavin-5′-phosphate (R5-P), a water-soluble, photosensitive antioxidant with many health-promoting effects, was selected as the model compound and then encapsulated into our multiple emulsion gels, to further evaluate their encapsulation and protection capacity for active cargos.

■

MATERIALS AND METHODS

Materials. Glycyrrhizic acid mono ammonium salt (GA, purity >98%) was purchased from Acros Organics, USA. Polyglycerol polyricinoleate (PGPR, purity >99%, Admul Wol 1403k) was purchased from Kerry Bio-Science (Norwich, New York, USA). Commercial sunflower oil was purchased from a local supermarket (Guangzhou, China). Nile Red (Technical grade) and Thioflavin T (ThT, dye content: 65−75%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Millipore water (18.2 MΩ·cm at 25 °C) was used throughout this work. All other chemicals used were of analytical grade. Preparation of Multiple W1/O/W2 Emulsion Gels. A two-step emulsification procedure was employed to produce W1/O/W2 double B

DOI: 10.1021/acs.jafc.7b04042 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry equipped with a Universal Peltier system and water bath (MultiTemp III, Amersham Biosciences) for temperature control. A parallel plate geometry of 27.83 mm diameter with a gap of 1.0 mm was used. All samples were carefully scooped onto the rheometer plate. Under the oscillatory mode, amplitude sweeps (stress = 0.1−500 Pa, frequency = 1 Hz) and frequency sweeps (0.1−100 Hz, stress = 10 Pa, within the linear viscoelastic region) were performed to measure the variations of the storage modulus (G′) and loss modulus (G″). For flow measurements, samples were subjected to increasing shear rate from 0.1 to 50 s−1. All the measurements were performed at 25 °C. Photosensitive Cargo Protection in Multiple Emulsion Gels. To evaluate the loading and protection capacity for water-soluble, photosensitive active cargos, the R5-P (0.2 wt %) was dissolved in the internal W1 phase or the external W2 phase or both prior to the homogenization step. Then, the gelled W1/O/W2 emulsions containing R5-P were produced according to the procedure described in the above section. To measure the degradation behavior of R5-P loaded in the emulsions, these emulsions were placed in unsealed vials and exposed to UV irradiation for 3 h from the top using a Philips 125 W mercury arc lamp operated at 365 nm. The color change of emulsions during irradiation was recorded using a colorimeter (ColorFlex EZ, Hunter lab). The tristimulus color coordinates (L*, a*, b*) of the emulsions were measured, and the total color difference (ΔE*) was then calculated a s f o l l o w s : ΔE* = (L* − L0*)2 + (a* − a0*)2 + (b* − b0*)2 , where L*, a*, and b* represent the color coordinates of the emulsions at a certain storage time, and L0*, a0*, and b0* are the initial values. Statistical Analysis. Unless specified otherwise, three independent trials were performed, each with a new batch of sample preparation. Analysis of variance (ANOVA) of the data was performed using the SPSS 19.0 statistical analysis system. Duncan’s test was used for comparison of mean values among three treatments using a level of significance of 5%.

Figure 1. (a) Photographs of multiple W1/O/W2 emulsion gels prepared with 4 wt % GA fibrils and different PGPR concentrations (0− 4 wt %). (b) The appearance of these multiple emulsion gels with 0, 1, and 4 wt % PGPR, extruded with a pipet.

observed, especially in Figure 2a, which demonstrates that the W1/O/W2 multiple emulsions were successfully fabricated. From Figure 2b, it was found that the compartmented structure remained apparently unchanged after 10 days, suggesting a good stability of our gelled multiple emulsions. It should be noted that even after storage of 30 days, the double emulsion droplets could still be clearly observed, although the fraction of inner water droplets decreased to some extent (see Figure S2, SI). Additionally, with increasing PGPR concentration, the double emulsion droplet size gradually decreased, and the inner droplet fraction seems to be increased (Figure 2). This can be explained by the fact that the higher PGPR concentration would lead to the formation of more inner droplets with smaller size, which may facilitate forming the smaller W1/O/W2 emulsion droplets stabilized by GA fibrils during the second emulsification. On the basis of our previous studies, the GA fibrils have a multilayer adsorption at the oil−water interface, yielding the multilayer fibril-covered emulsion droplets.30,31 Therefore, it can be concluded that the multiple W1/O/W2 emulsion droplets should be coated by the multilayer GA fibril shells. Herein, for observation of the interfacial GA fibril layer and the fibrillar network in bulk, we used the dye ThT to label the GA fibril solutions (4 wt %) prior to sample preparation, and then observed the microstructures of the gelled multiple emulsions as well as the individual multiple emulsion droplets after dilution. As seen from CLSM images in Figure 3, in all cases, strong ThT fluorescence enhancement was clearly observed, which demonstrates the presence of the continuous fibrillar network in bulk (Figures 3a−f and S3) as well as the GA fibril layer around the multiple emulsion droplets (Figure 3g,h). Furthermore, the size and size distribution of emulsion droplets (black pores) within the gel network (Figures 3a−f and S3) were found to be in line with the CLSM images of multiple emulsions dyed with Nile Red (Figure 2c). From Figure 3g,h, the thickness of the interfacial fibril shell can be estimated to be about 1 μm, which is significantly higher than that of a single self-assembled GA fibril (around 2.5 nm) in water,32 suggesting the formation of a multilayer fibril shell at the surface of multiple emulsion droplets. This is in good agreement with our previous studies.30,31 The size distribution and droplet size of multiple W1/O/W2 emulsion droplets were further determined by static light scattering, as shown in Figure 4a,b, respectively. As can be seen, all multiple emulsion droplets showed a homogeneous size

■

RESULTS AND DISCUSSION Formation and Stability of Gelled Multiple W1/O/W2 Emulsions. Our previous studies have demonstrated that the self-assembled GA nanofibrils can be used as building blocks to construct stable emulsion gels, which are based on the multilayer adsorption of GA fibrils at the oil−water interface as well as the formation of a fibrillar hydrogel network in the continuous phase.30,31 Herein, we used the GA fibril solution (4 wt %) as the external W2 phase to stabilize the water (W1)-containing oil droplets during the second emulsification. The subsequent cooling could strengthen the hydrogen-bond interactions between excess GA fibrils in the continuous phase (W2) as well as around the surfaces of oil droplets, self-assembling into a three-dimensional hydrogel network,30,31 and then a gelled multiple emulsion was obtained, as presented in Figure 1a. As can be seen, the formation of the self-standing multiple emulsion gels was not affected by PGPR concentration (0.5−4 wt %). Figure 1b shows the photographs of the appearance of multiple emulsions containing PGPR of 1 and 4 wt %, and it was found that these multiple emulsions could be extruded and shaped with a pipet, demonstrating that they exhibit a yield stress. The gel-like behavior of these W1/O/W2 multiple emulsions will be further studied in the following mechanical property measurements. To clearly visualize the structure of the multiple emulsions, the gelled samples were diluted 1:10 using water to obtain homogeneous double emulsion droplets, and their microstructure observations were further performed by using CLSM. Figure 2 shows the CLSM images of the freshly prepared multiple emulsions dyed with Nile Red under bright-field (Figure 2a,b) and confocal (Figure 2c) modes. As can be seen, the oil globules containing a number of small internal water droplets with relatively homogeneous size distribution were clearly C

DOI: 10.1021/acs.jafc.7b04042 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 2. CLSM images of the reconstituted multiple emulsions containing 0.4 wt % GA fibrils from the W1/O/W2 emulsion gels prepared with 4 wt % GA fibrils and different PGPR concentrations (0−4 wt %): (a, b) bright field images; (c) Nile Red fluorescence images (the oil phases appear in green). The samples were obtained at initial preparation (a, 0 day) and after 10 days of storage at 25 °C (b, c). All scale bars in panel a are 25 μm, and the scale bars for panels b and c are 7.5 μm.

Figure 3. CLSM images (a−f) of fresh multiple W1/O/W2 emulsion gels prepared with 4 wt % GA fibrils and PGPR concentrations of 0 (a), 1 (b, e, and f), 2 (c), and 4 wt % (d): (a−e) ThT fluorescence images (highlighting the GA fibrillar network in bulk); (f) bright field image. CLSM images (g, h) of the diluted multiple emulsions containing 0.4 wt % GA fibrils from the gelled sample stabilized by 4 wt % GA fibrils and 1 wt % PGPR: (g) ThT fluorescence image (showing the GA fibrillar assemblies at the oil−water interface); (h) bright field image. All scale bars in panels a−d are 25 μm, and the scale bars for panels e−h are 7.5 μm.

observed (Figure 4b), confirming the structural stability of our multiple emulsions. To further investigate the stability of the inner water phase within the oil droplets of multiple emulsions, we performed DSC measurements to determine the yields of our

distribution (Figure 4a), and with increasing PGPR concentrations (0−4 wt %), the average emulsion droplet diameter (d43) decreased from 17.2 to 7.5 μm (Figure 4a,b). Moreover, after storage of 10 days, no significant change in the d43 values was D

DOI: 10.1021/acs.jafc.7b04042 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Laplace pressure within the inner W1 phase is matched with the osmotic pressure difference between the inner W1 and outer W2 phases.33−35 These results (Figure 4) are in good agreement with the CLSM observations (Figures 2 and 3), indicating that gelled W1/O/W2 multiple emulsions having superior stability, high yield, and homogeneous size distribution can be successfully created by a synergistic combination of hydrophobic surfactant (PGPR) and GA fibrils as stabilizers. We then studied the mechanical properties of these gelled multiple emulsions by performing small-deformation oscillatory and flow measurements. The results of oscillatory stress sweeps (at a fixed frequency of 1 Hz) and frequency sweeps (at a fixed stress of 10 Pa) for the multiple emulsions with different PGPR concentrations are presented in Figure 5a,b, respectively. As can be seen in Figure 5a, in all cases, the storage modulus (G′) was always significantly larger than the loss modulus (G″) in their individual linear viscoelastic regions, suggesting that these multiple emulsions exhibit mostly an elastic solid-like behavior, in agreement with the previous observation (Figure 1b). With increasing PGPR concentrations (0−4 wt %), no obvious changes in the values of G′, G″, and yield stress (defined here when G″ > G′) were observed, which indicates that the presence of the inner W1 phase stabilized by PGPR did not significantly affect the network strength of GA fibrillar hydrogel in the outer W2 phase of multiple emulsions. This analysis is supported by the results of frequency sweeps (Figure 5b) and apparent viscosity curves (Figure 5c). Moreover, as seen from Figure 5b, both G′ and G″ for all the cases displayed a relatively weak frequency dependence, and the G′ curves had slightly positive slopes, suggesting that the rheological response of multiple emulsions is not largely influenced by the applied deformation rate even at high frequency (100 Hz). We further determined these mechanical parameters of the multiple emulsions after storage of 10 days. As shown in Figure S4 (SI), no obvious differences were detected in the results of amplitude and frequency sweeps, which confirm the excellent mechanical stability of these gelled multiple emulsions, in good agreement with the previous observations (Figures 2, 4, and S2). Role of GA Fibrillar Network on Improvement of the Multiple Emulsion Stability. It has been recognized that the osmotic pressure differences between inner and outer water phases can lead to the water diffusion in multiple emulsions, resulting in swelling or shrinkage of inner water droplets.18,19,34 This can change the structure of multiple emulsions during production and storage, thus affecting their yield and stability. Moreover, the relatively high Laplace pressure of the small inner water droplets can also promote the migration of water molecules toward the outer phase. On the basis of the above results (Figures 2−5), the gelled multiple emulsions due to the formation of GA fibrillar hydrogel network in the continuous W2 phase exhibited superior stability and high yield (especially at 1− 4 wt % PGPR), suggesting that the water diffusion due to osmotic pressure differences and high Laplace pressure might not obviously occur in our gelled multiple emulsions. Since the used PGPR concentration (1−4 wt %) is high enough to stabilize the inner W1 phase (15 wt %), we can conclude that in our gelled multiple emulsions, it seems likely that both the interfacial fibril films around the multiple emulsion droplets and the GA hydrogel in the continuous W2 phase are major factors in providing stability for the system, preventing the water diffusion between inner droplets and outer bulk water phase. We have previously demonstrated that the multilayer interfacial fibril shell with high electrostatic force can give

Figure 4. (a) Droplet size distributions of fresh multiple W1/O/W2 emulsion gels prepared with 4 wt % GA fibrils and different PGPR concentrations (0−4 wt %). (b) Changes in multiple droplet size (d43) of these samples (0 day) after 10 days of storage at 25 °C. (c) Yields of these multiple emulsion gels at initial (0 day) and after 10 days at 25 °C.

gelled multiple emulsions right after and 10 days after preparation, respectively. A very small amount of NaCl (0.05 wt %) was added into the inner W1 phase of multiple emulsions for DSC measurements to better observe and calculate the freezing enthalpy of W1 phase. The yield results are presented in Figure 4c. As can be seen, the initial yields (0 day) significantly increased with increasing PGPR concentrations (0.5−2 wt %), and the multiple emulsions containing higher PGPR concentrations (1−4 wt %) showed higher yield values (85.6−92.5%), suggesting their good formability. Moreover, it was found that the yields of the multiple emulsions (0.5−4 wt % PGPR) remained unchanged after 10 days, which demonstrates that the inner W1 phase was stable within the oil droplets of multiple emulsions, in line with the size results of multiple emulsion droplets (Figure 4b). In general, the stable yield means that the E

DOI: 10.1021/acs.jafc.7b04042 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 5. Amplitude (a) and frequency (b) sweeps for multiple W1/O/W2 emulsion gels prepared with 4 wt % GA fibrils and different PGPR concentrations (0−4 wt %). G′ and G″ are shown as filled and open symbols, respectively. (c) Viscosity curves of these multiple emulsion gels. All measurements were performed at 25 °C.

Figure 6. (a) CLSM images (scale bar = 25 μm) in bright filed mode for multiple W1/O/W2 emulsions prepared with 1 wt % PGPR and different GA fibril concentrations (0.5−4 wt %). (b) DSC curves of these multiple emulsion gels (1−4 wt % GA fibrils), obtained at a cooling rate of 5 °C/min (exothermal up).

rapidly trigger the formation of GA fibrillar hydrogel network in the W2 phase, trapping the water (W1)-containing oil droplets and thus keeping the droplets fixed in the gel matrix. This can contribute to the physical stability of multiple emulsions against flocculation and creaming (Figure 4). In addition, the highly viscoelastic fibrillar hydrogel in the bulk phase is also believed to be able to affect the water diffusion between inner and outer

superior stability to O/W emulsion droplets during storage and heating.30,31 In our multiple emulsion gels, such similar multilayer GA fibrils on the droplet surface (Figure 3g,h) also can prevent the coalescence of double emulsion droplets. Next, we discuss the contribution of the GA hydrogel in the continuous water phase (W2) to multiple emulsion stability. Based on our previous work,30,31 it is known that cooling in an ice bath can F

DOI: 10.1021/acs.jafc.7b04042 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 7. (a) Photographs of multiple W1/O/W2 emulsion gels containing R5-P (0.2 wt %) prepared with 4 wt % GA fibrils and 1 or 2 wt % PGPR, before (above) and after (below) 3 h of UV irradiation. (b) Total color change (ΔE*) of these multiple emulsion gels after UV irradiation for 3 h. Control, 4 wt % GA fibril-based O/W emulsion gels with R5-P in continuous phase; 1% PGPR-W2 and 2% PGPR-W2 designate multiple W1/O/W2 emulsion gels stabilized by 4 wt % GA fibrils with 1 and 2 wt % PGPR, respectively, with R5-P in the outer W2 phase; 1% PGPR−W1 + W2 and 2% PGPRW1 + W2 designate those multiple emulsion gels with R5-P in both W1 and W2 phases (50:50, w/w, respectively), while 1% PGPR-W1 and 2% PGPR-W1 designate those W1/O/W2 emulsion gels with R5-P in the inner W1 phase.

water phases. In Figure 5a, we showed that all gelled multiple emulsions had a high storage modulus G′, which can reach around 104 Pa. The high storage modulus should be easily enough to overcome the Laplace pressure of small inner water droplets, and accordingly the water diffusion from the inner to the outer water phase could be effectively slowed down. To further evaluate the role of GA fibrillar hydrogel network on multiple emulsion formation and stability, the impact of GA fibril concentrations (0.5−4 wt %) on the properties of multiple emulsions was investigated, and the PGPR concentration was fixed at 1 wt %. As seen from Figure S5a (SI), at a low fibril concentration (0.5 wt %), the prepared emulsion sample was fluid and did not show any gelation. Upon further increase of fibril concentrations (1−4 wt %), as expected, we were able to obtain the self-standing emulsion gels, probably due to the formation of the viscoelastic GA hydrogel network in the outer water phase. This is supported by the results of oscillatory amplitude and frequency sweeps applied to these gelled multiple emulsions (Figure S5b,c, SI), showing that the viscoelastic modulus of G′ and G″ significantly increased with increasing GA fibril concentration. Figures 6a and S6 (SI) show the CLSM

images of the freshly prepared multiple emulsions with different GA fibril concentrations. As can be seen, in all cases, the multiple emulsion droplets with compartmented structure can be clearly observed, independent of fibril concentration. However, with increasing GA fibril concentration, the fraction of inner water droplets seems to be increased, and the emulsion droplet size gradually decreased. We used DSC to detect instabilities due to osmotic pressure differences between inner and outer water phases for these multiple emulsion gels (1−4 wt % GA fibrils).33,34 As seen from Figure 6b, the DSC curves of the multiple emulsions with 1 and 2 wt % GA fibrils show that after the outer water phase was completely frozen, the peak of the outer water phase did not return to the baseline, as compared to that of the multiple emulsion gel containing 4 wt % GA fibrils. Another peak occurred that lasts to a temperature of around −30 °C (red arrows, Figure 6b), which is also not the same temperature for the freezing of inner water droplets. This can be explained by the fact that the water molecules may diffuse from the inner droplets to the outer water phase during the measurement, and thus when they reach the outer phase, the water crystallization can cause the G

DOI: 10.1021/acs.jafc.7b04042 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry peak observed.33,34 This indicates that the water diffusion process occurred in the multiple emulsions with 1 and 2 wt % GA fibrils. Therefore, we can conclude that compared to the multiple emulsion gel with 4 wt % GA fibrils, the network of fibrillar hydrogel in the multiple emulsions of 1 and 2 wt % GA fibrils is relatively weak with lower G′ and G″ values (Figure S5b,c, SI), which may be not sufficient to overcome the osmotic-driven water diffusion. This is in good agreement with previous analysis (Figures 2−5), suggesting that the highly viscoelastic GA hydrogel network in the continuous water phase (W2) plays an essential role in the formation and stability of multiple emulsions. Encapsulation and Protection of Photosensitive Cargos. Due to the compartmentalized internal structure, multiple W1/O/W2 emulsions have been considered as good candidates for encapsulation and protection of various active ingredients, especially water-soluble, environment-sensitive cargos. Herein, we fabricated functional multiple emulsion gels by dissolving the water-soluble R5-P (model cargo) into the inner (W1) or outer (W2) water phase or both, to evaluate the encapsulation and stabilization of photosensitive cargos in our gelled multiple emulsions. R5-P is a highly unstable active molecule in light environments and therefore tends to lose its yellow color during light illumination. Figure 7a shows the photographs of the colored multiple emulsion gels containing R5-P before and after UV irradiation. As can be seen, after 3 h of illumination, the multiple emulsion gels (1 and 2 wt % PGPR) with R5-P in single W1 phase showed no obvious color fading, as compared to those with R5-P in single W2 phase as well as in both W1 and W2 phases. Moreover, the color fading for the emulsion gels with R5-P in the outer W2 phase was more apparent than those with R5-P in both W1 and W2 phases. The above observations (Figure 7a) are supported by the results of total color change (ΔE*) of these R5-P loaded emulsion gels (Figure 7b). The control O/W simple emulsion gel and the multiple emulsion gels with R5-P in the outer W2 phase showed similar ΔE* values, which were significantly higher than those of the multiple emulsion gels with R5-P loaded in the inner W1 phase, as well as in both W1 and W2 phases (Figure 7b). This indicates that the water-soluble, photosensitive bioactive cargos can be effectively protected and stabilized by encapsulating them into the internal water droplets of our gelled multiple emulsions. Accordingly, the highly viscoelastic GA hydrogel network as well as both the dense interfaces stabilized by GA fibrils and PGPR molecules might provide an efficient wall to block light, thus reducing the light-driven molecular decomposition and improving the stability of photosensitizers like R5-P. These results suggest that our multiple emulsion gels could be used as stable delivery vehicles to encapsulate and protect various bioactive cargos in functional foods, cosmetics, and pharmaceutical applications. In conclusion, we have successfully fabricated a gelled multiple W1/O/W2 emulsion by utilization of the unique combination of emulsifying and gelation abilities of GA nanofibrils, assembling into a fibrillar hydrogel network in the continuous water phase (W2). The prepared multiple emulsion gels displayed a relatively homogeneous droplet size distribution, high yield (85.6−92.5%), as well as superior storage stability. We showed that the multiple emulsion stability is directly related to the multi-interfacial fibril shell and the GA hydrogel in the continuous W2 phase, which can effectively prevent flocculation, creaming and coalescence of double emulsion droplets. Of particular interest is the presence of viscoelastic GA fibrillar hydrogel in bulk, showing a high storage modulus, which is believed to be able to overcome the Laplace

pressure of small inner water droplets (W1), and thus strongly slow down the osmotic-driven water diffusion from the inner to the outer water phase. We demonstrated that these multicompartmentalized emulsion gels can be used to encapsulate and protect photosensitive bioactive ingredients by loading them into the internal water droplets. We expect that these stable multiple emulsion gels based on natural saponin nanofibrils could be used to deliver and stabilize a wide variety of photosensitive, watersoluble cargos, which allows us to find more practical applications in, for example, foods, pharmaceuticals, and cosmetics.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04042. Chemical structures of GA, PGPR, R5-P, and ThT molecules, CLSM images of multiple emulsion gels after 30 days of storage, overlay images of fluorescence and bright field for multiple emulsion gels with different PGPR concentrations, amplitude and frequency sweeps for multiple emulsion gels after 10 days, photographs, amplitude sweeps, and frequency sweeps for multiple emulsions with different GA fibril concentrations, and CLSM images for these multiple emulsions with different GA fibril concentrations (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*Zhili Wan. E-mail: [email protected]; [email protected]. *Xiaoquan Yang. E-mail: [email protected]. Fax: (086) 2087114263. Tel: (086) 20-87114262. ORCID

Zhili Wan: 0000-0002-5865-4301 Xiaoquan Yang: 0000-0002-4016-9834 Funding

This work is supported by grants from the Special and General Projects of China Postdoctoral Science Foundation (2017T100635 and 2016M600655), the Fundamental Research Funds for the Central Universities (2017BQ101), and the National Natural Science Foundation of China (31771923). Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Chu, L. Y.; Utada, A. S.; Shah, R. K.; Kim, J. W.; Weitz, D. A. Controllable monodisperse multiple emulsions. Angew. Chem., Int. Ed. 2007, 46, 8970−8974. (2) Garti, N. Double emulsions - scope, limitations and new achievements. Colloids Surf., A 1997, 123−124, 233−246. (3) Zhao, C. X. Multiphase flow microfluidics for the production of single or multiple emulsions for drug delivery. Adv. Drug Delivery Rev. 2013, 65, 1420−1446. (4) Jiménez-Colmenero, F. Potential applications of multiple emulsions in the development of healthy and functional foods. Food Res. Int. 2013, 52, 64−74. (5) McClements, D. J.; Decker, E. A.; Weiss, J. Emulsion-based delivery systems for lipophilic bioactive components. J. Food Sci. 2007, 72, R109−R124. (6) McClements, D. J. Advances in fabrication of emulsions with enhanced functionality using structural design principles. Curr. Opin. Colloid Interface Sci. 2012, 17, 235−245. (7) Bonnet, M.; Cansell, M.; Placin, F.; David-Briand, E.; Anton, M.; Leal-Calderon, F. Influence of ionic complexation on release rate H

DOI: 10.1021/acs.jafc.7b04042 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry profiles from multiple water-in-oil-in-water (W/O/W) emulsions. J. Agric. Food Chem. 2010, 58, 7762−7769. (8) Shima, M.; Morita, Y.; Yamashita, M.; Adachi, S. Protection of Lactobacillus acidophilus from the low pH of a model gastric juice by incorporation in a W/O/W emulsion. Food Hydrocolloids 2006, 20, 1164−1169. (9) Dickinson, E. Double emulsions stabilized by food biopolymers. Food Biophys. 2011, 6, 1−11. (10) Muschiolik, G. Multiple emulsions for food use. Curr. Opin. Colloid Interface Sci. 2007, 12, 213−220. (11) Norton, J. E.; Norton, I. T. Designer colloidstowards healthy every day foods? Soft Matter 2010, 6, 3735−3742. (12) Muschiolik, G.; Dickinson, E. Double emulsions relevant to food systems: preparation, stability, and applications. Compr. Rev. Food Sci. Food Saf. 2017, 16, 532−555. (13) Shum, H. C.; Zhao, Y. J.; Kim, S. H.; Weitz, D. A. Multicompartment polymersomes from double emulsions. Angew. Chem., Int. Ed. 2011, 50, 1648−1351. (14) Lee, D.; Weitz, D. A. Nonspherical colloidosomes with multiple compartments from double emulsions. Small 2009, 5, 1932−1935. (15) Garti, N.; Bisperink, C. Double emulsions: progress and applications. Curr. Opin. Colloid Interface Sci. 1998, 3, 657−667. (16) Ficheux, M. F.; Bonakdar, L.; Leal-Calderon, F.; Bibette, J. Some stability criteria for double emulsions. Langmuir 1998, 14, 2702−2706. (17) Zhao, C. X.; Chen, D.; Hui, Y.; Weitz, D. A.; Middelberg, A. P. J. Controlled generation of ultrathin-shell double emulsions and studies on their stability. ChemPhysChem 2017, 18, 1393−1399. (18) Mezzenga, R.; Folmer, B. M.; Hughes, E. Design of double emulsions by osmotic pressure tailoring. Langmuir 2004, 20, 3574− 3582. (19) Jiao, J.; Rhodes, D. G.; Burgess, D. J. Multiple emulsion stability: pressure balance and interfacial film strength. J. Colloid Interface Sci. 2002, 250, 444−450. (20) Perez-Moral, N.; Watt, S.; Wilde, P. Comparative study of the stability of multiple emulsions containing a gelled or aqueous internal phase. Food Hydrocolloids 2014, 42, 215−222. (21) Li, J.; Shi, Y.; Zhu, Y.; Teng, C.; Li, X. Effects of several natural macromolecules on the stability and controlled release properties of water-in-oil-in-water emulsions. J. Agric. Food Chem. 2016, 64, 3873− 3880. (22) Jiménez-Alvarado, R.; Beristain, C. I.; Medina-Torres, L.; RománGuerrero, A.; Vernon-Carter, E. J. Ferrous bisglycinate content and release in W1/O/W2 multiple emulsions stabilized by proteinpolysaccharide complexes. Food Hydrocolloids 2009, 23, 2425−2433. (23) Benichou, A.; Aserin, A.; Garti, N. W/O/W double emulsions stabilized with WPI-polysaccharide complexes. Colloids Surf., A 2007, 294, 20−32. (24) Delample, M.; Da Silva, F.; Leal-Calderon, F. Osmotically driven gelation in double emulsions. Food Hydrocolloids 2014, 38, 11−19. (25) Hattrem, M. N.; Dille, M. J.; Seternes, T.; Draget, K. I. Macro- vs. micromoleuclar stabilisation of W/O/W-emulsions. Food Hydrocolloids 2014, 37, 77−85. (26) Patel, A. R.; Dumlu, P.; Vermeir, L.; Lewille, B.; Lesaffer, A.; Dewettinck, K. Rheological characterization of gel-in-oil-in-gel type structured emulsions. Food Hydrocolloids 2015, 46, 84−92. (27) Mun, S.; Choi, Y.; Park, S.; Surh, J.; Kim, Y. R. Release properties of gel-type W/O/W encapsulation system prepared using enzymatically-modified starch. Food Chem. 2014, 157, 77−83. (28) Weiss, J.; Scherze, I.; Muschiolik, G. Polysaccharide gel with multiple emulsion. Food Hydrocolloids 2005, 19, 605−615. (29) Benna-Zayani, M.; Kbir-Ariguib, N.; Trabelsi-Ayadi, M.; Grossiord, J. L. Stabilisation of W/O/W double emulsion by polysaccharides as weak gels. Colloids Surf., A 2008, 316, 46−54. (30) Wan, Z.; Sun, Y.; Ma, L.; Guo, J.; Wang, J.; Yin, S.; Yang, X. Thermoresponsive structured emulsions based on the fibrillar selfassembly of natural saponin glycyrrhizic acid. Food Funct. 2017, 8, 75− 85. (31) Wan, Z.; Sun, Y.; Ma, L.; Yang, X.; Guo, J.; Yin, S. Responsive emulsion gels with tunable properties formed by self-assembled

nanofibrils of natural saponin glycyrrhizic acid for oil structuring. J. Agric. Food Chem. 2017, 65, 2394−2405. (32) Saha, A.; Adamcik, J.; Bolisetty, S.; Handschin, S.; Mezzenga, R. Fibrillar networks of glycyrrhizic acid for hybrid nanomaterials with catalytic features. Angew. Chem. 2015, 127, 5498−5502. (33) Schuch, A.; Kohler, K.; Schuchmann, H. P. Differential scanning calorimetry (DSC) in multiple W/O/W emulsions. J. Therm. Anal. Calorim. 2013, 111, 1881−1890. (34) Oppermann, A. K. L.; Renssen, M.; Schuch, A.; Stieger, M.; Scholten, E. Effect of gelation of inner dispersed phase on stability of (w1/o/w2) multiple emulsions. Food Hydrocolloids 2015, 48, 17−26. (35) Sameh, H.; Wafa, E.; Sihem, B.; Fernando, L.-C. Influence of the molecular transport on the evolution of W/O/W emulsions. Langmuir 2012, 28, 17597−17608.

I

DOI: 10.1021/acs.jafc.7b04042 J. Agric. Food Chem. XXXX, XXX, XXX−XXX