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Responsive Emulsion Gels with Tunable Properties Formed by SelfAssembled Nanofibrils of Natural Saponin Glycyrrhizic Acid for Oil Structuring Zhili Wan,† Yingen Sun,† Lulu Ma,† Xiaoquan Yang,*,†,‡ Jian Guo,† and Shouwei Yin† †

Research and Development Center of Food Proteins, Department of Food Science and Technology, 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: Saponin nanofibrils assembled from natural glycyrrhizic acid (GA) have been recently shown to be an effective structurant for edible oil structuring. This work showed that the microstructure and mechanical properties of the novel emulsion gels formed by GA fibrils could be well tuned by oil phase polarity. For more polar oils (algal oil), the GA fibrils had a higher affinity to the oil−water interface, showing a faster adsorption kinetics, thus leading to the formation of fine multilayer emulsion droplets with smaller droplet size. Accordingly, the emulsion gels had a denser network microstructure and higher mechanical strength, which should be attributed to the fact that the smaller emulsion droplets could be packed more tightly within the continuous network, providing stronger interdroplet interactions, and thereby contribute to reinforcing the gel matrix. In addition, all emulsion gels had interesting thermoresponsive behavior, independent of oil phase, which is probably due to the thermoreversibility of the hydrogen-bond fibrillar network in the continuous phase. KEYWORDS: edible oil structuring, emulsion gels, glycyrrhizic acid, saponin nanofibrils, oil phase polarity, thermoresponsive

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INTRODUCTION Edible oil structuring has received considerable interest in recent years due to its application potential in foods, cosmetics, and pharmaceuticals. In food processing, due to the rapidly growing demand for reducing the saturated or trans fatty acids in foodstuffs for health and nutritional considerations, much research has been focused on the development of novel alternative strategies to structure vegetable oils for replacing the solid fats and providing the required sensory and flavor properties in food products.1,2 Among these strategies, oleogelation and structured emulsions are the most commonly reported structuring approaches, and the formed soft solid-like structured oils are known as oleogels (or organogels) and emulsion gels (or gelled emulsions).3−7 Generally, the low molecular weight organic compounds are more commonly used as oil-structuring agents to create various structured oils via their self-assembly, such as 12-hydroxystearic acid,8,9 waxes,10,11 fatty acids or alcohols,12 lecithin−sorbitan tristearate,13 and the mixtures of phytosterol with oryzanol or monoglycerides.14−16 In comparison with the semisynthetic or even synthetic lipidic additives, the naturally occurring amphiphilic small molecules such as triterpenoid saponins are more highly desired to be used as novel structuring agents for edible oils due to their excellent bioavailability, biocompatibility, and biodegradability.17−20 Natural saponins such as glycyrrhizic acid (GA) exhibit interesting self-assembly behaviors and in most cases also show many biological activities for the human body. The unique combination of self-assembly properties and bioactivity makes them particularly attractive for food, pharmaceutical, and personal-care applications. GA, the main ingredient of licorice © XXXX American Chemical Society

root extract, is widely used in candies and sweets due to its intense sweetness (50 times sweeter than sucrose). GA also shows many biological effects, such as anti-inflammatory, antitumor, antivirus, and antifungal activities.21 Most recently, Mezzenga and co-workers reported that GA molecules possess a complex self-assembly behavior in water, forming long nanofibrils, which, upon increasing concentrations, can further lead to the formation of a supramolecular hydrogel with fibrillar network.17 In addition, work from the research groups of Bag and Ju demonstrated that GA fibrils and their derivatives conjugated with peptides were capable of self-assembling into thermoreversible supramolecular organogels in various organic solvents by forming fibrillar networks through noncovalent intermolecular interactions.18−20 Unfortunately, GA is unable to structure vegetable oils directly because it has a very limited solubility in oils, and as a consequence, no information is available about the applications of natural saponin GA in edible oil structuring. However, amphiphilic GA molecules or fibrillar assemblies do have affinity for the hydrophobic oil phase and thus can be used as natural emulsifiers for fabricating oil/water (O/W) emulsions. Thus, the synergistic combination of gelation and emulsifying behaviors gives GA fibrillar assemblies potential as natural structuring agents for constructing novel emulsion gel systems. As expected, our recent findings showed that the selfReceived: Revised: Accepted: Published: A

November 22, 2016 February 9, 2017 March 7, 2017 March 7, 2017 DOI: 10.1021/acs.jafc.6b05242 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

solution was obtained. O/W emulsions were prepared by first dispersing vegetable oils in hot GA fibril solutions (80 °C) under mild agitation for 2 min, and then the resulting dispersions were immediately sheared using an Ultra-Turrax T10 (IKA-Werke GmbH & Co., Germany) at 20,000 rpm for 2 min. The resultant emulsions were cooled and then stored overnight (12 h) at room temperature (25 °C) before further experiments. The dried emulsion gel samples were obtained by using lyophilization, where the samples were frozen at −40 °C, followed by drying for 24 h in a Christ DELTA 1-24 LSC freeze-dryer (Christ, Germany). The preparation of functional emulsion gels was performed similarly by dissolving β-carotene or αtocopherol (0.1 wt % of oil) into the oil phase prior to the homogenization step. Interfacial Properties Measurements. The interfacial tension and dilatational modulus of GA fibril solutions (0.025−0.1 wt %) at the oil−water interface were measured through the pendant drop method on an OCA20 tensiometer (Dataphysics Instruments GmbH, Germany) equipped with an oscillating drop accessory (ODG-20). The densities of different vegetable oils were determined using an Anton Paar DMA 35N density meter. A drop of sample solution (10 μL) was formed in an optical glass cuvette containing purified oil and monitored with a video camera. The interfacial tension (γ) was calculated from the shape analysis of a pendent drop according to the Young−Laplace equation. The dynamic interfacial tension of GA fibril solution was recorded for 30 min, by which time the interfacial tension has reached a constant value. After adsorption, quasi-equilibrium conditions of interface were obtained, and then dilatational amplitude sweeps from 1.5 to 10% deformation were performed at a frequency of 0.1 Hz to obtain interfacial dilatational parameters. The complex modulus was calculated from the intensity and phase of the first harmonic of a Fourier transform of the oscillatory surface pressure signal. All of the measurements were performed at 25 °C, and reported values represent the average of five to seven measurements. Droplet Size and Zeta Potential Measurements. The droplet size (surface area weighted mean diameter, d32) and zeta potential of the GA fibril-stabilized emulsions were measured using a Mastersizer 3000 and Nano ZS Zetasizer (Malvern Instruments Ltd., UK), respectively, after appropriate dilution with water. The refractive indices of vegetable oils and water were taken as 1.47 and 1.33, respectively. All measurements were carried out at 25 °C, and the results reported are averages of three measurements. Microstructure Observations. The microstructure of emulsions/ emulsions gels was studied using a confocal laser scanning microscope (CLSM, Leica Microsystems Inc., Heidelberg, Germany) and a polarized light microscope (PLM, Axioskop 40 Pol/40A Pol, ZEISS, Göttingen, Germany) equipped with a Power Shot G5 camera (Canon, Japan) and a Liakam hot stage (CI 94). For PLM, the samples were placed on a flat slide and covered by a coverslip. The magnification was 500× (50 × 10), and each image was acquired under normal and polarized light. For CSLM visualization, Nile Red (0.1 wt %), a fluorescent dye for oil, was first dissolved in vegetable oils, which were then used to prepare emulsion gels. The emulsion gels were placed on concave confocal microscope slides and covered with coverslips. They were examined using an argon krypton laser (ArKr, 488 nm) with a 100× oil immersion objective lens at room temperature (25 °C). ThT was used to label the GA fibrillar network of emulsion gels. ThT (0.01 wt %) was first dissolved in GA fibril solutions prior to the gel preparation. 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. Samples were imaged using a Leica TCS-SP5 confocal microscope (Leica Microsystems Inc., Heidelberg, Germany). The oil phase dyed with Nile Red is green, whereas the GA fibrillar network dyed with ThT is blue. To further visualize the gel structure, the vegetable oil was replaced with the volatile hexane as the oil phase. The homogenization time was reduced to 1 min to prevent hexane evaporation during emulsification. After the formation of emulsion gels, the hexane was removed by evaporation at ambient pressure and temperature (25 °C) for 12 h. The dried samples were mounted on a holder with double-sided adhesive tape and sputter-coated with gold (JEOL JFC-1200 fine

assembled GA nanofibrils could be used as a structuring material to transform liquid olive oil into a soft solid emulsion gel, which had a fibrillar network structure due to the enhanced noncovalent interactions among GA fibrils in the continuous phase as well as at the droplet surface.22 The GA fibril-stabilized emulsion gels with high oil fraction could be prepared using a facile one-step only emulsification, and they displayed many encouraging properties, such as thermal reversibility, high gel strength, shear sensitivity, and good thixotropic recovery. These results demonstrated that natural saponin GA has the capacity of constructing vegetable oil soft solid products by utilization of its interesting fibrillar self-assembly behavior.22 Despite the promising applications of the GA fibril-based emulsion gels in edible oil structuring, many questions remain in this new system, such as the influence of vegetable oil type and composition (i.e., oil phase polarity) as well as some processing conditions (e.g., emulsification rate), because these factors are expected to influence the average emulsion droplet size, which would change the interdroplet interactions and thus affect the final properties (e.g., microstructure and mechanical modulus) of emulsion gels.5,23,24 Therefore, in this work, we attempted to tune the microstructure and mechanical properties of the novel GA fibril-stabilized emulsion gels by controlling the emulsion droplet size, which was highly affected by the oil phase polarity and processing conditions. To achieve our aims, we first selected three edible vegetable oils, sunflower oil, flaxseed oil, and algal oil, which have significantly different fatty acid types and compositions, as oil phase to prepare the emulsion gels. The used sunflower oils have a high oleic acid (monounsaturated fatty acids, MUFAs) content of around 80%, flaxseed oils are rich in α-linolenic acid (polyunsaturated fatty acids, PUFAs), and algal oils are excellent sources of long-chain n-3 PUFAs with a percentage of DHA above 40%. In addition, on the basis of the practical consideration of health recommendations, the combination of oils rich in MUFAs (sunflower oil) and PUFAs (algal oil) was used as the mixed oil phase to further study the influence of oil phase composition. We then characterized the mechanical properties of emulsion gels by performing a small-deformation rheological test. The gel microstructure was studied by a combination of optical, confocal, and scanning electron microscopy techniques. The adsorption of GA fibrils at the oil−water interface was also determined to gain more insight into the link between the oil phase polarity and the final properties of emulsion gels. Finally, the capacity of these emulsion gels as natural delivery vehicles for oil-soluble functional ingredients was evaluated.

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MATERIALS AND METHODS

Materials. Glycyrrhizic acid mono ammonium salt (GA; purity > 98%) was purchased from Acros Organics, USA. Three vegetable oils, sunflower oil (STANDARD FOODS, China), flaxseed oil (Hongjingyuan Co., Ltd., China), and algal oil (Type II, Runke Bioengineering Co., Ltd., China), were purchased from commercial sources and were purified with Florisil (60−100 mesh, Sigma-Aldrich) to remove impurities as described elsewhere before any measurements. Nile Red, thioflavin T (ThT), β-carotene, and α-tocopherol (vitamin E) 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 GA Fibril-Stabilized Emulsions and Emulsion Gels. Stock solutions of GA fibrils were prepared by dissolving appropriate amounts of GA in water in a sealed vial; the vials were heated at 80 °C in a water bath under mild agitation until a clear B

DOI: 10.1021/acs.jafc.6b05242 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry coater, Japan) before images were recorded using field emission scanning electronic microscopy (FE-SEM) on a Zeiss Merlin FE-SEM. Rheological Measurements. The rheological measurements of emulsion gels were performed on a Haake RS600 rheometer (HAAKE Co., Germany) 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. A range of oscillatory experiments including amplitude sweeps (stress = 0.1−1000 Pa, frequency = 1 Hz) and frequency sweeps (0.1−100 Hz, stress = 10 Pa, within the linear viscoelastic region) were performed at 25 °C. Temperature sweeps including heating from 25 to 80 °C and cooling back to 25 °C at a rate of 5 °C/ min were carried out at a constant stress of 1 Pa and a frequency of 1 Hz. For flow measurements, samples were subjected to increasing shear rates from 0.1 to 50 s−1 at 25 °C. For thixotropy evaluation, the viscosity of emulsion gels with time was measured at alternating shear rates (0.1 and 10 s−1, respectively). Fourier Transform Infrared Spectroscopy (FTIR). The FTIR spectra of raw GA, vegetable oils, and emulsion gel samples were obtained within the range between 4000 and 400 cm−1 on a VERTEX 70 FTIR spectrometer (Bruker, Germany) equipped with a narrowband mercury cadmium telluride detector with a resolution of 2 cm−1. 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%.

interfaces after 30 min of adsorption. As can be seen, the interfacial tension value of purified algal oil against water (10.68 mN/m) was significantly lower than those of sunflower (22.76 mN/m) and flaxseed (18.74 mN/m) oils, suggesting that algal oil is more polar than both sunflower and flaxseed oils, which should be probably due to its higher content of long-chain n-3 PUFAs (DHA, >40%).25,26 The equilibrated interfacial tension (γ30 min) of 0.1% GA fibril at the algal oil−water interface was 4.38 mN/m, which is significantly lower than those at sunflower/flaxseed oil interfaces. This indicates that the GA fibril showed a higher affinity to more polar algal oil, thus decreasing the interfacial tension more effectively. In addition, the interfacial tension value of mixed sunflower/algal oil (50/ 50, w/w) was found to be closer to that of algal oil, suggesting the properties of the mixed oil with an identical oil content might be mostly dominated by the more polar algal oil. On the other hand, for all investigated cases, the interfacial layer formed from GA fibril after equilibrium adsorption displayed a fairly viscous response in dilatation deformations (see Figure S1Bd, SI) with very low values of surface elasticity (around 6− 10 mN/m, 1.5−10% amplitude), suggesting the formation of a relatively weak and viscous interfacial layer. The faster adsorption kinetics generally contribute positively to the emulsifying performance of surface-active agents, forming more homogeneous emulsion droplets with smaller droplet size.27,28 Herein, we prepared O/W emulsions with different oil phases (5 wt % oil) by using 0.25% GA fibril as emulsifiers. It can be seen from Figure 1A that, compared to other oil phases, the sunflower oil emulsion stabilized by 0.25% GA fibrils showed a larger d32 at around 4.2 μm, and the droplet size distribution was polydisperse and broad. The emulsions of algal oil and mixed oil phases had similar d32 values (2.2 and 2.6 μm, respectively) with relatively homogeneous size distributions, which can be explained by their similar interfacial tension values (Table 1). These results perfectly match the general trends seen in interfacial tension data (Table 1), further confirming the strong relationship between the emulsifying ability and the adsorption kinetics of emulsifiers (GA fibrils). The above analyses are supported by the morphology observations of emulsion droplets using PLM, as shown in Figure 1B. As can be seen, in all cases, the presence of a socalled “Maltese cross” interference figure was clearly observed (Figure 1B), especially in the PLM image of 0.5% GA fibrilstabilized emulsion droplets (10% sunflower oil) (Figure 1C). The presence of a radiant halo with a Maltese cross, a characteristic of anisotropic materials with two vibration directions, strongly indicates the formation of a multilamellar structure of the emulsion droplets’ shells.29 Thus, we can conclude that the GA fibrils may have a multilayer adsorption at the oil droplet surface probably due to the interfibrillar interactions (hydrogen bonds), yielding the multilayer interfacial structure. These results are in good agreement with our previous findings,22 and imply that the multilayer interfacial organization of GA fibrils is independent of oil phase type and composition. In addition, due to the adsorption of charged GA fibrils onto the droplet surface, all of the emulsion droplets (pH around 4.5) with different oils were highly negatively charged (−50.5 to −54.5 mV), which could provide high electrostatic forces for emulsion stabilization. Previous studies also demonstrated that the olive oil-in-water emulsions stabilized by GA fibrils had a superior stability during storage and heating, which is mainly attributed to the formation of multilayer fibril shells with high electrostatic repulsive forces, thereby protecting

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RESULTS AND DISCUSSION Multilayer Emulsion Droplets Stabilized by GA Nanofibrils. Natural saponin GA has a hydrophobic triterpenoid aglycon moiety (18β-glycyrrhetinic acid) attached to a hydrophilic diglucuronic unit. Owing to the amphiphilic structure, GA molecules show a complex self-assembly behavior in water, where the hydrophobic triterpenoid aglycon moieties interact laterally in a head-to-head way, and the hydrophilic diglucuronic units are left exposed to water.17 The anisotropic assembly of GA leads to the formation of long nanofibrils with a thickness of around 2.5 nm, independent of GA concentration.17 It has been demonstrated that all GA monomers assembled to form uniform fibrils in water when the GA concentration was above 0.1 wt % (Figure S1A, Supporting Information, SI). Amphiphilic GA monomers and fibrillar assemblies (fibrils) have affinity for the hydrophobic oil phase and thus can effectively reduce the interfacial tension (see Figure S1B, SI), in line with our previous studies.22 We then investigated the impact of oil phase type on the interfacial properties of GA fibrils (0.1%) at the oil−water interface. Table 1 shows the measured interfacial tension values (γ30 min) of GA fibril solution (0.1%) at different oil−water Table 1. Interfacial Tension Values (γ30 min) of GA Fibril Solution (0.1 wt %) at Different Oil−Water Interfaces after 30 min of Adsorptiona oil phaseb sunflower oil S:A 1:1 algal oil flaxseed oil

oil density (ρ, g/cm3)

oil interfacial tension (against water, mN/m)

interfacial tension (γ30 min, mN/m)

0.914

22.76 ± 0.14a

10.68 ± 0.18a

0.931 0.947 0.926

12.01 ± 0.13c 10.68 ± 0.12d 18.74 ± 0.20b

5.21 ± 0.23c 4.38 ± 0.11d 7.96 ± 0.12b

a Values are the mean and standard deviation of triplicates. Different letters indicate significant differences between groups (p < 0.05). bS, sunflower oil; A, algal oil.

C

DOI: 10.1021/acs.jafc.6b05242 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. (A) Droplet size distributions of O/W emulsions with different oil phases (5 wt %; S, sunflower oil; A, algal oil) stabilized by 0.25 wt % GA fibril. (B) PLM images (scale bar = 30 μm) of these emulsions, showing a Maltese cross. (C) PLM image (scale bar = 30 μm) of 0.5% GA fibrilstabilized emulsion droplets (10% sunflower oil). (D) Schematic illustration of the fibrillar self-assembly of amphiphilic GA molecules in water and the construction of emulsion droplets with a multilayer GA fibril shell.

Figure 2. Photographs of (A) emulsion gels with different oil phases (40 wt %; S, sunflower oil; A, algal oil; F, flaxseed oil) prepared at a constant GA fibril concentration (4 wt %) and the corresponding appearance of these emulsion gels (lower images). (B) Dried samples containing nearly 91 wt % vegetable oil obtained by freeze-drying of these emulsion gels.

D

DOI: 10.1021/acs.jafc.6b05242 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. PLM images (scale bar = 30 μm) of emulsion gels containing different oil phases (40 wt %; S, sunflower oil; A, algal oil) prepared at a constant GA fibril concentration (4 wt %). Oil phases: (a) sunflower oil; (b) S:A 7:3; (c) S:A 5:5; (d) S:A 3:7; (e) algal oil; (f) flaxseed oil.

the fibril-coated emulsion droplets against flocculation and coalescence (Figure 1D).22 On the basis of the above results, we can conclude that the oil phase polarity had a significant impact on the interfacial adsorption process of GA fibrils, which showed a higher affinity to more polar algal oil, forming an interface with a lower interfacial tension value (Table 1). Accordingly, the different adsorption kinetics resulted in the formation of multilayer emulsion droplets with different droplet sizes (Figure 1A,B). As the structural building blocks making up the emulsion gel, the size and the colloidal interactions of emulsion droplets are known to influence the structure and functional properties of emulsion gels drastically.5,23,24,30 Therefore, we expect that the properties of novel emulsion gels based on natural GA fibrils can be varied by changing the emulsion droplet size, which is shown to be highly affected by the oil phase polarity and processing conditions, and the results are described in the following sections. GA Fibril-Based Emulsion Gels with Tunable Properties. In this section, we examine the impact of oil phase polarity on the microstructure and mechanical properties of emulsion gels stabilized by GA fibrils. Previous studies have shown that the fine emulsion gels with homogeneous appearance could be successfully created when the GA fibril and olive oil concentrations were above 1 and 20 wt %, respectively.22 Herein, the emulsion gels stabilized by 4 wt % GA fibrils with a constant oil concentration of 40 wt % were prepared, and then the effect of oil phase type and composition on the gel formation was investigated, as shown in Figure 2. As can be seen from Figure 2A, for all studied vegetable oils, a series of fine emulsion gels with homogeneous appearance were obtained, suggesting that the formation of GA fibril-based emulsion gels should be independent of oil phase polarity. However, compared to sunflower oil, the light yellow emulsion gels of algal and flaxseed oils (especially the algal oil) showed a more solid appearance with a relatively dry surface (see below images of Figure 2A), which implies that these emulsion gels seem to have a more compact and stronger structure. Additionally, for the mixed sunflower/algal oil phase, at low

algal oil ratio (S:A 7:3), the emulsion gel showed an appearance with a wet surface similar to that of pure sunflower oil; however, upon further increase of algal oil concentration (S:A 5:5 and 3:7), the appearance of emulsion gels became very close to that of algal oil, indicating the gel properties may be mainly dominated by the algal oil phase. These results completely reproduce the general trends observed in the data from interfacial tension measurements (Table 1) and emulsion droplet characteristics (Figure 1). Therefore, it can be concluded that the average size of the constituent emulsion droplets may be correlated to the appearance and structural properties of final emulsion gels stabilized by GA fibrils.5,23,24,30 This will be further discussed in the following paragraphs of microstructure and rheological properties of emulsion gels. In addition, all of these emulsion gels could be easily spreadable (data not shown), showing their potential applications in foods, cosmetics, and pharmaceuticals, such as in spreads and ointments. These emulsion gels with three different oils were further subjected to water removal through lyophilization. As can be seen from Figure 2B, in all cases, the gel-like solids were obtained without any oil leakage, which suggests that the oil droplets coated by multilayer GA fibrils could maintain good stability during freeze-drying, independent of oil phase. Compared to the white sunflower oil gels, the dried emulsion gel of algal oil was found to be closer to a solid powder state, implying a more compact structure with better stability, in line with the appearance of fresh algal oil emulsion gel (Figure 2A). These results indicate that the GA fibril-based emulsion gels can be used as a template for the preparation of solid oil products (oil gels or powders) containing nearly 91 wt % vegetable oil. Microstructure of Emulsion Gels. To gain more insight into the link between emulsion droplet size and the final properties of emulsion gels, the microstructure observations of emulsion gels stabilized by GA fibrils were first performed by using PLM and CSLM. Figure 3 shows the impact of oil phase polarity on the gel microstructure observed under PLM. As can be seen, all emulsion gels showed a porous network microstructure, which E

DOI: 10.1021/acs.jafc.6b05242 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. CSLM images (scale bar = 25 μm) of emulsion gels with different oil phases (40 wt %; S, sunflower oil; A, algal oil) stabilized by 4 wt % GA fibrils: (a) ThT fluorescence image (highlighting the GA fibrillar network); (b) bright field image; (c) overlay image of fluorescence and bright field. Oil phases: (a, b, and c) sunflower oil; (d) S:A 5:5; (e) algal oil; (f) flaxseed oil.

with ThT. The dye ThT was used to label the GA fibrillar network of emulsion gels due to its specific binding to fibrillar structure.22,31 As can be seen, in all cases, the continuous fibrillar network structure was clearly observed, and the size and size distribution of emulsion droplets (black pores) within the gel network were in line with the PLM images (Figure 3). This is further supported by the CSLM images of emulsion gels dyed with Nile Red (Figure S3, SI). From Figure S3, the oil droplets were also found to be tightly packed, especially in the emulsion gel of algal oil (Figure S3e), which results in the distortion of the original round droplet shapes (marked by red arrows). The close packing and connection of emulsion droplets could allow them to interact strongly with each other, which thus contributes to the increase in the strength and stability of gel network.5,30 This may be a factor in the better appearance and stability during freeze-drying of the algal oil emulsion gel (Figure 2). It is known that the textural properties of emulsion gels are largely dependent on the emulsion droplet size, and a lowering of the droplet size can cause an obvious increase in the rigidity of emulsion gels, where the oil droplets function as active filler particles to reinforce the gel matrix.5,30 Here, on the basis of these observations of gel appearance and microstructure (Figures 2−4 and Figure S3), combined with the data from interfacial tension (Table 1) and emulsion droplet characteristics (Figure 1), we can conclude that the oil phase polarity (type and composition) could affect the size and size distribution of the constituent emulsion droplets significantly, which then tune the microstructure and mechanical properties of the GA fibril-based emulsion gels. For the more polar algal oil, the GA fibrils had a higher affinity to the algal oil−water

appears to be formed by the thickened planar adhesion junctions between emulsion droplets. Our previous studies have demonstrated that the GA fibril-based emulsion gels had a fibrillar network formed by noncovalent interfibrillar interactions (hydrogen bonds) in the continuous phase as well as at the droplet surface,22 thus showing strong birefringence under PLM, especially around the oil droplets due to the presence of the GA fibril shell (see Figure 3). Also from Figure 3, it can be clearly seen that for the emulsion gel of algal oil, the emulsion droplets showed a smaller droplet size and seem to be packed together more closely in the network, as compared to those of sunflower and flaxseed oils (especially sunflower oil). For the emulsion gels with mixed oil phase, with increasing algal oil concentration, the emulsion droplet size gradually decreased and the whole network structure started to resemble that of pure algal oil. To further confirm the microstructure of individual emulsion droplets within the gel matrix, these emulsion gels were diluted with water under mild stirring to give samples containing 0.25% GA fibril concentration. After dilution, all of the reconstituted emulsions could be obtained due to the destruction of noncovalent fibrillar network, and their morphologies were then observed under PLM (Figure S2, SI). As can be seen, the radiant halo with a Maltese cross in the PLM images of reconstituted emulsion droplets was clearly observed, indicating the multilayer fibril shell of emulsion droplets within the continuous network. This is in line with the PLM images of fresh emulsion droplets prepared by 0.25% GA fibrils (Figure 1B). We further used CSLM to observe the microstructure of emulsion gels with different oil phases (Figures 4 and S3, SI). Figure 4 shows the CSLM images of these emulsion gels dyed F

DOI: 10.1021/acs.jafc.6b05242 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. (A) Amplitude and (B) frequency sweeps for emulsion gels with different oil phases (40 wt %; S, sunflower oil; A, algal oil; F, flaxseed oil) prepared at 4 wt % GA fibrils. G′ and G″ are shown as solid and open symbols, respectively. (C) Viscosity curves and Herschel−Bulkley flow parameters (inset table) of these emulsion gels. All measurements were performed at 25 °C.

interface, showing a faster adsorption kinetics, which thus contributes to the formation of fine emulsion droplets with smaller droplet size. Accordingly, the algal oil emulsion gel displayed a more solid appearance and better stability due to the stronger colloidal interactions between emulsion droplets (active filler particles). In the following sections, we investigated the mechanical properties of these emulsion gel by small deformation rheological measurements to further confirm the above analyses. Mechanical Properties of Emulsion Gels. Here, the mechanical properties of the GA fibril-based emulsion gels were determined by performing a small-deformation rheology test, including oscillatory and flow measurements. Panels A and B of Figures 5 show the results of oscillatory amplitude (stress = 0.1−1000 Pa, frequency = 1 Hz) and frequency (0.1−100 Hz, stress = 10 Pa, within the linear viscoelastic region) sweeps applied to emulsion gels, respectively. As can be seen in Figure 5A, for all investigated cases, the elastic modulus (G′) was always much higher than the viscous modulus (G″) in their individual linear viscoelastic regions (LVR) (data not shown), indicating these 4% GA fibril-stabilized emulsion gels have mostly elastic solid-like behavior, in agreement with previous studies.22 Compared to the sunflower and flaxseed oils, the algal oil emulsion gel showed significantly broader LVR, higher critical stress (crossover point of G′ and G″), and higher G′ values over the applied amplitude range, suggesting a higher gel strength. For the emulsion gels with mixed sunflower/algal oil phase, the G′ values gradually increased with increasing algal oil

concentration, and at higher S:A ratios (5:5 and 3:7) the gel strength appears to be mainly dominated by the algal oil. Figure 5B shows the frequency dependence of the rheological response of these emulsion gels. As can be seen, the G′ curves had slightly positive slopes, and both G′ and G″ (data not shown) for all of the systems displayed a relatively weak frequency dependence. This indicates that the rheological response of emulsion gels is not markedly affected by the applied deformation rate even at high frequency (100 Hz).32,33 The loss tangent (G″/G′) values were always very low ( 1) of the material.32,34 The obtained flow parameters, including yield stress (σy), consistency coefficient (K), and flow index (n), are listed in the inset table of Figure 5C. As can be seen, all of the emulsion gels showed a prominent shear thinning behavior (n < 1), but the degree of shear thinning was lower for the emulsion gel of algal oil as compared to sunflower and flaxseed oils, indicating a weaker shear sensitivity. In addition, the higher values of σy and K of the algal oil emulsion gel indicate that it had a denser and stronger network structure, thus resisting the disruption of oil droplet clusters and the subsequent arrangement of these clusters in the flow direction.34,35 These results are in good agreement with the data from amplitude and frequency sweeps (Figure 5A,B). Taken together, all of the data of small-deformation rheology test (Figure 5) clearly suggest that the mechnical properties of the GA fibril-based emulsion gels can be tuned by varying the oil phase type and composition (oil phase polarity), which is

consistent with the observations of gel appearance and microstructure (Figures 2−4 and Figures S2 and S3). Combined with the data from interfacial tension (Table 1) and emulsion droplet characterization (Figure 1), it seems clear that the GA fibril-based emulsion gels with tunable microstructure and mechanical properties can be engineered by controlling the droplet size of the constituent emulsion droplets (active filler particles), which is considered to be a major factor affecting the structure and functional properties of emulsion gels.5,30 To further confirm the role of emulsion droplet size, the impact of homogenization conditions on the properties of the GA fibril-based emulsion gels was then investigated. For the specific sunflower oil emulsion gel, different homogenization conditions (rates and time) would result in the formation of emulsion droplets with different droplet sizes, which are thus expected to tune their microstructure and mechanical properties accordingly. As seen from Figure S4 (SI), as expected, these properties of emulsion gel were obviously affected by homogenization rates and time, showing that the emulsion gel with smaller droplet size and more homogeneous size distribution had a denser network as well as a higher mechanical strength. These results are in good agreement with the above analyses (Figures 1−5). H

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Journal of Agricultural and Food Chemistry Thermoresponsive and Thixotropic Properties. For future applications of these GA fibril-based emulsion gels, properties such as thermoreversibility and thixotropy are of importance. Therefore, these properties were further evaluated using rheological measurements in the current work. Previous studies have shown that the olive oil emulsion gel stabilized by GA fibrils had a thermoreversible behavior.22 Accordingly, the studied emulsion gels with different oil phases should also have interesting responsive behavior as a function of temperature. As expected, all of the emulsion gels displayed a clear gel-to-sol transition during heating and a reversible gelation during cooling (Figure S5, SI), suggesting a good thermoreversibility, which is probably due to the noncovalent GA fibrillar network in the continuous phase.22 Thus, by simply changing temperature, these emulsion gels can be switched reversibly between gel and liquid dispersion (sol), independent of oil phase. The thixotropic behavior of the GA fibril-based emulsion gels was also studied by subjecting them to a three-interval time test, wherein the viscosity of samples was monitored as a function of time under an alternate cycle of low and high shear rates (0.1, 10, and 0.1 s−1). As seen from Figure S6 (SI), for all investigated cases, there was no apparent change in the viscosity values over time at a low shear rate (0.1 s−1) in the first interval, but an evident drop in the viscosity was observed as the shear rate was increased from 0.1 to 10 s−1. In the third interval, the emulsion gels (especially the algal oil) showed an obvious recovery when the shear rate was changed back to 0.1 s−1. The recovery percentage for all emulsion gels was in the range of 60−80%, suggesting a good structure recovery at rest. Compared to sunflower (62.5%) and flaxseed (72.3%) oils, the algal oil emulsion gel showed a higher percentage (80.4%), which is in line with previous results (Figures 2−5). SEM and FTIR. To visualize the fibrillar network structure of emulsion gels more directly, the volatile hexane was used as oil phase for making emulsion gels, and the samples for SEM observations were prepared by evaporation under air-drying condition. The use of hexane as oil phase did not influence the emulsion gel formation (data not shown). Figure 6 shows the SEM images of the evaporated samples stabilized by GA fibrils (0.25, 1, and 4 wt %). As seen from Figure 6A, for the 0.25% GA fibril-stabilized emulsion, the evaporation of the hexane phase deformed the structure into a polyhedral morphology (Figure 6Aa), more commonly observed in foams, clearly confirming the formation of GA fibril layer at the emulsion droplet surface. The thickness of the interfacial fibril layer was shown to be around 100 nm, which is much higher than that of a single GA fibril (around 2.5 nm) assembled in water (see Figure S1A, SI).17 This indicates the possible formation of a multilayer fibril structure at the droplet surface, in good agreement with the previous observations of emulsion droplets under PLM (Figures 1 and S2, SI). At higher GA fibril concentrations of 1 and 4 wt %, the hexane emulsion gels were obtained, and the SEM images of their air-dried samples are shown in Figure 6, panels B and C, respectively. As can be seen, the porous continuous network was clearly observed, especially in the 4% GA fibril sample (Figure 6Cg), showing that the oil droplets (hexane) embedded in this network were packed together closely. This is again in good agreement with the images of PLM (Figure 3) and CSLM (Figure 4). In addition, it is interesting to note that these emulsion droplets were found to be covered by a layer of fibrous network (Figure 6Ce,h), appearing only in the dried samples of emulsion gels (1−4% GA fibrils), which is probably the GA fibrillar network in the

continuous phase (Figures 3 and 4). This is consistent with previous studies showing that the GA fibrillar network was formed through interfibrillar junctions and entanglements in aqueous phase.17,22 These observations provide more insight about the microstructure and mechanical properties of the GA fibril-based emulsion gels with different oil phases (Figures 1−5). The FTIR characterization of neat oils, raw GA, and emulsion gels highlighted the role of hydrogen-bonding interactions in the gel formation. As seen from Figure S7 (SI), the strong and broad O−H absorptions at 3650−3200 cm−1 were present only in both the samples of GA and emulsion gels (1−4% GA fibrils) and expectedly absent in the sunflower oil and fluid emulsion (0.5% GA fibrils). This indicates that the interfibrillar hydrogen bonding in the continuous phase is the main factor responsible for the fibrillar network formation of emulsion gels, in agreement with previous studies.22,36 The FTIR spectra of all emulsion gels with different oil phases were further studied in Figure 7 and

Figure 7. FTIR spectra of emulsion gels with different oil phases (40 wt %; S, sunflower oil; A, algal oil) prepared at 4 wt % GA fibrils.

showed that all of the emulsion gels had broad O−H absorptions at 3650−3200 cm−1, which is independent of oil phase type and composition. This indicates that the continuous network structure of emulsion gels was mainly formed by the interfibrillar hydrogen-bonding forces. Delivery Vehicle for Functional Ingredients. Owing to the multilayer fibril shell of emulsion droplets with high electrostatic forces, the emulsion gels formed by natural GA fibrils displayed good stability during storage and drying (Figure 1), suggesting their potential as novel delivery vehicles for functional ingredients and bioactive molecules in foods, pharmaceuticals, and cosmetics.22 Meanwhile, these edible vegetable oils (especially the algal oil) are prone to be oxidized due to the high levels of unsaturated fatty acids, and therefore, the loading of oil-soluble antioxidants into these GA fibril-based emulsion gels could also effectively enhance their oxidative stability during processing and storage. Herein, we further prepared the functional emulsion gels by dissolving oil-soluble antioxidants, such as β-carotene and α-tocopherol (model compound), in the oil prior to the emulsification step to evaluate the loading capacity of oil-soluble bioactives. As can be seen from Figures 8 and S8 (SI), the incorporation of βcarotene and α-tocopherol did not influence the formation and stabilization of all emulsion gels, independent of oil phase polarity. Moreover, after storage of 30 days, these colored and antioxidative emulsion gels showed an excellent stability I

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Figure 8. (A) Photographs of 4 wt % GA fibril-stabilized emulsion gels with different oil phases (40 wt %; S, sunflower oil; A, algal oil; F, flaxseed oil) containing β-carotene (0.1 wt % of oil) at initial (0 day) and after 30 days of storage at room temperature (25 °C). (B) Storage modulus (G′) versus frequency for these emulsion gels during storage at room temperature (25 °C). Oil phases: (a) sunflower oil; (b) S:A 5:5; (c) algal oil; (d) flaxseed oil.

tuned by oil phase polarity (oil type and composition), which has a significant impact on the droplet size of the constituent emulsion droplets, a main factor that is believed to be correlated strongly with the textural properties of emulsion gels. For more polar edible oils (algal oil), the GA fibrils showed a higher affinity to the oil−water interface and thus a faster adsorption kinetics, leading to the formation of fine multilayer emulsion droplets with smaller droplet size. These

without any obvious changes in the gel appearance (Figure 8A), which is further confirmed by the results of frequency sweeps (Figure 8B). These results indicate that the novel GA fibrilbased emulsion gels with tunable properties could be used as stable delivery vehicles in various functional food, pharmaceutical, and cosmeceutical products. In conclusion, the microstructure and mechanical properties of the GA fibril-based emulsion gels were shown to be well J

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(5) Dickinson, E. Emulsion gels: the structuring of soft solids with protein-stabilized oil droplets. Food Hydrocolloids 2012, 28, 224−241. (6) Jadhav, S. R.; Hwang, H.; Huang, Q.; John, G. Medium-chain sugar amphiphiles: a new family of healthy vegetable oil structuring agents. J. Agric. Food Chem. 2013, 61, 12005−12011. (7) Silverman, J. R.; John, G. Biobased fat mimicking molecular structuring agents for medium-chain triglycerides (MCTs) and other edible oils. J. Agric. Food Chem. 2015, 63, 10536−10542. (8) Rogers, M. A.; Wright, A. J.; Marangoni, A. G. Nanostructuring fiber morphology and solvent inclusions in 12-hydroxystearic acid/ canola oil organogels. Curr. Opin. Colloid Interface Sci. 2009, 14, 33− 42. (9) Wright, A. J.; Marangoni, A. G. Formation, structure, and rheological properties of ricinelaidic acid-vegetable oil organogels. J. Am. Oil Chem. Soc. 2006, 83, 497−503. (10) Hwang, H. S.; Kim, S.; Singh, M.; Winkler-Moser, J. K.; Liu, S. X. Organogel formation of soybean oil with waxes. J. Am. Oil Chem. Soc. 2012, 89, 639−647. (11) Patel, A. R.; Babaahmadi, M.; Lesaffer, A.; Dewettinck, K. Rheological profiling of organogels prepared at critical gelling concentrations of natural waxes in a triacylglycerol solvent. J. Agric. Food Chem. 2015, 63, 4862−4869. (12) Schaink, H.; Van Malssen, K.; Morgado-Alves, S.; Kalnin, D.; van der Linden, E. Crystal network for edible oil organogels: possibilities and limitations of the fatty acid and fatty alcohol systems. Food Res. Int. 2007, 40, 1185−1193. (13) Nikiforidis, C. V.; Scholten, E. Self-assemblies of lecithin and αtocopherol as gelators of lipid material. RSC Adv. 2014, 4, 2466−2473. (14) Bot, A.; Agterof, W. G. Structuring of edible oils by mixtures of γ-oryzanol with β-sitosterol or related phytosterols. J. Am. Oil Chem. Soc. 2006, 83, 513−521. (15) Sintang, M. D. B.; Rimaux, T.; van de Walle, D.; Dewettinck, K.; Patel, A. R. Oil structuring properties of monoglycerides and phytosterols mixtures. Eur. J. Lipid Sci. Technol. 2017, 118, 1500517. (16) Sawalha, H.; den Adel, R.; Venema, P.; Bot, A.; Flöter, E.; van der Linden, E. Organogel-emulsions with mixtures of β-sitosterol and γ-oryzanol: influence of water activity and type of oil phase on gelling capability. J. Agric. Food Chem. 2012, 60, 3462−3470. (17) 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. (18) Lu, J.; Hu, J.; Song, Y.; Ju, Y. A new dual-responsive organogel based on uracil-appended glycyrrhetinic acid. Org. Lett. 2011, 13, 3372−3375. (19) Bag, B. G.; Majumdar, R. Self-assembly of a renewable nanosized triterpenoid 18β-glycyrrhetinic acid. RSC Adv. 2012, 2, 8623− 8626. (20) Lu, J.; Gao, Y.; Wu, J.; Ju, Y. Organogels of triterpenoidtripeptide conjugates: encapsulation of dye molecules and basicity increase associated with aggregation. RSC Adv. 2013, 3, 23548−23552. (21) Asl, M. N.; Hosseinzadeh, H. Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds. Phytother. Res. 2008, 22, 709−724. (22) Wan, Z.; Sun, Y.; Ma, L.; Guo, J.; Wang, J.; Yin, S.; Yang, X. Thermoresponsive structured emulsions based on fibrillar selfassembly of natural saponin glycyrrhizic acid. Food Funct. 2017, 8, 75−85. (23) Ye, A.; Taylor, S. Characterization of cold-set gels produced from heated emulsions stabilized by whey protein. Int. Dairy J. 2009, 19, 721−727. (24) Kiokias, S.; Varzakas, T. Innovative applications of food related emulsions. Crit. Rev. Food Sci. Nutr. 2016, 1130017. (25) Binks, B. P.; Lumsdon, S. O. Effects of oil type and aqueous phase composition on oil-water mixtures containing particles of intermediate hydrophobicity. Phys. Chem. Chem. Phys. 2000, 2, 2959− 2967. (26) Schmidt, S.; Liu, T.; Rütten, S.; Phan, K.-H.; Möller, M.; Richtering, W. Influence of microgel architecture and oil polarity on stabilization of emulsions by stimuli-sensitive core-shell poly(N-

smaller emulsion droplets were found to be packed together more tightly within the continuous fibrillar network, providing stronger droplet−droplet interactions, and thereby contribute to reinforcing the gel matrix. Consequently, the obtained emulsion gels displayed a more solid appearance, a denser network microstructure, and a higher mechanical strength. All emulsion gels had interesting temperature-responsive behaviors, independent of oil phase, which is probably due to the thermoreversibility of the hydrogen-bond fibrillar network in the continuous phase. Additionally, these stable emulsion gels also showed potential as natural vehicles for loading and delivering functional ingredients. We hope that these findings could potentially aid in the design and construction of natural saponin fibril-based emulsion gel systems with specific textural properties for foods, cosmetics, and pharmaceutical applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05242. AFM images of GA fibril solutions, dynamic interfacial tension for GA fibril solutions at different oil−water interfaces, Lissajous plots of surface pressure versus deformation obtained during amplitude sweep of the sunflower oil−water interface, and PLM image of GA fibril-stabilized emulsion droplets; PLM images of the reconstituted emulsions from emulsion gels with different oil phases; CSLM images of emulsion gels with different oil phases; frequency sweeps and PLM images for sunflower oil emulsion gels prepared at different homogenization conditions; thermoresponsive and thixotropic properties of emulsion gels with different oil phases; FTIR spectra of raw GA, sunflower oil, and emulsion gels prepared at different fibril concentrations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(X.Y.) E-mail: [email protected], [email protected]. Fax: (086) 20-87114263. Phone: (086) 20-87114262. ORCID

Xiaoquan Yang: 0000-0002-4016-9834 Funding

This work is supported by grants from the General Project of China Postdoctoral Science Foundation (2016M600655) and the National Natural Science Foundation of China (31371744 and 31501425). Notes

The authors declare no competing financial interest.

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REFERENCES

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