Mengyue
Xu‡
a,
Lulu
Ma‡
a,
Qing
Li
a,
Jiahao
Wu
b,
Zhili
Wan
*abc,
To
Ngai
*b and
Xiaoquan
Yang
a
aLaboratory of Food Proteins and Colloids, School of Food Science and Engineering, Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou 510640, China
bDepartment of Chemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong, China. E-mail: zhiliwan@scut.edu.cn; tongai@cuhk.edu.hk; Fax: +(086) 20-87114263; Fax: +(852) 26035057; Tel: +(852)39431223
cOverseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111 Center), Guangzhou 510640, China
First published on 22nd November 2021
Herein, we report a new class of high internal phase gel emulsions (gel-HIPEs) that are mechanically robust, adaptable, and processable. They can be synthesized facilely by using the natural food-grade saponin glycyrrhizic acid (GA) as the sole stabilizer, which is shown to be versatile for various oils. The structural properties of these HIPEs including appearance, viscoelasticity and processability are well controlled by simply changing the concentration of GA nanofibrils. When the GA nanofibril concentration exceeds 0.3 wt%, the unique gel-HIPEs can be produced through the formation of fibrillar hydrogel networks in the continuous phase. When the nanofibril concentration only increases to 5 wt%, it is surprising to see that these gel-HIPEs display an extremely high mechanical strength, and the storage moduli as well as the yield stress values can reach 408.5 kPa and 3340 Pa (or even more), respectively. We conjecture that such remarkable mechanical performance is mainly attributed to the highly viscoelastic GA nanofibrillar networks in the continuous phase of gel-HIPEs, which can actively trap the nanofibril-coated emulsion droplets and thus strengthen the gel matrix. Consequently, the robust gel-HIPEs can be used as a solid template to fabricate stable porous materials without the need for crosslinking of the continuous phase, and the open- and closed-cell foam microstructures are controlled by the nanofibril concentration. Furthermore, the nanofibril-based HIPEs are promising long-term delivery vehicles with controlled-release properties for lipophilic active cargoes, since the strong fibrillar networks at the droplet surfaces and in the continuous phase can effectively retard the active release.
In view of their wide applications, the efficient and facile fabrication of stable HIPEs is highly required and remains an important issue in emulsion science due to their ultrahigh internal phase fraction and thermodynamic instability. Typically, the formation of HIPEs generally requires the addition of interfacial stabilizers, such as small surfactants (e.g., sorbitan monooleate and CTAB),5,6 amphiphilic polymers,7 or colloidal particles (e.g., silica and PNIPAM-based microgel particles).8,9 In comparison with these surfactant-stabilized HIPEs, the HIPEs stabilized by colloidal particles, known as Pickering HIPEs, are less susceptible to coalescence, Ostwald ripening, and creaming due to the irreversible adsorption of the particles at the interface, which makes them highly stable for months or even years.10 However, the preparation of Pickering HIPEs requires specially surface-modified particles with appropriate amphiphilicity,9,10 and the formed emulsions are sometimes unstable and may undergo phase inversion at a high volume fraction (around 0.7) of the dispersed droplets.11,12 In recent years, considering the environmental drawbacks and even the potential safety concerns of synthetic surfactants or inorganic particles, there is growing interest in the development of food-grade Pickering HIPEs by using particles of natural origin as stabilizers, such as particles made of animal and plant proteins,13–15 modified starch,16 cellulose,17 and chitin.18 Unfortunately, these natural building blocks are often restricted by relatively poor solubility and uncontrollable particle wettability, and thus require complicated and time-consuming fabrication and surface modification processes, which limit their use as efficient and robust stabilizers to make food-grade HIPEs. Therefore, it remains highly desirable to explore new building blocks from natural edible substances, for the facile synthesis of stable and sustainable HIPEs for their safe applications in food, pharmaceutical, and personal care products.
Recently, we reported a new kind of food-grade oil-in-water (O/W) gel emulsion which was solely stabilized by a naturally occurring triterpenoid saponin glycyrrhizic acid (GA).19,20 GA is the main active compound ingredient of licorice root extract and is widely used as a sweetening agent in candies and sweets or as drugs for treating liver and skin diseases due to its wide biological activities. As a chiral molecule, GA has a hierarchical self-assembly in water forming long semiflexible nanofibrils (few nm thickness), which then form a nematic supramolecular hydrogel with a hydrogen-bond 3D fibrillar network when the nanofibril concentration is above 0.3 wt%.19,21 These amphiphilic nanofibrils display spatially controllable self-assembly at liquid–liquid interfaces and in the aqueous phase,22–25 forming fibrillar gel networks in the continuous phase as well as on the surfaces of emulsion droplets after emulsification, which thus leads to the formation of oil-in-water gel emulsions. The most prominent feature of our GA nanofibril-based gel emulsions is that their gelation behaviors mainly rely on the assembled fibrillar hydrogel network in bulk, rather than the tight stacking of the dispersed droplets covered by GA nanofibrils; therefore, the volume fraction of the dispersed phase of our GA nanofibril gel emulsions can be varied continuously (below and above 0.74), which does not significantly affect the stability and structural properties of the whole emulsions. Recent studies have demonstrated that the gel emulsion strategy based on the use of suitable low-molecular-weight gelator molecules is a potential solution to overcome the common problems of traditional HIPEs.26,27 For example, the mechanical and long-term storage stabilities of HIPEs can be obviously increased by the gelation of the continuous phase as well as the compact stacking of the dispersed droplets, and their structural properties also can be highly controlled by tuning the amounts of gelators.26,27 Therefore, there is no doubt that this structuring strategy can broaden the practical applications of HIPEs.
Herein, we exploit the use of natural GA nanofibrils as a sole stabilizer for the facile and efficient synthesis of a new class of structured O/W HIPEs. Stable and processable gel-HIPEs can be rapidly fabricated by adopting a simple one-step emulsification at high temperature (80–90 °C) with subsequent cooling (Fig. 1a), which has the advantage of not requiring specialized equipment, the addition of any synthetic surfactants, particles, or polymers, or any surface modification processes. During the hot homogenization (80–90 °C), GA nanofibrils with favorable interfacial activity form multilayer interfacial networks to stabilize the generated emulsion droplets (Fig. 1a and c).19,20,22 The subsequent cooling can strengthen the interfibrillar hydrogen-bond interactions leading to the formation of viscoelastic hydrogel networks in the continuous phase and at the droplet surfaces, and then the gel-HIPEs can be created (Fig. 1). We then characterized the mechanical properties and microstructure of gel-HIPEs by performing a small-deformation rheological test and a range of microscopy techniques, respectively. Finally, the potential applications of these gel-HIPEs as templates for the synthesis of biosafe porous materials or as natural delivery vehicles for hydrophobic functional ingredients (e.g., β-carotene) were evaluated.
The structural rigidness and processability of HIPEs are found to be highly dependent on the concentration of GA nanofibrils (0.1–5 wt%). At low nanofibril concentrations (0.1 and 0.25 wt%), which are those below the critical gelation concentration of GA nanofibrils in water (0.3 wt%), the prepared HIPEs are fluid because the amount of free GA nanofibrils is not sufficient to gel the continuous phase (Fig. 1b). When the nanofibril concentration is above 0.3 wt% (0.5–5 wt%), as expected, the self-standing gel-HIPEs were obtained due to the formation of a viscoelastic GA hydrogel in the continuous phase (Fig. S1†). The increasing GA nanofibril concentrations significantly promote the structural rigidness and solid-like appearance of these gel-HIPEs (Fig. 1c). This can be explained by the formation of stronger GA fibrillar hydrogel networks in the continuous phase which thus entrap the emulsion droplets more compactly. A further increase in the volume fraction of internal phase (up to 90%) can also lead to stable and homogeneous gel-HIPEs (Fig. S3†). These results reveal that GA nanofibrils are natural and efficient building blocks to produce stable and processable gel-HIPEs, and their structural properties (e.g., appearance, viscoelasticity, and stability) can be highly controlled by simply changing the concentration of the stabilizer (GA nanofibrils), which is difficult to realize in traditional HIPEs, where the ratio of the two immiscible phases can only be adjusted within a narrow range due to the geometrical requirement, resulting in limited formulations for practical applications.4,5,8,27 This is beneficial for the applications of our GA nanofibril HIPEs in food, cosmetic, and pharmaceutical products.
To visualize the GA fibrillar networks and the arrangement of emulsion droplets within the HIPEs, we adopted polarized light microscopy (PLM) and confocal laser scanning microscopy (CLSM) to observe the microstructure of gel-HIPEs with 80 vol% sunflower oil. As shown in Fig. 2a–c, all gel emulsions showed strong birefringence under PLM, which is attributed to the presence of anisotropic GA assemblies including a multilamellar nanofibril shell at the surfaces of the droplets and a fibrillar hydrogel network in the continuous phase. Our previous studies have demonstrated that the multilayer adsorption of GA nanofibrils at the liquid interfaces and the formation of the viscoelastic GA hydrogel networks in the continuous phase can provide superior stability to the nanofibril-stabilized emulsions and foams.19,20,23,24 A porous honeycomb-like network formed by the thickened planar adhesion junctions between emulsion droplets can be clearly seen from these PLM images, and these nanofibril-coated droplets appear to be closely packed together in the continuous matrix. The polyhedral shape and size of these tightly packed oil droplets were observed more directly in the CLSM images of the gel-HIPEs dyed with Nile Red (Fig. 2d–f, and S4†), which is in good agreement with the PLM results. We further used volatile hexane as the oil phase (80 vol%) to produce the HIPEs, and then prepared the samples for SEM observations by evaporation under air-drying conditions. The use of hexane as the oil phase did not affect the formation of nanofibril HIPEs. As seen from Fig. 2g–i, a fibrous network in the continuous phase is clearly observed, and the networks become more evident and integrated with increasing GA nanofibril concentration (0.25–1 wt%). These results confirm the presence of the GA fibrillar network within the continuous phase of HIPEs, which is formed through the interfibrillar hydrogen-bond junctions and entanglements.19,20,23,24
We then studied the mechanical properties of the GA nanofibril-stabilized gel-HIPEs by performing oscillatory amplitude and frequency sweep measurements. Fig. 3a and c show the storage (G′) and loss (G′′) moduli as a function of stress and frequency for the gel-HIPEs (80 vol% sunflower oil) with different GA nanofibril concentrations (0.5–5 wt%), respectively. As seen in Fig. 3a, for all samples, the storage modulus, G′, is always much higher than the loss modulus G′′ in the linear viscoelastic regime (LVR), which indicates the elastic solid-like behaviors of these gel-HIPEs. With increasing nanofibril concentration from 0.5 to 5 wt%, the values of G′ increase from 688.5 Pa to 408.5 kPa (Fig. 3a, within the LVR), and the corresponding critical yield stress increases from 83.5 Pa to 3340 Pa (Fig. 3b, defined as the crossover point of G′ and G′′), which means that the mechanical strength and the elasticity of the gel-HIPEs strongly depend on the GA nanofibril concentration. This can be further confirmed by the frequency sweep measurements (Fig. 3c), which show that the values of G′ are higher than those of G′′ within the applied frequency range, and both G′ and G′′ display a weak frequency dependence, indicating that these gel-HIPEs have an excellent tolerance to external forces. The increase of the oil phase volume fraction leads to a further increase in the gel strength (Fig. 3d), and the values of G′ and yield stress for the 5 wt% GA nanofibril-stabilized gel-HIPE with 90 vol% oil can reach 860.1 kPa and 7069.5 Pa, respectively. Such surprising high storage moduli (within the LVR) are unprecedented, which are nearly 2–3 orders of magnitude larger than those for previously reported HIPEs with relatively obvious viscoelastic characteristics, such as the microgel-stabilized Pickering HIPEs (around 200 Pa),30 the HIPEs stabilized by linear homopolymers (7100 Pa),31 depletion attraction-induced Pickering HIPEs (<400 Pa),32 and Pickering HIPEs stabilized by proteins and protein-based particles (<3000 Pa).13,14,33,34 This can be explained by the fact that for those previously reported HIPEs, their solid-like viscoelastic properties are mainly dominated by the tight packing of the dispersed oil droplets,1,4,8,27 and the interdroplet interactions are also relatively weak for reinforcing the continuous matrix of HIPEs. In contrast, for our GA nanofibril-stabilized gel-HIPEs, the highly viscoelastic behaviors mainly rely on the nanofibrillar networks in the continuous phase and at the droplet surfaces (Fig. 1a), which are formed through the strong interfibrillar hydrogen-bond interactions.19,20,24 Additionally, the GA nanofibril-coated emulsion droplets are packed and connected very closely within the continuous network due to the interfibrillar interactions (Fig. 2), which can provide stronger droplet–droplet and droplet–matrix interactions, and thereby further contribute to strengthening the gel matrix. These factors therefore endow the nanofibril-stabilized gel-HIPEs with such spectacular mechanical elasticity. Due to their high structural viscoelasticity, the nanofibril-stabilized HIPEs are rigid and processable, and the processability appears to be largely dependent on the nanofibril concentration (Fig. 3b, inset image). These results taken together reveal that the GA nanofibril-stabilized gel-HIPEs are highly elastic, solid-like yield stress materials.
We demonstrated the universality of the GA nanofibrils as natural emulsifiers for producing stable and processable gel-HIPEs containing a variety of oil phases, such as sunflower oil, algal oil, MCT oil, castor oil, dodecane, and mineral oil. These liquid oils are commonly used in applications but have significantly different compositions and polarities. The same emulsification procedure as that used with sunflower oil gel-HIPEs was performed (Fig. 1a). As seen in Fig. 4, for all the cases, rigid and processable gel-HIPEs are formed, and their structural properties including appearance and mechanical viscoelasticity can be well tuned by the GA nanofibril concentrations (1 and 5 wt%). This indicates the versatility and robustness of the GA nanofibrils as a sole and highly efficient stabilizer for the facile synthesis of gel-HIPEs, and these soft solid oil products are expected to find a broad range of applications in various fields.
The applicability of our nanofibril-stabilized gel-HIPEs was investigated. We showed that the GA nanofibril-based porous foams can be obtained by using these robust gel-HIPEs as a precursor template, which were prepared by using volatile hexane (80 vol%) as the oil phase and then evaporated by freeze-drying. From the FE-SEM images (Fig. 5), we can see that the microstructure of these porous materials can be controlled by tuning the GA nanofibril concentrations. Note that at a low GA nanofibril concentration (0.1 wt%), no porous network structure can be observed, and the walls of the droplets appear to completely collapse after drying (Fig. 5a), due to the absence of the viscoelastic fibrillar networks in the precursor HIPEs (Fig. 1b). The gel-HIPEs prepared at 1 and 3 wt% GA nanofibrils show a typical open porous network, and the pore size is generally consistent with the droplet size of precursor HIPEs (Fig. 2), suggesting the structural integrity of the GA nanofibril-coated droplets during drying. Interestingly, with the increase of the nanofibril concentration to 5 wt%, the changes of the obtained porous foam from open- to closed-cell structures are clearly observed (Fig. 5 and S5†), and the solid foam shows a more homogeneous appearance (Fig. 5d and f, inset images). It is noted that there is no obvious pore throat within the pore walls, which are formed from the drying of the GA nanofibrillar networks around the droplets and in the continuous phase. This can be explained by the fact that the viscoelastic fibrillar networks inside the gel-HIPEs become stronger at higher GA nanofibril concentration (Fig. 3) and therefore do not rupture to form pore throats under freeze-drying. We thus show that our robust gel-HIPEs stabilized solely by natural GA nanofibrils can be used as a simple and solid template for the synthesis of a variety of porous materials without the crosslinking of the continuous phase. These results indicate that the highly viscoelastic GA nanofibrillar networks can convey the superior stability to these nanofibril-based gel-HIPE templates during drying, and thus are the key factors to produce stable and controllable porous structures.
Another important application for O/W HIPEs is their use as delivery systems for the encapsulation and release of oil-soluble bioactive ingredients in foods, pharmaceuticals, and cosmetics. Herein, we further fabricated functional HIPEs by encapsulating a model hydrophobic active cargo (i.e., β-carotene) into the sunflower oil phase and then investigated the in vitro release behavior of β-carotene from these nanofibril-stabilized HIPEs. As seen from Fig. 6a, the release kinetic profiles of β-carotene from the HIPEs strongly depend on the concentration of GA nanofibrils (0.1–5 wt%). At a low nanofibril concentration (0.1 wt%), the release kinetics of β-carotene from the obtained fluid HIPEs (Fig. 1b) can be described as a two-step biphasic process, that is, an initial fast release stage (within 1 h) followed by a subsequent slow and sustained release. Compared with the liquid-HIPE at 0.1 wt% GA nanofibrils, the gel-HIPEs prepared using 0.5–5 wt% nanofibrils (Fig. 1b) display significantly different release profiles, which show that the release rates and the accumulative amounts of β-carotene released largely decrease with increasing nanofibril concentration. Particularly, for these gel-HIPEs at higher GA nanofibril concentrations (3 and 5 wt%), the detectable amounts of β-carotene are very low (3.4–5.6%) during the entire release period (72 h). This can be explained by the fact that a higher concentration of GA nanofibrils results in a higher packing density of the multilayer fibril shell around emulsion droplets (Fig. 2), which thus can effectively inhibit the transport of β-carotene incorporated into the oil phase through the droplet surfaces (Fig. 6b). More importantly, at high GA nanofibril concentrations (0.5–5 wt%), the viscoelastic GA nanofibrillar networks in the continuous phase of these gel-HIPEs (Fig. 2) play a more dominant role in retarding the release. The stronger fibrillar networks at the surfaces of the droplets and in the continuous phase formed at higher nanofibril concentrations can generate higher diffusion resistance (Fig. 6b), and consequently the release of β-carotene is markedly decreased, especially in these gel-HIPEs with 3 and 5 wt% GA nanofibrils. These results indicate that the O/W HIPEs stabilized by GA nanofibrils are promising long-term delivery vehicles for hydrophobic active cargoes, and the cargo release properties can be controlled by simply tuning the concentration of GA nanofibrils in the HIPE formulations.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1fo01656c |
‡ These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2022 |