Article pubs.acs.org/JAFC
Gelatin-Based Nanocomplex-Stabilized Pickering Emulsions: Regulating Droplet Size and Wettability through Assembly with Glucomannan Weiping Jin,†,‡,§ Jieyu Zhu,§ Yike Jiang,§ Ping Shao,§,∥ Bin Li,*,†,‡ and Qingrong Huang*,§ †
College of Food Science and Technology, Huazhong Agricultural University, 1st Shizishan Road, Wuhan, Hubei 430070, P. R. China Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, 1st Shizishan Road, Wuhan, Hubei 430070, P. R. China § Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, New Jersey 08901, United States ∥ Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, P. R. China ‡
ABSTRACT: Particle size and surface wettability play leading roles in the distribution of particles on the oil−water interface and the stability of emulsions. This work utilized nanocomplexes assembled from gelatin and tannic acid to stabilize Pickering emulsions. The sizes and surface wettability of particles were further regulated by using a polysaccharide. The sizes of nanocomplexes ranged from 205.8 to 422.2 nm and increased with the addition of polysaccharide. Their contact angles decreased from 84.1° to 59.3°, revealing their hydrophilic nature. Results of fluorescence microscopy and cryo-scanning electron microscopy indicated that nanocomplexes were located at the oil−water interface. Interfacial shear and dilatational rheological data revealed a fast and irreversible adsorption behavior, which differed from rearrangement of gelatin molecules at the oil−water interface. The minimal concentration of nanocomplexes required to stabilize emulsions was 0.1 wt %. Our results demonstrated that protein−polyphenol−polysaccharide nanocomplexes had the potential to be applied to form stable surfactant-free food emulsions for the delivery of nutraceuticals. KEYWORDS: nanocomplexes, Pickering emulsion, interfacial tension, particle size, surface wettability
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nanoparticles,11 and protein-based complexes12,13 have been used to prepare stable Pickering emulsions. The high stability of Pickering emulsions can be explained by the high free energy of desorption for solid particles, as shown in eq 114
INTRODUCTION Interactions between proline-rich proteins and polyphenols have aroused interest for decades in addressing the problems of haze in beer, wine, and juice, which were mainly due to the formation of various colloidal particles.1 The compositions and storage conditions (such as pH, ethanol content, and the presence of polysaccharides) influence the structure and physical properties of colloidal particles.2 Some researchers reported that certain polysaccharides had a stabilizing effect on haze, whereas others found that polysaccharides increased haze due to a simple increase in the size of haze particles.3 Here, to obtain controllable colloidal particles, controlling the pH and adding polysaccharide must be taken into account. Pickering emulsions stabilized by colloidal particles of sizes from a few nanometers to hundreds of micrometers are of widespread interest in areas such as pharmaceutics, cosmetics, and food science.4 Advantages of Pickering emulsions include a high stability, a relatively well-controlled size distribution, a low toxicity, and being surfactant-free.5 Currently, because of the side effects of certain low-molecular weight synthetic surfactants,6 studies have been devoted to exploring new types of edible colloidal particles. The development of environmentally friendly nanoparticles, especially on the basis of food-grade natural resources, is desirable.7 Protein-based complexes, as promising delivery vehicles, have received an increasing level of attention because of their unique properties, like tunable sizes, interfacial activities, and an interior network for encapsulation of nutrients or drugs. Soy protein nanoparticles,8,9 zein colloidal particles,10 whey protein microgel © 2017 American Chemical Society
E = πr 2γOW(1 − |cos θ|)2
(1)
where r is the radius of solid particles, γOW is the interfacial tension of the aqueous/oil phase, and θ is defined as the threephase contact angle formed at the three-phase boundary where solid particles, the continuous phase, and the dispersed phase intersect. Thus, the desorption energy of solid particles from the interface indicated that the stability of formed emulsions would be affected by particle size and surface wettability. If θ < 90°, particles of a hydrophilic nature are preferred to stabilize oil-in-water emulsions, whereas if θ > 90°, they could stabilize water-in-oil emulsions because of hydrophobic surfaces. Binks’s research revealed that a relatively stable Pickering emulsion could be formed when the contact angles of particles were in the range of 30−150°.15 In addition, the shape and size of particles are key factors that affect the stability of Pickering emulsions. The large particle sizes with an affordable wettability can improve emulsion stability.16 Received: Revised: Accepted: Published: 1401
September 17, 2016 January 24, 2017 January 29, 2017 January 30, 2017 DOI: 10.1021/acs.jafc.6b04146 J. Agric. Food Chem. 2017, 65, 1401−1409
Article
Journal of Agricultural and Food Chemistry
alternating electrical field by the probe used in its dip cell configuration. The static contact angle (θ) was measured using the VCA optima setup (AST Product Inc.). Complex solutions (1.0 wt %) were deposited onto cleaned glass substrates by drying at 40 °C. The θAW values were measured by dropping water droplets (2 μL) onto complex films. Contact angle values were obtained by calculating the imaged droplets with Laplace−Young’s function. Every sample took at least six replicates, and the average value was utilized. Transmission electron microscopy (TEM) images of three kinds of nanocomplexes were obtained on a JEM-2100F instrument (JEOL). Fabrication and Characterization of GGTN-Stabilized Pickering Emulsions. The following typical procedure for preparing nanoparticle-stabilized Pickering emulsions was used. The MCT oil phase and the 1.0 wt % (calculated by gelatin concentration) GGTN solutions were mixed with 50 and 50 wt % ratios into a glass vial (inner volume of 20 mL). The mixture was homogenized with a vortex (up to 3000 rpm) for 1 min. Emulsions were then kept at room temperature (∼25 °C) for 72 h to observe the stability. Optical Microscopy and Fluorescence Microscopy. Microscopic photographs of Pickering emulsions were visualized using the 40× objective microscope (Nikon TE2000), and digital images were captured. The adsorption of nanocomplexes at oil−water interfaces was taken using a fluorescence microscope (Nikon TE2000). After emulsions were formed, GGTNs were stained with rhodamine B, and then emulsions were washed with water to ensure no color in the lower layer. Samples were imaged 3 days after preparation. A drop of the emulsion was diluted 2-fold with water on a microscopic slide. Cryo-Scanning Electron Microscopy (Cryo-SEM). To directly visualize the arrangement of GGTNs located at the oil−water interfaces, freeze-fracture scanning electron microscopy (Cryo-SEM) was performed. Droplets were placed on the specimen held and rapidly frozen in situ by immersion into liquid nitrogen. Then, the frozen sample was transferred to the preparation chamber (Alto-CryoTransfer systems, GATAN) under vacuum, which was precooled to −130 °C with a vacuum pressure of 10−6 mbar. After a cooled knife had fractured the sample, the fractured surface sublimated extra water at a temperature of −95 °C for 4 min. A nanothin Ag film was coated before samples were inserted into a LEO 1530VP field emission scanning microscope chamber, where the temperature was also held at −130 °C. After the rapid freeze, fracture, coating, and sublimation steps, the surface structure of emulsion droplets was preserved and observed.22 Interfacial Tension Measurements. The oil-in-water interfacial tension (γ) was measured versus time for gelatin and GGTN solutions by an automated drop tensiometer (Tracker Teclis-IT Concept). Measurements of interfacial tension (γ) were based on axisymmetric drop shape analysis of an oil droplet in the aqueous phase. The droplet volume was set as 5 μL, and measurements were taken for 3500 s until a stable adsorption layer formed on the surface of the oil droplet.23 The interfacial tension of clean water and MCT oil was 26 mN m−1.24 Oscillating Droplet Measurements. Oscillations were produced by a position-encoded motor and transmitted through a piston coupled to the syringe carrying the capillary. For the sinusoidal volume fluctuations, the chosen surface amplitude was in the linear domain and fixed at 10%. The controlled frequency ranged from 0.01 to 0.1 Hz. All measurements were taken at 25 ± 0.02 °C and repeated three to five times. The relative area variation imposed by the apparatus and the interfacial tension response was recorded to calculate dilational interfacial elasticity E with eq 2
Gelatin consists of partially hydrolyzed collagen, which has been widely used in the food industry as a stabilizer because of its high surface active capacity, but gelatin-stabilized emulsions always showed poor long-term stability against aggregation and coalescence of dispersed droplets.17 Gelatin is also the prolinerich protein commonly used as a protein model for binding with polyphenols. Tannic acid (TA) has a strong interaction with proline-rich protein and is commonly used in clarifying the undesirable haze in beverages.1 TA is geared to a group of hydrolyzable tannins and has diverse biological properties, including antioxidant and antibacterial properties.18 Glucomannan is a neutral polysaccharide that can be applied to improve the stability of a beverage because of its good water solubility. It also possesses health benefits like lowering the blood cholesterol level, aiding in weight loss, and working as a prebiotic.19 Interactions between gelatin and TA have been proved to be mainly hydrophobic interaction.20 Glucomannan is one of the polysaccharides with a gel-like structure and could interact with TA through co-aggregation effects. In addition, hydrogen bonding among hydroxyl groups could affect the formation of hydrophobic area. Therefore, the interactions between ternary complexes would be dominated by hydrophobic interactions and hydrogen bonding. They formed coaggregates together within the nanometer range, which can be considered nanocomplexes. Therefore, this study aimed to fabricate colloidal particles through the binding between gelatin and TA under suitable pH conditions and to tune the particle size and surface wettability of colloidal particles using glucomannan. Dynamic interfacial adsorption and properties of the interfacial layer were recorded with an automated drop tensiometer. Microsurface morphologies of those colloidal particle-stabilized Pickering emulsions were investigated using fluorescence microscopy and cryo-scanning electron microscopy (Cryo-SEM). Physical stabilities of Pickering emulsions were evaluated under various pH or ionic strength conditions.
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MATERIALS AND METHODS
Materials. Gelatin granules (type B, G108396, ∼240 g of bloom) and tannic acid (TA, 98% HPLC-grade purity) were purchased from Aladdin Industrial Corp. (Shanghai, China). Glucomannan powders (Konjac, molecular weight of ∼900 kDa) were provided by Konson Co. (Wuhan, China). MCT (medium chain triacylglycerol oil, Neobee@M-5, 66% C8:0 and 32% C10:0) was a gift from Stepan Co. (Northfield, IL) and used as the oil phase. Other analytical-grade chemicals, including NaCl and HCl, were purchased from SigmaAldrich Chemical Co. (St. Louis, MO) and used without further purification. Water (γ = 71.8 ± 0.2 mN m−1 at 25 °C, resistivity of 18.2 MΩ cm) used to prepare solutions was purified through a Milli-Q water purification system (Millipore, Milford, MA). Preparation and Characterization of Gelatin−Glucomannan−Tannic Acid (GGT) Self-Assembled Nanocomplexes. For the preparation of gelatin−glucomannan−tannic acid selfassembled nanocomplexes (GGTNs), the gelatin granules were dispersed at a concentration of 2.0 wt % at 40 °C for 1 h. The 0.2 wt % konjac glucomannan dispersion was obtained by magnetic stirring for 2 h. Tannic acid (20 mg/mL) was dissolved in Milli-Q water with an ultrasound bath for 5 min. Ten microliters of gelatin, gelatin−glucomannan blended solutions [10:1 (w/w) and 5:1 (w/w), respectively] and a tannic acid solution (70 μL) were mixed together while being continuously stirred at 25 °C. Final GGTN aqueous dispersions were created after the pH had been adjusted to 4.88 ± 0.02 and nanosized colloidal complexes formed.21 Detailed compositions of three kinds of GGTN dispersions are listed in Abbreviations Used. Nano ZS (Malvern Instruments) was used to measure the hydrodynamic diameter of GGTNs at 25 °C. The ζ potentials of samples were determined by light scattering upon the application of an
E = dγ /d ln A
(2)
where γ is the interfacial tension and A is the area of the drop. Then one can evaluate the real part (E′) and the imaginary part (E″) of the dilatational interfacial elasticity
E = E′ + iE″
(3)
For an insoluble monolayer, E′ characterizes the elastic behavior of the interface film while E″ is related to the viscous property of interface film. Dilatational elastic modulus is given by 1402
DOI: 10.1021/acs.jafc.6b04146 J. Agric. Food Chem. 2017, 65, 1401−1409
Article
Journal of Agricultural and Food Chemistry
Figure 1. (A) Hydrodynamic diameter distributions, (B) ζ potentials, and (C) TEM images of GGTN0, GGTN1, and GGTN2.
Figure 2. Static contact angles of (A) GGTN0, (B) GGTN1, and (C) GGTN2.
E = (E′2 + E″2 )1/2
The morphology and size distribution of GGTNs were observed by TEM (Figure 1C; the scale bar represents 500 nm). GGTN0, GGTN1, and GGTN2 are shown from left to right, respectively. All the nanocomplexes displayed spherical shapes with different sizes. The sizes of particles were 100−200, 200−400, and 300−400 nm for GGTN0, GGTN1, and GGTN2, respectively, presenting an increasing trend. Sizes observed by TEM were always slightly smaller than that of DLS, resulting from a shrinking effect of TEM sample preparation. Researchers reported polysaccharides had an inhibitory effect on astringency resulting from aggregates assembled by proteins and polyphenols. This effect was ascribed to two mechanisms.25 The first one claimed that polysaccharides participated in molecular self-assembly to form a ternary complex. The second one claimed that polysaccharides might compete with proteins to bind with polyphenol. In the case presented here, when glucomannan was added to those systems, ternary nanocomplexes with increasing particle size were formed. Because of the introduction of most hydroxyl groups, adding glucomannan not only increased the particle size of nanocomplexes but also might change the surface wettability. Thus, the air−water contact angle was monitored to evaluate the surface hydrophobic−hydrophilic properties. The contact angle, conventionally measured at the air, aqueous, and oil interfaces, reflected the wettability of the surface and was calculated by Laplace−Young function. As for air−water or oil− water interfaces, when the contact angle was >90°, it represented a hydrophobic surface. When the contact angle was GGTN2 > GGTN0 > gelatin. That means complex particle-stabilized emulsions are easy to achieve. Influence of GGTN Solution Concentration on Emulsion Stability. The comparison of interfacial tension at equilibrium as a function of complex particle concentration can be observed in Figure 6. With an increase in the concentration of the GGTN solution, the equilibrium tension decreased slightly from ∼18.5 to 17.5 mN·m−1 except for GGTN1. When the concentration reached 0.25 wt %, the tendency in tension was to plateau and was independent of concentration, indicating that was the equilibrium tension at saturation (σeqsat). Among all the GGTN solutions, the GGTN1 solution presented a unique interfacial property, which yielded the lowest σeqsat value, suggesting that GGTN1 complexes were favored to absorb on the oil−water interface and reduce the surface tension. Stable emulsions were achieved when the GGTN concentration was >0.1 wt %. With an increase in GGTN concentration, the droplet size decreased slightly, and the droplet size distribution of emulsions tended to be even. Diameters of GGTN-stabilized emulsions ranged from 80 to 1405
DOI: 10.1021/acs.jafc.6b04146 J. Agric. Food Chem. 2017, 65, 1401−1409
Article
Journal of Agricultural and Food Chemistry
distribution from 20 to 150 μm, but when the φ was 0.6, droplet size tended to be homogeneous in the range of 100− 150 μm. All the freshly prepared Pickering emulsions went through a fast creaming process within the first several hours. The creaming index (CI%), defined as the height of the lower serum (Hs) divided by the height of the entire emulsion (Ht), revealed the creaming instability of emulsions. The rate of creaming mainly depends on differences in density between the oil and aqueous phase, droplet size, and interactions among droplets. Large differences of oil−water density and droplet size would promote creaming. The higher particle concentration, indicating sufficient aggregation on interfacial areas, allowed droplets to connect into a network structure.8 The creaming instability of emulsions stabilized by GGTNs (0.5 wt %) as a function of time with different oil fractions was studied in Figure 10. The rate of creaming increased with a decreasing oil fraction in all emulsions, resulting in a relatively small droplet size. GGTN1and GGTN2-stabilized emulsions effectively inhibited the creaming process in the first half-hour, especially for GGTN1stabilized emulsions. The reason might be the strong contacts between neighboring adsorbed nanocomplexes leading to inhibition of creaming. Influence of pH and Ionic Strength on Emulsion Stability. The stability of Pickering emulsions versus changes in the external conditions has been studied. The influence of pH and ionic strength is shown in Figure 11. The emulsion system was diluted 5-fold with pH buffers or NaCl solutions to yield the final set conditions. After samples had been held for 24 h, digital pictures and microstructures were observed simultaneously. Values of pH settings are basically used to mimic some physical environments, such as pH 1.5 (near that of gastric juice), pH 3.0−4.5 (close to those of many actual food systems), and pH 7.5 (near that of intestinal juice). Microscopic images of droplets under different pH conditions showed that the sizes that decreased slightly with pH were far from those of the original emulsions (nearly pH 5.0−5.5). In fact, those results indicated that GGTN-stabilized emulsions would remain stable under various pH conditions and emulsions were likely to be applied in the delivery of hydrophobic nutraceuticals. Meanwhile, the appearance of emulsion samples did not show any significant changes within the set of salt concentrations. The microscopic images also revealed no obvious changes in oil droplets, but when the salt concentration reached 100 mM, the amount of smaller oil droplets seemed to increase in the GGTN1- and GGTN2-stabilized Pickering emulsions. This phenomenon can be explained by the possibility that fewer surface charges were neutralized by ions at certain salt concentrations, resulting in bridging effects among droplet interfaces and leading to coalescence of emulsions. In summary, three kinds of protein−polyphenol selfassembled nanocomplex-stabilized Pickering emulsions have been investigated. Adding glucomannan to protein−polyphenol systems controlled the particle sizes and surface wettability of self-assembled nanocomplexes. The more glucomannan was added, the larger and more hydrophilic the nanocomplexes that were obtained. Fluorescence microscopy and Cryo-SEM showed that those nanocomplexes or their aggregates were located on the oil−water interfaces to serve as a stabilizer. Dynamic interfacial rheology showed interfacial films at the triglyceride oil−water interface that belonged to elastic films.
Figure 7. Surface dilational viscoelastic modulus of (A) GGTN0, (B) GGTN1, and (C) GGTN2 solutions as a function of dilational frequency.
of E′ presented the same increasing trend as the oscillational frequency, but the E″ values decreased slightly. Influence of Oil Ratio on Emulsion Stability. First, the effect of the oil phase ratio (φ) is depicted in Figure 9. The volume of the emulsified layer was increased when the oil phase ratio increased from 0.1 to 0.6. The concentration of GGTN solutions was set at 0.5 wt %. Stable emulsions were obtained over the whole range of oil phase ratios in this study, but with different creaming behavior (Figure 9a). At the same time, the size and distribution of oil droplets were observed by microscopy (Figure 9b). When the oil phase ratio was
