Eugenol Nanoemulsion Stabilized with Zein and Sodium Caseinate by

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Eugenol Nanoemulsion Stabilized with Zein and Sodium Caseinate by Self-Assembly Lei Wang and Yue Zhang* Department of Food Science and Technology, University of NebraskaLincoln, 1901 North 21st Street, Lincoln, Nebraska 68588, United States ABSTRACT: Eugenol-loaded nanoemulsion by zein and sodium caseinate (NaCas) was prepared without using specific equipment or organic solvents. The deprotonated eugenol in hot alkaline was added to NaCas/zein mixtures with different mass ratios at pH 11.5 and then neutralized to pH 7.0. The nanoemulsions showed a well-defined diameter (around 109−139 nm) and a negative surface potential (from −28.5 to −35.8 mV) with spherical morphology. The entrapment efficiency (EE) of 1% (v/v) eugenol reached 84.24% by 2% (m/v) NaCas/zein at a mass ratio of 1:1. This formulation also showed the narrowest size distribution and extraordinary stability during ambient storage (22 °C) up to 30 days and retained good redispersibility after spray- or freeze-drying. The current study showed a promising clean and low-cost strategy to deliver lipophilic compounds containing the hydroxyl group. KEYWORDS: eugenol, zein, sodium caseinate, self-assembly, nanoemulsion

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INTRODUCTION Essential oils (EOs) are the volatile fraction extracted from various parts of edible and medicinal plants, including buds, barks, flowers, leaves, fruits, and seeds. Many EOs have been known to possess various excellent antibacterial, antioxidant, and antimycotic properties, are classified as generally recognized as safe (GRAS) food additives, and thus are used as natural preservatives (e.g., DMC Base Natural, a commercial preservative) in the food industry.1 2-Methoxy-4-(2-propenyl)phenol (eugenol), a primary component in the clove EO, exhibits a broad spectrum of antifungal and antibacterial activities and, hence, has attracted increasing research interest to replace traditional antimicrobial agents. However, the high volatility and water insolubility have limited its applications as a preservative in the food industry. Emulsification can be an effective approach to improve physicochemical properties of EOs (e.g., increase the water solubility and lower the volatility).2 Nanoemulsions have attracted considerable attention in recent years as a result of their extremely small droplet size, higher kinetic stability, and better bioavailability than those of conventional emulsions.3 Improved optical transparency and antimicrobial activity of EOs, such as carvacrol and eugenol, have been observed after being incorporated into various nanoemulsions.4−6 Generally speaking, the emulsion characteristics, such as the particle size, shape, and releasing mechanism, as well as encapsulation efficiency and stability are governed by the types of emulsifiers and emulsifying techniques.2 On one hand, the high-energy emulsifying techniques, such as high-pressure/speed homogenization, microfluidization, and ultrasonication, have several drawbacks, such as the limit of size reduction and high capital and operating costs that can be a big challenge for scale-up,7 while low-energy methods with no specialized or costly equipment are more appealing for some applications. On the other hand, some high-performance emulsifiers, such as synthetic surfactants (e.g., Tweens and Spans), may have © XXXX American Chemical Society

regulatory considerations, including potential toxicity and safety, that limit their applications in food and pharmaceutical industries.8,9 Therefore, preparing EO nanoemulsions with food-grade biopolymers, such as plant or animal proteins, through a low-energy method can be a promising strategy for the food industry.3 Among food-grade emulsifiers, zein, corn prolamine, is one of the most extensively investigated ingredients to encapsulate hydrophobic compounds, such as curcumin,10,11 thymol,12−14 and fish oil.15,16 In comparison to many animal-source proteins, zein is less expensive and possesses higher hydrophobicity and thermal resistance. In addition, zein has a very special brick-like shape in aqueous phase, thus overcoming the drawback of hydrophilic protein-based delivery systems that may need chemical or physical treatment to harden the nanoparticles to achieve sustained drug release.2,12 Because of these promising characteristics, including biocompatibility, biodegradability, unique hydrophobic property, and relatively slow digestibility, zein can be an ideal vehicle for hydrophobic materials. However, because zein is water-insoluble and its isoelectric point is around 6.8, the utilization of zein nanoparticles in neutral pH is limited and these particles are usually prepared in 70−80% (v/v) alcohol solutions or other organic solvents.17 Although the alcohol or other organic solvents can be removed by anti-solvent precipitation or spray-/freeze-drying, it is more desirable to prepare zein nanoparticles without the use of alcohol and other organic solvents for “green consumption”. Meanwhile, the redispersibility and stability of zein nanoparticles in aqueous systems are still great challenges, limiting their applications as delivery carriers.10,18 Received: Revised: Accepted: Published: A

January 13, 2017 March 17, 2017 March 20, 2017 March 20, 2017 DOI: 10.1021/acs.jafc.7b00194 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

being stirred at 600 rpm. The emulsions were further freeze-dried at −50 °C for 48 h (Freezone, Labconco Corporation, Kansas, MO) or spray-dried using a laboratory-scale spray-dryer (Buchi Mini Spray Dryer, B-290, Switzerland) at a feed rate of 15% and an aspirator setting of 100% with the inlet temperature of 150 °C. The freeze- or spray-dried powders were collected and stored at −20 °C before further study. Particle Size and ζ-Potential. The particle size parameters, including the hydrodynamic diameter (Dh), weight mean diameter (d4,3), and polydispersity index (PDI), and ζ potential at pH 7.0 were measured using a dynamic light-scattering (DLS) Nano-ZS Zetasizer (Malvern Instruments, Worcestershire, U.K.). Before measurement, samples were centrifuged at 5000 rpm for 5 min to remove large insoluble aggregates and then diluted to the same protein concentration (0.1%, w/v) using distilled water (pH 7.0). The volume mean diameter (d4,3) for freshly prepared nanoemulsions with 1% (v/ v) eugenol was calculated using eq 1

Caseins, the major phosphoproteins of mammalian milk, have good emulsifying and stabilizing properties. Sodium caseinate (NaCas), a commercially available casein-rich ingredient, has been commonly used as a food-grade emulsifier in food and beverage industries.18,19 The adsorption of NaCas on zein colloidal particles has recently been studied for the purposes of improving the stability of zein particles.14,20 Zein nanoparticles showed high dispersibility and stability at pH 7.0 after complexation with NaCas by electrostatic and steric stabilization.10,18 In another study, a pH-cycle method, by controlling dissociation at alkaline pH and reassembly of NaCas during acidification back to neutral pH, was reported to fabricate co-assembled zein/NaCas nanoparticles without using organic solvents and specialized equipment.20 Because the alkaline treatment duration was short, no hydrolysis or change of primary structure was observed on both proteins. Because some EOs containing hydroxyl groups (such as eugenol and thymol) can be deprotonated at alkaline pH, in which condition zein is soluble and casein micelles are dissociated, a wellblended solution of three components can be prepared to interact with each other. During neutralization, reassociation of casein micelles may enable the co-assembling of zein with NaCas and entrapment of eugenol into the NaCas/zein complex probably through hydrophobic attraction and potentially electrostatic attraction.20 We hypothesize that this self-assembly method can provide a new solution to incorporate EOs, such as eugenol, into the NaCas/zein complex and, thus, form EO nanoemulsions without alcohol or other organic solvents. Therefore, the objective of this work was to prepare EO nanoemulsions using zein and NaCas as co-emulsifiers through a low-energy self-emulsifying technique. Eugenol was selected as a model compound containing hydroxyl groups in this study, so that this technique can be applied to other EOs, such as thymol and carvacrol. The physical properties including the droplet (particle) size, ζ-potential, and redispersibility of nanoemulsions were characterized.

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d4,3 =

∑ nidi 4 ∑ nidi 3

(1)

where ni is the number of droplets with a diameter of di. Entrapment Efficiency (EE). The EE was determined by subtracting the free oil content from the total oil content. The free oil concentration in the samples was measured according to the method of Luo et al., with modifications.21 After centrifugation, 0.5 mL of the serum phase was gently mixed with 2 mL of petroleum ether for 10 min. After another centrifugation at 3000 rpm for 2 min, 0.1 mL of the upper phase was transferred to a 15 mL tube and the solvent was evaporated in a fume hood. The extracted oil was diluted by 4 mL of ethanol, and the absorbance was measured at 280 nm (Abs280 nm) using an ultraviolet−visible (UV−vis) spectrophotometer (Evolution 201/220, Thermo Fisher Scientific, Inc., Waltham, MA). A standard curve plotting the Abs280 nm value versus eugenol concentration was established. The EE was then calculated using eq 2. EE (%) =

total oil amount − free oil amount × 100% total oil amount

(2)

Surface Hydrophobicity (S0). S0 of individual emulsifiers and their combinations was measured using a fluorescence probe [(8anilinonaphthalene-1-sulfonic acid (ANS)] according to the method of Luo et al., with modifications.22 Briefly, a total of 4 mL of sample with different protein concentrations (0.01−0.1%, w/w) in phosphatebuffered saline (PBS, 10 mM sodium phosphate at pH 7.0) was mixed with 20 μL of 8 mM ANS solution that was previously prepared by dissolving ANS in 10 mM PBS. After the mixtures were vortexed, they were incubated at room temperature (21 °C) in the dark for another 2 h. The fluorescence of the supernatant was measured using a LS55 fluorescence spectrometer (PerkinElmer, Waltham, MA). The emission spectra were recorded from 400 to 600 nm with excitation wavelength set at 350 nm. The slope of fluorescence intensity at 485 nm against the protein concentration after linear regression (R2 > 0.99) was used as an index for S0. Transmission Electron Microscopy (TEM). Freshly prepared nanoemulsions (pH 7.0) were diluted to the appropriate concentration by deionized water (pH 7.0), dropped onto a plasma-treated (glowdischarged) carbon-filmed grid, and then dried in the air. The observations were performed on a JEM-2200FS microscope (JEOL, Ltd., Japan) with an acceleration voltage of 80 kV. Scanning Electron Microscopy (SEM). The morphology of freshly prepared and dried samples was observed using SEM (SU-70, Hitachi, Pleasanton, CA). The fresh emulsions were dripped on the polycarbonate membrane filters (Sterlitech Corporation, Kent, WA) with a pore size of 200 nm, which were mounted on the double-side sticky tape, while the spray- or freeze-dried powders were directly adhered to a conductive carbon tape, followed by drying in a vacuum oven at 3000 Pa at room temperature (21 °C). Then, the samples were mounted onto specimen stubs and coated with a conductive gold layer (99%) was purchased from Sigma-Aldrich (St. Louis, MO). NaCas was purchased from American Casein Company (Burlington, NJ). Eugenol (>98%) was obtained from Acros Organics (Morris Plains, NJ). Other chemicals obtained from Fisher Scientific, Inc. (Pittsburgh, PA) were of analytical grade and used without further purification. Preparation of Stock Solutions. A total of 2 g of zein or NaCas was suspended in 100 mL of double-distilled water at 21 °C. The pH was brought to 11.5 using 3 M NaOH, which was determined previously to be the lowest pH to solubilize zein.20 The aqueous solutions were stirred at 600 rpm for 2 h. Deprotonation of Eugenol. The deprotonation of eugenol was accomplished according to the method of Luo et al., with modifications.21 A total of 1 mL of eugenol was mixed with 9 mL of 3 M NaOH in a glass vial, and then the mixture was heated at 120 °C for 10 min in a glycerol bath to obtain a transparent solution. Preparation of Eugenol Emulsions. The stock solutions of zein and NaCas at 2% (w/v) were mixed at different volume ratios to obtain the aqueous phase with mass percent ratios of 0.5:0.5, 0.5:1, 1:0.5, 1:1, and 2:1 of NaCas/zein. After magnetic stirring at 600 rpm for 30 min, 1 mL of deprotonated eugenol was added to 9 mL of emulsifier aqueous solution to obtain mixtures containing 1% (v/v) eugenol. In another set, different volumes of eugenol were added to 9 mL of zein/NaCas solution at a mass ratio of 1:1 to obtain mixtures with the eugenol concentration ranging from 0.5 to 2% (v/v). Then, the pH of mixtures was acidified to 7.0 using 3 M citric acid while B

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Journal of Agricultural and Food Chemistry Redispersibility of Spray- and Freeze-Dried Nanoemulsions. A total of 30 mg of spray- and freeze-dried sample powders was redispersed in 1 mL of distilled water for 1 h, and the redispersibility was studied by measuring the turbidity, particle size, and ζ potential of the dispersions, with a comparison of the sample before spray-/freezedrying. After centrifugation at 5000 rpm for 10 min, the serum phases were diluted and 100 μL of diluted sample was gently mixed with 5 mL of Coomassie reagent (Thermo Fisher Scientific) at room temperature (21 °C) for 15 min. The absorbance at 595 nm was measured using the above UV−vis spectrophotometer. The protein contents were calculated on the basis of a pre-established standard curve using bovine serum albumin. The redispersibility was then determined by the following equation (eq 3):

redispersibility (%) =

than that of 0.5:1, indicating that a relatively stable zein emulsion with a narrower size distribution could be achieved with a greater amount of NaCas. The intensity and volume mean particle size distributions of nanoemulsions are shown in Figure 2. For the intensity particle size distributions, only the nanoemulsion at a NaCas/zein mass ratio of 1:1 was monomodal, while other nanoemulsions showed the bimodal patterns with a majority of the particles around 100 nm and a smaller population less than 100 nm. The sample with NaCas/ zein at 1:2 showed a noticeable broader particle size distribution than other samples with a second peak rightshifted, which was in accordance with its higher PDI and Dh values. As reported in the previous literature,20,24 the reassociation property and negative charge of casein micelles facilitated the caseins to successfully adsorb on the surface of zein nanoparticles. The multimodal distribution of the sample with NaCas/zein at 1:2 might be due to the fact that the amount of NaCas was not sufficient to saturate the surfaces of zein nanoparticles and stabilize the nanoemulsions. Consequently, the droplets were prone to aggregate with a broader size distribution.25 In comparison, the volume distribution of nanoemulsions at a NaCas/zein mass ratio of ≥1:1 was unimodal, while it showed a more bimodal behavior at a NaCas/zein mass ratio of < 1:1. Hence, the variation of average d4,3 for emulsions was significant, ranging from 50.8 to 140.9 nm. Overall, d4,3 increased with increasing emulsifier concentrations, except samples with NaCas/zein at 0.5:1 or 1:2, indicating that more emulsifiers adsorbed at the interface in a higher concentration. Contrary to the intensity particle size observation, samples with NaCas/zein at 0.5:1 and 1:2 showed much smaller d4,3 than other samples and their volume distribution was dominated by small droplets (Figure 2). The results indicate that nanoemulsions with NaCas/zein at a mass ratio lower than 1:1 were unstable, which were in agreement with the severe precipitation observed in these samples. The ζ potential of eugenol nanoemulsions prepared by NaCas/zein at different mass ratios is also shown in Table 1. The values of freshly prepared nanoemulsions ranged from −28.5 to −36.8 mV. Colloidal particles with a ζ potential magnitude of ≥30 mV could typically be stabilized by repulsive electrostatic interactions, which may have been the case for stable emulsions, as observed in Figure 1.26 The sample with a NaCas/zein mass ratio of 1:0.5 showed a more negative surface charge than that of 0.5:1, which was in accordance with the lower PDI observation. A most negative charge of −36.8 mV was found at a mass ratio of 1:1, indicating that this formulation may exhibit the best stability by preventing the aggregation tendency of zein in the aqueous phase. Overall, these results show that increasing the NaCas content in blends decreased the visible precipitates, induced a smaller particle size with monomodal size distribution, increased the ζ potential magnitude of eugenol emulsions, and hence exhibited a better performance on emulsion stabilization. During storage at ambient conditions up to 7 days, no significant changes of the mean Dh and PDI of samples of NaCas/zein at 0.5:0.5, 1:0.5, and 1:1 were observed (p < 0.05). With regard to NaCas/zein mass ratios of 0.5:1 and 1:2, they exhibited a significant increase in the particle size and PDI after 7 days of storage (p < 0.05). Eugenol EE. The EE and its impact on the particles are vital for the delivery system.27 As shown in Figure 3, the EE value increased with the increase in the mass of total proteins, except the sample with a NaCas/zein mass ratio of 0.5:1. This

protein concentration in the supernatant protein concentration before centrifugation × 100%

(3)

Statistical Analysis. All of the data were recorded in triplicates and reported as the mean ± standard deviation (SD). The statistical analyses were performed using the Statistical Analysis System software 9.4 (SAS Institute, Cary, NC). One-way analysis of variance (ANOVA) was employed to compare the differences between pairs of means using a Tukey’s test.23 The significance level was set at p < 0.05.

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RESULTS AND DISCUSSION Self-Assembly of Eugenol Nanoemulsion. The appearance of 1% (v/v) eugenol nanoemulsions prepared by NaCas and zein with different mass ratios is recorded (Figure 1). All

Figure 1. Appearance of 1% (v/v) eugenol encapsulated in NaCas/ zein with mass percent ratios of (A) 0.5:0.5, (B) 0.5:1, (C) 1:0.5, (D) 1:1, and (E) 1:2 at pH 7.0.

nanoemulsions were dispersed in water in a homogeneous manner, with a uniform milky appearance, as shown in panels A−E of Figure 1. However, the emulsions with NaCas/zein at mass ratios of 0.5:1 and 1:2 had visible precipitates probably as a result of the lack of sufficient NaCas to stabilize zein. The storage stability of nanoemulsions was evaluated by measuring Dh, PDI, and ζ potential, as shown in Table 1. The mean Dh of all of the freshly prepared samples were in the range of 109− 140 nm. The PDI for all samples were less than 0.4, showing a relatively narrow distribution of particle sizes. The nanoemulsion prepared by NaCas/zein at 0.5:1 exhibited the smallest Dh of 109.65 nm, while the largest Dh (139.4 nm) was obtained with NaCas/zein at 1:2. The particle size increased with the increase of the emulsifier, except the sample with NaCas/zein at 0.5:1. The relatively low value of Dh of this sample may be attributed to the fact that large aggregates of zein particles were removed from this dispersion during acidification and the actual amount of zein in remained dispersion may be lower. Using emulsifiers at the same solid content, a steadier storage stability with a lower PDI was observed in the sample with a NaCas/zein mass ratio of 1:0.5 C

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Table 1. Dh, PDI, and ζ-Potential of 1% (v/v) Eugenol Emulsions with Different Mass Ratios of NaCas and Zein during 7 Days of Storage at 21 °Ca mass ratio (NaCas/zein) parameter size

PDI

ζ potential

day 0 1 3 5 7 0 1 3 5 7 0 1 3 5 7

0.5:0.5 115.2 113.5 114.1 116.8 114.6 0.209 0.213 0.209 0.211 0.215 −28.5 −28.1 −28.2 −28.2 −28.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7.53 a 9.32 a 8.10 a 3.28 a 8.58 a 0.017 a 0.006 a 0.016 a 0.019 a 0.006 a 2.51 a 2.66 a 2.58 a 2.72 a 0.65 a

0.5:1 109.65 115.7 124.8 130.7 136.6 0.285 0.283 0.323 0.357 0.395 −30.1 −31.0 −29.1 −31.2 −30.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1:0.5

12.7 c 6.28 bc 10.1 b 9.46 b 7.41 a 0.017 d 0.049 d 0.033 c 0.054 b 0.036 a 3.52 b 2.06 a 4.16 bc 1.08 a 2.95 b

122.7 118.6 126.2 123.4 120.5 0.228 0.227 0.233 0.218 0.226 −35.8 −35.6 −34.5 −36.0 −35.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

9.36 a 10.12 b 4.25 a 4.59 a 8.79b a 0.039 a 0.061 a 0.017 a 0.071 ab 0.015 a 3.50 a 4.40 a 3.10 ab 1.44 a 2.29 a

1:1 116.4 110.6 108.7 109.6 112.0 0.212 0.208 0.213 0.209 0.205 −36.8 −36.6 −35.9 −36.0 −35.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1:2

7.22 a 10.82 b 8.9b a 5.82 a 8.15 a 0.016 a 0.018 a 0.009 a 0.005 a 0.009 a 2.95 a 4.18 a 2.06 b 3.21 b 3.37 b

139.4 140.8 147.4 154.7 158.4 0.390 0.390 0.408 0.419 0.455 −30.7 −31.6 −30.4 −30.0 −30.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.05 c 10.30 c 8.60 b 16.56 a 13.52 a 0.016 c 0.028 c 0.021 b 0.015 b 0.011 a 1.95 b 1.39 a 2.16 b 3.37 cb 2.87 b

a Values are expressed as the mean and SD of at least three measurements. Letters (a−d) mean significant (p < 0.05) difference within the same column.

Figure 2. Particle size distribution of 1% (v/v) eugenol nanoemulsions with NaCas/zein mass percent ratios of (A) 0.5:0.5, (B) 0.5:1, (C) 1:0.5, (D) 1:1, and (E) 1:2 at pH 7.0.

values varied from 45.8 to 90.9% depending upon the eugenol concentrations (Figure 4A). The highest EE, reaching 90.9%, was obtained when 0.5% (v/v) eugenol was emulsified. The reason for the lower EE with an increased amount of eugenol could be a more porous polymer matrix structure filled by eugenol, through which eugenol could easily escape to the outer phase.29 Dh increased significantly with an increased eugenol concentration (Figure 4B) and ζ potential magnitude (p < 0.05). A similar trend of an increased droplet diameter with the increased oil concentration in emulsions was also reported previously that may be attributed to the deficiency of the protein.11 When the concentration of oil in the emulsion increased, the protein could not completely cover the surface of the emulsion, and thus, the collision of molecules led to oil

treatment had the lowest EE (46.6%), possibly as a result of its relatively lower protein content after acidification, as discussed earlier. The colloidal particles exhibited the highest EE value of 84.24% at a NaCas/zein mass ratio of 1:1. This EE value was comparable to or higher than those of zein-stabilized emulsions using an aqueous ethanol solution (EE = 16%) or high-speed homogenization (EE of around 65−75%).12,14,28 The emulsion prepared by NaCas/zein with a mass percent ratio of 1:1 showed the best stability and highest EE value and, hence, was used for the rest of this study. Influence of the Oil-Phase Concentration. The influence of different concentrations of eugenol on the EE value, Dh, and ζ-potential of nanoemulsion with a NaCas/zein mass ratio of 1:1 is shown in panels A−C of Figure 4. The EE D

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Figure 3. EE of 1% (v/v) eugenol nanoemulsions with NaCas/zein with mass percent ratios of 0.5:0.5, 0.5:1, 1:0.5, and 1:1 (pH 7.0). Different letters above bars indicate significant differences (p < 0.05).

droplet aggregation, which eventually brought about a larger droplet size. By loading eugenol, the ζ-potential of zein/NaCas nanoparticles became more negative but was increased from −35 to −29 mV with an increased amount of eugenol (Figure 4C), indicating that the electrostatic repulsion between droplets became less effective to prevent the particles from coming together and flocculating.30 Dh and ζ potential of eugenol emulsions after storage for 30 days are also shown. Overall, freshly prepared nanoemulsions exhibited Dh in the range of 100−170 nm. The average Dh of NaCas/zein nanoparticles without eugenol as well as the samples with 0.5 and 1.0% eugenol encapsulated remained stable with a slight increase (p > 0.05) during 30 days of storage at room temperature (Figure 4B), indicating that the NaCas-stabilized zein emulsion had excellent stability at neutral pH. However, the stability decreased with a high eugenol content at 1.5 and 2.0% because Dh increased significantly (p < 0.05) after 30 days. The increase in the hydrodynamic diameter with an increasing oil content could be attributed to the deficiency of surface-active agents (proteins), as described above. This phenomenon has also been reported by others that an increasing percentage of sunflower oil in the emulsions resulted in a larger mean droplet diameter for the same conditions.31 In the emulsification process, the available proteins on the surface of droplets decrease along with the increasing oil content, thus favoring oil droplet coalescence and, consequently, increasing Dh.31 S0. S0 is an index for the protein to adsorb to the oil side of the interface that greatly influences its emulsifying capacity.32,33 To better understand potential synergistic effects between NaCas and zein, the S0 values of the blends of NaCas and zein at different mass ratios were measured (Figure 5). The highest S0 value was obtained for the sample of zein alone that was dispersed in PBS buffer where a high amount of hydrophobic surface was available for the fluorescence probe (ANS). However, the fluorescence intensity of ANS decreased significantly when zein was dissolved in water solution as a result of the tendency of zein to aggregation (data not shown). In comparison of the nanoparticles with the same solid content in the water phase, S0 increased with an increase of zein in the NaCas/zein blends (988.2 of 0.5:1 versus 526.1.1 of 1:0.5). A higher S0 value indicated the accessibility of the probe ANS to hydrophobic zein, which may result from the increased surface area of NaCas/zein nanoparticles.20 As reported earlier, more exposed hydrophobic residues would promote the protein arrangements with better hydrophilic−lipophilic balances to

Figure 4. (A) EE, (B) droplet dimension, and (C) ζ-potential of nanoemulsions prepared with 1% NaCas, 1% zein, and 0−2% eugenol (pH 7.0) at five levels: 0% (□), 0.5% (■), 1% (△), 1.5% (▼), and 2% (◇). Means marked with asterisks indicate significant difference (p < 0.05) within emulsion type with its previous storage time.

interact at the water−oil interface, thereby giving more stable emulsions.35 Therefore, the combination of NaCas with zein may promote the entanglement of zein across the oil−water interface and enhance the emulsifying property.34 However, with more hydrophobic surface exposed, the aggregation process may occur and decrease the stability of particles. As evidence, the sample with a NaCas/zein mass ratio of 0.5:1 exhibited the highest S0 among these blends but showed relatively lower EE (Figure 3) and broader PDI (Table 1) than NaCas/zein at a ratio of 1:1. Morphology. The TEM images could directly show the interior morphology of liquid core structures of emulsions.36 Figure 6 showed the TEM images of freshly prepared E

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Figure 5. S0 of NaCas/zein dispersions with different mass ratios. Different letters above bars show a significant difference (p < 0.05).

Figure 6. TEM images of freshly prepared NaCas/zein dispersions at 1:1 (A) without eugenol and (B) with 1.0% (v/v) eugenol. The images were taken at 20000× magnification.

Figure 7. SEM images of freshly prepared emulsion with NaCas/zein at 1:1 and eugenol at (a) 0%, (B) 0.5%, (C) 1.0%, (D) 1.5%, and (E) 2.0%. The images were taken at 20000× magnification.

nanoemulsions with NaCas/zein at a mass ratio of 1:1 with or without eugenol. All samples showed spherical structures within the nanoscale that a few elongated nanowires deposited on the surface or cross-linked the particles, showing that the hydrophilic caseins were present around hydrophobic zein nanospheres and stabilized them. More particles with a larger size can be observed with the encapsulation of eugenol, which was in accordance with the trend of Dh. The absence of aggregation demonstrated that these emulsions were welldispersed in the aqueous phase. This observation also confirmed the successful self-emulsification of eugenol emulsions during the subsequent neutralization to pH 7.0. The size of emulsions obtained from TEM was somewhat smaller than that determined by DLS because TEM measures size in the dry state, while DLS measures the hydrodynamic size in the aqueous phase. The morphology of NaCas/zein nanoparticles with different concentrations of eugenol was investigated by SEM (Figure 7). All of the samples showed a spherical shape and smooth surface. A higher eugenol content significantly increased the size of nanoparticles (panels D and E of Figure 7), further confirming the Dh and TEM observations. However, some particles with a small size can also be seen in emulsion samples with 2% eugenol. The morphology of these eugenol-loaded NaCas/zein particles by our method is similar to those prepared using alcohol solution with liquid−liquid dispersion methods.14,37 Because eugenol is hydrophobic and believed to be encapsulated into the core of zein particles via strong hydrophobic interactions, a smooth and compact structure could be formed upon the phase separation.14 In our case, the reprotonation of ionized eugenol during neutralization enables eugenol to interact with the zein protein and be incorporated into the NaCas/zein spheres by selfassembly. This mechanism is discussed in a later section.

Redispersibility of Eugenol-Loaded Nanocapsules. To be incorporated into food products more easily and extend the shelf life, liquid nanoemulsions can be transformed into dry powders, of which good redispersibility is demanded for many food applications.38 The redispersibility and morphology of spray- and freeze-drying powders were characterized by DLS and SEM, as shown in Table 2 and Figure 8. Both powders were easy to completely redisperse in water up to 30 mg/mL of solid contents after gentle stirring, with a homogeneous translucent appearance at 15 mg/mL (Figure 8). The PDI of both dried samples was around 0.22 with low turbidity, indicating an excellent stability of the nanoemulsions against dehydration. The particle characteristics of the redispersed emulsions were fairly similar to those of freshly prepared emulsions that only spray-drying increased Dh and PDI slightly. The solubility of free eugenol in water at room temperature was 1.35 mg/mL.39 After encapsulation, eugenol showed significantly enhanced solubility (8.92 mg/mL), as shown in Table 2. There is a slight decrease in the eugenol content in redispersions, attributed mainly to the loss of eugenol in the spray- or freeze-dried process. This result agreed with previous studies.40,41 The freeze-dried powder had a slighter Dh, narrower size distribution, and higher eugenol content than those by spray-drying, which provided a better performance of redispersed emulsion. Figure 8 showed the SEM photographs of the encapsulated powders containing 1% eugenol after sprayand freeze-drying. The eugenol encapsulated powders prepared by spray-drying showed hollow-wall structures with collapsed, red-blood-cell-shaped, and smooth surfaces. The size of capsules was also heterogeneous. A drastic change in the morphology and shape of particles was observed in freeze-dried powders that a flake-like structure formed by small agglomF

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Journal of Agricultural and Food Chemistry Table 2. Characterization of Fresh Eugenol Nanoemulsion and the Redispersions after Spray- and Freeze-Dryinga treatment

absorbance at 600 nm

Dh (nm)

PDI

ζ-potential (mV)

dispersed eugenol (mg/mL)

freshly prepared spray-dried freeze-dried

0.26 a 0.22 b 0.27 a

123.5 b 137.2 a 127.2 ab

0.220 b 0.227 a 0.219 b

−33.5 a −33.2 a −33.9 a

8.92 a 7.23 c 8.15 b

a

Nanoemulsion was prepared using 1% (v/v) eugenol with 1:1 NaCas/zein. With and without drying, the dispersions were diluted to 0.3% (w/w) solid mass before measurement. For the same parameter, numbers sharing different letters (a−c) are significantly different (p < 0.05).

Figure 9. Schematic illustration for encapsulation of eugenol by selfassembly of NaCas and zein.

providing a combination of electrostatic repulsion and steric stabilization forces against zein aggregation.43,44 As a consequence, the repulsive electrostatic and steric interactions provided by hydrophilic NaCas would prevent eugenol-loaded zein colloidal particles from aggregation or precipitation, which is evidenced by the observation of stripped NaCas around zein nanospheres in the TEM images (Figure 6). In this study, eugenol-encapsulated nanoparticles were produced using a self-emulsification process with naturally occurring emulsifiers NaCas and zein. The self-emulsification was performed by deprotonation and neutralization procedures without organic solvents and specific equipment. Eugenol nanoemulsions with NaCas/zein at a mass ratio of 1:1 showed the highest EE with a small average diameter and excellent stability during 30 days of storage at ambient conditions and could be used directly with translucent appearance. The sprayand freeze-dried nanocapsules exhibited excellent redispersibility without significant changes on the physical properties of nanoemulsion. This study suggested that a low-energy selfassembly method could potentially be used to prepare zeinbased delivery systems for EOs and other bioactive compounds in food, pharmaceutical, and agricultural applications.

Figure 8. SEM image of (A) spray-dried and (B) freeze-dried powders prepared using 1% (v/v) eugenol with 1:1 NaCas/zein. (Inset) Appearance of rehydrated nanoemulsions at 10 mg/mL (total solid mass).

erated spheres, which was also observed by Quispe-Condori et al.42 Thus, this study demonstrated the potential of dehydrating the NaCas/zein nanoemulsions for utilization as powdered functional ingredients in foods and beverages. Proposed Mechanism for Eugenol Nanoemulsion Formation. The self-assembly mechanism of eugenol nanoemulsion stabilized with NaCas and zein is proposed in Figure 9. At pH 11.5, zein is negatively charged and becomes soluble, while casein micelles are dissociated. The interactions between these biopolymers allow for the inner diffusion of zein in the opened casein network. The addition of deprotonated eugenol into casein/zein dispersion results in a well-blended aqueous phase containing these three components. When the alkaline dispersion is gradually neutralized to pH 7.0, both zein and eugenol become protonated and progressively lose their solubility but the hydrophobic interaction between them allows eugenol to be encapsulated into hydrophobic regions of the zein protein. Meanwhile, caseins around zein reassemble and encompass insoluble zein gradually during the acidification,

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 402-472-2766. Fax: 402-472-1693. E-mail: yue. [email protected]. ORCID

Yue Zhang: 0000-0002-6140-3757 G

DOI: 10.1021/acs.jafc.7b00194 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Funding

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This work is supported by startup funding from the University of NebraskaLincoln. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. You Zhou from the Center for Biotechnology at the University of NebraskaLincoln for his assistance in TEM and SEM measurements.

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DOI: 10.1021/acs.jafc.7b00194 J. Agric. Food Chem. XXXX, XXX, XXX−XXX