Curcumin encapsulation and protection based on lysozyme nanoparticles

Abstract Curcumin possesses antioxidant, anti‐inflammatory, and other properties. However, this compound exhibits low bioavailability because of its poor solubility and stability. In this paper, lysozyme nanoparticles were fabricated through solvent evaporation, and then, the solubilization and protection capability of curcumin were investigated. Lysozyme nanoparticles were characterized by dynamic light scattering technique, atomic force microscopy, transmission electron microscopy, and Fourier transform infrared spectroscopy. The load capacity and stability in thermal environment were further explored. Results showed that the lysozyme nanoparticle displayed a spherical structure (127.9 ± 2.12 nm) with favorable distribution. The solubility of curcumin can increase to 22 μg/mL. After encapsulation by lysozyme nanoparticles, the retentive curcumin can reach up to 67.9% and 30.25% at 25°C and 50°C, respectively, significantly higher than that of free curcumin. Meanwhile, experiments on DPPH free radicals indicated the curcumin loaded by lysozyme nanoparticle possessed higher free radical scavenging activity than that of free curcumin with same treatments. The results confirmed that lysozyme nanoparticles exhibit potential applications in solubilizing and protecting the environment‐sensitive hydrophobic functional components.

functional components, especially for the compounds being sensitive to the environment, such as Cur and folic acid (Das, Kasoju, & Bora, 2010;Sneharani, Karakkat, Singh, & Rao, 2010). A carrageenan/lysozyme soluble complex was prepared by one-step self-assembly and was found to be a suitable vehicle for solubilizing and protecting curcumin in heating and ultraviolet radiation environment (Xu et al., 2014).
In this paper, lysozyme nanoparticles (Ly NPs) were prepared by desolvation method. Ly NPs were subsequently characterized through particle size and morphology. Therefore, the encapsulation and protection of Cur were explored, and the antioxidant activity was further verified using DPPH. This endeavor attempted to provide a simple and feasible strategy for solubilization and protection of the hydrophobic functional components.

| Materials
Curcumin (95.0% purity), as well as lysozyme (Ly, 14.3 kDa) from chicken egg white, was purchased from Sinopharm Chemical Reagent Co., Ltd. Other chemicals were of reagent grade and used without purification. All solutions used in the experiments were prepared using ultrapure water through a Millipore (Millipore) Milli-Q water purification system.

| Preparation of Ly NPs
Ly NPs were prepared by desolvation (Jahanban-Esfahlan, Dastmalchi, & Davaran, 2016). Ly was dissolved in purified water with gentle magnetic stirring for 2 hr at room temperature at the concentration of 1.0 mg/mL. Ethanol was added to Ly solutions at the volume ratio of 15:5 with further stirring for 1 hr. After becoming turbid, the solution was incubated at 80°C for 15 min. Subsequently, Ly NPs were prepared by removing ethanol through rotary evaporation at 30°C under vacuum, and the NPs were stored at 4°C for further use.

| Characterization of Ly NPs
The particle size and zeta potential (ζ) of Ly NPs were measured with Nano-ZS90 at 25°C (Melvin Instruments) according to Xu (Xu, Ge, et al., 2018). The Fourier transform infrared (FT-IR) spectra were recorded using a Nicolet Nexus 470 spectrometer with 32 scans and resolution of 4 cm −1 in the range of 400-4000 cm −1 . The morphology of the complex was investigated with a JEOL transmission electron microscope (TEM) Hitachi). During the TEM test, a drop of Ly NPs solution was dispensed directly onto a carbon-coated copper grid and allowed to dry spontaneously.
Morphology of the particles was determined using atomic force microscopy (AFM) (Wei et al., 2015). Tapping mode measurements were performed in air using a Dimension Icon with a Nanoscope IV controller (Bruker Corporation) and silicon cantilevers with tetrahedral tips (OMCL-AC160TS-Olympus) with a nominative force constant of 42 Nm −1 and a resonance frequency of around 300 kHz. For sample preparation, undiluted nanosuspensions were dropped onto a freshly cleaved mica surface (Plano GmbH). After 5 min, the liquid was removed with a tissue and the samples were completely dried before imaging.

| Cur protection by Ly NPs
To evaluate the effect of encapsulation on the stability of Cur against heat treatment, Cur retention rate was comparatively studied. Free Cur solution and Cur-loaded NPs solution at 10 μg/mL (in water) shared 25°C and 50°C treatment for 30 min. Free Cur was prepared equally to the encapsulated Cur in which water was replaced by the Ly NPs solution. For the Cur-encapsulated Ly NPs, ethanol was added equally, stirred for 4 hr, and then evaporated overnight at 40°C under vacuum (Kumar & Ahuja, 2013). The retention rate of Cur was calculated through the ratio of absorbance at 428 nm.  Figure 2 shows that the average particle size of the prepared Ly

| RE SULTS AND D ISCUSS I ON
NPs was 127.9 ± 2.12 nm. The size distribution index (PDI) of Ly NPs was 0.29 ± 0.03. The microscopic morphology of Ly NPs was observed through atomic force microscopy (AFM) and TEM ( Figure 3). AFM and TEM results showed that Ly NPs displayed elliptical appearance with a diameter probably around 90 nm and good dispersivity. The measured particle size was smaller than the hydrate particle size.
Cur is recognized as a physiological and pharmacological functional nutritional component. Figure 4 illustrates that the hydrophobic  For free Cur, the degradation behavior with different rates occurred at 25°C and 50°C. The survival rates were 58.6% and 15.4% after 30 min of treatment. The heat-induced Cur degradation behavior has been confirmed by other reports (Ko, Chang, Wang, Wang, & Hsieh, 2015;Refat, 2013). Figure 7 shows that in all cases the antioxidant activity of Cur decreased after heat treatment, indicating its sensibility in thermal environment (Paramera, Konteles, & Karathanos, 2011). The free Cur exhibited high radical scavenging activity at 56.3% and decreased to 50% after heat treatment at 50°C for 30 min. The DPPH radical scavenging activity of packaged Cur increased to 71.9% and 76.5% after treatment at 25°C and 50°C for 30 min, respectively.

| D ISCUSS I ON
Ly NPs displayed a single favorable peak distribution with the PDI less than 0.3 and good dispersivity of the prepared Ly NPs (Zhu et al., 2013). The size distribution of Ly NPs was resulted from swelling behavior in the water environment and dehydration during drying. The size of Ly NPs became smaller as measured by TEM. This phenomenon was also widely observed in other similar studies .
Cur offers potential applications in the fields of functional food and medicine, and has attracted considerable attention and research in recent years. However, its low water solubility and stability result in low bioavailability, which notably limits its wide application. Entrapment of Cur based on Ly NPs indicated that 72% (7.3 ± 0.13 μg/mL, 5.07 × 10 −5 mol/L) of Cur was loaded in Ly NPs.

Solubility improved by approximately 664 times compared with that
in the water (11 ng/mL) (Kaminaga et al., 2003). The improvement in Cur water solubility was an order of magnitude higher than those in previous reports based on beta-casein micelles and hydrophobic modified starch (7.5 × 10 −5 mol/L, 1 × 10 −5 mol/L) (Letchford & Burt, 2007;Yu & Huang, 2010). This phenomenon may be induced by the exposure of the hydrophobic group in the hydrophobic cavity of F I G U R E 5 TEM of free Ly NPs (a) and Cur-loaded Ly NPs (b NPs. Ly NPs were difficult to aggregate and exhibited improved elliptic morphology. FT-IR further illustrated that the structure of Ly was partially damaged. These results indicated that Cur was loaded driven by thermodynamics and self-assembled by physical interaction. This phenomenon was also confirmed by Cur-encapsulated zein nanoparticle, which indicates that hydrophobic interaction was the main force between the Cur and Ly NPs .
Temperature and other factors affect the structure and activity of Cur. However, heating is a conventional technique in food processing, especially for sterilization. Figure 8 shows that the Ly

ACK N OWLED G EM ENT
This work was financially supported by the Young Backbone Teachers Program (Grant No. 2018GGJS-13) and Nanhu Scholars Program for Young Scholars of XYNU.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.