Preparation and characterization of gamma oryzanol loaded zein nanoparticles and its improved stability

Abstract Gamma oryzanol (GO), a bioactive ingredient found in rice bran oil, performs a variety of biological effects such as antioxidant activity, reduction of total cholesterol, anti‐inflammation, and antidiabetes. However, GO is water‐insoluble and normally degrades through oxidation. Thus a nano‐encapsulation technique was investigated to improve its stability and quality. In this research, gamma oryzanol was successfully encapsulated into zein nanoparticles. The fabrication parameters including pH, zein concentration (0.3, 0.4, and 0.5% w/v), and % GO loading (30, 40, and 50% by weight) were investigated. Particle size, zeta potential, yield, encapsulation efficiency and the stability or GO retention during the storage were determined. The morphology of gamma oryzanol loaded zein nanoparticles (GOZNs) was observed by scanning electron micrographs and transmission electron microscope. The increase of zein concentration and % GO loading resulted to an increase of yield, encapsulation efficiency, and particle size. The particle size of the GOZNs ranged from 93.24–350.93, and 144.13–833.27, and 145.27–993.13 nm for each zein concentration with 3 loading levels, respectively. Nano‐encapsulation exhibited higher % GO retention compared with nonencapsulated GO during 60 days storage both at 4°C and −18°C. In vitro study indicated the sustained release of GO in the simulated gastric fluid followed by simulated intestinal fluid. This finding indicated a high potential for the application of insoluble GO with improved stability by encapsulation with the hydrophobic zein protein.


| INTRODUC TI ON
Rice is a significant food resource and widely cultivated in many parts of the world. Thailand is one of the world leaders for rice exporter.
Rice bran and germ, by-products from the milling process into white rice, also provide beneficial nutrition such as vitamins B, and E, beta-carotene, anthocyanin, gamma oryzanol (GO), and gamma-aminobutyric acid (GABA). GO can be extracted as a mixture of ferulic acid (4-hydroxy-3-methoxy cinnamic acid) esters with phytosterols and triterpene alcohols (Kim et al., 2013). The ferulic acid ester in rice is well known for its biological effects, including anti-inflammation, antidiabetes, antiaging, anticholesterol actions, and relieving menopausal symptoms (Khalid et al., 2017). GO is a potent inhibitor of iron-driven hydroxyl radical formation and associated with antioxidant activity in stabilizing lipids (Suh et al., 2007). However, GO is a hydrophobic, water-insoluble compound with low absorption and poor bioavailability (Kim et al., 2013) which limit its application. The enhancement of its solubility would widen the application of GO in food, cosmetics, and related products.

Encapsulation has been proposed as an effective technique
for maintaining the quality and stability of active food ingredients (Padua & Wang, 2009) against oxidation and provide better solubility (Raharjo et al., 2019). The small particle size could help the delivery of the bioactive compounds within the body. Nanotechnology indicates a high potential for the development of functional foods, without affecting consumer perception and improving the uptake of individual components. Recently, there are considerable interests in developing high-performance delivery vehicles to protect bioactive compounds, improve bioactive compound bioavailability for improving drug delivery to the target, and enhancing the stability of active chemicals for drug formulation (Huang et al., 2020).
Various techniques were used for the nano-encapsulation of bioactive compounds such as spray drying and coacervation, including solid-lipid nanoparticle, and liquid-liquid dispersion method used for encapsulation of bioactive compounds by zein protein (Zhong & Jin, 2009). For microencapsulation of GO, native rice starch and pregelatinized rice starch were used using spray drying (Moongngarm et al., 2016), while Khalid et al.(2017) used microchannel emulsification with medium-chain triglyceride oil to encap the GO. The particle sizes obtained ranged around 5-60 µm and 26-28 µm, respectively. However, it is wellknown that reducing the particle size of encapsulated particle to nano-size would improve the uptake of encapsulated bioactive compound in cells (Luo et al., 2011).
Zein is the prolamine protein from corn endosperm and a significant by-product in the corn starch industry and generally recognized as safe by the US Food Drug Administration. The current production of corn starch sources in Thailand is estimated to be 2.26 million tons (Win, 2017). Zein has several properties allowing it to self-assemble into different structures such as film, sponges, and spheres (Wang & Padua, 2010). In the pharmaceutical and food areas, zein shows a high potential for application as a biomaterial for the nano-encapsulation of bioactive compounds with the ability to provide moisture, an oxygen barrier, and high thermal resistance (Huang et al., 2020).
Zein can assist the control release of encapsulated hydrophobic compound such as polyphenols, vitamins, and omega-three fatty acids into nonaqueous environment and prolong its shelf life (Donsì et al., 2017;Wu et al., 2012).
Though zein nanoparticles have been prepared and characterized for over a decade, to the best of our knowledge, there has been no report on the use of zein for nano-encapsulation of GO. In addition, using both zein and GO would add value to the abundant by-products from corn and rice, which contain highly functional ingredients.
In present study, GO loaded zein nanoparticles (GOZNs) were fabricated and characterized in order to improve GO dispersibility and stability during a long storage. The effects of zein concentration, and % GO loading on particle size, zeta potential, encapsulation yield, and efficiency were determined. Long-term storage stability of GO in the nanoparticles was measured during 60 days. In vitro release of GO from the nanoparticles was measured. The nanoparticle structure was also examined.

| Materials
GO was purchased from Tsuno Rice Fine Chemicals Co., Ltd.

| Preparation of ZNs: Effect of pH and zein concentration
A liquid-liquid dispersion was modified from the method used by Luo et al., (2013). The effect of the surfactant ratio between tween 80 and lecithin on the particle size and zeta potential of the particles obtained was studied (data not shown), and the ratio at 1:2 (w/w) was selected. pH stability of the zein suspension was studied. The surfactant was dissolved at 0.025% (w/v) in 0.1 M citrate solution, and the pH was adjusted to pH 5, 7, 7.4, 8, and 9 to evaluate the pH stability of the ZN dispersion. Twenty ml of 0.4% zein solution (w/v) in 85% ethanol was mixed with the surfactant solution (60 ml) to prepare 0.1% (w/v) zein dispersion at a various pH level and stirred using a homogenizer (Ultra-Turrax T25, IKA, Germany) operated at 15,000 rpm for 10 min in an ice bath. The ethanol in the dispersion was removed using a vacuum rotary evaporator and centrifuged.
The particle size and zeta potential of freshly prepared zein nanoparticles (ZNs) and those of 24 hr storage at 25°C ± 2°C were measured.
In order to increase the yield of ZNs and GOZNs, the zein concentration in the dispersion was increased from 0.1% to 0.25, 0.5, 0.6, and 0.7% (w/v) with pH 8 following the above method. The dispersions were stored at room temperature (25°C ± 1°C) for 24 hr and observed for their stability.

| Preparation of GOZN with different zein concentrations and % GO loading
To study the effect of zein concentrations and % GO loading on the properties of the nanoparticles obtained, GOZNs were fabricated as follows: GO was dissolved in 1% isopropanol (w/v) at room temperature and added dropwise into 20 ml of zein solution in ethanol using a magnetic stirrer at 450 rpm for 10 min. Then, the sample was added to 60 ml of 0.1 M citrate solution containing the surfactant (tween 80/lecithin at 0.025%, w/v), and the final concentration of zein dispersion was 0.3, 0.4, and 0.5% (w/v), respectively (with pH of the aqueous phase between 8 and 9). The mixture was homogenized and evaporated as described above. Three % levels of GO loading at 30, 40, and 50% of zein (w/w) were studied. Each condition was

| Particle size and Zeta potential
Particle size and distribution of the nanoparticles, as well as polydispersity index (PDI), were measured by dynamic light scattering (DLS) using a DelsaTM nanoparticle analyzer (Beckman Coulter, Fullerton, CA). Surface charges of different samples were measured using a Laser Doppler Velocimeter (Zetasizer Nano ZS90, Malvern, UK) with a folding capillary cuvette. Zeta potential of the samples was converted from the measured electrophoretic mobility.

| Colloidal stability of ZNs and GOZNs
After preparation for GOZNs, 10 ml of each dispersion was poured into a glass bottle and sealed with a plastic cap and stored overnight at 25°C ± 1°C. The stability of the dispersion prepared at different pH and from different zein concentration was observed for changes in particle size distribution which could result to phase separation after 24 hr.

| Encapsulation efficiency and yield
The encapsulation efficiency (EE) of GO in GOZNs was defined as the GO content encapsulated in ZNs following Sakulkhu et al., (2007).

| Long-term storage ability of GO
The dried GOZNs from 0.3/30 (particle size 127 nm) and free GO were sealed in the glass vial (20 ml) and stored at 4°C and −18°C in the refrigerator for 60 days. The samples were taken out every 15 days to determine for the retention amount of GO by HPLC following the methods described above.

| In vitro release of GO from GOZNs and free GO
The GO release profile of GOZNs and remaining free GO in SGI tract containing enzymes was evaluated following Luo et al. (2011). In vitro release was studied with 10 mg of GOZNs and remaining free GO in simulated gastric fluid (SGF) for 0.5 hr followed by simulated intestinal fluid (SIF) for 6 hr. The samples were first mixed in 30 ml SGF pH 1.2 with 0.1% pepsin (w/v) and incubated in a shaking water bath (37 °C, 100 rpm). The digestion was stopped by raising pH to 7.5 (1 M NaOH) and centrifuged (9,000 g, 50 min) to separate the supernatant. The amount of GO from GOZNs released, and remaining free GO was determined by HPLC. Then, 30 ml SIF pH 6.8 with 1% pancreatin (w/v) was added to the precipitate and incubated for 6 hr (37 °C, 100 rpm). The GO release medium and remaining free GO (5 ml) were collected and analyzed for GO content at predetermined times (1, 2, 4, and 6 hr).

| Statistical analysis
The result is shown as the mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to analyze the statistical significance using SPSS 22.0 for Windows (SPSS Inc., Chicago, III, USA). The data were subjected to ANOVA, and a comparison of means was carried out by Duncan's Multiple Range Test (DMRT). Table 1 shows that the particle size (118-129 nm) of ZNs prepared at pH 7.4 and 8 was not significantly different after the preparation and also after 30 days storage. However, a minor change of their zeta potential was observed after the storage. An increase of pH to 9 resulted to significantly smaller ZNs at 58.0 ± 2.0 nm which changed to larger particle (87.9 ± 0.9 nm) after 30 days. Protein coagulation occurred rapidly after the mixing at pH 5 and precipitation of the zein particles was observed after some hours with pH 7.

| Preparation of ZNs: effect of pH and zein concentration
Zein protein contains roughly hydrophobic and hydrophilic amino acid residues. Zein was denatured at pH 5 and 7 which closed to its isoelectric point (pH 6.8). Shukla and Cheryan (2001) reported that zein is rich in glutamic acid (26%) and can be dissolved well in alkaline solution. Deprotonated carboxylic groups in glutamate occurs when pH of the aqueous phase increased to a basic condition, resulting in a negative charge on the zein molecule surface led to smaller zein particles (Zhong & Jin, 2009). The particle size of zein depends on many factors such as zein concentrations, percentage of ethanol-water solvent, pH of the aqueous solution (Zhong & Jin, 2009), and phase separation based on differential solubility (Podaralla & Perumal, 2012). Under acidic conditions, zein tends to form larger particles and polydispersity than in neutral and base solutions. Zein has been reported to have a monomeric form at pH > pI, and particle size decreased with an increase of pH (Podaralla & Perumal, 2012).
Zeta potential is an essential parameter for understanding the nanoparticle surface and for predicting the stability of the nanoparticles in the dispersion. ZNs dispersion prepared at pH 7.4, 8, and 9 indicated surface charge at around −31 mV which exhibited stable colloid (Kumar & Dixit, 2017). The ZNs prepared at pH 9 presented high degree of colloidal stability and the surface charge did not change even after the storage at 4 O C for 30 days as indicated in Table 1 and Figure 1.
When zein concentration was increased from 0.1% to 0.25, 0.5, 0.6, and 0.7% (w/v), a precipitation ( Figure 2) was observed in the ZNs dispersion of zein concentration higher than 0.5% (w/v) thus 0.5% zein concentration was selected for further study. An increase of pH of the aqueous phase from 7.4 to 9 caused similar results that a measurable decrease in the final mean particle size of zein in the dispersion, from 213.80 ± 0.82 nm to 141.40 ± 0.61 nm, which were larger than the particle size obtained with the use of 0.1% zein (w/v) F I G U R E 1 Dispersion of zein nanoparticles, prepared at different pH, after the preparation (a) and 24 hr storage at 25°C (b). Precipitation in the square (pH 7) could be observed  58.0 ± 2.0 aA −31.0 ± 3.4 aA 87.9 ± 0.9 aB −31.0 ± 1.3 bA Note: Values were expressed as means ± SD (n = 3). Different superscript letters (a-e) in the same column for particle size, and zeta potential, and letters (A-B) in the same row for particle size, and zeta potential mean significantly different (p < .05).
as shown in Table 2. The results were corresponding with the study of Zhong and Jin (2009), who reported the monotonic increase of particle sizes and the viscosity of zein, resulting in a larger size.
During liquid-liquid dispersion, stronger inertia occurred with higher zein concentrations, leading to droplet deformation. Larger droplets may be formed during the dispersion process and, thus resulting to larger particles (Liu et al., 2019;Zhong & Jin, 2009).
The zeta potential of the nanoparticle ranged from −24.33 ± 3.51 to −33.43 ± 02.93. The reduction of zein nanoparticle size was corresponding with the zeta potential and the polydispersity index (PDI). Danaei et al., (2018) reported that PDI values lower than 0.05 were mainly seen with highly monodisperse standards, while PDI values higher than 0.7 indicated an extensive particle size distribution of the sample.

| Effect of zein concentrations and % GO loading
3.2.1 | Particle size, zeta potential, encapsulation yield and efficiency, and colloidal stability The increase of zein concentration and % GO loading tended to increase the particle size, encapsulation yield, and efficiency as presented in Note: Values were expressed as means ± SD (n = 3). Different superscript letters in the same column mean significantly different (p < .05).
Reducing the particle size of encapsulated particle to nano-size would improve the uptake of encapsulated bioactive compound in cells (Luo et al., 2011). The encapsulation efficiency ranged from 61.46%-77.97%, 67.16%-71.90%, and 60.32%-90.99% for each zein concentrations (0.3, 0.4, and 0.5% w/v) with three % GO loadings, respectively, while the highest yield of nanoparticles, at 75.03%, was obtained with 0.5% zein concentrations at 50% GO loading. Similar phenomena were reported with the encapsulation of α-tocopherol by zein protein, where the encapsulation efficiency increased with the increase of zein protein (Luo et al., 2011).
The poor aqueous solubility of GO often limits its application in functional food and beverage (Kim et al., 2013). The encapsulation of GO in the zein nanoparticle will help the dispersion of this bioactive compound in a food system (Padua & Wang, 2009;Raharjo et al., 2019). Determining the colloidal stability of the micro/nano-dispersion is necessary since food processing is usually a complex instability resulted when attractive interparticulate forces predominated over repulsive interparticulate forces, which caused an increase in particle size and particle aggregation, respectively (Guo et al., 2008). The results indicated that the size of the particle had more influence on the dispersion stability during the storage than the surface charge shown.

| SEM, TEM, and FTIR
SEM images of the micro/nanoparticles are shown in Figure 4. Most particles are spherical and present the size in accordance with the particle sizes measured in the dispersion (Table 3). However, microparticles larger than 2 µm with textured surface were observed in the samples from 0.5/40 to 0.5/50. This could be the formation of larger self-assembled micro/ nanosphere of the zein with increased zein concentration (Luo et al., 2011) during the centrifugation and freeze drying.

| GO retention during storage
Generally, GO is susceptible to oxygen in the air and degraded during food processing and storage (Moongngarm et al., 2016). The retention of GO decreased to 87.12 and 79.62% at 30 days and 60 days storage at 4 o C, which was higher than that of free GO at 83.21, and 69.32%, respectively (Figure 7). The results showed that encapsulation in zein nanoparticles could improve the stability of GO inside. Shin et al. (1997) reported that GO was degraded through oxidation resulting from oxygen in the air and sunlight (Moongngarm et al., 2016) and the loss of GO was around 16.4% and 62.7% after 35 days and one year storage, respectively. Comparing to this, the retention of GO in GOZNs reduced to 90.13, and 90.12 at 30 days, and 60 days storage at −18 o C while the retention of free GO was 87.53 and 82.32%, respectively. At lower temperatures, degradation of the bioactive compound could be prolonged, and the antioxidant activity could be preserved because of the reduced activity of related enzymes (Qiu et al., 2014). The results confirmed that the nano-encapsulation could delay the oxidation of GO by protecting the core material from the environment.

| In vitro release of GO from GOZNs and free GO
The release profile of GOZNs and remaining free GO over time in a simulated gastrointestinal fluid under with pepsin and pancreatin at 37°C for 6 hr are expressed in Figure 8. Both studies were characterized by different two-step phases. The high GO release of GOZNs in SGF 30 min, at 46.79% was mainly due to the collapse of a sample from zein enzymatic breakdown (Luo et al., 2011). Adding of SIF at 0.5 hr and incubated further showed that the GO release was slowed hr. Comparing to this, remaining free GO was only 8.18% at 0.5 hr of SGF and increased rapidly to 100% after the incubation with SIF with pancreatin for 6 hr. The low amount of remaining free GO from unencapsulated GO or free GO was very low in the first step since GO is water-insoluble. Further incubation with pancreatin could solubilize the hydrophobic GO into the liquid. The sustained release effect by the encapsulation may be attributed to the strong hydrophobic interaction of GOZNs. Similar observation was also reported by Liu et al. (2019), pointing out that coating with a protein-based delivery zein nanoparticles could control the release of the bioactive compound inside.

| CON CLUS ION
This preparation of ZNs demonstrated that the pH of the aque-