Improving Physicochemical Stability of Quercetin-Loaded Hollow Zein Particles with Chitosan/Pectin Complex Coating

Hollow nanoparticles are preferred over solid ones for their high loading capabilities, sustained release and low density. Hollow zein particles are susceptible to aggregation with a slight variation in the ionic strength, pH and temperature of the medium. This study was aimed to fabricate quercetin-loaded hollow zein particles with chitosan and pectin coating to improve their physicochemical stability. Quercetin as a model flavonoid had a loading efficiency and capacity of about 86–94% and 2.22–5.89%, respectively. Infrared and X-ray diffraction investigations revealed the interaction of quercetin with zein and the change in its physical state from crystalline to amorphous upon incorporation in the composite particles. The chitosan/pectin coating improved the stability of quercetin-loaded hollow zein particles against heat treatment, sodium chloride and in a wide range of pH. The complex coating protected quercetin that was encapsulated in hollow zein particles from free radicals in the aqueous medium and enhanced its DPPH radical scavenging ability. The entrapment of quercetin in the particles improved its storage and photochemical stability. The storage stability of entrapped quercetin was enhanced both at 25 and 45 °C in hollow zein particles coated with chitosan and pectin. Therefore, composite hollow zein particles fabricated with a combination of polysaccharides can expand their role in the encapsulation, protection and delivery of bioactive components.


Introduction
Hollow zein particles have been fabricated by wrapping sodium carbonate (Na 2 CO 3 ) nanoprecipitate as sacrificial templet with zein in ethanol-water binary mixture followed by antisolvent precipitation [1]. Hollow particles in the loading and controlled release of bioactive components were preferred over their solid counterpart for more surface area and low density [2], but proteinaceous nature limits their utility as an efficient delivery system due to destabilization around pI (5-6.5), presence of counterion and high temperatureinduced denaturation [3,4]. Numerous strategies have been adopted to overcome instability issues of zein particles, for instance, coating with proteins [5,6], polysaccharides [7] and lipids [8]. Pectin is an anionic biodegradable polymer found in the plant cell wall and mainly made up of methyl esterified 1-4 linked α-D-galacturonic acid and 1-2-linked α-Lrhamnopyranose. Composite hollow zein particles with casein and pectin were developed by heating at 80 • C and 6.2 pH for 1 h to attain outstanding stability under simulated gastrointestinal conditions. Still, these composite particles were limited only to encapsulate and deliver heat-sensitive bioactives [9].
Chitosan-coated solid zein particles were fabricated through hydrophobic, hydrogen and van der Waals interactions at pH 4, improving the entrapment, photo/thermal Hollow zein (HZ) particles were fabricated with sodium carbonate sacrificial templet by mixing 1.75 mL of absolute ethanol and 2.5 mL of 50 mg/mL zein in 70% (v/v) ethanolwater binary mixture with 0.75 mL of 0.5, 1 and 2 (w/v%) Na 2 CO 3 aqueous solution under magnetic stirring at 1000 rpm for 1 min followed by adding to a 20 mL ultra-pure water [1,21]. The HZ particles were mixed with 0.5, 1 and 2 mg/mL chitosan in 1% (v/v) acetic acid at a 1:1 volume ratio under stirring at 1000 rpm for 30 min. Ethanol in the particle dispersion was removed under vacuum with a rotary evaporator RE-52C (Shanghai Tianheng Instrument Co. Ltd., Shanghai, China) at 35 • C for 30 min. Unstable particles were separated by centrifugation the particle dispersion at 2000× g for 15 min; the supernatant was then centrifuged at 15,000× g for 30 min to remove unabsorbed chitosan, and the precipitate was redispersed in an equal volume of distilled water to obtained chitosan-coated hollow zein (HZ-chi) particles. The aqueous solutions of pectin at 0.01, 0.025, 0.05 and 0.1 mg/mL were added to the dispersion of HZ-chi particles under stirring for 30 min at pH 4-4.5 to obtain pectin-and chitosan-coated hollow zein (HZ-chi/pec) particles. The quercetin-loaded hollow particles were prepared by adding 2, 3, 4 and 5 mg of quercetin in 50 mg/mL zein stock solution in ethanol-water binary mixture corresponding to 100, 150, 200 and 250 µg/mL of quercetin in the final particle dispersion.

Characterization of Particles 2.3.1. Particle Size and ζ-Potential
Samples were diluted by 200 folds with distilled water and measured at 25 • C and analyzed on a NanoBrook Omini particle size analyzer (Brookhaven Instrument, New York, NY, USA) at a scattering angle of 90 • . NNLS function was used to acquire the size distribution, while phase analysis light scattering (PALS) was employed to estimate the ζ-potential. Samples were prepared in triplicates for each measurement.

Loading Efficiency and Loading Capacity of Quercetin
Quercetin-loaded hollow zein and hollow zein-chitosan particles were centrifuged at 2000× g for 15 min to remove unencapsulated quercetin, and the supernatant and samples were diluted 50-fold in ethanol for the measurement of quercetin absorption at a λ max of 373 nm using a UV1800 UV-Vis spectrophotometer (Shimadzu Corporation, Tokyo, Japan) with a standard curve constructed from 1 to 20 µg/mL of quercetin dissolved in ethanol (a correlation coefficient of 0.999, Figure S4), adopting the method of Wang et al. [22]. Samples were prepared in triplicates for each measurement. Loading efficiency and capacity of quercetin in the particles were determined by the following equations.

Microstructural Analysis
Lyophilized particles were mounted to the surface of double-sided carbon tape and coated with a thin layer of gold. The morphology of particles was observed with a Hitachi SU8010 FE-SEM (Hitachi, Co., Tokyo, Japan) operated at an accelerating voltage of 8 kV.

Infrared Spectroscopy (IR)
Samples were freeze-dried and pressed into a transparent pellet with KBr, Infrared spectra of particles and raw materials were collected in the range of 400-4000 cm −1 on Nicolet™ iS™ 10 FT-IR Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) at a 4 cm −1 resolution and 16 scans.

X-ray Diffraction (XRD)
A Bruker D2 PHASER (Brucker, Odelzhausen, Germany) X-ray diffractometer, operated at 30 kV, 10 mA was used to obtain X-ray diffractograms of quercetin, zein, chitosan, pectin and quercetin-loaded hollow zein particles coated with chitosan and pectin. The data were collected over an angular range from 5 • to 40 • 2θ in continuous mode using step size and time of 0.02 • and 5 s, respectively.

Sodium Chloride Stability
Freshly prepared quercetin-loaded HZ, HZ-chi and HZ-chi/pec particles were exposed to 50, 100, 200, 300, 400, 500 mM sodium chloride under continuously mixing for 30 min followed by a 5 min rest [8]. Particle size and ζ-potential were measured, as mentioned above in Section 2.3.1.

pH Stability
The pH values of quercetin-loaded HZ, HZ-chi and HZ-chi/pec particles were adjusted from 2 to 9 with 0.1 mM hydrochloric acid and sodium hydroxide and then continuously stirred for 30 min [8]. Particle size and ζ-potential were analyzed by diluting them in pH-adjusted Milli-Q water [23].

Antioxidant Activity
ABTS and DPPH assays of quercetin-loaded HZ and HZ-chi/pec particles were estimated according to the method of Dong et al. and Pan et al. [24,25]. In brief, equal volumes of potassium persulfate (2.6 mM) and ABTS (7.4 mM) were mixed and allowed to react and generate ABTS + for 12 h in the dark. After the diluted ABTS + solution with an absorbance of 0.7 at 734 nm was mixed with samples at a volume ratio of 2:1 in the dark for 6 min, the absorbance was measured at 734 nm on Synergy H1 Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). Likewise, after 0.1 mM DPPH in ethanol was added to samples in equal volumes and allowed to react for 30 min in the dark, the absorbance was recorded at 517 nm. The ABTS + and DPPH scavenging capacity was calculated with the help of the following equation, where A Control and A Sample are the absorbances of the free radical solution without and with samples, respectively.
2.6. Functional Characteristics of Particles 2.6.1. Photochemical Stability of Quercetin Photochemical stability of pristine and encapsulated quercetin was evaluated by the procedure presented by Sun et al. [26]. Quercetin dispersed in water and encapsulated in HZ and HZ-chi/pec particles at 5 µg/mL were irradiated up to 120 min with a 365 nm ultraviolet lamp (VWR International Inc., West Chester, PA, USA). Samples were collected at 0, 15, 30, 60, 90 and 120 min, and the content of quercetin was analyzed with the help of UV-Vis spectrophotometer mentioned above in Section 2.3.2.

Storage Stability of Quercetin and Particles
Samples were stored at 25 and 45 • C for 28 days inside an LRH-250F incubator (Yiheng Scientific Instrument Co., Ltd. Shanghai, China). The stability of quercetinloaded particles was analyzed in terms of particle size and ζ-potential during storage. The retention of quercetin was expressed as percent retention and calculated by using the following equation: Quercetin retention (%) = Q t /Q i × 100 (4) where Q i and Q t are the content of quercetin at the beginning and specific time intervals during storage.

Statistical Analysis
All experiments were done in triplicates. The results were expressed in mean plus standard deviation and analyzed for a significant difference (p < 0.05) with IBM SPSS statistics 20.0 software package (IBM, Armonk, NY, USA).

Effect of Na 2 CO 3 and Chitosan on Hollow Zein Particles
The size of hollow zein particles is greatly influenced by the concentration of Na 2 CO 3 used for the preparation of sacrificial templet [1,27]. The smallest particles of 76 nm were fabricated with 1% Na 2 CO 3 ( Figure 1A). At 0.5 and 2% Na 2 CO 3, the size of HZ particles was 145 and 210 nm, respectively. The reason for the bigger particles is due to the formation of a thicker zein shell around all the available sodium carbonate nuclei formed in ethanolic conditions at a Na 2 CO 3 concentration of 0.5% but the formation of bigger Na 2 CO 3 nanocrystal in size due to excessive aggregation and crystal growth at 2% [27]. The pH values of the HZ particle dispersions prepared with 0.5, 1 and 2% Na 2 CO 3 were 9.04, 10.31 and 10.77, respectively, which is above the pI of zein [28]. The ζ-potential of HZ particles was −8, −26 and −24 mV when prepared with 0.5, 1 and 2% Na 2 CO 3 ( Figure 1B), receptively. There was a slight increase in the particle size of HZ particles upon the addition of chitosan ( Figure 1A), except that precipitation was observed at 0.05% chitosan and 2% Na 2 CO 3 . The increase in the particle size was the most obvious when the chitosan concentration was 0.1% at 1% and 2% Na 2 CO 3 . In the presence of chitosan, the pH of particle dispersions shifted to 4.0-4.5. Chitosan interacts with proteins below their pI through hydrophobic, electrostatic, van der Waals and hydrogen bonding [12,29]. The ζ-potential of HZ-chi particles ranged between +30 and +58 mV ( Figure 1B). The amine groups in chitosan contribute to the positive surface charge of composite zein particles [30,31]. These results suggest the formation of a chitosan shell.

Statistical Analysis
All experiments were done in triplicates. The results were expressed in mean plus standard deviation and analyzed for a significant difference (p < 0.05) with IBM SPSS statistics 20.0 software package (IBM, Armonk, NY, USA).

Effect of Na2CO3 and Chitosan on Hollow Zein Particles
The size of hollow zein particles is greatly influenced by the concentration of Na2CO3 used for the preparation of sacrificial templet [1,27]. The smallest particles of 76 nm were fabricated with 1% Na2CO3 (Figure 1A). At 0.5 and 2% Na2CO3, the size of HZ particles was 145 and 210 nm, respectively. The reason for the bigger particles is due to the formation of a thicker zein shell around all the available sodium carbonate nuclei formed in ethanolic conditions at a Na2CO3 concentration of 0.5% but the formation of bigger Na2CO3 nanocrystal in size due to excessive aggregation and crystal growth at 2% [27].
The pH values of the HZ particle dispersions prepared with 0.5, 1 and 2% Na2CO3 were 9.04, 10.31 and 10.77, respectively, which is above the pI of zein [28]. The ζ-potential of HZ particles was −8, −26 and −24 mV when prepared with 0.5, 1 and 2% Na2CO3 ( Figure  1B), receptively. There was a slight increase in the particle size of HZ particles upon the addition of chitosan ( Figure 1A), except that precipitation was observed at 0.05% chitosan and 2% Na2CO3. The increase in the particle size was the most obvious when the chitosan concentration was 0.1% at 1% and 2% Na2CO3. In the presence of chitosan, the pH of particle dispersions shifted to 4.0-4.5. Chitosan interacts with proteins below their pI through hydrophobic, electrostatic, van der Waals and hydrogen bonding [12,29]. The ζ-potential of HZ-chi particles ranged between +30 and +58 mV ( Figure 1B). The amine groups in chitosan contribute to the positive surface charge of composite zein particles [30,31]. These results suggest the formation of a chitosan shell.

Fabrication of Pectin-Coated Composite Particles
HZ-chi particles prepared with 1% Na2CO3 were further coated with pectin to form a second layer. The addition of pectin did not influence particle PDI (Table 1). When the concentration of chitosan was 0.5 mg/mL, the size of HZ-chi particles increased from 87 to 170 and 180 nm upon adding 0.01 and 0.025 mg/mL of pectin, respectively. When the

Fabrication of Pectin-Coated Composite Particles
HZ-chi particles prepared with 1% Na 2 CO 3 were further coated with pectin to form a second layer. The addition of pectin did not influence particle PDI (Table 1). When the concentration of chitosan was 0.5 mg/mL, the size of HZ-chi particles increased from 87 to 170 and 180 nm upon adding 0.01 and 0.025 mg/mL of pectin, respectively. When the concentration of chitosan was 2 mg/mL, the size of HZ-chi particles increased from 84 to 194 and 192 nm upon adding 0.01 and 0.025 mg/mL of pectin, respectively. Meanwhile, a reduction in the ζ-potential positive values was observed, showing insufficient pectin to fully cover the surface of particles. With further increasing the pectin concentration to 0.05 and 0.1 mg/mL in the presence of 0.5 mg/mL chitosan and to at 0.05 mg/mL pectin in the presence of 2 mg/mL, the particle dispersion became unstable with the formation of precipitates, due to charge neutralization [7]. The neutralization was observed at 0.01 and 0.025 mg/mL pectin in the presence of 1 mg/mL chitosan (Table 1). However, in the presence of 1 mg/mL chitosan, an increment in size to 248 and 219 nm was observed Antioxidants 2021, 10, 1476 6 of 16 at 0.05 and 0.1 mg/mL pectin, respectively, and ζ-potentials changed to negative values, suggesting the formation of pectin surface layer. An absolute value of ζ-potential above 20 mV is so high enough to ensure the physical stability of biopolymer-based particles [32]. Therefore, the hollow zein particles prepared with 1 mg/mL chitosan and 0.1 mg/mL pectin were used for further study on their physicochemical and functional attributes.
Values with different letters (upper case A and B for the concentration of chitosan; lower case a, b and c for the concentration of pectin) are significantly different in rows (p < 0.05); -represents the aggregation followed by precipitation of nanoparticles.

Encapsulation of Quercetin
Quercetin was used as a model bioactive flavonoid to be encapsulated in hollow zein particles. The loading efficiencies of quercetin were 72.71-79.86% in HZ particles, while the loading efficiencies increased to 90.58-93.86% at the flavonoid concentrations of 100-200 µg/mL and 85.84% at 250 µg/mL in HZ-chi particles ( Table 2). The higher loading efficiency of quercetin in the presence of chitosan is attributed to its interaction with chitosan through electrostatic and hydrogen bonding, in addition to hydrophobic interaction with zein [33,34]. The loading capacity of quercetin in the absence and presence of chitosan remained similar and increased from about 2% to 6%. This is different from a decreasing trend that was commonly observed in composite protein particles with increasing the content of the wall materials [35,36]. SEM images showed that quercetin-loaded HZ, HZ-chi and HZ-chi/pec were spherical with a smooth surface ( Figure 2). The loading of quercetin increased the size of HZ particles without and with chitosan and/or pectin coating ( Figure S1A). A similar trend was previously reported for the encapsulation of resveratrol and curcumin in composite hollow zein particles [11,37]. The loading of quercetin did not affect the ζ-Potential of HZ particles but increased ζ-potential absolute values of HZ-chi and HZ-chi/pec particles (Figure S1C), possibly due to rearrangement and exposure of more charged groups upon flavonoid inclusion [33]. particles without and with chitosan and/or pectin coating ( Figure S1A). A similar trend was previously reported for the encapsulation of resveratrol and curcumin in composite hollow zein particles [11,37]. The loading of quercetin did not affect the ζ-Potential of HZ particles but increased ζ-potential absolute values of HZ-chi and HZ-chi/pec particles (Figure S1C), possibly due to rearrangement and exposure of more charged groups upon flavonoid inclusion [33].

XRD
Quercetin is a crystalline solid in its pristine form [38], as evident from sharp X-ray diffraction patterns at 8, 10, 12, 13.01, 15, 17 and 27 of diffraction angle (2θ) (Figure 3). The distinct peaks of quercetin with lower intensities were apparent in its mixture of zein, chitosan and pectin. In the diffractograms of qHZ, qHZ-chi and qHZ-chi/pec particles, the crystalline peaks of quercetin disappeared, advocating the transformation from crystalline to an amorous state upon encapsulation in particles [10,39]. Pure zein and chitosan showed mild crystallinity with moderate peaks at 9.56 and 20.24 2θ because of respective α-helixes and crystal lattice structure [40,41]. When zein was structured into HZ, HZ-chi, HZ-chi/pec, qHZ, qHZ-chi and qHZ-chi/pec particles, the diffraction pattern raised from α-helixes in zein and crystal lattice of chitosan became flattened due to interaction between the polymers during the formation of the composite particles [42,43].

XRD
Quercetin is a crystalline solid in its pristine form [38], as evident from sharp X-ray diffraction patterns at 8, 10, 12, 13.01, 15, 17 and 27 of diffraction angle (2θ) (Figure 3). The distinct peaks of quercetin with lower intensities were apparent in its mixture of zein, chitosan and pectin. In the diffractograms of qHZ, qHZ-chi and qHZ-chi/pec particles, the crystalline peaks of quercetin disappeared, advocating the transformation from crystalline to an amorous state upon encapsulation in particles [10,39]. Pure zein and chitosan showed mild crystallinity with moderate peaks at 9.56 and 20.24 2θ because of respective α-helixes and crystal lattice structure [40,41]. When zein was structured into HZ, HZ-chi, HZ-chi/pec, qHZ, qHZ-chi and qHZ-chi/pec particles, the diffraction pattern raised from α-helixes in zein and crystal lattice of chitosan became flattened due to interaction between the polymers during the formation of the composite particles [42,43].
particles without and with chitosan and/or pectin coating ( Figure S1A). A similar trend was previously reported for the encapsulation of resveratrol and curcumin in composite hollow zein particles [11,37]. The loading of quercetin did not affect the ζ-Potential of HZ particles but increased ζ-potential absolute values of HZ-chi and HZ-chi/pec particles ( Figure S1C), possibly due to rearrangement and exposure of more charged groups upon flavonoid inclusion [33].

XRD
Quercetin is a crystalline solid in its pristine form [38], as evident from sharp X-ray diffraction patterns at 8, 10, 12, 13.01, 15, 17 and 27 of diffraction angle (2θ) (Figure 3). The distinct peaks of quercetin with lower intensities were apparent in its mixture of zein, chitosan and pectin. In the diffractograms of qHZ, qHZ-chi and qHZ-chi/pec particles, the crystalline peaks of quercetin disappeared, advocating the transformation from crystalline to an amorous state upon encapsulation in particles [10,39]. Pure zein and chitosan showed mild crystallinity with moderate peaks at 9.56 and 20.24 2θ because of respective α-helixes and crystal lattice structure [40,41]. When zein was structured into HZ, HZ-chi, HZ-chi/pec, qHZ, qHZ-chi and qHZ-chi/pec particles, the diffraction pattern raised from α-helixes in zein and crystal lattice of chitosan became flattened due to interaction between the polymers during the formation of the composite particles [42,43].   [34,44]. The absorption bands were clearly seen with lower intensities in the physical mixture of quercetin, zein, chitosan and pectin ( Figure 4A). However, the characteristic absorption bands of quercetin were invisible in HZ, HZ-chi and HZ-chi/pec particles, indicating the entrapment of quercetin in the particles and limited starching and bending of various bonds [42]. The encapsulation of quercetin resulted in the variation of OH stretching of HZ, HZ-chi and HZ-chi/pec particles ( Figure 4A,B), suggesting the flavonoid interaction with wall materials [45]. Upon encapsulation of quercetin in hollow zein particle, the amide I peak of zein was unchanged, whereas the amide II showed a redshift to 1537, 1543 and 1539 cm −1 in HZ, HZ-chi and HZ-chi/pec particles ( Figure 4A). These shifts in the amide II result from hydrophobic, electrostatic and hydrogen bonding of quercetin with wall materials [46,47]. changed from 1658 and 1541 cm −1 to 1656 and 1536 cm −1 in HZ particles, to 1658 and 1542 cm −1 in HZ-chi particles, and to 1654 and 1635 cm −1 in HZ-chi/pec particles. The variations of stretching and bending vibrations of C=O and N-H indicate that hydrophobic and electrostatic interactions occurred during the fabrication of HZ, chitosan and chitosan/pectin coated HZ particles [29]. The distinct absorption peaks of quercetin IR spectrum at 3386, 1655, 1599, 1373, 1257 and 1160 cm −1 ( Figure 4A) signify O-H, C=O, C=C, C-OH, C-O-C and C-OH (B ring), respectively [34,44]. The absorption bands were clearly seen with lower intensities in the physical mixture of quercetin, zein, chitosan and pectin ( Figure  4A). However, the characteristic absorption bands of quercetin were invisible in HZ, HZchi and HZ-chi/pec particles, indicating the entrapment of quercetin in the particles and limited starching and bending of various bonds [42]. The encapsulation of quercetin resulted in the variation of OH stretching of HZ, HZ-chi and HZ-chi/pec particles ( Figure  4A,B), suggesting the flavonoid interaction with wall materials [45]. Upon encapsulation of quercetin in hollow zein particle, the amide I peak of zein was unchanged, whereas the amide II showed a redshift to 1537, 1543 and 1539 cm −1 in HZ, HZ-chi and HZ-chi/pec particles ( Figure 4A). These shifts in the amide II result from hydrophobic, electrostatic and hydrogen bonding of quercetin with wall materials [46,47].

Salt Endurance
Colloidal stability against salt was assessed by exposing particles to various concentrations of NaCl. Quercetin-loaded hollow zein particles without and with chitosan were highly unstable and precipitated out even at 50 mM NaCl ( Figure S2A,B), attributed to the screening effect and charge neutralization by counterions [7,43]. Quercetin-loaded HZchi/pec showed a remarkable endurance to precipitation up to 500 mM NaCl ( Figure S2C). The size of quercetin-loaded HZ-chi/pec particles kept a single peak and increased gradually as the concentration of NaCl increased up to 200 mM ( Figure 5A). Meanwhile, their ζpotential changed to −29 mV ( Figure 5B). It is possible that the NaCl ions screen the repulsion among the polysaccharide chain, leading to a greater adsorption of pectin on the particle surface [48,49]. These results are different from the sedimentation of pectin-coated zein particle at 70 mM NaCl reported by Huang et al. [16]. It can be thus speculated that the complex coating of chitosan and pectin provided better stability against salt than did by pectin alone. The size of quercetin-loaded HZ-chi/pec particles became bigger and had two

Salt Endurance
Colloidal stability against salt was assessed by exposing particles to various concentrations of NaCl. Quercetin-loaded hollow zein particles without and with chitosan were highly unstable and precipitated out even at 50 mM NaCl ( Figure S2A,B), attributed to the screening effect and charge neutralization by counterions [7,43]. Quercetin-loaded HZchi/pec showed a remarkable endurance to precipitation up to 500 mM NaCl ( Figure S2C). The size of quercetin-loaded HZ-chi/pec particles kept a single peak and increased gradually as the concentration of NaCl increased up to 200 mM ( Figure 5A). Meanwhile, their ζ-potential changed to −29 mV ( Figure 5B). It is possible that the NaCl ions screen the repulsion among the polysaccharide chain, leading to a greater adsorption of pectin on the particle surface [48,49]. These results are different from the sedimentation of pectin-coated zein particle at 70 mM NaCl reported by Huang et al. [16]. It can be thus speculated that the complex coating of chitosan and pectin provided better stability against salt than did by pectin alone. The size of quercetin-loaded HZ-chi/pec particles became bigger and had two peaks ( Figure 5A), and their ζ-potential absolute values significantly decreased upon further increasing the concentration of NaCl. These results suggest the salt at high concentrations reduced the electrostatic repulsion between particles, resulting in their aggregation [46]. peaks ( Figure 5A), and their ζ-potential absolute values significantly decreased upon further increasing the concentration of NaCl. These results suggest the salt at high concentrations reduced the electrostatic repulsion between particles, resulting in their aggregation [46].

pH Stability
Protein-based particles are liable to aggregation around pI [6], limiting their application. Quercetin-loaded hollow zein particles were sable with similar size distribution at pH 2, 3 and 4 ( Figure 6A) but precipitated at pH 5 around pI, due to charge neutralization and lack of repulsive forces [50,51]. It is difficult to re-disperse the precipitated zein particles above the pI of zein. Quercetin-loaded hollow zein-chitosan particles showed two size distributions above and below pH 4 ( Figure 6A). The change in the particle size below pH 4 is attributed to swelling and dissolution of the polymers with higher charge density [18], while the formation of bigger aggregates is at pH 5, close to the pI of zein [7]. The particles with chitosan were unstable at pH 6-9 due to the deprotonation of chitosan amine groups at pka and above [52,53]. It is noted that quercetin-loaded HZ-chi/pec particles showed good stability across the investigated pH range of 2-5 ( Figure 6A). The ζ-potential of quercetin-loaded HZ-chi/pec was the highest at pH 5 and decreased as pH decreased ( Figure  6B), due to deionization of the carboxylic groups of pectin below its pka (3.5) [16]. At pH 6-9, the size of quercetin-loaded HZ-chi/pec particles was around 220 nm and less than those at lower pH. Their ζ-potential also decreased slightly as the pH increased from pH 5. Karim et al. also reported a decline in the ζ-potential and size of pectin/chitosan-coated nanoliposome at pH 8 compared with that of pH 5 [18]. These changes might be due to the detachment of loosely adsorbed pectin molecules from the surface, since chitosan possesses less charged groups as the pH increases [54]. These findings suggest that the double coating with chitosan and pectin provides excellent protection against aggregation, especially around the pI of zein and pka of chitosan.

pH Stability
Protein-based particles are liable to aggregation around pI [6], limiting their application. Quercetin-loaded hollow zein particles were sable with similar size distribution at pH 2, 3 and 4 ( Figure 6A) but precipitated at pH 5 around pI, due to charge neutralization and lack of repulsive forces [50,51]. It is difficult to re-disperse the precipitated zein particles above the pI of zein. Quercetin-loaded hollow zein-chitosan particles showed two size distributions above and below pH 4 ( Figure 6A). The change in the particle size below pH 4 is attributed to swelling and dissolution of the polymers with higher charge density [18], while the formation of bigger aggregates is at pH 5, close to the pI of zein [7]. The particles with chitosan were unstable at pH 6-9 due to the deprotonation of chitosan amine groups at pk a and above [52,53]. It is noted that quercetin-loaded HZ-chi/pec particles showed good stability across the investigated pH range of 2-5 ( Figure 6A). The ζ-potential of quercetin-loaded HZ-chi/pec was the highest at pH 5 and decreased as pH decreased ( Figure 6B), due to deionization of the carboxylic groups of pectin below its pk a (3.5) [16]. At pH 6-9, the size of quercetin-loaded HZ-chi/pec particles was around 220 nm and less than those at lower pH. Their ζ-potential also decreased slightly as the pH increased from pH 5. Karim et al. also reported a decline in the ζ-potential and size of pectin/chitosan-coated nanoliposome at pH 8 compared with that of pH 5 [18]. These changes might be due to the detachment of loosely adsorbed pectin molecules from the surface, since chitosan possesses less charged groups as the pH increases [54]. These findings suggest that the double coating with chitosan and pectin provides excellent protection against aggregation, especially around the pI of zein and pk a of chitosan.

Temperature Stability
Bioactive-component-loaded carrier systems for food application might be exposed to different temperature treatments during the production cycles. Figure 7 shows the effect of temperature on the size distribution and ζ-potential of quercetin-loaded HZ, HZchi and HZ-chi/pec particles. Quercetin-loaded HZ particles had ζ-potential values be-

Temperature Stability
Bioactive-component-loaded carrier systems for food application might be exposed to different temperature treatments during the production cycles. Figure 7 shows the effect of temperature on the size distribution and ζ-potential of quercetin-loaded HZ, HZ-chi and HZ-chi/pec particles. Quercetin-loaded HZ particles had ζ-potential values between +41-+48 mV ( Figure 7B). Size of quercetin-loaded HZ particles increased as temperature increased and showed two peaks above 40 • C ( Figure 7A), possibly attributed to rearrangement of zein molecules and exposure of nonpolar groups followed by a collapse of hollow structure [55]. This is different from solid zein particles, those were colloidally stable when heated at pH 4 and 80 • C for 120 min [43]. Likewise, Figure 7A shows that size of quercetin-loaded HZ-chi particles increased with increasing temperature, but two peaks were observed above 70 • C, suggesting that the chitosan coating improves the particle stability against heat treatment. Their ζ-potential was +32 mV at 30 • C and increased to a range of +45 and +48 mV at 40-80 • C and to +56 mV at 90 • C. The changes might be due to that the realignment of zein and chitosan at higher temperatures possibly leads to inter/intramolecular interactions [5,56]. The quercetin-loaded HZ-chi/pec particles were stable when the temperature was increased to 40 • C ( Figure 7A). Then, their size gradually increased upon further increase in temperature and showed two peaks at 90 • C. Their ζ-potential was kept between −25 and −32 mV at all the investigated temperatures. These results indicate that the pectin coating further improves the colloidal stability of quercetin-loaded HZ-chi particles. It is presumed that steric stabilization of pectin coating inhibits the collision of zein particles with more exposed reactive functional groups at the higher temperature, thus preventing increment in size and particle aggregation [57]. Figure 6. Size distribution (A) and ζ−potential (B) of quercetin−loaded hollow zein particles without (blue) and with chitosan (red) and chitosan/pectin (black) coating at various pH values.

Temperature Stability
Bioactive-component-loaded carrier systems for food application might be exposed to different temperature treatments during the production cycles. Figure 7 shows the effect of temperature on the size distribution and ζ-potential of quercetin-loaded HZ, HZchi and HZ-chi/pec particles. Quercetin-loaded HZ particles had ζ-potential values between +41-+48 mV ( Figure 7B). Size of quercetin-loaded HZ particles increased as temperature increased and showed two peaks above 40 °C (Figure 7A), possibly attributed to rearrangement of zein molecules and exposure of nonpolar groups followed by a collapse of hollow structure [55]. This is different from solid zein particles, those were colloidally stable when heated at pH 4 and 80 °C for 120 min [43]. Likewise, Figure 7A shows that size of quercetin-loaded HZ-chi particles increased with increasing temperature, but two peaks were observed above 70 °C, suggesting that the chitosan coating improves the particle stability against heat treatment. Their ζ-potential was +32 mV at 30 °C and increased to a range of +45 and +48 mV at 40-80 °C and to +56 mV at 90 °C. The changes might be due to that the realignment of zein and chitosan at higher temperatures possibly leads to inter/intramolecular interactions [5,56]. The quercetin-loaded HZ-chi/pec particles were stable when the temperature was increased to 40 °C ( Figure 7A). Then, their size gradually increased upon further increase in temperature and showed two peaks at 90 °C. Their ζpotential was kept between −25 and −32 mV at all the investigated temperatures. These results indicate that the pectin coating further improves the colloidal stability of quercetinloaded HZ-chi particles. It is presumed that steric stabilization of pectin coating inhibits the collision of zein particles with more exposed reactive functional groups at the higher temperature, thus preventing increment in size and particle aggregation [57].

Storage Stability
After storage at 25 and 45 • C for 7 days, quercetin-loaded HZ and HZ-chi particles precipitated, while no precipitation was observed for quercetin-loaded HZ-chi/pec particles ( Figure S3). Figure 8 shows size distribution and ζ-potential of quercetin-loaded HZ-chi/pec particles during storage. Their size distribution remained a single peak during storage at 45 • C and increased to around 325 nm after 6 days. At 25 • C, the size distribution kept a single peak around 220 nm after 6 days and then showed two peaks. These results indicate quercetin-loaded HZ-chi/pec particles are more stable at 45 • C than 25 • C, possibly due to the high temperature facilitates adsorption of pectin to the particles more effectively and improves its complex formation capacity [58,59]. At 25 • C, a smaller size, around 90 nm was observed after storage for 13 days (Figure 8), probably due to the detachment, depolymerization and hydrolysis of galacturonic acid glycan chains of pectin at pH < 5 [60]. At 25 • C, a larger size around 900-1500 nm was observed after 20 days (Figure 8). The ζ-potential of HZ-chi/pec particles became more negative over time, both at 25 and 45 • C. These changes indicate the realignment of pectin during storage and exposure of more charged COOgroups at the interface, leading to an upsurge in ζ-potential [18,54]. Pectin coating substantially enhances the colloidal stability of quercetin-loaded HZ-chi/pec compared with that of qHZ and qHZ-chi ( Figure S3A,B) by preventing aggregation of particles through electrostatic repulsions and steric stabilization as well [61].
around 90 nm was observed after storage for 13 days (Figure 8), probably due to the detachment, depolymerization and hydrolysis of galacturonic acid glycan chains of pectin at pH < 5 [60]. At 25 °C, a larger size around 900-1500 nm was observed after 20 days (Figure 8). The ζ-potential of HZ-chi/pec particles became more negative over time, both at 25 and 45 °C. These changes indicate the realignment of pectin during storage and exposure of more charged COOgroups at the interface, leading to an upsurge in ζ-potential [18,54]. Pectin coating substantially enhances the colloidal stability of quercetin-loaded HZ-chi/pec compared with that of qHZ and qHZ-chi ( Figure S3A,B) by preventing aggregation of particles through electrostatic repulsions and steric stabilization as well [61].

Antioxidant Activity
The antioxidant activity of encapsulated quercetin was investigated by ABTS and DPPH radical scavenging assays (Figure 9). The ABTS + scavenging capacity of quercetinloaded HZ was lower than the quercetin dispersed in water and ethanol. The reason for the lower scavenging capacity of encapsulated quercetin is being embedded in the hydrophobic pockets of protein and inaccessible to ABTS + [30]. Furthermore, the hydroxyl groups in the B ring of quercetin are the main contributor of H + and are involved in hydrogen bonding in the composite particles, thus unavailable to scavenge the free radicals. Earlier, quercetin encapsulated in SPI and solid zein particles showed a similar reduction in the ABTS + savaging capacity. On the other hand, the interfacial chitosan-pectin coating in quercetin-loaded HZ-chi/pec particles may increase the accessibility of hydrophilic ABTS + to quercetin, demonstrating higher scavenging of ABTS + compared with that of qHZ ( Figure 9). DPPH scavenging capacity of quercetin was improved upon encapsulation in HZ and HZ-chi/pec particles by compared with that of dispersed in ethanol and water ( Figure 9). Earlier, it has been reported that both zein and DPPH being soluble in

Antioxidant Activity
The antioxidant activity of encapsulated quercetin was investigated by ABTS and DPPH radical scavenging assays (Figure 9). The ABTS + scavenging capacity of quercetinloaded HZ was lower than the quercetin dispersed in water and ethanol. The reason for the lower scavenging capacity of encapsulated quercetin is being embedded in the hydrophobic pockets of protein and inaccessible to ABTS + [30]. Furthermore, the hydroxyl groups in the B ring of quercetin are the main contributor of H + and are involved in hydrogen bonding in the composite particles, thus unavailable to scavenge the free radicals. Earlier, quercetin encapsulated in SPI and solid zein particles showed a similar reduction in the ABTS + savaging capacity. On the other hand, the interfacial chitosan-pectin coating in quercetin-loaded HZ-chi/pec particles may increase the accessibility of hydrophilic ABTS + to quercetin, demonstrating higher scavenging of ABTS + compared with that of qHZ ( Figure 9). DPPH scavenging capacity of quercetin was improved upon encapsulation in HZ and HZ-chi/pec particles by compared with that of dispersed in ethanol and water ( Figure 9). Earlier, it has been reported that both zein and DPPH being soluble in ethanol-water binary medium facilitate scavenging of DPPH by hydrophobic antioxidants encapsulated in the zein particles [62,63]. The ABTS and DPPH radical scavenging assay revealed that by encapsulating quercetin in composite hollow zein particles, it could be better protected from free reactive radicals in the surrounding medium along with an improved or sustained antioxidant activity. ethanol-water binary medium facilitate scavenging of DPPH by hydrophobic antioxidants encapsulated in the zein particles [62,63]. The ABTS and DPPH radical scavenging assay revealed that by encapsulating quercetin in composite hollow zein particles, it could be better protected from free reactive radicals in the surrounding medium along with an improved or sustained antioxidant activity.

Photochemical Stability
Quercetin is prone to degradation upon exposure to ultraviolet light due to oxidative decarboxylation of the C ring [22]. The retention of unencapsulated quercetin sharply decreased to 25% after 120 min of irradiation ( Figure 10A). The quercetin loaded in HZ and HZ-chi/pec particles was more stable, with retention of about 80% after 120 min of irradiation. These results suggesting the hollow particle provide the excellent stability of quercetin against ultraviolet light. The protection results from the physical barrier and lightscattering effect of the particles [61]. The similar retention of quercetin in HZ and HZchi/pec particles ( Figure 10A) indicates that the protection was fundamentally attributed to zein. A comparable protective effect has previously been reported by encapsulating quercetin in WPI/lotus root amylopectin, pea protein-isolated and zein/soluble soybean polysaccharide composite nanoparticles due to hydrogen and hydrophobic interaction between quercetin and proteins [39,43,64].

Photochemical Stability
Quercetin is prone to degradation upon exposure to ultraviolet light due to oxidative decarboxylation of the C ring [22]. The retention of unencapsulated quercetin sharply decreased to 25% after 120 min of irradiation ( Figure 10A). The quercetin loaded in HZ and HZ-chi/pec particles was more stable, with retention of about 80% after 120 min of irradiation. These results suggesting the hollow particle provide the excellent stability of quercetin against ultraviolet light. The protection results from the physical barrier and light-scattering effect of the particles [61]. The similar retention of quercetin in HZ and HZchi/pec particles ( Figure 10A) indicates that the protection was fundamentally attributed to zein. A comparable protective effect has previously been reported by encapsulating quercetin in WPI/lotus root amylopectin, pea protein-isolated and zein/soluble soybean polysaccharide composite nanoparticles due to hydrogen and hydrophobic interaction between quercetin and proteins [39,43,64].
ethanol-water binary medium facilitate scavenging of DPPH by hydrophobic antioxidants encapsulated in the zein particles [62,63]. The ABTS and DPPH radical scavenging assay revealed that by encapsulating quercetin in composite hollow zein particles, it could be better protected from free reactive radicals in the surrounding medium along with an improved or sustained antioxidant activity.

Photochemical Stability
Quercetin is prone to degradation upon exposure to ultraviolet light due to oxidative decarboxylation of the C ring [22]. The retention of unencapsulated quercetin sharply decreased to 25% after 120 min of irradiation ( Figure 10A). The quercetin loaded in HZ and HZ-chi/pec particles was more stable, with retention of about 80% after 120 min of irradiation. These results suggesting the hollow particle provide the excellent stability of quercetin against ultraviolet light. The protection results from the physical barrier and lightscattering effect of the particles [61]. The similar retention of quercetin in HZ and HZchi/pec particles ( Figure 10A) indicates that the protection was fundamentally attributed to zein. A comparable protective effect has previously been reported by encapsulating quercetin in WPI/lotus root amylopectin, pea protein-isolated and zein/soluble soybean polysaccharide composite nanoparticles due to hydrogen and hydrophobic interaction between quercetin and proteins [39,43,64].

Storage Stability
Liu and coworkers reported a drastic decrease in the content of quercetin to around 38% in 3 days of storage at room temperature [39]. The degradation of quercetin was faster at 45 • C than 25 • C, with the retention of quercetin being 42% and 16% after 2 days ( Figure 10B), respectively. Its complete loss was observed at 25 • C and 45 • C after 4 days. The degradation of quercetin is attributed to its auto-oxidation in the aqueous medium, which is much pronounced at elevated temperature [19,65]. The retention of quercetin was significantly greater in HZ-chi/pec particles during storage, being 84% and 67% at 25 and