Preparation, characterization and stability of curcumin-loaded zein-shellac composite colloidal particles

Food Chemistry 228 (2017) 656–667

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Preparation, characterization and stability of curcumin-loaded zein-shellac composite colloidal particles Cuixia Sun, Chenqi Xu, Like Mao, Di Wang, Jie Yang, Yanxiang Gao ⇑ Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Laboratory for Food Quality and Safety, Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, PR China

a r t i c l e

i n f o

Article history: Received 4 September 2016 Received in revised form 6 January 2017 Accepted 1 February 2017 Available online 3 February 2017 Keywords: Zein Shellac Curcumin Composite particles Stability Release

a b s t r a c t Curcumin-loaded zein-shellac composite particles were prepared by the antisolvent co-precipitation method. The encapsulation efficiency of curcumin was significantly improved from 82.7% in zein particles to 93.2% in zein-shellac complex particles. The result of differential scanning calorimetry suggested that curcumin in the polymeric matrix was in an amorphous state. Fourier transform infrared spectroscopy analysis revealed that curcumin had non-covalently interacted with zein and shellac, mainly through hydrogen bonding and hydrophobic interaction. Aggregates in irregular shapes, with large sizes, were found by atomic force microscopy, and conglutination, integration or fusion of different entities into network structures occurred at a high level of shellac. At the mass ratio of zein to shellac of 1:1, curcumin in the complex particles exhibited improved photochemical and thermal stability. Curcumin-loaded zeinshellac complex particles allowed the controlled release of curcumin in both PBS medium and simulated gastrointestinal fluids. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Curcumin (Cur) (1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-hep tadiene-3,5-dione) is a natural lipophilic polyphenol found in the rhizomes of turmeric (Curcuma longa), which has been used as a food seasoning ingredient and dietary supplement, as well as traditional Asian medicine for centuries (Hu et al., 2015; Li, Shin, Lee, Chen, & Park, 2016; Mehanny, Hathout, Geneidi, & Mansour, 2016). Cur is notable for the diversity of its biological activities, e.g. antioxidant, anti-inflammatory and anticancer, as well as inhibition ability against cardiovascular, neurodegenerative and metabolic diseases (Sharma, Gescher, & Steward, 2005). Despite its potential health benefits, the application of Cur in food and pharmaceutical products is limited, due to its low water solubility and poor oral bioavailability. Besides, Cur is sensitive to alkaline environments, UV irradiation and heating (Patel, Hu, Tiwari, & Velikov, 2010). In order to improve the stability and oral bioavailability of Cur, various delivery systems have been designed, e.g. hydrogels, dendrimers, liposome, and micro/nano-particles. Compared with other delivery systems, nanoparticles have been widely investigated due to their distinct advantages, e.g. high encapsulation efficiency, ⇑ Corresponding author at: Box 112, No. 17 Tsinghua East Road, Haidian District, Beijing 100083, PR China. E-mail address: [email protected] (Y. Gao). http://dx.doi.org/10.1016/j.foodchem.2017.02.001 0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.

slower degradation rate, small particle size (10–1000 nm), effective penetration ability and targetability (Wang, Sun, et al., 2016). Polymer nanoparticles can be fabricated from a variety of ingredients, mainly including natural polymers such as bcyclodextrin (Moorthi, Senthil Kumar, Mohan, & Kathiresan, 2013), chitosan (Facchi et al., 2016), alginate (Hu & McClements, 2015), gelatin (Gómez-Estaca, Gavara, & Hernández-Muñoz, 2015), soy protein (Chen, Zhang, & Tang, 2016) and lactoferrin (Bollimpelli, Kumar, Kumari, & Kondapi, 2016). Protein-based particles are usually fabricated from watersoluble proteins, and they can also be formed with alcoholsoluble proteins. Zein, the major storage protein of corn, is composed of lipophilic amino acid residues. Due to its inherent hydrophobic property, zein can be easily converted into spherical colloidal nanoparticles by the method of anitsolvent coprecipitation for the encapsulation of Cur (Patel et al., 2010). Compared with zein particles, composite particles formed by zein and polysaccharides can provide a better protection for Cur (Liang et al., 2015). Hu et al. (2015) fabricated core-shell biopolymer nanoparticles, using zein as the core and pectin as the shell at pH 4.0 and the nanoparticles showed a higher loading efficiency (>86%) of Cur. However, most complex systems have consisted of zein and hydrophilic polysaccharides. Any investigation into interactions between zein and hydrophobic components has been scarcely reported.

657

C. Sun et al. / Food Chemistry 228 (2017) 656–667

Shellac, a resin secreted by the female lac beetle, has received much attention for its biomedical applications, especially for use in colon-targeted drug delivery (Wang, Yu, Li, Bligh, & Williams, 2015). Shellac is insoluble in water, but highly soluble in anhydrous ethanol. It has a good film-forming capacity, high gloss, and poor permeability to gases, acid and water vapour. Phan, Debeaufort, Luu, and Voilley (2008) fabricated an edible bilayer membrane composed of agar and/or cassava starch as a cohesive structural layer and ethanol-cast shellac layer as a moisture barrier layer and the system was suitable for food preservation. Messaoud et al. (2016) combined shellac with alginate as the coating to form composite capsules so as to encapsulate riboflavin. However, any information about the formation of complexes between shellac and prolamins, such as zein (a typical plant protein from corn), is not available to date. In the present work, the method of antisolvent co-precipitation was used to fabricate the Cur-loaded zein-shellac composite particles. Interactions among the different components of Cur, zein and shellac were explored. The encapsulation efficiency, photochemical and thermal stability of Cur, as well as the micro-morphology of the Cur-loaded zein-shellac composite particles were evaluated. Furthermore, the ability of zein-shellac binary complexes to control the release of Cur was also investigated in this work, which might be useful for the development of potential carriers for bioactive compounds.

at 765g for 10 min to remove larger aggregates and unloaded Cur. The final dispersions were then stored at 4 °C, and parts of the dispersions were freeze-dried for 48 h with an Alpha 1-2 D Plus freeze-drying apparatus (Marin Christ, Germany) to acquire dry particles for solid state characterization analysis. Control samples (free Cur solutions) were also prepared by the above method. In this work, samples of zein-shellac binary complexes with different mass ratios of 5:1, 2:1, 1:1 and 1:2 were termed as Z-S5:1, ZS2:1, Z-S1:1 and Z-S1:2, respectively. Cur-loaded zein-shellac complex particles were termed as Z-S/Cur. And complex particles with Cur at the percentage of 10, 20, 50, 100 and 200 were termed as ZS/Cur10, Z-S/Cur20, Z-S/Cur50, Z-S/Cur100 and Z-S/Cur200, respectively. Samples of Cur-loaded individual zein or shellac solutions were termed Z/Cur and S/Cur, respectively. 2.3. Colloidal particles characterization

2. Materials and methods

2.3.1. Encapsulation efficiency (EE) The quantity of Cur in the dispersion was determined by the method of Patel et al. (2010) with some modifications. Briefly, freshly prepared colloidal dispersions were diluted by aqueous ethanol solution (80%, v/v), and the absorption at 426 nm was recorded using a UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The content of Cur was quantified against a standard curve of Cur dissolved in aqueous ethanol solution (80%, v/v). Encapsulation efficiency was calculated by the following equation:

2.1. Materials

Encapsulation efficiency ð%Þ ¼

Cur (>95% purity) was purchased from Hebei Food Additive Co., Ltd. (Hebei, China). Zein with a protein content of 95% (w/w) was obtained from Gaoyou Group Co. Ltd. (Jiangsu, China). Shellac (S) (wax-free), pepsin (600 U/mg, Sigma 77160), bile salts (Sigma B8631) and pancreatin (8
USP, Sigma P7545) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Absolute ethanol (99.9%) was purchased from Eshowbokoo Biological Technology Co., Ltd (Beijing, China). 2.2. Preparation of Cur-loaded zein-shellac complex particles Zein, shellac and Cur were separately dissolved in aqueous ethanol (80%, v/v) solution containing 0.1 M phosphate buffer saline (PBS) to reach concentrations of 6 mg/ml, 6 mg/ml and 12 mg/ ml, respectively, with magnetic stirring at 600 rpm at room temperature for 2 h. The Cur solution was kept in the dark during dissolution and storage. Cur solution was diluted by aqueous ethanol solution (80%, v/v) to acquire five different concentrations (0.6, 1.2, 3.0, 6.0 and 12.0 mg/ml), and the solutions were then adjusted to pH 8.0, using 0.1 M NaOH. Individual zein and shellac solutions were mixed at different volume ratios (5:1, 2:1, 1:1, 1:2) within a consistent total volume of 6.0 ml on a vortex shaker at 2200 rpm for 30 s to acquire the zein-shallac binary complex solutions which were allowed to stand for 2 h at room temperature. Then Cur solutions at five different concentrations were added to zein-shellac solutions at the volume ratio of 1:1 and mixed on a vortex shaker at 2200 rpm for 30 s. Cur solutions were also added to individual zein or shellac solutions by the aforementioned process and used as the controls. Afterwards, the formation of composite colloidal particles was induced by the antisolvent co-precipitation method described in the previous literature with some modifications (Yadav & Kumar, 2014). Briefly, the mixed solutions were slowly injected into PBS (pH 8.0) to get a 7-fold volume dilution, and the solutions were stirred at 600 rpm for 20 min. The suspensions were centrifuged

the weight of Cur in dispersion total weight of Cur input
100

2.3.2. Particle size and zeta-potential measurement Particle size of Cur-loaded zein-shellac ternary complex colloidal dispersions was determined by dynamic light scattering (DLS), using a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK). The intensity of light scattered was monitored at a 90° angle. All the liquid samples were equilibrated for 60 s at 25 °C inside the instrument before dynamic light back scattering, then data were collected over 10 sequential readings. Each sample was analyzed in triplicate and the results were collected as cumulative mean diameter (size, nm) for particle size. Zeta-potential of the colloidal dispersions was determined by measuring the direction and velocity of particle movement in a well-defined electric field. The data were calculated by the instrument using the Smoluchowski model. All measurements were performed in triplicate. 2.3.3. Differential scanning calorimetry (DSC) The thermal characteristics of Cur-loaded zein-shellac ternary complex colloidal particles were studied, using differential scanning calorimetry (DSC-60, Shimadzu, Tokyo, Japan) as described by Pan and Zhong (2016) with some modifications. Briefly, 5.0 mg of each freeze-dried sample were weighed in an aluminium pan and hermetically sealed. Samples were heated from 30 °C to 300 °C at a heating rate of 10 °C /min. An empty aluminium pan was applied as the reference. 2.3.4. Fourier-transform infrared (FTIR) spectroscopy The infrared spectra of samples were measured by the potassium bromide (KBr) pellet method, using a Spectrum 100 Fourier transform spectrophotometer (PerkinElmer, U.K.) in 400– 4000 cm1 range, with a resolution of 4 cm1. Potassium bromide was used as a reference. For each measurement, 11 scans were

658

C. Sun et al. / Food Chemistry 228 (2017) 656–667

taken. The data were analyzed using Omnic v8.0 (Thermo Nicolet, USA). 2.3.5. Atomic force microscopy (AFM) observation AFM was used to examine the morphology of samples. The measurements were performed according to the method described by Wang, Crofts, and Padua (2003) with slight modifications. The freeze-dried samples were dissolved in 80% aqueous ethanol solution to reach a concentration of 10–15 lg/ml. About 2.0 ll of the solution were dripped onto the mica slide and allowed to dry for 1 h in a dark place before AFM scans were taken. AFM measurements were carried out using NTEGRA Solaris AFM (NT-MDT, Russia) with operation under non-contact mode. The measurement was set at a resonance frequency of 300 kHz, a scan rate of 0.7 Hz and a force constant of 40 N/m.

Briefly, 80 ml of base media are filled into the dissolution vessel, and incubated in a shaker at a speed of rotation of 150 rpm with the attached water bath at 37 ± 0.5 °C. Briefly, 5 ml of SGF were added to the medium, followed by acidification, using 1 M HCl until a constant end-point value of pH 2.0 was reached. Cur powder (1 mg) and freeze-dried samples (equivalent to 1 mg Cur) were weighed and put into the dissolution vessel (in triplicate) to start the gastric incubation. After one hour of gastric incubation, the reaction mixture was neutralized by the addition of 10 ml of SIF and 1 ml of 1 M NaOH to reach an end-point pH of 7.4. Samples were collected at the following time points: 5, 15, 30, 60, 90, 120 and 180 min. After centrifugation at 10000g for 5 min to remove insoluble materials, the Cur concentration was determined by the aforementioned method in Section 2.3.1. A similar experiment was carried out in 50 mM PBS (pH 7.4) containing 0.05% Tween 80 without any enzyme or pH adjustment.

2.4. Stability evaluation 2.4.1. Physical stability The stability of the colloidal dispersions was evaluated, using a Turbiscan Lab (Formulaction, Toulouse, France) according to Yuan, Gao, Zhao, and Mao (2008) with some modifications. The colloidal dispersions were sampled immediately after preparation and each sample was transferred to a glass cylindrical cell and analyzed by a light beam emitted in the near infrared (880 nm) wavelength. Two synchronous optical sensors received, respectively, light transmitted through the sample and light backscattered by the sample. The backscattering was directly dependent on the particle mean diameter. The results were expressed as Turbiscan stability index (TSI) which reflects changes in the intensity of backscattering light. The sample in the cell was scanned every 30 min for 8 h at 25 °C. 2.4.2. Photochemical stability In order to determine photochemical stability, the solutions of free Cur and Cur-loaded colloidal dispersions were subjected to illumination in a light cabinet (Q-Sun, Q-Lab Corporation, Ohio, USA) for 60 min. The light intensity was set at 0.35 W/m2 and temperature was set at 35 °C (Wang, Liu, & Gao, 2016). The sampling was carried out at time points of 5, 10, 15, 30, 45 and 60 min. The amount of Cur in the samples was determined according to the aforementioned method in Section 2.3.1. Each sample was analyzed in triplicate and results were expressed as means ± standard deviation. 2.4.3. Thermal stability The thermal stability of Cur in composite colloidal particles was analyzed according to the method described by Liu, Liu, Zhu, Gan, and Le (2015) with some modifications. Briefly, the freshly prepared dispersions were placed in a thermostatic water bath at temperatures of 30, 40, 50, 60, 70, 80 and 90 °C for 20 min. After the thermal treatment, the samples were immediately cooled in ice water to room temperature. The amount of Cur in the samples was then determined according to the aforementioned method in Section 2.3.1. 2.5. Release property An in vitro kinetic release test of Cur in simulated gastrointestinal fluids was performed, following the method of Li et al. (2016) with some modifications. As the solubility of Cur in water is very low, the water containing 0.05% Tween 80 was selected as the medium for release profile testing (Patel et al., 2010). Simulated gastric fluid (SGF) was prepared by adding 9 mg/ml of pepsin to 0.1 M HCl aqueous solution. For the preparation of simulated intestinal fluid (SIF), 7.2 mg/ml of pancreatin and 22.5 mg/ml of bile extracts were added to 0.1 M NaHCO3 aqueous solutions.

2.6. Statistical analysis The whole experiment was conducted in triplicate with data reported as the mean ± standard deviation. The analysis of variance (ANOVA) was performed by using the SPSS 12.0 package. Differences between pairs of means were compared using a Tukey test. The significance level (P) was set at 0.05. 3. Results and discussion 3.1. Formulation optimization: Particle size, zeta-potential and EE Both the particle size and zeta-potential are key parameters when obtaining stable nanoparticles as a delivery system for bioactive compounds. As shown in Fig. 1a), when the incorporated amount of Cur was increased, the particle size of Cur-loaded zein nanoparticles was changed from 118.3 ± 0.5 (Z/Cur20) to 240.2 ± 2.6 nm (Z/Cur100), and that of Cur-loaded shellac particles was varied from 62.9 ± 13.2 (S/Cur20) to 4280 ± 302.1 nm (S/ Cur100), respectively. The size of Z-S/Cur100 composite particles was larger than that of Z/Cur binary particles but smaller than that of S/Cur particles, indicating that the binary complex between zein and shellac was formed at pH 8.0 with a more compact structure than single shellac. The results suggested that Cur content strongly affected the particle size. The size of almost all nanoparticles was first increased and then decreased with increase of Cur concentration, except for the size of Z-S1:2/Cur. This result may be due to the fact that Cur inclusion, at a low level, induced the formation of a relatively loose structure between zein and shellac, and further increase in the concentration of Cur resulted in a compact structure, due to strong interactions among zein, shellac and Cur (Sun, Dai, & Gao, 2016). Z-S1:2/Cur exhibited a consistent increase in particle size with increase in the levels of Cur. A possible explanation was that, at a higher proportion of shellac, a network structure was formed between zein and shellac, and aggregation gradually occurred with increased level of Cur, which also indicated that excessive Cur could not be embedded in the network (Luo, Teng, & Wang, 2012). A similar result was reported by Sarika and Nirmala (2016), who found that the presence of Cur inside gum arabic aldehyde-gelatin nanogel increased the particle size. The zeta-potential of zein nanoparticles (Fig. 1g) was 19.7 ± 1.9 mV and remained almost constant as the content of Cur was increased. Zein was reported to have an isoelectric point (pI) of pH 6.2 (Rishi & Munir, 2001) and, at pH 8.0, zein molecules should therefore have a net negative charge. This result was in accordance with Hu et al. (2015) who reported that the zeta potential of Cur-fortified zein nanoparticles was not strongly affected by Cur content. As shown in Fig. 1h, the zeta-potentials of the individ-

659

C. Sun et al. / Food Chemistry 228 (2017) 656–667 Z-S5:1 5000

Z-S2:1

(a)

Z-S1:2

4000

3000

60

2000

1000

0

Z-S 5:1 Z-S 2:1 Z-S 1:1 Z-S 1:2 Zein Shellac

(b)

80

Zein Shellac

EE (%)

Particle size (nm)

100

Z-S1:1

40

20

Z-S Z-S /C

ur 10

Z-S /C

ur 20

Z-S /C

ur 50

Z-S /C

ur 10

0

Z-S /C

ur 20

0

0

10 Z-S/Cur

20 Z-S/Cur

50 Z-S/Cur

-15

-15

-20

-20

-25

-25

-potential (mV)

-potential (mV)

(C)

-30

100 Z-S/Cur

200

Z-S/Cur

Samples

Samples

-35

(d)

-30

-35

-40

-40 Z-S5:1

10 Z-S/Cur

20

Z-S/Cur

50 100 200 Z-S/Cur Z-S/Cur Z-S/Cur

Z-S2:1

10 Z-S/Cur

50 100 200 Z-S/Cur Z-S/Cur Z-S/Cur

20 Z-S/Cur

Samples

Samples

-15

(e) -15

(f)

-20

-potential (mV)

-potential (mV)

-20

-25

-30

-35

-25

-30

-35

-40

-40

Z-S1:1 Z-S1/Cur10 Z-S1/Cur20 Z-S1/Cur50Z-S1/Cur100Z-S1/Cur200

Z-S1:2

Samples

-15

10 Z-S/Cur

20 Z-S/Cur

50 100 S/Cur200 Z-S/Cur Z-S/Cur Z-

Samples

(g)

-10

(h)

-15

-20

-potential (mV)

-potential (mV)

-20

-25

-30

-25 -30 -35

-35 -40 -45

-40

Z

Z/Cur10

Z/Cur20

Z/Cur50

Samples

0 Z/Cur10

0 Z/Cur20

Cur

S

S/Cur10

S/Cur20

0 0 S/Cur50 S/Cur10 S/Cur20

Samples

Fig. 1. Particle size (a), entrapment efficiency of curcumin (b) and zeta-potential (c-h) of samples with different zein to shellac mass ratios of 5:1 (c), 2:1 (d),1:1 (e), 1:2 (f), zein (g) and shellac (h).

660

C. Sun et al. / Food Chemistry 228 (2017) 656–667

ual shellac and curcumin solutions were 12.4 ± 0.8 and 24.4 ± 0.7 mV, respectively. The incorporation of Cur led to an increase of the absolute value of the zeta-potential of Curentrapped shellac colloidal particles from 12.4 ± 0.8 to 40.1 ± 0.9 mV, which might be attributed to the fact that the addition of Cur led to more intensive cross-linking between shellac molecules, and excessive Cur was absorbed onto the surface of shellac particles. The zeta-potentials for samples of Z-S5:1/Cur, ZS2:1/Cur, Z-S1:1/Cur and Z-S1:2/Cur are shown in Fig. 1c, d, e and f, respectively. The changing tendency for the absolute values of zeta-potential of Cur-loaded Z-S composite particles was that they all first declined but then increased with the rise of Cur proportion, except for those of Z-S1:2/Cur which exhibited a constant increased absolute value of zeta-potential. These results suggested that the change in zeta-potential was dependent on the amount of Cur in the ternary complex, due to the fact that Cur alone had a charge of 24.4 ± 0.7 mV, as shown in Fig. 1f. A small amount of Cur (ZS/Cur10 and Z-S/Cur20) was well distributed inside the particles while the excessive Cur (Z-S/Cur50, Z-S/Cur100 and Z-S/Cur200) was adsorbed on the surface of particles. The effects of different formulations on EE are shown in Fig. 1b). As expected, there was a gradual decrease in EE with increase of Cur concentration. When the percentage of Cur to zein or shellac was changed from 10 to 20, the EE of Cur in zein nanoparticles was decreased from 82.7% to 42.5% (P < 0.05), which was lower than that in shellac particles (decreased from 91.5% to 80.5%). The EE of Cur in Z-S1:2 (Z-S1:2/Cur20) was decreased from 93.2% to 87.5% (P < 0.05). This finding suggested that the cross-link among Cur, zein and shellac, through non-covalent interactions, improved the embedding ability of colloidal particles. There are three hydroxyl groups and one carbonyl group in a Cur molecule, which may interact with the hydroxyl groups and carbonyl groups of shellac and tyrosyl of zein to form hydrogen bonds (Mehanny et al., 2016; Soradech, Limatvapirat, & Luangtana-Anan, 2013), resulting in an increase of EE. These findings indicated that zein and shellac showed a synergistic effect on EE of Cur, which may be ascribed to the increased viscosity induced by the incorporation of shellac, leading to strong entrappable ability of Cur (Vandenberg, Drolet, Scott, & De la Noüe, 2001). Liang et al. (2015) found that N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) resulted in a higher EE compared to the single zein nanoparticles, as the longer chain of the HTCC molecule could entrap more Cur. On the basis of the aforementioned results, it is believed that ZS2:1/Cur, Z-S1:1/Cur and Z-S1:2/Cur showed favourable EE of Cur among the Z-S/Cur systems. In addition, when the percentage of Cur in the complexes of Z-S2:1, Z-S1:1 and Z-S1:2 was 20, particle sizes of all samples were on a micron scale, with a relatively higher EE of Cur. As a result, the samples of Z-S/Cur20 were selected for further study, including the samples of Z-S2:1/Cur20, Z-S1:1/Cur20and Z-S1:2/Cur20, which are termed as Z-S2:1/Cur, Z-S1:1/Cur and Z-S1:2/Cur in the following descriptions. 3.2. FTIR analysis Fourier transform infrared measurements were performed to obtain some information about the nature of the possible interactions inside the colloidal particles. Fig. 2a) shows the FTIR spectra of single zein, shellac and Cur, and Fig. 2b) shows those of Curloaded zein-shellac complex particles. In the spectrum of Cur, the most obvious peak, at 1627.44 cm1, was attributed to the stretching of C@O, and the peak at 1602.41 cm1 is stretching vibrations of aromatic rings. No peak was observed in the carbonyl region (1800–1650 cm1), suggesting that Cur existed in the keto-enol tautomeric form (Mangolim et al., 2014). The peaks at 1428.72, 1183.39, 1154.04 cm1 were related to the vibrations of CACAC,

CACAH and CAOAC of aromatic rings and the inter-ring chain of Cur (Liu et al., 2015; Yadav & Kumar, 2014), which was not observed in Cur-loaded zein and/or shellac colloidal particles. The characteristic peak of Cur, at 1513.27 cm1, was due to the vibrations of CAO and CAC (Li et al., 2016). Interestingly, this peak was masked by amide II bands from zein, which still existed in the samples of Cur-loaded Z/S composite particles, suggesting that hydrophobic interaction between the Cur and Z/S complex may occur. The peak at 1276.51 cm1was shifted to 1273.64, 1283.52, 1282.08, 1284.36 cm1 in the spectra of Z/Cur, Z-S2:1/Cur, Z-S1:1/ Cur, Z-S1:2/Cur complex particles, which may result from the stretching and bending vibrations of different C-O groups (Mohammed-Ziegler & Billes, 2002). A similar result was reported by Hu et al. (2015) who found that the peak at 1273.0 cm1, observed in the spectrum of Cur, was shifted to 1278.0 cm1 in the spectrum of the Cur-loaded zein-pectin nanoparticles. The peak at 1116.74 cm1 was assigned to m(O-CH3), d(CCH) of aromatic rings (Kolev, Velcheva, Stamboliyska, & Spiteller, 2005), which was shifted to 1110.34 cm1 in the spectrum of Z/Cur binary complex particles due to the interaction between Cur and Z after Cur encapsulation. The characteristic peak of Cur at 1232.99 cm1 was not observed in the spectra of Cur-loaded Z-S complex colloidal particles, indicating that other intermolecular forces, e.g. hydrogen bonding, might exist among Cur, zein and shellac due to the interactions among hydrophilic groups. These results were in accordance with previous reports that the interactions between zein and curcumin consisted of hydrogen bond and hydrophobic effects (Zou, Li, Percival, Bonard, & Gu, 2012). Zein and shellac exhibited broad characteristic peaks at 3313.40 and 3415.76 cm1, respectively. According to the study of Cerqueira, Souza, Teixeira, & Vicente (2011), the broad band at 3100– 3500 cm1 was due to the OH stretching vibration of hydroxylsbound water. However, after the formation of the Cur-loaded Z-S complex, a shift of hydrogen bands occurred, further confirming that strong hydrogen bonds were formed among zein, shellac and Cur because hydrogen bonds could be generated between amide groups of glutamine in zein and hydroxyl groups in shellac and Cur. 3.3. Physical stability and thermal property Fig. 3(a) shows the TSI profiles as a function of time for 8 h at an interval of 0.5 h to characterize the phase separation kinetics of colloidal dispersions. Each point of the graph represents the backscattering variation from 4 cm height of the colloidal dispersions which were placed in glass cylindrical cells. The slopes indicate the rate of colloidal particle aggregation, sinking to the lower part of the cell. The colloidal dispersion was stabilized by the steric hindrance and electrostatic properties of the repulsive forces that could prevent flocculation and coalescence (Panaras, Moatsou, Yanniotis, & Mandala, 2011). A lower slope signified stronger resistance of colloidal particles against the aggregation and gravitational separation. TSI for the samples of Z-S2:1/Cur, Z-S1:1/Cur, Z/ Cur and S/Cur at 8 h were 1.54, 2.18, 14.17 and 8.43, respectively, indicating that the physical stability of ternary composite colloidal dispersions was better than that of binary ones. However, the TSI of the sample of Z-S1:2/Cur was 12.92, implying that shellac, at a high proportion in Z-S/Cur, may disrupt the physical stability, due to its large particle size as confirmed in Fig. 1a. Fig. 3b) shows the DSC thermograms for zein, shellac, Cur, Curloaded zein-shellac composite colloidal particles. In the curve of Cur, there was a sharp endothermic peak around 166.5 °C, which was attributed to the melting of crystalline regions of Cur. Jasim and Talib (1992) reported that the melting point of Cur was around 170 °C. However, this melting peak was not observed in the curves of samples for Z-Cur, S-Cur and Z-S/Cur, indicating that Cur was

661

3511.07

1232.99

3390.73

1576.28

Curcumin

Shellac

1657.81

3313.40

1642.14

3415.76

Absorbance (a.u.)

1116.74

1276.51 1183.39 1154.04

1627.44 1602.41 1513.27

(a)

1428.72

C. Sun et al. / Food Chemistry 228 (2017) 656–667

Zein 4000

3500

3000

2000

1500

1000

500

-1

1116.98 1115.29 1116.57

Z-S1:1/Cur

1116.36

1282.08

Z-S1:2/Cur

Z-S2:1/Cur

1110.34

3261.35

1273.64

1513.77

1283.52

1514.67

3417.46

Absorbance (a.u.)

1514.92

1514.96

(b)

1284.36

Wavenumber (cm )

S/Cur

Z/Cur 4000

3500

3000

2000

1500

1000

500

-1

Wavenumber (cm ) Fig. 2. FTIR spectra of individual zein, shellac and curcumin (a), and the complexes of zein-curcumin, shellac-curcumin and curcumin-loaded zein-shellac (b).

well dispersed in the polymeric matrix and encapsulated in the amorphous state. A similar observation has been reported by Shaikh, Ankola, Beniwal, Singh, and Kumar (2009) who confirmed that the crystalline Cur was converted to the amorphous state after being entrapped in a nanoscale polymeric matrix. Zein and shellac exhibited broad endothermic peaks at 108.5 and 97.7 °C, respectively. These characteristic endotherms could be associated with the evaporation of bound water from the polymer (Parveen, Mitra, Krishnakumar, & Sahoo, 2010), and were shifted towards lower temperatures at 92.7 (Z-Cur), 95.7 (Z-S2:1/ Cur), 93.1 (Z-S1:1/Cur) and 76.3 °C (Z-S1:2/Cur) after Cur incorpora-

tion. Similarly, the melting peaks of Cur-loaded composite colloidal particles were shifted to lower temperatures than those of zein. The decrease in melting temperatures may be attributed to intermolecular interactions among Cur, zein and shellac, as confirmed by the FTIR analysis in this work, which resulted in the formation of Cur-loaded zein-shellac complexes with a loose network structure and large particle size, leading to weak stability against the thermal treatment. Nevertheless, zein was found as typical spherical particles, with compact structure and small size, which required higher temperature to destroy its molecular structure (Wang & Padua, 2010).

662

C. Sun et al. / Food Chemistry 228 (2017) 656–667

(a) Z-S2:1/Cur Z-S1:1/Cur

14

Z-S1:2/Cur Z/Cur S/Cur

12

TSI (%)

10 8 6 4 2 0 0

1

2

3

4

5

6

7

8

9

Time (h)

(b) Z-S1:2/Cur

Z-S1:1/Cur

Heat flow endo up (mW)

Z-S2:1/Cur S/Cur Z/Cur Cur Shellac Zein

50

100

150

Temperature (

200

250

)

Fig. 3. Turbiscan stability index (TSI) values as a function of time (a) and DSC thermograms (b) of samples, including individual zein, shellac and curcumin, and the complexes of zein-curcumin and shellac-curcumin, and curcumin-loaded zein-shellac.

3.4. Atomic force microscopy AFM is usually applied to characterize the three-dimensional structure of food materials with the dual advantage of being a nondestructive process and involving minimal sample preparation. In the present study, we took advantage of the unique properties of AFM to explore the microstructure of zein, shellac, zein-shellac binary complex and Cur-loaded zein-shellac composite particles. As shown in Fig. 4, zein exhibited typical nanospheres, as reported in our previous study (Sun, Dai, He, et al., 2016). Shellac showed diverse shapes, including approximately 31.3% nanospheres, and 18.8% oval and other irregular shapes. When shellac was mixed with zein at the mass ratio of 2:1, the morphology of zein-shellac

binary composites presented inhomogeneous spherical shapes with diameters decreasing from 0.91 to 0.62 lm, suggesting that zein and shellac could cross link to form smaller particles with more compact structures. A similar phenomenon was also reported for zein-chitosan: composite particles remained spherical (Luo, Zhang, Whent, Yu, & Wan, 2011), but the coating by shellac increased the particle size of bare magnetic nanoparticles (Gong et al., 2012). Distinctly different morphologies were observed in the AFM images when Cur was embedded in zein-shellac complex particles; these were dependent on the mixing mass ratio of zein to shellac. It was found that the sample of Z-S2:1/Cur exhibited a network structure containing several microspheres with diameters ranging from

C. Sun et al. / Food Chemistry 228 (2017) 656–667

0.64 to 1.89 lm, indicating that the incorporation of Cur led to the formation of aggregates of irregular shape and with a large size. The network structure may well explain the higher EE of Cur in zein-sheallc composite colloidal particles than that of individual zein or shellac particles. When the amount of shellac was increased, the samples of Z-S1:1/Cur and Z-S1:2/Cur exhibited random geometry. It was also observed that the phenomenon of conglutination, with integration or fusion of different entities, occurred at a high proportion of shellac. The observation may be explained by the FTIR analysis because Cur could interact with zein and shellac by hydrogen bonding and hydrophobic effects, which may enhance the intermolecular cross linking. 3.5. Photochemical and thermal stability The applications of Cur are limited because of its sensitivity to ultraviolet radiation and thermal treatment. The present work explored the thermal and ultraviolet light stability of the encapsulated Cur in comparison to the free Cur (Fig. 5). The data from the

663

UV irradiation are summarized in Fig. 5a). Cur loaded in colloidal dispersions was found to be more stable against UV irradiation than was free Cur. At the end of UV irradiation for 1 h, more than 88% of Cur was retained in the colloidal dispersion (Z-S1:1/Cur) as compared to less than 24% in the free Cur solution. The rate of Cur degradation was significantly (P < 0.05) decreased in the presence of a polymer matrix by provision of protection against the attack. This finding was consistent with the report of Patel et al. (2010). Thermal treatment at various temperatures (30, 40, 50, 60, 70, 80 and 90 °C) for 20 min incubation was performed to investigate the thermal stability of Cur, as shown in Fig. 5b). It was observed that after the treatment at 60 and 90 °C for 20 min, the content of Cur in the free Cur solution was reduced to 30.0% and 12.2%, respectively. When Cur was loaded onto colloidal particles, the retention of Cur was significantly (P < 0.05) enhanced. Particularly, the protective effect of Cur in zein-shellac composite particles was more obvious when the mass ratio of zein to shellac was 1:1 since the retained amounts of Cur after 60 and 90 °C treatment for

Fig. 4. AFM images of samples, including zein, shellac and the complexes of zein- shellac and curcumin-loaded zein-shellac.

664

C. Sun et al. / Food Chemistry 228 (2017) 656–667

Fig. 4 (continued)

20 min were 94.0% and 34.4%, which were thus enhanced by 3.1fold and 2.8-fold, respectively. This result was in line with the report of Liang et al. (2015) who found that zein/quaternized chitosan nanoparticles (at the weight ratio of 1:1) provided more protection for Cur against thermal treatment at 60 °C treatment for 30 min and 80 °C treatment for 1 min. Overall, the encapsulation of Cur into the zein-shellac complex matrix will be a potential way to provide protection against a harsh environment. One possible protection mechanism is that the binary complexation between zein and shellac induces more compact structure, as confirmed by AFM, which resulted in the improved thermal stability of Cur.

3.6. Release profiles The cumulative release of Cur in PBS medium and under simulated gastro-intestinal (SGI) conditions was evaluated by monitoring the concentration of Cur as a function of time (0–180 min). Fig. 6a) shows the detected percentage of Cur in PBS medium containing 0.05% Tween 80. All formulations presented a two-step released profile, including a burst release within 90 min, followed by a controlled and sustained release for up to 180 min. This finding was similar to the report by Zhong, Jin, Davidson, and Zivanovic (2009). Free Cur showed a burst release since less than 44% of Cur was detected in the medium after incubation for 180 min. The col-

C. Sun et al. / Food Chemistry 228 (2017) 656–667

665

Fig. 5. Curcumin retention during UV irradiation as a function of time (a) and during thermal treatment as a function of temperature (b) in free curcumin solution and in the colloidal dispersions.

loidal dispersion of zein-shellac complex exhibited an obvious sustained release and almost 90% of the Cur was retained when the mass ratio of zein to shellac was 1:1 after 180 min. According to the report of Patel et al. (2010), more Cur in the colloidal dispersion of zein was maintained compared to the free Cur solution. Fig. 6b) shows the digestion of Cur in simulated gastric fluids (SGF) and simulated intestinal fluids (SIF) in the presence of pepsin and pancreatin, respectively. The changing trend of release profile of Cur in simulated gastrointestinal fluids was similar to that in PBS medium. It was found that, after a digestion for 180 min, the detected level of Cur was 20.9% in free Cur solution, while the content of Cur was 49.10% in the sample of Z-S1:1/Cur. The findings indicated that zein-shellac complex particles provided a controlled and sustained release of Cur in both PBS medium and simulated gastrointestinal condition, which may be ascribed to the compact structure of Z-S/Cur induced by the interactions among zein, shel-

lac and Cur. Similar results were also revealed by Luo et al. (2011) and Somchue, Sermsri, Shiowatana, and Siripinyanond (2009), who suggested that the release of a-tocopherol in protein-based particles could be significantly retarded due to the polysaccharide coating.

4. Conclusions Curcumin could be interacted with zein and shellac to form a ternary complex at pH 8.0 as confirmed by FTIR analysis. The properties of Cur-loaded zein-shellac complex particles were dependent on the mass ratios of different components. Zein-shellac binary matrix provided a higher encapsulation efficiency of Cur than that of individual components. Cur entrapment induced distinctly different morphologies of zein-shellac complex particles,

666

C. Sun et al. / Food Chemistry 228 (2017) 656–667

(a) 90

Z-S1:1/Cur Z-S2:1/Cur S/Cur Z/Cur Cur

80

Curcumin detected (%)

Z-S1:2/Cur

70 60 50 40 30 20 10 0 0

20

40

60

80

100

120

140

160

180

120

140

160

180

Time (min) Z-S1:2/Cur

(b)

Z-S1:1/Cur

Curcumin detected (%)

50

Z-S2:1/Cur S/Cur Z/Cur Cur

40

30

20

10

0 0

20

40

60

80

100

Time (min) Fig. 6. Curcumin released profiles as a function of time in PBS medium (a) and simulated gastrointestinal tract condition.

dependent on the mixing mass ratio of zein to shellac. When the mass ratio of zein to shellac was 1:1, Cur-loaded zein-shellac composite particles showed the most effective prevention against curcumin degradation induced by thermal treatment and UV light radiation, and exhibited a great ability to sustain the release of curcumin in both PBS medium and simulated gastrointestinal tract conditions. The formation of zein-shellac complexes could provide a new insight into the development of delivery systems for bioac-

tive components in food systems, which may largely broaden the application of alcohol-soluble proteins and hydrophobic components in the food and beverage industries. Acknowledgements The research was funded by the National Natural Science Foundation of China (No. 31371835).

C. Sun et al. / Food Chemistry 228 (2017) 656–667

References Bollimpelli, V. S., Kumar, P., Kumari, S., & Kondapi, A. K. (2016). Neuroprotective effect of curcumin-loaded lactoferrin nano particles against rotenone induced neurotoxicity. Neurochemistry International, 95, 37–45. Cerqueira, M. A., Souza, B. W., Teixeira, J. A., & Vicente, A. A. (2011). Effect of glycerol and corn oil on physicochemical properties of polysaccharide films–A comparative study. Food Hydrocolloids, 27(1), 175–184. Chen, S., Zhang, N., & Tang, C. H. (2016). Influence of nanocomplexation with curcumin on emulsifying properties and emulsion oxidative stability of soy protein isolate at pH 3.0 and 7.0. Food Hydrocolloids, 61, 102–112. Facchi, S. P., Scariot, D. B., Bueno, P. V. A., Souza, P. R., Figueiredo, L. C., Follmann, H. D. M., ... Follmann, A. F. (2016). Preparation and cytotoxicity of N-modified chitosan nanoparticles applied in curcumin delivery. International Journal of Biological Macromolecules, 87, 237–245. Gómez-Estaca, J., Gavara, R., & Hernández-Muñoz, P. (2015). Encapsulation of curcumin in electrosprayed gelatin microspheres enhances its bioaccessibility and widens its uses in food applications. Innovative Food Science & Emerging Technologies, 29, 302–307. Gong, J. L., Chen, L., Zeng, G. M., Long, F., Deng, J. H., & He, X. (2012). Shellac-coated iron oxide nanoparticles for removal of cadmium (II) ions from aqueous solution. Journal of Environmental Sciences, 24, 1165–1173. Hu, K., Huang, X., Gao, Y., Huang, X., Xiao, H., & McClements, D. J. (2015). Core-shell biopolymer nanoparticle delivery systems: Synthesis and characterization of curcumin fortified zein-pectin nanoparticles. Food Chemistry, 182, 275–281. Hu, K., & McClements, D. J. (2015). Fabrication of biopolymer nanoparticles by antisolvent precipitation and electrostatic deposition: Zein-alginate core/shell nanoparticles. Food Hydrocolloids, 44, 101–108. Jasim, F., & Talib, T. (1992). Some observations on the thermal behaviour of curcumin under air and argon atmospheres. Journal of Thermal Analysis, 38, 2549–2552. Kolev, T. M., Velcheva, E. A., Stamboliyska, B. A., & Spiteller, M. (2005). DFT and experimental studies of the structure and vibrational spectra of curcumin. International Journal of Quantum Chemistry, 102, 1069–1079. Li, J. L., Shin, G. H., Lee, I. W., Chen, X. G., & Park, H. J. (2016). Soluble starch formulated nanocomposite increases water solubility and stability of curcumin. Food Hydrocolloids, 56, 41–49. Liang, H. S., Zhou, B., He, L., An, Y. P., Lin, L. F., Li, Y., ... Li, B. (2015). Fabrication of zein/quaternized chitosan nanoparticles for the encapsulation and protection of curcumin. RSC Advances, 5, 13891–13900. Liu, Y. J., Liu, D. D., Zhu, L., Gan, Q., & Le, X. Y. (2015). Temperature-dependent structure stability and in vitro release of chitosan-coated curcumin liposome. Food Research International, 74, 97–105. Luo, Y., Teng, Z., & Wang, Q. (2012). Development of zein nanoparticles coated with carboxymethyl chitosan for encapsulation and controlled release of vitamin D3. Journal of Agricultural and Food Chemistry, 60, 836–843. Luo, Y., Zhang, B., Whent, M., Yu, L. L., & Wan, Q. (2011). Preparation and characterization of zein/chitosan complex for encapsulation of alphatocopherol, and its in vitro controlled release study. Colloids and Surfaces BBiointerfaces, 85, 145–152. Mangolim, C. S., Moriwaki, C., Nogueira, A. C., Sato, F., Baesso, M. L., & Neto, A. M. (2014). Curcumin-beta-cyclodextrin inclusion complex: Stability, solubility, characterisation by FT-IR, FT-Raman, X-ray diffraction and photoacoustic spectroscopy, and food application. Food Chemistry, 153, 361–370. Mehanny, M., Hathout, R. M., Geneidi, A. S., & Mansour, S. (2016). Exploring the use of nanocarrier systems to deliver the magical molecule; Curcumin and its derivatives. Journal of Controlled Release, 225, 1–30. Messaoud, G. B., Sánchez-González, L., Probst, L., Jeandel, C., Arab-Tehrany, E., & Desobry, S. (2016). Physico-chemical properties of alginate/shellac aqueouscore capsules: Influence of membrane architecture on riboflavin release. Carbohydrate Polymers, 144, 428–437. Mohammed-Ziegler, I., & Billes, F. (2002). Vibrational spectroscopic calculations on pyrogallol and gallic acid. Journal of Molecular Structure: Journal of Theoretical and Computational Chemistry, 618, 259–265. Moorthi, C., Senthil Kumar, C., Mohan, S., & Kathiresan, K. (2013). SLS/bCD-curcumin nanosuspension: Preparation, characterization and pharmacological evaluation. Journal of Pharmacy Research, 7, 219–223.

667

Pan, K., & Zhong, Q. X. (2016). Low energy, organic solvent-free co-assembly of zein and caseinate to prepare stable dispersions. Food Hydrocolloids, 52, 600–606. Panaras, G., Moatsou, G., Yanniotis, S., & Mandala, I. (2011). The influence of functional properties of different whey protein concentrates on the rheological and emulsification capacity of blends with xanthan gum. Carbohydrate Polymers, 86, 433–440. Parveen, S., Mitra, M., Krishnakumar, S., & Sahoo, S. K. (2010). Retraction notice to enhanced antiproliferative activity of carboplatin loaded chitosan-alginate nanoparticles in retinoblastoma cell line. Acta Biomaterialia, 6, 3120–3131. Patel, A., Hu, Y., Tiwari, J. K., & Velikov, K. P. (2010). Synthesis and characterisation of zein-curcumin colloidal particles. Soft Matter, 6, 6192–6199. Phan, T. D., Debeaufort, F., Luu, D., & Voilley, A. (2008). Moisture barrier, wetting and mechanical properties of shellac/agar or shellac/cassava starch bilayer biomembrane for food applications. Journal of Membrane Science, 325, 277–283. Rishi, S., & Munir, C. (2001). Zein the industrial protein from corn. Industrial Crops and Products, 13, 171–192. Sarika, P. R., & Nirmala, R. J. (2016). Curcumin loaded gum arabic aldehyde-gelatin nanogels for breast cancer therapy. Materials Science and Engineering, 65, 331–337. Sharma, R. A., Gescher, A. J., & Steward, W. P. (2005). Curcumin: The story so far. European Journal of Cancer, 41, 1955–1968. Shaikh, J., Ankola, D. D., Beniwal, V., Singh, D., & Kumar, M. R. (2009). Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when compared to curcumin administered with piperine as absorption enhancer. European Journal of Pharmaceutical Sciences, 37, 223–230. Somchue, W., Sermsri, W., Shiowatana, J., & Siripinyanond, A. (2009). Encapsulation of a-tocopherol in protein-based delivery particles. Food Research International, 42, 909–914. Soradech, S., Limatvapirat, S., & Luangtana-Anan, M. (2013). Stability enhancement of shellac by formation of composite film: Effect of gelatin and plasticizers. Journal of Food Engineering, 116, 572–580. Sun, C., Dai, L., & Gao, Y. (2016). The Binary complex based on zein and propylene glycol alginate for delivery of quercetagetin. Biomacromolecules, 17, 3973–3985. Sun, C., Dai, L., He, X., Liu, F., Yuan, F., & Gao, Y. (2016). Effect of heat treatment on physical, structural, thermal and morphological characteristics of zein in ethanol-water solution. Food Hydrocolloids, 58, 11–19. Vandenberg, G. W., Drolet, C., Scott, S. L., & De la Noüe, J. (2001). Factors affecting protein release from alginate-chitosan coacervate microcapsules during production and gastric/intestinal simulation. Journal of Controlled Release, 77, 297–307. Wang, Y., & Padua, G. W. (2010). Formation of zein microphases in ethanol-water. Langmuir, 26, 12897–12901. Wang, Q., Crofts, A. R., & Padua, G. W. (2003). Protein-lipid interactions in zein films investigated by surface plasmon resonance. Journal of Agricultural and Food Chemistry, 51, 7439–7444. Wang, W., Liu, F., & Gao, Y. (2016). Quercetagetin loaded in soy protein isolate-jcarrageenan complex: Fabrication mechanism and protective effect. Food Research International, 83, 31–40. Wang, W., Sun, C., Mao, L., Ma, P., Liu, F., Yang, J., et al. (2016). The biological activities, chemical stability, metabolism and delivery systems of quercetin: A review. Trends in Food Science & Technology, 56, 21–38. Wang, X., Yu, D. G., Li, X. Y., Bligh, S. W. A., & Williams, G. R. (2015). Electrospun medicated shellac nanofibers for colon-targeted drug delivery. International Journal of Pharmaceutics, 490, 384–390. Yadav, D., & Kumar, N. (2014). Nanonization of curcumin by antisolvent precipitation: Process development, characterization, freeze drying and stability performance. International Journal of Pharmaceutics, 477, 564–577. Yuan, Y., Gao, Y., Zhao, J., & Mao, L. (2008). Characterization and stability evaluation of b-carotene nanoemulsions prepared by high pressure homogenization under various emulsifying conditions. Food Research International, 41, 61–68. Zhong, Q., Jin, M., Davidson, P. M., & Zivanovic, S. (2009). Sustained release of lysozyme from zein microcapsules produced by a supercritical anti-solvent process. Food Chemistry, 115, 697–700. Zou, T., Li, Z., Percival, S. S., Bonard, S., & Gu, L. (2012). Fabrication, characterization, and cytotoxicity evaluation of cranberry procyanidins-zein nanoparticles. Food Hydrocolloids, 27, 293–300.