Fabrication and characterization of binary composite nanoparticles between zein and shellac by anti-solvent co-precipitation

Fabrication and characterization of binary composite nanoparticles between zein and shellac by anti-solvent co-precipitation

Food and Bioproducts Processing 1 0 7 ( 2 0 1 8 ) 88–96 Contents lists available at ScienceDirect Food and Bioproducts Processing journal homepage: ...

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Food and Bioproducts Processing 1 0 7 ( 2 0 1 8 ) 88–96

Contents lists available at ScienceDirect

Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp

Fabrication and characterization of binary composite nanoparticles between zein and shellac by anti-solvent co-precipitation Shuai Chen, Chenqi Xu, Like Mao, Fuguo Liu, Cuixia Sun, Lei Dai, 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 and Nutritional Engineering, China Agricultural University, 100083, China

a r t i c l e

i n f o

a b s t r a c t

Article history:

The anti-solvent co-precipitation method was applied to fabricate zein (Z) and shellac (S)

Received 19 April 2017

composite nanoparticles with different mass ratios (Z:S, 50:1, 10:1, 5:1, 2.5:1, 1:1, 1:1.5 and

Received in revised form 13 October

1:2.5) at pH 8.0. Measurement of particle size and turbidity, in combination with analy-

2017

ses of Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), differential

Accepted 13 November 2017

scanning calorimetry (DSC), fluorescence spectroscopy, and atomic force microscope (AFM)

Available online 21 November 2017

were performed to characterize Z–S composite nanoparticles. Results showed that hydrogen bonding and hydrophobic attraction were involved in the interactions between zein and

Keywords:

shellac, leading to the changes in secondary structure and thermal stability of zein. At low

Zein

levels of shellac (Z:S, from 50:1 to 2.5:1), a compact structure of Z–S composite nanoparti-

Shellac

cles was formed, which had smaller particle sizes, higher turbidity value and better thermal

Composite nanoparticles

stability. At high levels of shellac (Z:S, from 2.5:1 to 1:2.5), a cross-linked structure of Z–S

Interaction

composite nanoparticles was generated, which exhibited larger particle sizes, lower turbid-

Characterization

ity value, and poorer thermal stability. The potential mechanism of a two-step process was

Anti-solvent co-precipitation

proposed to explain the formation of Z–S composite nanoparticles. Findings in the present work will help further understand the interaction between alcohol-soluble biopolymers (e.g. zein and shellac) and provide a new insight into the development of potential carriers for bioactive compounds. © 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1.

Introduction

Nanoparticles have particular physicochemical properties and functional attributes. They are increasingly being applied as delivery systems in the food industry in order to improve the stability and oral bioavailability of bioactive components, e.g. curcumin, ˇ-carotene, and quercetin. Nanoparticles have an advantage over hydrogels, organogels, liposome, and microparticles due to their smaller particle size, higher encapsulation efficiency, more effective penetration ability and targetability (Wang et al., 2016). Nanoparticles are usually fabricated from varieties of natural polymers, mainly including food-



grade proteins and polysaccharides, because they are biocompatible, biodegradable, and non-toxic properties (Joye and McClements, 2016), such as soy protein (Chen et al., 2016), lactoferrin (Bollimpelli et al., 2016), gelatin (Gómez-Estaca et al., 2015), chitosan (Facchi et al., 2016), alginate (Hu and McClements, 2015), and ˇ-cyclodextrin (Moorthi et al., 2013). Protein-based nanoparticles are usually formed with water-soluble proteins, such as soy protein and lactoferrin, as well as alcohol-soluble proteins. Zein, the main storage protein in corn seeds, contains over 50% hydrophobic amino acids, and it can be easily converted into spherical colloidal nanoparticles by the method of anti-solvent precipitation

Corresponding author. E-mail address: [email protected] (Y. Gao). https://doi.org/10.1016/j.fbp.2017.11.003 0960-3085/© 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Food and Bioproducts Processing 1 0 7 ( 2 0 1 8 ) 88–96

(ASP) (Joye and Mcclements, 2013). Compared with zein nanoparticles, the composite nanoparticles prepared by zein and biopolymers have some advantages. Liang et al. (2015) revealed that zein-quaternized chitosan composite nanoparticles provided a better protection for curcumin than zein nanoparticles. Hu and McClements (2015) reported that zein–alginate composite nanoparticles had good stability to pH (from pH 3.0 to 8.0) and temperature (90 ◦ C, 120 min). Hu et al. (2015) prepared core–shell 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 curcumin. Nevertheless, most previous studies focused on the development of complexes using zein and hydrophilic biopolymers, such as alginate (Hu and McClements 2015), chitosan (Liang et al., 2015), pectin (Hu et al., 2015), and caseinate (Pan and Zhong, 2016). Because of many bioactive components, e.g. curcumin, ˇ-carotene, and quercetin were hydrophobic, they might tend to be encapsulated in the hydrophobic delivery system. Yet little information was available on the composite nanoparticle systems consisting of zein and hydrophobic biopolymers. Shellac is a resinous secretion of the female insect Kerrialacca, which is a natural hydrophobic biopolymer, and mainly contains the polyesters of hydroxy fatty acid and sesquiterpene acids. It has a good film-forming capacity, high gloss, and low permeability to gases, acid and water vapor, which allow it for coating purposes in foods and drugs (Wei et al., 2015a). Al-Gousous et al. (2015) studied the disintegration properties of shellac-based enteric coatings, and regarded shellac as a promising coating material, especially for use in colon-targeted drug delivery since it could resist the stomach acid environment and achieve a timed enteric or colonic release (Wang et al., 2015). Therefore, shellac was chosen to fabricate composite nanoparticles with zein in the present study. Previous studies mainly focused on the formation of protein and biopolymer composite nanoparticles commonly resulting from elec-

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saline (PBS). The zein solution (10 mg/mL) and shellac solution (10 mg/mL) were stirred by a constant magnetic stirring at 600 rpm for 2 h at room temperature, and then were adjusted to pH 8.0. Shellac solution was diluted by 80% ethanol aqueous solution to acquire seven different concentrations, and 10 mL of each shellac solution was mixed with zein solution at a fixed concentration in an equivalent volume on a vortex shaker at 1500 rpm for 30 s, which formed the resulting solutions with seven Z:S mass ratios (50:1, 10:1, 5:1, 2.5:1, 1:1, 1:1.5, 1:2.5). The mixed solutions were held for 2 h at room temperature. Afterwards, Z–S composite nanoparticles were formed by the ASCP method described in the previous literature with some modifications (Sun et al., 2017). Briefly, 20 mL zein and shellac ethanol aqueous solution was slowly injected into 60 mL PBS (pH 8.0) using a syringe with a constant stirring at 600 rpm for 20 min. To acquire aqueous dispersions, three quarters of ethanol aqueous solution were removed under reduced pressure (−0.1 MPa) by rotary evaporation at 45 ◦ C. The composite nanoparticle dispersions of Z–S were acquired. Individual zein or shellac dispersion was prepared by the same way as controls. The colloidal nanoparticle dispersions were stored in the refrigerator at 4 ◦ C for further analysis, and part of the dispersions were freeze-dried for 48 h with Alpha 1–2 D Plus freeze-drying apparatus (Marin Christ, Germany) to acquire dry particles for solid state characterization analysis.

2.3.

Particle size and zeta-potential measurements

ticles, and investigated the interaction mechanism between zein and amphiphilic biopolymer (e.g. PGA). Nevertheless, shellac is a hydropho-

Particle size of Z–S composite nanoparticles 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 analysis, and 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 values of zein and shellac dispersions were determined in triplicate, and the data were calculated by the instrument using the Smoluchowski model.

bic biopolymer, the interaction mechanism between zein and shellac, and characteristics of Z–S binary composite nanoparticles prepared by

2.4.

trostatic interactions between oppositely charged macromolecules (De Kruif et al., 2004). It is worthy to mention that zein and shellac, have similar electric charges in acidic or alkaline environment. A hypothesis is proposed that zein and shellac might form a special structure. The method of anti-solvent co-precipitation (ASCP) was applied to prepare Z–S composite nanoparticles, which was obviously different from the traditional anti-solvent precipitation method (Joye and Mcclements, 2013; Davidov-Pardo et al., 2015; Luo et al., 2011; Yadav and Kumar, 2014). Sun et al. (2017) used the ASCP method to fabricate zein and propylene glycol alginate (PGA) composite nanopar-

ASCP method were investigated. The purpose in present study was to explore the formation mechanism and the physical, structural, thermal and morphological characteristics of Z–S composite nanoparticles. Results from present work might play a promoting role in developing novel carriers for bioactive compounds, which would have potential application for nutraceutical delivery systems.

2.

Materials and methods

2.1.

Materials

Shellac (wax-free) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Zein with a protein content of 95% (w/w) was obtained from Gaoyou Group Co., Ltd. (Jiangsu, China). Ethanol (99.9%) was purchased from Eshowbokoo Biological Technology Co., Ltd. (Beijing, China). All other chemicals were analytical grade unless stated otherwise.

2.2.

Preparation of Z–S composite nanoparticles

Zein and shellac were separately dissolved in 500 mL 80% ethanol aqueous solution containing 0.1 M phosphate buffer

Turbidity measurement

Nephelometry measurements were performed in a HACH 2100N laboratory turbidimeter (Loveland, USA), and the turbidity of Z–S composite nanoparticle dispersions was evaluated as described by Yang et al. (2014a). The optical apparatus was equipped with a tungsten-filament lamp with three detectors: a 90◦ scattered-light detector, a forward-scatter light detector and a transmitted light detector. The calibration was performed using a Gelex Secondary Turbidity Standard Kit (HACH, Loveland, USA), which consists of stable suspensions of a metal oxide in a gel. All measurements were performed in triplicate.

2.5.

Fourier transform infrared (FTIR) spectroscopy

The infrared spectra of samples were measured with the potassium bromide (KBr) pellet method described by Sun et al. (2015) using a Spectrum 100 Fourier transform spectrophotometer (PerkinElmer, U.K.) in the range of 400–4000 cm−1 , with a resolution of 4 cm−1 . Potassium bromide was used as a reference. For each measurement, 11 scans were taken. All the samples analyzed were freeze-dried from evaporated disper-

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sions, including zein, shellac and Z–S composite particles. The data were analyzed using Omnic v8.0 (Thermo Nicolet, USA).

2.6.

Circular dichroism (CD) measurement

CD spectra were performed using a CD spectropolarimeter (Pistar ␲-180, Applied Photophysics Ltd., U.K.) at a far-UV (190–260 nm) region under constant nitrogen flush. Path length was 0.1 cm for the far-UV region. Ellipticity of Z–S composite nanoparticle dispersions was recorded at a speed of 100 nm/min, 0.2 nm resolution, 20 accumulations, and 2.0 nm bandwidth. The collected data were analyzed using Dichroweb: the online Circular Dichroism Website http://dichroweb.cryst.bbk.ac.uk (Whitmore and Wallace, 2004). The fractions of ˛-helix, ˇ-sheet, and unordered coil were estimated by K2D-SET4. Each spectrum was the average of three consecutive measurements.

2.7.

Differential scanning calorimetry (DSC) analysis

The thermal characteristics of Z–S composite nanoparticles were studied using differential scanning calorimetry as described by Pan and Zhong (2016) with some modifications. Briefly, 3.0 mg of each freeze-dried sample was weighed in an aluminium pan and hermetically sealed. The lids were pinned by a syringe needle to exclude the interference of moisture. Samples were heated from 30 ◦ C to 150 ◦ C at a heating rate of 10 ◦ C/min. The peak denaturation temperature (Td ), the onset denaturation temperature (Tonset ) and the enthalpy of denaturation (H) were computed using a DSC analyzer (DSC-60, Shimadzu, Tokyo, Japan) from each thermal curve.

Fig. 1 – The size (A) and turbidity (B) of composite nanoparticles of zein and shellac at different mass ratios.

2.10. 2.8.

Fluorescence measurements were carried out using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) as described by Liu et al. (2017). Spectra of Z–S composite nanoparticle dispersions were collected at 290–450 nm with a scanning speed of 100 nm/min. The fluorescence emission spectrum of zein was recorded with the excitation wavelength at 280 nm. Both excitation and emission slit widths were set at 10 nm. Protein intrinsic fluorescence was measured at a constant zein concentration (0.25 mg/mL) in the presence of shellac at different mass ratios. Each individual emission spectrum was the average of three runs. All data were collected at room temperature.

2.9.

Statistical analysis

Fluorescence spectroscopy analysis

Atomic force microscope (AFM) analysis

AFM was used to observe the morphology of zein, shellac and Z–S composite nanoparticles. The measurements were performed according to the method described by Wang et al. (2004). The freeze-dried samples were dissolved in PBS (pH 8.0) to reach a concentration of 10–15 ␮g/mL. About 2.0 ␮L of the solution was dripped onto the mica slide and allowed to dry for 1.0 h 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 340 kHz, a scan rate of 0.7 Hz and a force constant of 40 N/m.

All experiments were performed in triplicate and the results were expressed as mean value ± standard deviation (SD) in this study. Data were subjected to analysis of variance (ANOVA) using the software package SPSS 12.0 for Windows (SPSS Inc., Chicago, IL). Significant differences (p < 0.05) between means were identified using the Duncan procedure.

3.

Results and discussion

3.1. Particle size and turbidity of Z–S composite nanoparticles Particle size distribution is a key factor for obtaining stable nanoparticles as delivery systems of functional components. As shown in Fig. 1A, when the Z:S mass ratio was decreased from 50:1 to 2.5:1, the particle size of Z–S composite nanoparticles was significantly (p < 0.05) decreased from 94 nm to 57 nm, and these binary composite nanoparticles were much smaller (d < 100 nm) than individual zein colloidal particles (107 nm). These results revealed that a low concentration of shellac led to the formation of binary composite nanoparticles with a more compact structure. It might be due to the fact that zein and shellac are both hydrophobic biopolymers and negatively charged (−2.87 mV and −4.44 mV at pH 8.0, respectively). Thus, the zein molecule might be intervolved by shellac at a low concentration, and the hydrophobic interactions and electrostatic repulsion between zein and shellac could prevent the

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Fig. 2 – (A) FTIR spectra of native shellac, zein and composite nanoparticles of zein and shellac at mass ratios of 10:1, 2.5:1, 1:2.5. (B) CD spectra of native zein and composite nanoparticles of zein and shellac at mass ratios of 50:1, 10:1, 5:1, 2.5:1, 1:1, 1:1.5, 1:2.5. formation of large-size zein molecular aggregates (Kim and Xu, 2008). When the Z:S mass ratio was below 2.5:1, the composite particle size was significantly (p < 0.05) increased from 57 nm (Z:S, 2.5:1) to 245 nm (Z:S, 1:2.5). When the Z:S mass ratio was below 1:1.5, the composite particle size became larger (d > 146 nm) than individual zein nanoparticles (107 nm), indicating that a high shellac concentration resulted in the formation of large Z–S composite nanoparticles. The result could be explained by the fact that the cross-linking occurred between hydroxyl groups and carboxylic groups of shellac molecules (Soradech et al., 2013). A similar observation was reported by Luo et al. (2011) that the concentration of chitosan could affect the size of ˛-tocopherol-loaded zein particles when coated with chitosan. As shown in Fig. 1B, the turbidity of Z–S binary composite nanoparticle dispersions was significantly (p < 0.05) decreased from 130.0 to 9.0 NTU as the mass ratio of Z:S was decreased from 50:1 to 1:1, and gradually increased from 9.0 to 19.6 NTU as the mass ratio of Z:S was decreased from 1:1 to 1:2.5. It should be noted that a similar tendency was found between the particle size and turbidity, which was already reported in our previous study (Sun et al., 2015). The decrease in turbidity could be interpreted by the formation of Z–S binary composite nanoparticles with a more compact structure than individual

zein nanoparticles, and the increase in turbidity might be generated by the cross-linking of excessive shellac molecules at higher shellac concentration. It found that the particle size and turbidity were dependent on the level of shellac.

3.2.

FTIR analysis

The molecular interaction between zein and shellac was characterized by FTIR. The spectra of freeze-dried zein, shellac and Z–S composites at the ratios of 10:1, 2.5:1, 1:2.5 are shown in Fig. 2A. In the spectra of zein and shellac, the characteristic peaks at 3313.40 and 3415.76 cm−1 were ascribed to the stretching vibration of hydrogen bonds (Cerqueira et al., 2012). However, after the formation of binary composite nanoparticles, the peaks of hydrogen bonds were red-shifted to 3317.50, 3330.97 and 3383.31 cm−1 in the spectra of Z–S composite nanoparticles at mass ratios of 10:1, 2.5:1 and 1:2.5, respectively. The results might be explained by the fact that the generation of hydrogen bonding between amide groups of glutamine in zein and hydroxyl groups in shellac (Luo et al., 2011). As shown in Fig. 2A, zein had two typical amide peaks, the amide I (1657.81 cm−1 ) was mainly referred to C O and C N stretching; the amide II (1534.51 cm−1 ) was mainly governed by the bending vibration of N H groups and stretching vibration of C N groups (Sun et al., 2015; Tang et al., 2012). Compared

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with the spectra of zein, the amide I peaks of Z S composite nanoparticles (Z:S, 10:1, 2.5:1 and 1:2.5) had no obvious changes in location but decreased in peak intensity. The amide II peaks were shifted to 1535.72, 1535.45 and 1562.70 cm−1 in the spectra of Z S composite nanoparticles at Z:S mass ratios of 10:1, 2.5:1 and 1:2.5, respectively. These results suggested that the interaction between zein and shellac was consisted of not only hydrogen bonding but also hydrophobic effects which was ascribed to the higher content (>50%) of hydrophobic amino acids of zein and the hydrophobic groups of shellac. These findings were similar to the study of Sun et al. (2017) that there existed the hydrophobic interaction between zein and propylene glycol alginate, as well as the report of Luo et al. (2011) that the hydrophobic attraction occurred during the formation of zein and ˛-tocopherol nanoparticles.

3.3.

CD analysis

In the present study, Far-UV CD spectroscopy was used to measure the influence of shellac addition on the secondary structure of zein. As shown in Fig. 2B, the CD spectrum of zein showed two negative peaks at 210 and 224 nm, and a positive peak at 195 nm, with a zero crossing around 203 nm, which were characteristic of ˛-helical rich secondary structure (Selling et al., 2007). Significant (p < 0.05) changes were observed in the absolute values of mean residue ellipticities at both 210 and 224 nm, suggesting a great change in the secondary structure of zein in the presence of shellac. Alteration in protein secondary structure upon the interaction with polymer was regarded as a common phenomenon (Czarnik-Matusewicz et al., 2000; Mekhloufi et al., 2005). The secondary structure contents were estimated using the DICHROWEB procedure (Whitmore and Wallace, 2004). The fractions of ˛-helix, ˇ-sheet, and unordered coil were estimated by K2D-SET4 and the data are shown in supplementary material (Table 1 in Supplementary material). The native zein secondary structural contained 27.0% ˛-helix, 23.0% ˇ-sheet, and 51.0% unordered coils. It could be observed that the percentage of ˇ-sheet was significantly (p < 0.05) reduced from 23.0% to 13.0% as zein was mixed with shellac (Z:S, 5:1), which was accompanied with the slight increase of ˛-helix (from 27.0% to 30.0%) and random coil (from 51.0% to 57.0%). The changes in the proportion of ˛-helix and ˇ-sheet were similar to our previous study (Sun et al., 2017). Extended ˇ-sheet was commonly found in aggregated proteins (Lefevre and Subirade, 2000). The lower content of ˇ-sheet in Z–S composite nanoparticles than that in individual zein further confirmed that the formation of aggregated zein molecular clusters was inhibited at lower content of shellac. It should be noted that ˇ-sheet content of what was significantly (p < 0.05) increased from 19.0% to 51.0%, when the Z:S mass ratio was decreased from 2.5:1 to 1:2.5. Meanwhile, ˛-helix and random coil contents of Z–S composite nanoparticles were significantly (p < 0.05) decreased. Similar result was also reported that ˇ-sheet content of zein was increased when propylene glycol alginate was added (Sun et al., 2017). This result could be explained by the fact that a high concentration of shellac led to the protein unfolding and restructuring, which might be induced by the interaction between zein and shellac and the cross-linking of shellac molecules, in accordance with particle size and turbidity data (Fig. 1A and B). Sun et al. (2015) also found the similar phe-

Fig. 3 – DSC thermograms of native shellac, native zein and composite nanoparticles of zein and shellac at mass ratios of 50:1, 10:1, 5:1, 2.5:1, 1:1, 1:1.5, 1:2.5. nomenon in the investigation into intermolecular interaction between zein and quercetagetin.

3.4.

DSC

The thermograms of zein, shellac, and Z–S composite nanoparticles were monitored by DSC (Fig. 3, Supplementary material Table 2). Zein showed a broad endothermic peak at 89.5 ◦ C, which could be associated with the evaporation of bound water from polymers (Luo et al., 2011; Sun et al., 2017). Shellac exhibited an endothermic peak at 79.9 ◦ C. When shellac level was increased, the endothermic peaks of Z–S composite nanoparticles were shifted from 85.3 ◦ C (Z:S, 50:1) to 93.4 ◦ C (Z:S, 5:1). These results indicated that the presence of shellac improved anti-disintegration properties of Z–S composite nanoparticles. In particular, Z–S composite nanoparticles (Z:S, 5:1) exhibited the best thermal stability. It could be explained by that a more compact structure and small particle size of Z–S composite nanoparticles were formed when shellac was at a low concentration, as confirmed by FTIR, CD and particle size analyses in this work. These phenomena was also observed in the nanoparticles based on zein and hydrophobic material (˛-tocopherol) (Luo et al., 2011). The endothermic peaks of Z–S composite nanoparticles were gradually shifted down from 93.4 ◦ C to 80.5 ◦ C when the Z:S mass ratio was decreased (5:1 to 1:1.5), and these Z–S composite nanoparticles required less energy to unfold (Wei et al., 2015b). The decrease in endothermic peak temperatures might be attributed to the cross-linking of shellac molecules at a high level, leading to the loose structure and large particle size of Z–S composite nanoparticles, which was confirmed by FTIR, CD and particle size analyses in this work. It was interesting that the endothermic peak temperature of Z–S composite nanoparticles (Z:S, 1:1) was approximate to that of individual zein nanoparticles. This result might be explained by that the effect of shellac on Z–S composite nanoparticles could be divided into two aspects. On the one

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tryptophan (Trp) residues (Souza et al., 2011). Due to the aromatic character, Trp residues are commonly found to be fully or partially buried inside the hydrophobic core of proteins (Semenova and Dickinson, 2013). Yang et al. (2014b) suggested that the protein aggregation led to the shielding of Trp residues while the protein unfolding led to the exposure of Trp residues. It might be used to explain the increase of fluorescence intensity induced by low shellac concentrations (Z:S mass ratios from 10:1 to 5:1), which prevented the formation of aggregated zein molecular clusters though electrostatic repulsion and steric hindrance. The fluorescence intensity of Z–S composite nanoparticles (Z:S, 50:1) was lower than that of individual zein, which might be explained by that the level of shellac was too low to have an influence on the aggregation of zein molecular clusters. Continuously increasing the shellac concentration, the fluorescence intensity of zein in the composite nanoparticles of Z:S (2.5:1), Z:S (1:1), Z:S (2.5:1), Z:S (1:1.5), and Z:S (1:2.5) was steeply reduced and lower than that of individual zein (Fig. 4B). This result was ascribed to the fact that the crosslinking of shellac molecules occurred, and zein molecules were entangled in the cross-linked shellac molecules, therefore the tryptophan residues of zein were buried inside. It was similar to that the fluorescence intensity of lactoferrin was decreased with the rise of epigallocatechin gallate concentration (Yang et al., 2014b).

3.6.

Fig. 4 – Fluorescence emission spectra of zein and shellac composite nanoparticles. (A) native zein and composite nanoparticles of zein and shellac at mass ratios of 50:1, 10:1, 5:1; (B) native zein and composite nanoparticles of zein and shellac at mass ratios of 2.5:1, 1:1, 1:1.5, 1:2.5.

hand, a low level of shellac could prevent the formation of zein molecular clusters, and make a more compact structure of Z–S composite nanoparticle, leading to the increase of endothermic peak temperature. On the other hand, a high level of shellac might promote the cross-linking of shellac molecules, and make a loose structure of Z–S composite nanoparticles, leading to the decrease of endothermic peak temperature. The two impacts were offset each other. Thus, the mass ratio of Z:S (1:1) was a critical point of the two effects for endothermic peak temperature.

3.5.

Fluorescence spectra

The fluorescence spectra can be applied to analyze the microenvironment changes of proteins, and the interactions between proteins and hydrophobic compounds (Barik et al., 2003). Fluorescence spectra of Z–S composite nanoparticles at different mass ratios are shown in Fig. 4. It was observed that zein exhibited a fluorescence emission peak at 305 nm after being excited at 280 nm, which was in accordance with the previous report (Sun et al., 2016). No significant change in the wavelength of emission maximum was observed but the fluorescence intensity of zein was increased as the decrease of Z:S mass ratios from 50:1 to 5:1 (Fig. 4A). Conformational changes of proteins can be monitored by the fluorescence of

Atomic force microscopy (AFM)

In present study, the microstructure of zein, shellac and Z–S composite nanoparticles were investigated by AFM. It was reported that zein could form different structures as it holds different sides to either adsorb to the surface or expose itself because it has hydrophilic and hydrophobic surfaces (Wang et al., 2004; Gezer et al., 2015). As shown in Fig. 5A, native zein exhibited typical nanospheres with a diameter ranging from 0.58 to 1.93 ␮m. The sphericity and non-uniform sizes of zein were reported in previous literature (Luo et al., 2011; Sun et al., 2015). In general, the diameter measured by AFM was apparently larger than that by Zetasizer, which was observed by Wang et al. (2004). As shown in Fig. 5B, shellac exhibited the non-uniform microstructure, including nanosphere (31.3%), oval (18.8%) and other irregular shapes. When the Z:S mass ratio was 2.5:1, the morphology of Z–S composite nanoparticles showed inhomogeneous spherical shapes with diameters ranging from 0.62 to 0.91 ␮m (Fig. 5C), and it was smaller than individual zein nanoparticles (0.58–1.93 ␮m), suggesting that zein and shellac had an interaction to form smaller particles with more compact structures. Similar result was also reported by Luo et al. (2011), who found that zein and chitosan composite particles at a low level of chitosan were more uniform and smaller than that of native zein. As shown in Fig. 5D and E, Z–S composites showed an irregular geometry (Z:S, 1:1), and microspheres with a diameter of 4.13 ␮m (Z:S, 1:1.5). This might be caused by the intermolecular cross-linking induced by excessive shellac molecules (Wang et al., 2015). These results indicated that the presence of shellac led to the morphological alteration of Z–S composite nanoparticles (Wei et al. 2015a), and the change in the diameter of Z–S composites along with increasing shellac concentration was in agreement with the result of particle sizes analyzed by Zetasizer.

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Fig. 5 – AFM images of native zein, native shellac and composite nanoparticles of zein and shellac. Left and right columns represent the two-dimensional and three-dimensional images, respectively. (A) native zein; (B) native shellac; (C) composite nanoparticles of zein and shellac at mass ratio of 2.5:1; (D) composite nanoparticles of zein and shellac at mass ratio of 1:1; (E) composite nanoparticles of zein and shellac at mass ratio of 1:1.5.

3.7.

Interaction mechanism

From the above analyses and discussion, it could be concluded that the presence of shellac induced characteristic changes of

zein, which was dependent on the mass ratio of zein to shellac. The interaction mechanism between zein and shellac in the anti-solvent co-precipitation process at pH 8.0 was proposed as Fig. 6.

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References

Fig. 6 – Proposed interaction mechanism between zein and shellac.

At low levels of shellac (Z:S, from 50:1 to 2.5:1), Z–S composite nanoparticles showed smaller particle size, higher turbidity value, and higher thermal stability. On the one hand, shellac could prevent formation of zein molecular clusters through the electrostatic repulsion and steric hindrance. On the other hand, zein was assembled with shellac through hydrophobic interactions and hydrogen bonding to form a more compact structure during the process of ASCP. At high levels of shellac (Z:S, from 2.5:1 to 1:2.5), Z–S composite nanoparticles exhibited a large particle size, low turbidity, and less thermal stability. What’s more, it is worthy to mention that the secondary structure of zein in composite nanoparticles was significantly changed. ˛-helix content was decreased from 25.0% to 7.0%, while ˇ-sheet content was increased from 19.0% to 51.0%. The reason was that the cross-linking was formed by hydrogen bonding between hydroxyl groups and carboxylic groups of shellac molecules, and zein molecules were entangled in the cross-linked shellac molecules.

4.

Conclusion

Zein and shellac could self assemble to form dispersible composite nanoparticles by anti-solvent co-precipitation method. The formation of Z–S composites led to the fluorescence quenching and change in the secondary structure of zein, which was mainly attributable to the hydrogen bonding and hydrophobic effects. A potential two-step process mechanism was proposed to explain the formation of Z–S binary composite nanoparticles, with a more compact structure and smaller particle size at low levels of shellac (Z:S, from 50:1 to 2.5:1), and the generation of the cross-linking interaction induced by high levels of shellac (Z:S, from 2.5:1 to 1:2.5). These findings in present work might be beneficial to further understand the theory of interactions between alcohol-soluble proteins and polyesters, as well as the negatively charged proteins and biopolymers.

Acknowledgement This work was supported by the National Natural Science Foundation of China [No. 31371835].

Appendix A. Supplementary data Supplementary data associated with cle can be found, in the online https://doi.org/10.1016/j.fbp.2017.11.003.

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