Development of protein-polysaccharide-surfactant ternary complex particles as delivery vehicles for curcumin

Accepted Manuscript Development of protein-polysaccharide-surfactant ternary complex particles as delivery vehicles for curcumin Lei Dai, Yang Wei, Cuixia Sun, Like Mao, David Julian McClements, Yanxiang Gao PII:

S0268-005X(18)30652-0

DOI:

10.1016/j.foodhyd.2018.06.052

Reference:

FOOHYD 4529

To appear in:

Food Hydrocolloids

Received Date: 10 April 2018 Revised Date:

28 June 2018

Accepted Date: 30 June 2018

Please cite this article as: Dai, L., Wei, Y., Sun, C., Mao, L., McClements, D.J., Gao, Y., Development of protein-polysaccharide-surfactant ternary complex particles as delivery vehicles for curcumin, Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.06.052. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Graphic abstract: Cur12.5 Cur25 Cur50 Cur100 Cur125

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EE(%)

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Z-P-Cur

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Z-Cur

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Development of protein-polysaccharide-surfactant ternary complex

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particles as delivery vehicles for curcumin

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Lei Dai1, Yang Wei1, Cuixia Sun1, Like Mao1, David Julian McClements2, Yanxiang

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Gao1* Beijing Advanced Innovation Center for Food Nutrition and Human Health,

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Beijing Laboratory for Food Quality and Safety, Beijing Key Laboratory of

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Functional Food from Plant Resources, College of Food Science & Nutritional

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Engineering, China Agricultural University, Beijing, 100083, P. R. China 2

Department of Food Science, University of Massachusetts Amherst, Amherst, MA

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01003, USA

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*Corresponding author.

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Tel.: + 86-10-62737034

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Fax: + 86-10-62737986

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Address: Box 112, No.17 Qinghua East Road, Haidian District, Beijing 100083,

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China

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E-mail: [email protected]

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Abstract In this study, protein/polysaccharide/surfactant complexes were prepared by

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anti-solvent co-precipitation using zein, propylene glycol alginate (PGA), and either

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rhamnolipid or lecithin. The potential of using these ternary complexes as delivery

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systems for increasing the stability and bioaccessibility of curcumin (Cur) was

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investigated.

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zein-PGA-lecithin complexes are abbreviated as Z-Cur, Z-P-Cur, Z-P-R-Cur and

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Z-P-L-Cur, respectively. The presence of polysaccharides and surfactants in the

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complexes increased the encapsulation efficiency of the curcumin compared to zein

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nanoparticles alone: Z-Cur (21%); Z-P-Cur (67%); Z-P-R-Cur (92%); and, Z-P-L-Cur

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(94%).

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state inside the complexes. Fourier transform infrared and fluorescence spectroscopy

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indicated that electrostatic interactions, hydrogen bonding, and hydrophobic effects

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were the main forces involved in complex formation. Light scattering and

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electrophoresis measurements showed that the particle size and charge of the

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complexes depended on their composition. The presence of the surfactants in the

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complexes significantly improved the photo-stability and bioaccessibility of curcumin.

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Our results suggest that the ternary complexes developed in this study might be a

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promising means of encapsulating, protecting, and delivering hydrophobic

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nutraceuticals for applications in foods, supplements, and pharmaceuticals.

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Curcumin-loaded zein, zein-PGA, zein-PGA-rhamnolipid, and

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X-ray diffraction indicated that curcumin was present in an amorphous

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Keywords: Zein; Propylene glycol alginate; Surfactant; Complex particles; Curcumin;

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Bioaccessibility

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1. Introduction Curcumin (Cur) is a natural polyphenolic compound obtained from the rhizome

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of turmeric (Curcuma longa Linn). Curcumin has been used as a spice, pigment, and

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nutraceutical in traditional Indian and Chinese medicine for centuries (Mehanny,

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Hathout, Geneidi, & Mansour, 2016). Recently, many researchers have focused on

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utilizing curcumin as a nutraceutical in foods, supplements, and drugs due to its

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potentially beneficial health effects, such as antioxidant, anti-cancer, and

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anti-inflammatory activities (Wilken, Veena, Wang, & Srivatsan, 2011; Lv et al., 2014;

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Hu et al., 2015; Bordoloi & Kunnumakkara, 2018).

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curcumin into commercial products is often challenging because of its relatively low

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water-solubility, poor chemical stability, and low bioavailability (Mehanny et al.,

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2016; Xiao, Nian, & Huang, 2015).

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when exposed to light, alkaline pH, elevated temperatures, and gastrointestinal

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enzymes. Furthermore, the dissolution of curcumin under simulated gastrointestinal

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tract conditions is very low, resulting in a low bioaccessibility (Xiao et al., 2015; Yu,

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Shi, Liu, & Huang, 2012). The challenges associated with incorporating curcumin

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into commercial products can often be overcome by utilizing carefully designed

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colloidal delivery systems, such as liposomes (Gómez-Mascaraque, Sipoli, de La

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Torre, & López-Rubio, 2017), nanoemulsions (Li, Hwang, Chen, & Park, 2016; Liu,

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Ma, McClements, & Gao, 2016), Pickering emulsions (Marefati, Bertrand, Sjöö,

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Dejmek, & Rayner, 2017), solid lipid nanoparticles (Xue, Wang, Hu, Zhou, & Luo,

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2018), microgels (Zhang et al., 2016) and polymer nanoparticles (Dai, Sun, Li, Mao,

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Liu, & Gao, 2017; Xiao et al., 2015).

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colloidal delivery systems found that zein-based nanoparticles were particularly

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effective due to their ease of preparation, high loading capacity, and good

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physicochemical stability (Zou et al., 2016). In the current study, we examined the

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potential of extending the functional attributes of this kind of delivery system by

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forming binary and ternary complexes with polysaccharides and surfactants.

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However, the incorporation of

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In particular, curcumin tends to rapidly degrade

A recent comparison of different kinds of

Zein is a relatively hydrophobic protein derived from corn that is suitable for 3

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food applications.

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that it is insoluble in water, however it can be dissolved in concentrated aqueous

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ethanol solutions (60-90%) or alkaline solutions (pH > 11) (Patel, Hu, Tiwari, &

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Velikov, 2010a).

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zein nanoparticles can be formed using a liquid-liquid dispersion (antisolvent

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precipitation) method (Wu, Luo, & Wang, 2012).

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mixed together, and then injected into an antisolvent, which causes the zein molecules

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to spontaneously self-assemble into nanoparticles or microparticles.

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has been used to fabricate zein nanoparticles loaded with a variety of different

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lipophilic bioactive compounds, such as lutein (Chuacharoen & Sabliov, 2016),

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curcumin (Dai et al., 2017; Dai et al., 2018; Patel et al., 2010; Hu et al., 2015; Liang

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et al., 2015; Zou et al., 2016), resveratrol (Huang et al., 2017), vitamin D3 (Luo, Teng,

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& Wang, 2012), α-tocopherol (Luo, Zhang, Whent, Yu, & Wang, 2011) and

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quercetagetin (Sun, Dai, & Gao, 2016).

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substance is mixed with the zein and solvent solution prior to carrying out the

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antisolvent precipitation step.

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nanoparticles has been shown to improve their stability to light, pH changes, and

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heating, as well as to increase their oral bioavailability (Liang et al., 2015; Penalva et

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al., 2015; Chuacharoen & Sabliov, 2016).

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The different solubility of zein in different solvents means that

A solution of zein and solvent are

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This approach

In these cases, the lipophilic bioactive

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Encapsulation of bioactive compounds within zein

Despite the above advantages, the utilization of zein nanoparticles in functional

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Over half of the amino acids in zein are non-polar, which means

foods is often limited because they are highly susceptible to aggregation near their

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isoelectric point (pI ≈6.2), at high ionic strengths, at elevated temperatures, and in

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certain regions of the gastrointestinal tract (Chang et al., 2017a; Patel, Bouwens, &

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Velikov, 2010b).

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biopolymers, such as lecithin, Tween 80, caseinate, pectin, and chitosan, can be used

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to improve the aggregation stability of zein nanoparticles by forming a protective

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coating around them (Patel et al., 2010b; Hu et al., 2015; Luo et al., 2012, Hu, &

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McClements, 2014). The coatings formed by these surfactants and biopolymers

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increase the electrostatic and/or steric repulsion between the zein nanoparticles,

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thereby improving their aggregation stability.

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that surfactant-coatings could also improve the chemical stability of encapsulated

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Previous research has shown that food-grade surfactants and

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Chuacharo & Sabliov (2016) reported

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bioactives (lutein).

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In a previous study, we fabricated zein-polysaccharide and zein-surfactant binary

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complexes using an anti-solvent co-precipitation technique (ASCP), and showed that

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they had a better encapsulation efficiency and loading capacity then simple zein

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nanoparticles (Sun et al., 2016; Dai et al., 2017).

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the potential of ternary zein-polysaccharide-surfactant complexes for encapsulation,

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protecting, and release curcumin.

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reported that zein/caseinate/pectin ternary complexes had a better stabilization effect

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under simulated gastrointestinal tract conditions than zein/caseinate binary complexes.

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In the current study, we used propylene glycol alginate (PGA) as a model

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polysaccharide and either rhamnolipid or lecithin as model surfactants to form the

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ternary complexes.

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In the present study, we examined

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Indeed, Chang et al. (2017a, 2017b) recently

PGA is a high molecular weight linear polysaccharide with 50–85% of its

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carboxyl groups esterified, which makes it an amphiphilic molecule with good

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surface-activity (Sarker & Wilde, 1999).

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thickener, or stabilizer in foods.

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synergistic effect between zein and PGA in improving the encapsulation efficiency

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and loading capacity of quercetagetin (Sun et al., 2016). Lecithin is a natural

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surfactant with a polar head and non-polar tail, which has been shown to enhance the

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physical and chemical stability of bioactive-loaded delivery systems (Chuacharoen &

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Sabliov, 2016; Luo et al., 2017).

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antisolvent precipitation have been reported to have a higher stability against

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environmental stresses than simple zein nanoparticles (Dai, Sun, Wang, & Gao, 2016).

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Rhamnolipid is produced by microbial fermentation and consists of one or two polar

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rhamnose units attached to a non-polar fatty acid chain, which gives it good surface

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activity (Bai & McClements, 2016). Rhamnolipids have previously been used to

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improve the stability of both inorganic (Dwivedi et al., 2015) and organic (Dai et al.,

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2018) nanoparticles.

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It can therefore be used as an emulsifier,

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In our previous work, we found that there was a

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Indeed, zein-lecithin nanoparticles formed by

The primary objectives of this study were to determine whether

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zein/PGA/surfactant ternary complexes could be successfully fabricated using the

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antisolvent precipitation method, and to determine whether curcumin could be

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successfully encapsulated, protected, and released using them. In particular, we

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hypothesized that these ternary complexes would give better performance than either

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secondary complexes or simple zein nanoparticles. 5

To this end, we characterized the

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particle size, charge, encapsulation efficiency, loading efficiency, and stability of the

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curcumin-loaded complexes using a variety of analytical tools.

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the impact of passing the complexes through a simulated gastrointestinal tract on the

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stability and bioaccessibility of the encapsulated curcumin.

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therefore provide valuable information that can be used to develop more effective

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delivery systems for lipophilic bioactive agents.

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2. Materials and methods

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2.1. Materials

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This study should

Zein (91.3% protein, w/w) was obtained from the Sigma-Aldrich Chemical

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We also examined

Company (St. Louis, MO, USA). Propylene glycol alginate (PGA) with 87.9%

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esterified carboxyl groups was kindly supplied by Hanjun Sugar Industry Co. Ltd.

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(Shanghai, China). Rhamnolipid (>90% purity) containing a mixture of di-and

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mono-rhamnolipids was purchased from Shaanxi Parnell Biological Technology Co.

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Ltd (Shaanxi, China). Soy lecithin (S-100, 94% of phosphatidylcholine content) was

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purchased from Lipoid (Ludwigshafen, Germany). Curcumin (purity > 98%) was

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obtained from Tianjin Guangfu Fine Chemical Industry Research Institute (Tianjin,

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China). Pepsin from porcine gastric mucosa (600 U/mg, Sigma 77160), bile salts

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(Sigma B8631) and pancreatin with 8×USP specification (Sigma P7545) were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemical reagents

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(sodium hydroxide and hydrochloric acid) and solvents used in this work were of

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analytical grade.

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2.2. Fabrication of curcumin-loaded complex particles

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Curcumin-loaded ternary complexes were prepared using the antisolvent

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co-precipitation method according to the method described in our previous study with

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some modifications (Dai et al., 2017).

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zein/PGA/rhamnolipid complexes is described.

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prepared by dissolving zein (1.0 g) and PGA (0.2 g) in 100 mL of 70 % ethanol-water

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solution (v/v), and then rhamnolipid powder (1.0 g) was added into this solution and

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the mixture was stirred at 600 rpm for 2 h (25 °C).

Initially, the method for preparation of the

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First, a zein-PGA solution was

Second, a curcumin solution was

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formed by dispersing different levels of curcumin (25, 50, 100, 200 and 250 mg) into

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a 70 % ethanol-water solution (v/v) and then the mixtures were stirred (600 rpm) to

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ensure full dissolution.

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with the same volume of the curcumin solution with constant stirring (600 rpm) for 2

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h at 25 °C. This led to a series of solutions with zein-to-curcumin levels of 40:1, 20:1,

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10:1, 5:1, and 4:1 (w/w). Then, 20 mL of the above mixture was slowly injected into

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60 mL of distilled water using a syringe. The resulting systems were continually

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stirred (600 rpm) for 30 min at room temperature. Finally, the ethanol remaining in

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the dispersions was removed using a vacuum rotary evaporator with a vacuum of -0.1

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MPa at 45 °C for 20 min. The final dispersions were then adjusted to pH 4.0 using

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100 mM NaOH or HCl solutions, and centrifuged at 2000 rpm for 10 min to remove

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any large precipitates. Part of final dispersions were stored at 4 °C for further analysis

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and the other part was freeze-dried to obtain powders.

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After that, the zein/PGA/rhamnolipid solution was mixed

The curcumin-loaded zein, zein-PGA, and zein-PGA-lecithin complexes were

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prepared using a similar process but omitting or switching certain components. For

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the sake of brevity, curcumin-loaded zein, zein-PGA, zein-PGA-rhamnolipid, and

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zein-PGA-lecithin complexes are abbreviated as Z-Cur, Z-P-Cur, Z-P-R-Cur and

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Z-P-L-Cur, respectively.

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according to the following scheme: Z-P-R-Cur50 contained 50 mg of curcumin in 100

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mL of zein/PGA/rhamnolipid/curcumin ethanol solution.

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2.3. Measurement of particle size and charge

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Samples containing different curcumin levels were labelled

The particle size and ζ-potential of the curcumin-loaded complexes were

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determined at room temperature using an instrument that combines dynamic light

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scattering and electrophoresis (Zetasizer Nano-ZS90, Malvern Instruments, and

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Worcestershire, UK). The particle diameter of each sample was calculated by the

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instrument based on the Stokes-Einstein equation, while the ζ-potential was

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calculated based on the Smoluchowski model. Prior to measurement, the samples

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were diluted with pH 4.0 buffer solution to avoid multiple scattering effects.

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2.4. Encapsulation efficiency (EE) and loading capacity (LC)

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The EE and LC of the curcumin in the complexes were measured according to 7

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the description of Xiao et al. (2015) with some slight modifications. Freshly prepared

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dispersions were diluted using 80% ethanol-water solution (v/v), and then curcumin

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was extracted with the aid of sonication. The curcumin concentration was determined

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by measuring the absorbance at a wavelength of 426 nm using a UV-visible

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spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan).

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LC values of curcumin were calculated as follows:

( ) × 100 ( )



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ℎ



(

2.5. Photochemical stability of curcumin

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(

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× 100

The photochemical stability of curcumin in the complexes was determined using

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LC (%) =

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EE (%) =

The EE and

a controlled light cabinet (Q-Sun, Q-Lab Corporation, Ohio, USA). The freshly

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prepared curcumin-loaded complexes were transferred into transparent glass bottles,

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which were then placed into a light cabinet set at an intensity of 0.35 W/m2 and a

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temperature of 35 °C. Samples were collected at specific time intervals, 10, 30, 60, 90

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and 120 min, for analysis. The amount of curcumin remaining in the complexes was

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determined as described in Section 2.4.

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2.6. Fluorescence spectroscopy

The impact of curcumin level on the fluorescence behavior of zein was measured

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by a fluorescence spectrophotometer (F-7000, Hitachi, Japan). Briefly, the samples

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were first diluted to a constant zein concentration of 0.2 mg/mL. The fluorescence

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spectra were collected by scanning the emission wavelength from 290 to 450 nm

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using a fixed excitation wavelength of 280 nm.

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Additionally, the encapsulation and binding status of the encapsulated curcumin

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within the complexes were also determined using fluorescence spectrophotometry

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(F-7000, Hitachi, Japan). The emission spectra of samples were recorded between 450

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and 700 nm with an excitation wavelength of 420 nm using a quartz cell with 10 mm

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path length. Besides, 1 mg of curcumin dissolved in 1 mL of 70% ethanol-water

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solution and then dropped into 3 mL of water was used as a control sample. 8

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2.7. Fourier transform infrared spectroscopy (FTIR) The inter- and intra-molecular interactions among zein, PGA, surfactants (rhamnolipid and lecithin), and Cur were studied using Fourier transform

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spectrophotometry (Spectrum 100, Perkin-Elmer, Warrington, UK). FTIR spectra

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were acquired by scanning the samples across a range of wavenumbers (4000 to 400

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cm-1) at a resolution of 4 cm−1. The data obtained were then analyzed using the

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instrumental software (Omnic 8.0, Thermo Nicolet, USA).

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2.8. X-Ray diffraction (XRD)

The molecular arrangement of the curcumin within the complexes was

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determined using an X-ray diffractometer (Brucker D8, Odelzhausen, Germany) with

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a Cu anode, 40 kV voltage, and 40 mA current. The scanning angle ranged from 5° to

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55° (2θ) with a step size of 0.02°and a step time of 5 s.

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2.9. SEM

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The microstructures of the samples were determine using scanning electron microscopy (SEM, SU8010, Hitachi). The freeze-dried samples were put on a

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double-sided adhesive coated with a thin layer of gold and measured under10.0 kV

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acceleration voltage.

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2.10. In vitro gastrointestinal digestion

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The samples were passed through a simulated gastrointestinal tract according to the description of Sun et al. (2017) with some modifications. All the samples were

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placed in a glass beaker and then stored in a 37 °C shaking incubator at a rotation

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speed of 100 rpm for 15 min for preheating.

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2.10.1. Stomach phase

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30 mL of samples were mixed with 30 mL of simulated gastric fluid (SGF),

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which contained 3.2 mg/mL pepsin and was preheated to 37 °C. Then, the mixture

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was adjusted to pH 2.5 and incubated in the shaker at 37 °C for 1 h with continuous

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swirling at 100 rpm.

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2.10.2. Small Intestine phase

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30 mL of samples from the stomach phase were transferred into a 100 mL glass

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beaker and then adjusted to pH 7.0 (37 °C). Then, 30 mL of simulated intestinal fluid

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(SIF) containing 2.0 mg/ mL of pancreatin and 12.0 mg/ mL of bile salts were added. 9

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mixtures were incubated at 37 °C for 120 min with continuous stirring at 100 rpm.

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The digesta were collected for analysis at designed time intervals (30, 60, 90, and 120

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min).

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2.11. Curcumin bioaccessibility

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The bioaccessibility of curcumin was calculated after the samples had been

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passed through the simulated gastrointestinal tract based on the method described by

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Zou et al. (2016). 20 mL of raw digesta from each sample was centrifuged at 15,000

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rpm for 1 h. The clear supernatant was collected and assumed to be the “micelle”

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fraction in which the curcumin was solubilized. The curcumin concentrations in the

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raw digesta and mixed micelle phases were then determined. And the bioaccessibility

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of curcumin was calculated based on this data using the following equation: ./01233 Bioaccessibility = 100 × .456 70829:5

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Here, CMicelle and CRaw digesta are the concentrations of curcumin in the micelle fraction

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and in the raw digesta.

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2.12. Statistical Analysis

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All experiments were performed on at least two samples. The data obtained were analyzed using the analysis of variance (ANOVA) with Duncan’s test in a statistical

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software package (SPSS 18.0, SPSS Inc., Chicago, USA). P<0.05 was considered to

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be statistically different.

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3. Results and discussion

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3.1. Particle size and ζ-potential

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The effect of initial curcumin concentration on the particle size and charge of the

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curcumin-loaded complexes was measured (Fig. 1). The mean particle diameter of the

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zein nanoparticles depended on the level of curcumin present (Fig. 1A).

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mean particle diameter decreased when the curcumin concentration was increased

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from 0 to 50 mg (from 171 to 136 nm), but then it increased when the curcumin level

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was further increased to 125 mg (to 151 nm). This result is in agreement with earlier 10

Initially, the

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as the level of curcumin present increased (Patel et al., 2010). The ζ-potential of the

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zein nanoparticles decreased from +35.4 to +23.6 mV as the curcumin level was

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increased from 0 to 125 mg. This change in electrical characteristics may have

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occurred because curcumin has a negative charge (about -11.7 mV), which partially

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neutralized the positive charge on the cationic zein nanoparticles.

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also reported that the ζ-potential of zein nanoparticles gradually decreased after the

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incorporation of curcumin.

Patel et al. (2010a)

The unloaded Z-P complexes (821 nm) were considerably larger than the

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equivalent zein nanoparticles (171 nm) (P < 0.05).

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of the polysaccharide (PGA) interfered with the formation of the nanoparticles during

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the antisolvent co-precipitation process, or that it promoted their aggregation after

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formation.

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diameter with increasing curcumin level in the Z-P systems, however the smallest

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complexes (656 nm) were obtained at 100 mg curcumin (Fig 1B). The electrical

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charge on the Z-P complexes was slightly negative (around -7.5 mV) and did not

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depend strongly on curcumin concentration.

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carboxylic acid groups (-COO-) on the PGA molecule dominated the overall charge

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on the Z-P complexes.

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anionic polysaccharide (alginate) dominated the overall electrical characteristics of

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zein-polysaccharide complexes (Hu & McClements 2015).

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magnitude of the charge on the Z-P complexes resulted in relatively weak electrostatic

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repulsion between the complexes would account for the large measured particle

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diameter observed, since some aggregation may have occurred.

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In general, there was a trend towards a decrease in mean particle

This suggests that the anionic

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Other studies have also reported that the presence of an

The fact that the small

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This suggests that the presence

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The unloaded Z-P-R complexes (228 nm) were considerably smaller than the

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unloaded Z-P complexes (821 nm) (P < 0.05).

This suggests that the presence of the

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rhamnolipids inhibited the particle aggregation caused by the PGA, possibly by

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forming a protective coating around the complexes that increased the electrostatic and

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steric repulsion between them.

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decreased as the curcumin concentration increased: from around 228 to 178 nm as the

The size of the Z-P-R complexes gradually 11

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curcumin level went from 0 to 125 mg (Fig. 1C).

The electrical charge on the Z-P-R

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complexes was moderately negative (around -12.5 mV) and did not depend strongly

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on curcumin concentration.

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molecules were trapped in the inner core of Z-P-R complexes and so had little effect

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on the ζ-potential, which only detects changes in the surface potential of the particles.

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The unloaded Z-P-L complexes (417 nm) were considerably smaller than the

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This phenomenon may be ascribed to that the curcumin

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unloaded Z-P complexes (821 nm) (P < 0.05), but appreciably larger than the

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unloaded Z-P-R complexes (228 nm) (P < 0.05).

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also inhibit the aggregation of the Z-P particles caused by the PGA, but that it was

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less effective than the rhamnolipids.

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systems may have been due to the relatively low net charge on the Z-P-L complexes

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(around -8.3 mV).

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on the mean particle diameter or charge of the Z-P-L complexes (Fig. 1D), which may

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again because it is mainly located inside the zein nanoparticles that form the core of

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the ternary complexes.

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3.2. Encapsulation efficiency, loading capacity and photochemical stability

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This suggests that lecithin could

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The higher particle size observed in these

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The amount of curcumin present did not have a major influence

The encapsulation efficiency (EE) and loading capacity (LC) of the different

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complexes were also measured to evaluate their suitability as delivery systems for

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curcumin (Fig. 2). The EE of curcumin-loaded zein nanoparticles gradually decreased

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from 98.2% to 20.8% with increasing curcumin concentration (Fig. 2A), which

339

suggests that most of curcumin (almost 80%) was unable to bind to the proteins at

340

high levels. The Z-P complexes had a higher EE than that of the zein nanoparticles,

341

suggesting that the amphiphilic PGA molecules facilitated the incorporation of

342

curcumin into the complexes (P < 0.05).

343

PGA improved the EE of quercetagetin in zein nanoparticles (Sun et al. 2016). The

344

inclusion of the surfactants (rhamnolipid or lecithin) into the complexes led to a

345

further improvement in their EE at all curcumin levels.

346

curcumin level (125 mg/mL), the encapsulation efficiency of the Z-P-R (92.1%) and

347

Z-P-L (94.3%) ternary complexes was appreciably higher than both the zein

348

nanoparticles (20.8%) and Z-P binary complexes (67.0%) (P < 0.05). This effect

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In our previous study, it also found that

12

In particular, at the highest

ACCEPTED MANUSCRIPT 349

probably occurred because the surfactants were able to bind to hydrophobic groups on

350

the curcumin molecules, thereby keeping them trapped inside the complexes.

351

expected, the total amount of curcumin loaded (LC) into the various formulations

352

increased significantly with increasing curcumin concentration (Fig. 2B). The loading

353

capacity of the Z-P binary complexes was higher than that of the Z-P-R and Z-P-L

354

ternary complexes. This may have been because the surfactants increased the total

355

mass of the carriers, while the amount of curcumin present remained similar.

356

3.3. Photochemical stability

RI PT

As

As mentioned earlier, curcumin is highly sensitive to degradation in the presence

358

of ultraviolet light, and so we examined the ability of the different delivery systems to

359

protect curcumin from photo-degradation. Curcumin loaded into the complexes

360

exhibited a significantly higher (P<0.05) stability when exposed to UV than free

361

curcumin (Fig. S1), especially for the samples containing surfactants. For instance,

362

after 90 min irradiation, the fraction of curcumin retained by the different delivery

363

systems was 3.5%, 7.5%, 18.3%, 36.4%, and 58.4% for Cur, Z-Cur, Z-P-Cur,

364

Z-P-L-Cur, and Z-P-R-Cur, respectively. Previous researchers have also reported that

365

trapping curcumin in biopolymer complexes (kafirin-carboxymethyl chitosan)

366

protected it from photo-degradation (Xiao et al., 2015).

367

the ability of the structures formed by the biopolymers and/or surfactants within the

368

complexes to absorb or scatter UV light, so that less light was incident upon the

369

curcumin molecules.

370

molecular complexes with the curcumin, thereby altering the chemical stability of its

371

reactive groups.

372

and physicochemical mechanisms responsible for these effects.

373

3.4. Fluorescence spectroscopy

374

These effects may be due to

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357

AC C

Alternatively, the biopolymers or surfactants may have formed

Clearly, further work is required to identify the precise molecular

Measurements of the intrinsic fluorescence of proteins can provide valuable

375

information about changes in their conformation and molecular interactions.

376

Therefore, we determined the effects of curcumin on the fluorescence spectra of zein

377

(Fig. S2).

378

it was excited at 280 nm, which is in agreement with earlier studies (Joye et al., 2016).

Pure zein exhibited a strong fluorescence emission peak at 304 nm when 13

ACCEPTED MANUSCRIPT The peak fluorescence intensities of all the complexes decreased with increasing

380

curcumin concentration, suggesting that the curcumin quenched the fluorescence of

381

the zein molecules within the complexes. Joye et al. (2015) also reported a similar

382

phenomenon when resveratrol was incorporated into zein nanoparticles, which was

383

attributed to interactions between the protein and polyphenol molecules.

384

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379

Curcumin is a naturally fluorescent compound that produces a fluorescence emission peak when excited at 420 nm. To better understand the nature of the

386

molecular interactions between the curcumin and the other components in the

387

complexes we therefore measured changes in the curcumin fluorescence emission

388

spectra (Fig. 3). Pure curcumin exhibited a relatively small emission peak at 550.4 nm.

389

After incorporation into the complexes, the height of the emission peak due to

390

curcumin increased appreciably, by an amount that depended on the nature of the

391

complexes: Z-Cur > Z-P-Cur > Z-P-L-Cur > Z-P-R-Cur. This phenomenon is in

392

agreement with previous studies of the interactions of curcumin with zein, which

393

suggested that the observed increase in fluorescence intensity was due to hydrophobic

394

interactions between the non-polar zein and curcumin molecules (Chang et al., 2017a).

395

Presumably, the presence of non-polar regions on the surfaces of the amphiphilic

396

proteins, polysaccharides, and/or surfactant molecules used to construct the

397

complexes altered the nature of the hydrophobic interactions in the system, leading to

398

alterations in the fluorescence emission from curcumin (Chang et al., 2017a).

399

Furthermore, compared to pure curcumin (λmax = 550.4 nm), the wavelength where

400

the maximum in the emulsion peak occurred decreased significantly (P < 0.05) when

401

the curcumin was incorporated into the complexes: 520.2, 523.8, 535.2, and 510.8 nm,

402

for Z-Cur, Z-P-Cur, Z-P-R-Cur, and Z-P-L-Cur, respectively.

403

suggests that the presence of the proteins, polysaccharides and/or surfactants within

404

the complexes altered the local molecular environment of the curcumin. This result is

405

consistent with earlier studies, where a blue shift in λmax was observed when curcumin

406

was loaded into either kafirin or kafirin/carboxymethyl chitosan complexes (Xiao et

407

al., 2015).

408

bonding, hydrophobic attraction, and electrostatic interactions between the curcumin

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385

This blue shift again

The origin of this effect was attributed to a combination of hydrogen 14

ACCEPTED MANUSCRIPT 409

and surrounding biopolymer molecules.

410

3.5. XRD

411

X-ray diffraction was used to obtain information about the physical state of the curcumin in the delivery systems.

413

complexes with or without curcumin were therefore measured (Fig. 4). The XRD

414

pattern for pure curcumin exhibited sharp peaks (2θ) at 8.96o, 12.20o, 14.55o, 17.10o,

415

23.48o, 24.58o and 25.52o, confirming that it was in a crystalline form (Patel et al.,

416

2010). In contrast, zein exhibited two broad peaks at 9.6o and 19.9o, while PGA

417

exhibited two broad peaks at 6.8o and 20.04o, which suggested that both the pure

418

protein and polysaccharide samples were in an amorphous form. The binary and

419

ternary complexes had quite different XRD patterns to the pure zein or pure PGA

420

samples.

421

polysaccharide were observed, while for Z-P-L only the peak at 19.6° remained.

422

These results were attributed to changes in the interactions and organization of the

423

zein, PGA, and surfactant molecules within the different systems.

424

surfactant used to fabricate the complexes significantly affected the XRD patterns.

425

Interestingly, the sharp peaks characteristics of the crystalline form of curcumin were

426

not present in any of the complexes, which suggested that curcumin was trapped in

427

the complexes in an amorphous form. Similar results have also been reported in

428

earlier studies where curcumin was encapsulated in a polymeric matrix (Patel et al.,

429

2010; Liang et al., 2015).

430

3.6. FTIR

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For Z-P and Z-P-R, none of the peaks observed for the pure protein or

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The type of

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431

The XRD patterns of pure curcumin, and

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412

Fourier Transform Infrared (FTIR) analysis provides valuable insights into the

432

nature of the interactions between molecules.

433

provide information about the interactions among the curcumin, protein,

434

polysaccharide, and surfactant molecules in the complexes. The FTIR spectra of the

435

individual components and of the complexes were therefore measured (Fig. 5).

436

curcumin exhibited a sharp peak at 3511 cm-1, which is characteristic of this molecule

437

(Xiao et al., 2015). The pure zein, PGA, rhamnolipid, and lecithin samples showed

438

broad peaks around 3230, 3450, 3404 and 3430 cm−1, respectively. 15

For this reason, FTIR was used to

Pure

According to

ACCEPTED MANUSCRIPT previous studies, a broad band in the wavenumber range from 3500−3000 cm−1

440

corresponded to O-H stretching vibration (Luo et al., 2011). Compared to zein (3230

441

cm−1), the O-H vibration peaks in the Z-P-Cur, Z-P-R-Cur, and Z-P-L-Cur samples

442

were shifted to higher wavenumbers of 3311, 3356 and 3309 cm−1, respectively.

443

This suggests that there was an increase in the amount of hydrogen bonding in the

444

complexes. The FTIR spectrum of pure zein also exhibited two characteristic peaks at

445

1658 and 1533 cm-1, which represent amide I and amide II bands, respectively (Luo et

446

al., 2011). The amide I band is mainly associated with the stretching vibration of the

447

carbonyl group (C=O), while the amide II band is mainly associated with N-H

448

in-plane bending, C–N stretching, and C–C stretching vibrations (Xiao et al., 2015). It

449

is notable that there was a major change in the amide II band after formation of the

450

complexes. Compared to the spectrum of pure zein, the amide II peaks changed from

451

1533 to 1516, 1514, 1514, 1542 cm-1 for the Z-Cur, Z-P-Cur, Z-P-R-Cur, and

452

Z-P-L-Cur samples, respectively, and the intensity of the peaks was reduced. These

453

results suggest that electrostatic interactions were also important in the formation of

454

the complexes, which would be expected since the protein used has both positive and

455

negative charges on its surface, whereas the surfactants and polysaccharides used both

456

have negative charges. Liang et al. (2015) also reported a change in the amide I and

457

amide II bands in zein/chitosan nanoparticles, indicating that electrostatic interactions

458

were also important in this system. No peaks were present from 1800 to 1650 cm-1 for

459

the pure curcumin, suggesting that it was in a keto-enol tautomeric form (Yin, Lu, Liu,

460

& Lu, 2015).

461

and 1151 cm-1 were not observed in the spectra of the Z-Cur, Z-P-Cur, Z-P-R-Cur,

462

Z-P-L-Cur samples, indicating that Cur was successfully embedded in the

463

nanoparticles. Our results are in agreement with earlier studies that have also shown

464

that the characteristic FTIR peaks associated with crystalline curcumin are lost when

465

it is encapsulated within zein nanoparticles (Hu et al., 2015).

466

3.7. Morphological observation

467 468

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439

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The characteristic peaks observed for pure curcumin at 1510, 1280

The morphology of the complexes was determined using scanning electron microscopy (Fig. 6). The pure zein nanoparticles had a spherical shape and a fairly 16

ACCEPTED MANUSCRIPT 469

uniform size. The Z-Cur nanoparticles still had a spherical shape, but their surface

470

morphology appeared rougher, which was in agreement with previous reports (Liang

471

et al., 2015).

472

crystals at the surfaces of the protein nanoparticles.

473

the Z-P and Z-P-Cur systems was quite different from that of the Z and Z-Cur systems.

474

Both the Z-P and Z-P-Cur systems had a branch-like structure, which consisted of

475

many individual complexes linked together into chains. However, the thickness of

476

these chains became thicker in the presence of curcumin.

477

occurred because of the relatively low electrical charge on these complexes, which

478

meant there was not a strong electrostatic repulsion keeping them apart.

479

also a major change in the morphology of the complexes when the surfactants were

480

present.

481

was observed. For the Z-P-R-Cur sample, it was still possible to observe the

482

individual complexes, but they were still present in a highly aggregated network

483

containing many relatively large holes.

484

was evidence of spherically shaped complexes present, which suggests that they were

485

less likely to merge together.

486

changes in the morphology of the samples during their preparation for the SEM

487

measurements.

488

3.8. Influence of gastrointestinal conditions

RI PT

Interestingly, the morphology of

There was

M AN U

SC

This effect may have

For the Z-P-R sample, a coarse network of highly aggregates complexes

TE D

For the Z-P-L and Z-P-L-Cur systems, there

It should be noted that there may have been some

EP

489

This effect is probably because of the presence of some curcumin

The potential gastrointestinal fate of the different delivery systems was determined using a simulated gastrointestinal tract (GIT), which consisted of stomach

491

and small intestine phases. The mouth phase was omitted because the delivery

492

systems would only be expected to spend a short time there.

493

delivery systems were placed in simulated gastric fluids (SGF) for 60 min, and then in

494

simulated intestinal fluids (SIF) for another 120 min. The change in particle size in

495

the colloidal dispersions was measured as a function of digestion time (30, 60, 90,

496

120,150 and 180 min) and the results are shown in Fig. 7. As mentioned earlier, the

497

mean particle diameter of the initial Z-Cur complexes was around 150 nm. However,

498

after exposure to the SGF, the size of these complexes increased appreciably (P <

AC C

490

17

In summary, the

ACCEPTED MANUSCRIPT 499

0.05). These changes can be attributed to the low pH, high ionic strength, and high

500

enzymatic (protease) activity of the SGF (Parris, Cooke, & Hicks, 2005).

501

instance, zein has been shown to be hydrolyzed by pepsin under simulated stomach

502

conditions (Parris et al., 2005).

503

the Z-Cur nanoparticles decreased with increasing digestion time. This decrease is

504

probably due to the presence of bile salts, which are strong emulsifying agents that

505

can inhibit protein aggregation, and due to hydrolysis of the protein by proteases.

For

RI PT

During incubation in the SIF, the mean diameter of

The size of the complexes in the Z-P-Cur system also increased after exposure to

507

the stomach phase, but then they remained relatively constant during incubation in the

508

small intestine phase (Fig. 7), suggesting that the presence of PGA improved the

509

stability of the complexes.

510

remained fairly steady throughout the simulated GIT, only showing a slight increase

511

during incubation in the SIF (Fig. 7). In contrast, the Z-P-Cur complexes decreased in

512

size after exposure to the SGF, but then increased in size after exposure to the SIF.

513

The impact of surfactant type (lecithin or rhamnolipid) on the gastrointestinal fate of

514

the delivery systems can be attributed to their different molecular characteristics.

515

Lecithin contains a mixture of phospholipids that have a polar head attached to two

516

non-polar hydrocarbon tails, whereas rhamnolipid has a more plate-like structure with

517

one face being polar and the other non-polar.

518

reported that emulsions stabilized by lecithin contained relatively large individual oil

519

droplets, compared to other types of emulsifiers.

Chang & McClements (2016) also

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The mean particle diameter of the Z-P-R-Cur complexes

SEM images of the samples showed that exposure to simulated gastrointestinal

AC C

520

SC

506

521

conditions had a major influence on the morphology of the curcumin-loaded

522

complexes (Fig. 8).

523

spherical shape. After digestion in SGF for 30 min, the Z-Cur particles changed from

524

spherical to rectangular. After further digestion in SGF for 60 min, the Z-Cur particles

525

became highly aggregated. The morphology of the Z-Cur particles changed to a

526

plate-like structure after digestion in SIF. These findings are consistent with the

527

results of the particle size reported in Fig.7. Previous researchers also reported that

528

zein particles formed aggregates after exposure to simulated gastric juices (Parris et

As mentioned in the previous section, Z-Cur particles had a

18

ACCEPTED MANUSCRIPT 529

al., 2005). For the Z-P-Cur composite particles, irregular aggregates appeared after

530

introduction into the SGF for 30 min.

531

Z-P-Cur composite particles to form a network structure with holes. With further

532

digestion in SIF, an anomalous sheet structure was generated. Interestingly, for the

533

sample of Z-P-R-Cur composite particles, the network structure still remained after

534

digestion in SGF for 30 min and became more compact after 60 min of SGF digestion.

535

While in the SIF, the network structure disappeared and formed large aggregates.

536

Additionally, small squares appeared when Z-P-L-Cur composite particles were

537

exposed to SGF. However, the small squares changed to non-uniform aggregates after

538

digestion for 180 min in SIF. These effects may be attributed to various factors,

539

including changes in pH, ionic strength, enzyme activity, and bile salt concentrations

540

in the different regions of the simulated GIT.

541

attractive/repulsive interactions in the systems, thereby leading to changes in the

542

dissociation or agglomeration of the complexes.

543

(proteases) in the SGF and SIF may have partially hydrolyzed the zein molecules in

544

the system, thereby changing the structure of the complexes.

545

3.9. Bioaccessibility

RI PT

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These changes may have altered the

Moreover, the digestive enzymes

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546

Notably, digestion in SIF for 60 min caused

After being exposed to the small intestine phase, the samples were centrifuged and the micelle phases were collected to determine curcumin bioaccessibility (Fig. 9).

548

As expected, the presence of the surfactants (rhamnolipid and lecithin) significantly

549

increased curcumin bioaccessibility. In the absence of surfactants, the bioaccessibility

550

of curcumin in the zein nanoparticles and Z-P complexes was 29.1% and 48.0%,

551

respectively. However, in the presence of rhamnolipid and lecithin the bioaccessibility

552

increased to 82.5% and 87.6%, respectively. These results indicated that the presence

553

of the surfactants increased the solubilization capacity of the mixed micelles in the

554

small intestine fluids. In our previous study, it has shown that the bioaccessibility of

555

curcumin is relatively low in zein nanoparticles but can be increased by mixing them

556

with digestible lipid droplets (Zou et al., 2016).

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19

ACCEPTED MANUSCRIPT 557 558

4. Conclusions In summary, ternary protein/polysaccharide/surfactant complexes were

559

successfully prepared using an antisolvent co-precipitation method and were shown to

560

be promising colloidal delivery systems for curcumin.

561

a higher encapsulation efficiency than simple zein nanoparticles or binary

562

zein/polysaccharide complexes. The encapsulated curcumin was shown to be in an

563

amorphous form that interacted with the surrounding matrix through a combination of

564

hydrogen bonding, electrostatic interactions, and hydrophobic effects. The ternary

565

complexes also enhanced the photostability and bioaccessibility of curcumin.

566

summary, the ternary complexes developed in this study may be suitable for

567

efficiently encapsulating, protecting, and delivering curcumin in oral formulations.

568

Nevertheless, further work is required to determine whether they can be fabricated in

569

a cost-effective manner at a scale suitable for commercial applications, and to

570

establish whether they can be incorporated into real food and beverage products

571

without causing undesirable changes in quality attributes.

572

or human feeding studies are needed to determine their efficacy under more realistic

573

gastrointestinal conditions.

574

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EP

675

zein-propylene glycol alginate composite particles induced by calcium ions: Structural comparison between colloidal dispersions and lyophilized powders

after in vitro simulated gastraintestinal digestion. Journal of Functional Foods, 37, 25-48.

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of anti-cancer properties and therapeutic activity in head and neck squamous cell

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essential oils encapsulated in zein nanoparticles prepared by liquid–liquid

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nanoparticles to enhance the cellular uptake of curcumin. Food Hydrocolloids, 51,

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of a novel biodegradable macromolecule: Carboxymethyl zein. International

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Hydrocolloids, 58, 160-170.

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707

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AC C

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TE D

705

24

ACCEPTED MANUSCRIPT Figure captions Fig. 1. Effect of curcumin (Cur) concentration on the particle size and zeta-potential of zein (A), zein-PGA (Z-P; B), zein-PGA-rhamnolipid (Z-P-R; C) and

RI PT

zein-PGA-lecithin (Z-P-L; D) complex particles. (Different letters mean significant differences (P < 0.05).)

Fig. 2. Effect of curcumin (Cur) concentration on the encapsulation efficiency (EE; A)

SC

and loading capacity (LC; B) of zein, zein-PGA (Z-P), zein-PGA-rhamnolipid (Z-P-R)

differences (P < 0.05).)

M AN U

and zein-PGA-lecithin (Z-P-L) complex particles. (Different letters mean significant

Fig. 3. Florescence spectra of native curcumin (Cur), Cur-loaded in zein (Z-Cur), zein-PGA (Z-P-Cur), zein-PGA-rhamnolipid (Z-P-R-Cur) and zein-PGA-lecithin (Z-P-L-Cur) complex particles.

TE D

Fig. 4. XRD of native curcumin (Cur), curcumin loaded in zein (Z-Cur), zein-PGA (Z-P-Cur), zein-PGA-rhamnolipid (Z-P-R-Cur) and zein-PGA-lecithin (Z-P-L-Cur) complex particles.

EP

Fig. 5. FTIR spectra of native curcumin (Cur), curcumin loaded in zein (Z-Cur),

AC C

zein-PGA (Z-P-Cur), zein-PGA-rhamnolipid (Z-P-R-Cur) and zein-PGA-lecithin (Z-P-L-Cur) complex particles. Fig. 6. SEM images of curcumin loaded in zein (Z-Cur), zein-PGA (Z-P-Cur), zein-PGA-rhamnolipid (Z-P-R-Cur) and zein-PGA-lecithin (Z-P-L-Cur) complex particles. Fig.7. Influence of in vitro digestion time on the particle size of curcumin loaded in (Z-Cur),

zein-PGA

(Z-P-Cur),

zein-PGA-rhamnolipid

(Z-P-R-Cur)

and

zein-PGA-lecithin (Z-P-L-Cur) complex particles. (Different letters mean significant 1

ACCEPTED MANUSCRIPT differences (P < 0.05).)

Fig. 8. Influence of in vitro digestion time on the SEM images of curcumin loaded in zein (Z-Cur; A), zein-PGA (Z-P-Cur; B), zein-PGA-rhamnolipid (Z-P-R-Cur; C) and

RI PT

zein-PGA-lecithin (Z-P-L-Cur; D) complex particles. Fig. 9 Bioaccessibity of curcumin loaded in zein (Z-Cur), zein-PGA (Z-P-Cur), zein-PGA-rhamnolipid (Z-P-R-Cur) and zein-PGA-lecithin (Z-P-L-Cur) complex

AC C

EP

TE D

M AN U

SC

particles. (Different letters mean significant differences (P < 0.05).)

2

ACCEPTED MANUSCRIPT B

50 particle size zeta-potential

C

30 CD

D

20

10

0

c

-5

150

100

-10 AB

B AB

A

50

A

0 -R -C -R Z-P

ab

2.5 ur1 Z-P

-C -R

5 ur2

0 00 25 ur5 ur1 ur1 -C -C -C -R -R -R P P P Z ZZ-

400

300

200

100

-15

Z-P

zeta-potential

bc

a

200

AB

A

0

Z-

P-L

Z-P

AC C

EP

TE D

Fig. 1

3

-15

particle size

600

M AN U

Particle size (nm)

D

0

0 5 00 25 2.5 ur5 ur2 ur1 ur1 ur1 -C -C -C -C -C Z-P Z-P Z-P Z-P Z-P

500 b

BC

-10

Z-P

b

BC

AB

A

400

a b

AB

C

C

0

Particle size (nm)

a

-5 600

25 ur1 Z-C

particle size zeta-potential

300

250

00

Zeta potential (mV)

C

ur1 Z-C

b

200

0 0 ur5 Z-C

5 ur2 Z-C

a

a

RI PT

BC

2.5 ur1 Z-C

a

a

800

Particle size (nm)

Particle size (nm)

B

40

Zeta-potential (mV)

a c

c b

b

0

zeta-potential

a

A

in Ze

particle size

1000

bc

C -L-

2 .5 ur1

AB

AB

40

30

bc

c

SC

a

20

10

0 BC

C

5 0 r25 r50 r1 2 r10 Cu Cu Cu Cu -L-L-L-LP P Z-P Z-P Z Z

-10

Zeta-potential (mV)

200

Zeta-potential (mV)

A

ACCEPTED MANUSCRIPT Cur12.5 Cur25 Cur50 Cur100 Cur125

A 100

m m

k i

f

h g

80

j l

mm m m

j

hi

e

RI PT

60

c

40

b a

20

0

Z-P-Cur

Z-P-R-Cur

B

Cur12.5 Cur25 Cur50 Cur100 Cur125

TE D

20

15

j

10

EP

LC(%)

Z-P-L-Cur

M AN U

Z-Cur

SC

EE (%)

d

fg

AC C

5

ef

ef

c

k

i

i

i h

h

g

e d b a

b

d a

b

0

zein

Z-P

Z-P-R

Fig. 2

4

Z-P-L

ACCEPTED MANUSCRIPT

Cur Z-Cur100 Z-P-Cur100 Z-P-R-Cur100 Z-P-L-Cur100

1600

RI PT

1400

Intensity

1200 1000 800

SC

600

200 0 450

500

M AN U

400

550

600

Wavenumber (nm)

AC C

EP

TE D

Fig. 3

5

650

700

ACCEPTED MANUSCRIPT

Z-P-R-Cur100

RI PT

Z-P-L-Cur100 Z-P-Cur100 Z-Cur100 Intensity

Z-P-R Z-P-L

SC

Z-P PGA Zein

Cur

10

20

30

M AN U

2θ (°)

AC C

EP

TE D

Fig. 4

6

40

ACCEPTED MANUSCRIPT

1738 1660 1542

3309

Z-P-L-Cur100

1655 1514

3311

1658 1514

3300

1655 1516

Z-P-R-Cur100 Z-P-Cur100

1736

3430

Z-Cur100

1245

Lecithin

3404 1608

1715 3450

Rhamnolipid

1746 1619

PGA

1658 1533

3300

Zein

1510 1280

SC

1151 3511

4000

RI PT

Absorbance (a. u.)

3356

1240

Curcumin

3500

3000

2500

2000

1500 -1

M AN U

Wavenumber (cm )

1000

AC C

EP

TE D

Fig. 5

7

500

RI PT

ACCEPTED MANUSCRIPT

Z-Cur100

M AN U

SC

Zein

TE D

Z-P

Z-P-R-Cur100

AC C

EP

Z-P-R

Z-P-Cur100

Z-P-L

Z-P-L-Cur100

Fig. 6

8

ACCEPTED MANUSCRIPT Z-Cur Z-P-Cur Z-P-R-Cur Z-P-L-Cur

5000

4000 m m

3000

i h

RI PT

m

150

180

m

2000

lm kl

1000

k ik j f

j ab c

a

d e

h

h

ef

e

b

0 0

30

60

90

120

M AN U

Time (min)

k

AC C

EP

TE D

Fig. 7

9

i i

SC

Particle size (nm)

m

ACCEPTED MANUSCRIPT

90 min

60 min

SC

RI PT

30 min

120 min

180 min

M AN U

150 min

TE D

A. Z-Cur100

EP

30 min

AC C

120 min

60 min

90 min

150 min

180 min

B. Z-P-Cur100

10

ACCEPTED MANUSCRIPT

60 min

120 min

150 min

90 min

180 min

M AN U

C. Z-P-R-Cur100

SC

RI PT

30 min

EP

TE D

30 min

AC C

120 min

60 min

90 min

150 min

180 min

D. Z-P-L-Cur100 Fig. 8

11

ACCEPTED MANUSCRIPT 100 c c

60 b

RI PT

Bioaccessibility (%)

80

40 a

20

Z-P-Cur100 Z-P-R-Cur100 Z-P-L-Cur100

M AN U

Z-Cur100

SC

0

AC C

EP

TE D

Fig. 9

12

ACCEPTED MANUSCRIPT Highlights Protein/polysaccharide/surfactant complexes were prepared by anti-solvent co-precipitation

efficiency of curcumin

RI PT

Polysaccharide and surfactant in the complexes increased the encapsulation

forces involved in complex formation.

SC

Electrostatic attraction, hydrogen bonding and hydrophobic effects were the main

M AN U

The particle size and charge of the complexes were depended on their composition

The surfactant in the complexes improved the photo-stability, bioaccessibility of

AC C

EP

TE D

curcumin.