Enhancing the aqueous solubility of curcumin at acidic condition through the complexation with whey protein nanofibrils

Accepted Manuscript Enhancing the aqueous solubility of curcumin at acidic condition through the complexation with whey protein nanofibrils

Mehdi Mohammadian, Maryam Salami, Shima Momen, Farhad Alavi, Zahra Emam-Djomeh, Ali Akbar Moosavi-Movahedi PII:

S0268-005X(18)31110-X

DOI:

10.1016/j.foodhyd.2018.09.001

Reference:

FOOHYD 4638

To appear in:

Food Hydrocolloids

Received Date:

18 June 2018

Accepted Date:

03 September 2018

Please cite this article as: Mehdi Mohammadian, Maryam Salami, Shima Momen, Farhad Alavi, Zahra Emam-Djomeh, Ali Akbar Moosavi-Movahedi, Enhancing the aqueous solubility of curcumin at acidic condition through the complexation with whey protein nanofibrils, Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.09.001

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Enhancing the aqueous solubility of curcumin at acidic condition through the

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complexation with whey protein nanofibrils

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Mehdi Mohammadian1, Maryam Salami1*, Shima Momen1, Farhad Alavi1, Zahra Emam-

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Djomeh1, Ali Akbar Moosavi-Movahedi2

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1Department

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Resources, University of Tehran, Karaj, Iran.

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2Institute

of Food Science and Engineering, University College of Agriculture & Natural

of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

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*Corresponding author.

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Email address: [email protected]

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Abstract

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In the present study, whey protein nanofibrils (WPN) were used as carriers to improve the

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aqueous solubility of curcumin at acidic conditions (pH 3.2) for expanding its applications in

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functional beverages and drinks. Nanofibrils with nanometric diameter (less than 15 nm) and

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micrometric length were produced by 5 h heating (85°C) of whey protein isolate (WPI) solution

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at pH 2.0. WPN showed a higher surface hydrophobicity compared to the native parental

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proteins which improved its ability to form a soluble complex with curcumin. The water

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solubility of curcumin was increased by about 1200-folds through the complexation with WPN,

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whereas the complexation with WPI increased its solubility by approximately 180-folds.

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Fluorescence measurements and Fourier transform infra-red spectroscopy indicated that the

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hydrogen bonding and hydrophobic interactions were mainly contributed to the formation of

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curcumin-WPN complexes. Circular dichroism spectroscopy revealed that the secondary

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structures of WPN were not significantly affected by binding to curcumin. The structural phase

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of nanocomplexes also was studied using X-ray diffraction analysis. Complexation of whey

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protein nanofibrils with curcumin improved their apparent viscosity and surface activity which

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makes them appropriate candidates to design new functional food emulsions and beverages. The

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results of antioxidant activity measurements (DPPH radical scavenging activity and reducing

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power) also showed that the antioxidant capacity of curcumin was drastically improved through

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the complexation with WPN. Generally, this study suggests that the whey protein nanofibril

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could be employed as a material to enhance the food applications of curcumin as a water-

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insoluble bioactive compound.

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Keywords: Curcumin, Whey protein nanofibril, Nanocomplexes, Solubility, Structural

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properties, Antioxidant activity.

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1. Introduction

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Curcumin is a natural phenolic compound belonging to the group of curcuminoids derived from

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the rhizome of turmeric (Curcuma longa) (Patel, Hu, Tiwari, & Velikov, 2010). According to the

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extensive recent studies on the biological functionalities of curcumin, it is well-known that

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curcumin has the ability to improve the human health owing to its antioxidant, antibacterial,

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anticancer, anti-inflammatory, and antiviral activities (Yi et al., 2016; Li, Ma, & Ngadi, 2013).

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Despite having a wide range of health-promoting effects, the applications of curcumin as a

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bioactive and pharmacological agent in different functional products and formulations have been

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limited due to its extremely low water solubility, poor oral bioavailability, low chemical stability,

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and rapid metabolism (Li et al., 2013; Huang et al., 2016). Encapsulation within or complexation

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with proteins has been reported as a promising approach for improving the solubility of curcumin

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in aqueous solutions and increasing its bioavailability. In this regard, different food proteins such

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as soy proteins (Tapal & Tiku, 2012; Chen, Li, & Tang, 2015), whey proteins (Li et al., 2013; Yi

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et al., 2016; Liu, Chen, Cheng, & Selomulya, 2016; Liu et al., 2017), zein (Huang et al., 2016;

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Chang et al., 2017), and casein (Pan, Zhong, & Baek, 2013) have been used as carriers for

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enhancing the aqueous solubility and antioxidant activity of curcumin. For example, it was

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reported that the curcumin aqueous solubility was drastically increased by 812-fold through the

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complexation with soy protein isolate (Tapal & Tiku, 2012). Hydrophobic interactions play the

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most important role in the formation of food proteins-curcumin complexes. In fact, curcumin

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binds to the hydrophobic patches of protein molecules especially the aromatic amino acid

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residues (Liu et al., 2016). With less importance, hydrogen bonds also contribute to the

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generation of proteins-curcumin binary systems (Liu et al., 2017).

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Whey proteins have been widely employed in different food products and also used to fabricate

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various types of nutraceutical-carrying systems thanks to their superb nutritional properties and

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diverse technological functionalities (Nourbakhsh, Madadlou, Emam-Djomeh, Wang, &

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Gunasekaran, 2016). Whey proteins can form different structures like randomly shaped

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aggregates, nanofibrils, nanotubes, flexible strands, microgels, and particles (nano and micro)

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depending on the processing conditions such as primary protein concentration, temperature, pH

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value, and ionic strength (Nicolai, Britten, & Schmitt, 2011).

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Whey proteins especially β-lactoglobulin can self-assemble to nanofibrillar aggregates with a

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micrometric (1-10 μm) length and nanometric (1-10 nm) diameter through the prolonged heating

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at acidic condition (commonly at pH values about 2.0) and low ionic strength (Akkermans, van

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der Goot, Venema, van der Linden, & Boom, 2008). Whey protein nanofibrils are especially

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interesting because of their improved techno-functional properties compared to the non-

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fibrillated proteins such as the ability to form self-supporting hydrogels at very low protein

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concentrations, better emulsifying and foaming properties, and higher capacity to enhance the

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bulk viscosity (Ng et al., 2016; Mantovani, de Figueiredo Furtado, Netto, & Cunha, 2018).

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Moreover, it was investigated that fibrillated WPI possesses a higher in vitro antioxidant and

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radical scavenging activity compared to the parental proteins due to the generation of bioactive

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peptides during the fibrillation process (Mohammadian & Madadlou, 2016a; Feng et al., 2018).

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Thanks to their improved functional and biological attributes, edible protein-based nanofibrils

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were used as delivery bodies for different bioactive molecules such as limonene (Humblet-Hua,

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Scheltens, van der Linden, & Sagis, 2011) and micronutrients (Mohammadian & Madadlou,

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2016b).

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During the fibrillation process of whey proteins, peptides generated by heat and acid hydrolysis

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with high hydrophobicity have the ability to form nanofibrils (Gao, Xu, Ju, & Zhao, 2013). In

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this regard, Feng et al. (2018) reported that the surface hydrophobicity of whey proteins was

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significantly increased during the formation of fibrils. Accordingly, we hypothesized that whey

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protein nanofibrils with high hydrophobic nature can be used as a promising system for

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improving the aqueous solubility of curcumin at acidic conditions which enhances the

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applications of this bioactive molecule in food products especially functional acidic beverages

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and drinks. To the best of our knowledge, there was no study in the literature on enhancing the

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aqueous solubility and antioxidant activity of curcumin using food protein nanofibrillar

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structures. Therefore, in the present original work, our challenge was to employ whey protein

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nanofibrils as novel nanocarriers for enhancing the solubility of curcumin as a bioactive

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compound and this study is the first report dealing with this matter. In this regard, whey protein

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fibril-curcumin nanocomplexes were fabricated at an acidic condition (pH 3.2) and their

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structural, functional, in vitro antioxidant properties, and release behavior were investigated

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using different techniques.

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2. Materials and methods

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2.1. Materials

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Whey protein isolate (WPI) with more than 90 % protein content was purchased from Gallo

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Global Nutrition (Atwater, CA, USA). Curcumin was obtained from Merck (Darmstadt,

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Germany). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 8-Anilinonaphthalene-1-sulfonic acid

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(ANS) were purchased from Sigma-Aldrich (Sa. Louis, MO, USA). Enzyme pepsin (with

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activity more than 3000 units mg-1) and pancreatin (200 units mg-1) were purchased from Bio

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Basic (Bio Basic Inc., Canada). All of the other chemicals used in this research also were of

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analytical grade and purchased from Sigma-Aldrich and Merck.

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2.2. Formation of whey protein nanofibrils (WPN)

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Whey protein isolate was nanofibrillated according to the method described by Akkermans et al.

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(2008) with slight modifications. For this purpose, whey protein solution (50 mg mL-1) was

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prepared by dissolving an appropriate amount of WPI powder in distilled water containing

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sodium azide as an antimicrobial agent. Resulting solution was magnetically stirred (200 rpm)

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for 2 h and kept for 12 h at 4°C to fulfill the protein hydration. After that the pH of WPI solution

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was adjusted to 2.0 using HCl 8 M and then heated for 5 h at 85°C under mild condition of

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stirring. Finally, the fibrillation process was stopped by cooling down the solution by cold tap

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water. The resultant fibrillated WPI solution was stored at 4°C until the subsequent uses.

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2.3. Surface hydrophobicity measurement

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The surface hydrophobicity of WPI and WPN was determined according to the method of Gao et

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al. (2013) with some modifications using ANS as a fluorescent probe. For this purpose, 20 μL of

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ANS solution (8 mM) was added to 2 mL of WPI or WPN solution (pH 3.2) with concentrations

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of 0.01 to 0.2 mg mL-1, vortexed, and kept for 15 min in dark. After that, the fluorescence

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intensity of resulting solutions were measured using a Varian fluorescence spectrofluorometer

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(Cary Eclipse, Palo Alto, CA) at excitation and emission wavelengths of 390 nm and 470 nm,

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respectively. The entrance and exit slits were set at 2.5 nm. Finally, the fluorescence intensity

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was plotted versus protein concentration and its initial slope was used as an index of protein

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surface hydrophobicity (S0).

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2.4. Complexation of curcumin with WPI and WPN

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The pH values of fully-hydrated WPI and WPN solutions with protein concentrations of 50 mg

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mL-1 were adjusted to 3.2 using NaOH 5 M or HCl 8 M to simulate the conditions of common

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food beverages and drinks. After that, the curcumin which was dissolved in ethanol added to the

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protein solutions or distilled water under magnetically-stirred conditions. The final concentration

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of curcumin in mixtures was 0.5 mg mL-1 (curcumin to protein ratio of 1:100). This was

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followed by 12 h stirring at a dark condition and room temperature. The resultant complexes (C-

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WPI and C-WPN) were stored at 4°C or freeze-dried for subsequent analyses. It should be noted

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that the final concentration of ethanol in mixtures never exceeded 0.2% (v/v) which no

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significant effect on the structure of whey proteins was reported for ethanol at this concentration

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by a previous study (Liu et al., 2017).

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2.5. Determination of curcumin solubility

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The samples including WPI-curcumin and WPN-curcumin complexes as well as aqueous

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solution of curcumin were centrifuged at 5000 × g for 10 min to remove the undissolved

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curcumin (Tapal & Tiku, 2012). The resulting supernatant was diluted with ethanol to extract the

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curcumin and its absorbance was measured spectrophotometrically at 420 nm. Finally, the

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concentration of curcumin in supernatant was determined with a standard curve of curcumin

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(0.1-10 μg mL-1 dissolved in ethanol, R2= 99.99%) and the curcumin solubility in different

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medium (i.e. distilled water, WPI, and WPN) was calculated using the following equation:

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Curcumin solubility (%) =

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The sedimentation of curcumin in different samples (distilled water, WPI, and WPN) during one

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month of storage was also evaluated by monitoring their appearances.

amount of curcumin in supernatant × 100 total amount of added curcumin

(Eq. 1)

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2.6. Characterization of nanocomplexes

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2.6.1. Atomic force microscopy (AFM)

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The morphology of WPI, WPN, and their complexes with curcumin was studied using an atomic

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force microscope (Nanowizard II instrument, JPK, Germany) equipped with a HYDRA-

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Cantilever. Different specimens were diluted in distilled water with same pH (pH 3.2) to a final

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concentration of 0.05 mg mL-1 prior the microscopy. After that, 20 μL of diluted samples was

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deposited on a glass slide and air-dried. Finally, images were taken at a scan rate of 1.2 Hz. The

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JPK Data Processing software (version 3.4.15) was used to process the acquired AFM images.

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The morphology of curcumin-WPN nanocomplexes also was studied at pH value of 7.0 for

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studying their stability at neutral conditions.

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2.6.2. Fluorescence spectroscopy

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The binding of curcumin to WPI and WPN was studied by measuring the changes of protein and

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curcumin fluorescence intensities with the help of a fluorescence spectrofluorometer. At first,

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samples were diluted with distilled water (pH 3.2) to a protein concentration of 0.2 mg mL-1 and

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curcumin concentration of 2 μg mL-1 and then employed for fluorescence measurements. For the

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evaluation of protein intrinsic fluorescence, the emission spectra of samples were recorded from

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300 to 400 nm with an excitation wavelength of 280 nm. The emission spectra of curcumin in

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different mediums also were measured from 450 to 700 nm with an excitation wavelength of 420

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nm. The entrance and exit slits were set at 5 nm.

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2.6.3. Zeta-potential measurements

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The ζ-potential of different sample solutions including WPI, WPN, and their complexes with

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curcumin at pH value of 3.2 was determined with a dynamic light scattering (DLS) apparatus

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(Brookhaven Instruments Corp., Holtsville, NY, USA) in a ζ-potential measurement mode using

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a specialized zeta cell. All of the specimens were diluted with distilled water (pH 3.2) before the

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zeta-potential measurements.

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2.6.4. Fourier transform infra-red (FT-IR) spectroscopy

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The molecular attributes of pure curcumin, WPI, WPN and protein/curcumin complexes were

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investigated using FT-IR spectroscopy using a Bruker FT-IR spectrometer (Billerica,

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Massachusetts, United States). The freeze-dried samples were powdered and pressed to

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potassium bromide disks and scanned at a wavenumber range of 4000-500 cm-1 in a

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transmittance mode.

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2.6.5. Circular dichroism (CD) spectroscopy

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The secondary structural changes of WPI and WPN due to the complexation with curcumin were

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monitored by CD spectroscopy. For this purpose, the CD spectra of different samples with a final

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protein concentration of 0.2 mg mL-1 were recorded in Far-UV (190-260 nm) region using a

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Jasco spectropolarimeter (model J-810, Jasco, Japan) at room temperature by means of 1.0 mm

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path length cells. Finally, the secondary structure contents of different specimens were predicted

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using the CDNN program (version 2.1.0.233).

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2.6.6. X-ray diffraction (XRD) analysis

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The XRD patterns of freeze-dried samples including pure curcumin, WPI, WPN, WPI-curcumin

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complexes, and WPN-curcumin nanocomplexes were recorded on a Philips PW1730 X-ray

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diffractometer (PANalytical, Netherlands) equipped with a Cu Kα radiation source in the 2θ

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range of 5° to 50° with a step size of 0.05° and scan rate of 1° s-1. The operating voltage and

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filament current were set at 40 KV and 30 mA, respectively.

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2.6.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

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SDS-PAGE under reducing mode using a 12% polyacrylamide gel was used to study the

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composition of different samples including WPI, WPN, and their complexes with curcumin. For

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this purpose, 100 μL of protein sample solutions was mixed with 50 μL of SDS-PAGE sample

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buffer containing β-mercaptoethanol followed by incubating for 2 min in a boiling water bath.

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Afterward, 35 μL of the resulting mixtures was loaded into the wells and separated.

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2.6.8. Viscosity measurement

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The apparent viscosity of different sample solutions at pH 3.2 as a function of shear rate was

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determined using a rotational programmable viscometer (Rheo 3000, Brookfield Engineering,

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Inc., USA) at room temperature (25 ± 2°C). For this purpose, about 5 mL of each sample was

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poured into the cylinder of viscometer and sheared from 3 to 200 s-1 using a CC14 stainless steel

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cylindrical spindle.

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2.6.9. Surface tension

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The surface tension of different sample solutions as a function of time was evaluated using the

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Du Nouy ring method by a Kruss K100 tensiometer (KRÜSS GmbH, Hamburg, Germany) at

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25°C (Tomczynska-Mleko et al., 2014).

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2.6.10. Antioxidant properties

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2.6.10.1. DPPH radical scavenging activity

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DPPH radical scavenging capacity of different samples with protein concentration of 5 mg mL-1

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and curcumin concentration of 50 μg mL-1 was determined according to the method that

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described by Lin, Tian, Li, Cao, and Jiang (2012) with some modifications. One mL of each

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sample or distilled water (control) was mixed with 1 mL of ethanolic DPPH solution (0.2 mM)

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and stored for 30 min at room temperature in the dark. Thereafter, the absorbance was read at

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517 nm using a UV-visible spectrophotometer. The DPPH radical scavenging activity was

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calculated using the following equation:

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Radical scavenging activity(%) =

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where AC and AS are the absorbance of control and sample solutions, respectively.

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2.6.10.2. Reducing power

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Reducing power of different specimens was measured according to the method of Lin et al.

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(2012) with some adjustments. For this purpose, 0.5 mL of samples (protein concentration of 5

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mg mL-1 and curcumin concentration of 50 μg mL-1) was charged with 1.25 mL of 0.2 M

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phosphate buffer with pH value of 6.6 and 1.25 mL of potassium ferricyanide (10 g L-1) and then

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was incubated for 20 min at 50°C. After that, 1.25 mL of TCA (10%) was added to the samples

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and the resulting mixtures were centrifuged for 10 min at 1500 × g. 1.25 mL of supernatant was

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mixed with 1.25 mL of distilled water and 0.25 mL of FeCl3 (1 g L-1). Finally, after 10 min

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incubation at room temperature, the absorbance was measured spectrophotometrically at 700 nm.

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The higher absorbance indicated higher antioxidant activity.

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2.6.11. In vitro release behavior

AC ‒ AS AC

× 100

(Eq. 2)

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The in vitro release behavior of curcumin from nanocomplexes based on WPI and whey protein

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nanofibrils under simulated gastrointestinal conditions was studied according to the method of

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Xue et al. (2018) with some modifications. Briefly, 3 mL of C-WPI or C-WPN solutions were

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placed into a dialysis bag (molecular cut off of 12000 Da) and then was charged with 3 mL of

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simulated gastric fluid (SGF) containing sodium chloride, hydrochloric acid, and 0.32% enzyme

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pepsin with a final pH value of 1.2. Subsequently, the dialysis bag was immersed in a beaker

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containing 150 mL of enzyme-free SGF-ethanol (50% v/v) solution as the release medium. After

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2 h incubation at 37°C and shaking of 100 rpm, the mixture was mixed with 6 mL of simulated

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intestinal fluid (SIF, consisted of monobasic potassium phosphate, sodium hydroxide, and 10 g/L

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of pancreatin with the final pH value of 7.5) and the dialysis bag also was transferred to a beaker

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containing 150 mL of enzyme-free SIF-ethanol (50% v/v) solution and incubated for 4 h at 37°C

244

and continuous shaking of 100 rpm. It should be noted that the ethanol was used in the release

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medium to achieve the sink condition due to the poor aqueous solubility of curcumin. Aliquots of

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the outer release medium were collected at the selected time intervals (0, 1, 2, 3, 4, 5, and 6 h)

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and replaced with an equal volume of fresh medium. The content of the released curcumin was

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measured spectrophotometrically at 420 nm and its concentration was calculated using a

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curcumin standard curve which was prepared using the same release medium.

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2.7. Statistical analysis

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Statistical analysis of the data was carried out using one-way analyses of variance (ANOVA) and

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significant differences (p < 0.05) between the samples were determined by Duncan's multiple

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range procedure using SPSS software (version 23, IBM software, NY, USA). Each experiment

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was replicated at least three times.

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3. Results and discussion

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3.1. Surface hydrophobicity of WPI and WPN

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ANS as a fluorescence probe was employed to measure the surface hydrophobicity of WPI and

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WPN. The results showed that the surface hydrophobicity of WPI was increased drastically

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through the fibrillation process (S0 WPI= 50.93 ± 0.36 and S0 WPN= 174.28 ± 2.6). It was

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investigated that the specific peptides with high surface hydrophobicity and ability to form β-

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sheets are building blocks of whey protein nanofibrils which assemble to fibrillar structures

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during the heating at pH 2.0 (Kroes-Nijboer, Venema, & van der Linden, 2012). In accordance

263

with our results, Gao et al. (2013) also reported a higher surface hydrophobicity for whey protein

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nanofibrils compared to the non-fibrillated counterpart attributing to the partial unfolding of

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whey proteins during the fibrillation which exposes more hydrophobic groups. This higher

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surface hydrophobicity could be accounted for the superior curcumin binding capacity of whey

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protein nanofibrils compared to the native WPI which observed in the present study.

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3.2. Curcumin solubility and sedimentation

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The results of curcumin solubility measurements are shown in Fig. 1. The water dispersibility of

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curcumin in the presence of WPI and WPN was higher compared to the non-complexed

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curcumin. The solubility of curcumin-WPN complexes was also significantly more than the

272

complexes made of curcumin and native WPI. In fact, the solubility of curcumin in the present

273

study was enhanced by about 1200-folds through the complexation with WPN, whereas the

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complexation with WPI increased the water solubility of curcumin by approximately 180-folds

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(vs free curcumin in water with final curcumin and ethanol concentration of 0.5 mg mL-1 and

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0.2%, respectively). In agreement with these observations, Tapal and Tiku (2012) also reported

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that the solubility of curcumin in aqueous solutions was significantly increased (812-folds)

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through the complexation with soy proteins. Encapsulation of curcumin in WPI by spray drying

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also improved its solubility (Liu et al., 2016). Curcumin forms soluble complexes with proteins

280

(here WPI and WPN) through the hydrophobic interaction which leads to enhance its water

281

dispersibility. Sedimentation of curcumin in different states was also visually monitored during

282

one month of storage and the results are shown in Fig. 2. The aqueous solution of curcumin

283

became completely colorless after one month of storage due to the complete precipitation of

284

curcumin. No significant sedimentation was observed for curcumin-WPN nanocomplexes at the

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bottom of the test tube during the storage time, whereas a thin layer of curcumin sediment was

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observed in the case of curcumin-WPI complexes at the end of the storage period which

287

confirms the results of solubility measurements. Therefore, complexation of curcumin with whey

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protein nanofibrils can be used to overcome the low aqueous solubility of curcumin which limits

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its delivery by dietary formulations, supplements, and food beverages.

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3.3. Atomic force microscopy

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The shape and size of different samples including WPI, WPN, and their complexes with

292

curcumin were imaged using atomic force microscopy and the results are displayed in Fig. 3. For

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native WPI, nanometric spherical particles were observed (Fig. 3A) confirming the nature of

294

major whey protein components. In the case of WPI-curcumin complexes (Fig. 3B), larger

295

particles (heights up to 8 nm) with a homogenous morphology were investigated. These

296

observations are in a good agreement with those of Chen et al. (2015) who studied the

297

morphology of soy protein isolate (SPI)-curcumin nanocomplexes using AFM. They also

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reported a larger size for SPI-curcumin complexes (up to 6 nm) compared to the curcumin-free

299

counterpart (2-4 nm). As can be seen in Fig. 3C, 5 h heating of WPI solution at pH 2.0 resulted

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in the formation of fibrillar aggregates with nanometric diameter (up to 15 nm) and micrometric

301

length which is in accordance with previous studies (Gao et al., 2013; Mantovani et al., 2018; Ng

302

et al., 2016). AFM imaging also demonstrated that the morphology of nanofibrils was not

303

significantly affected through the complexation with curcumin (Fig. 3D). These results indicated

304

that the whey protein nanofibrils survived during the treating with curcumin and have a high

305

stability. The morphology of curcumin-whey protein nanofibrils complexes also was imaged at

306

pH 7.0 using AFM for assessing the stability of fibrillar structures at neutral condition in addition

307

to the acidic condition. The AFM imaging (Fig. 3E) showed that the curcumin-WPN

308

nanocomplexes survived during pH adjustment to 7.0 and were stable at this neutral condition.

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This observation suggested that the curcumin-whey protein nanofibrils complexes also can be

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used for enriching of food products and beverages with neutral pHs in addition to the acidic

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beverages and drinks. Mantovani, Fattori, Michelon, and Cunha (2016) also studied the pH-

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stability of whey protein nanofibrils in a pH range from 3 to 7 and reported a low susceptibility

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to pH changes for whey protein nanofibrillar structures. In accordance with our results,

314

Mohammadian and Madadlou (2016b) also showed same morphology for whey protein

315

nanofibrils at pH values of 2.0 and 7.0 which was investigated by atomic force microscopy.

316

3.4. Chemistry of nanocomplexes

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Fluorescence spectroscopy was used to study the interaction between curcumin as a ligand and

318

different forms of whey proteins (i.e. native and nanofibrillated). Fluorescence spectra of

319

different samples at an excitation wavelength of curcumin (i.e. 420 nm) are shown in Fig. 4A. It

320

was found that the free curcumin in water in the absence of WPI and WPN showed a low-

321

intensity broad peak at 493 nm which is in agreement with the results of Li et al. (2013). When

322

curcumin bound to WPI and WPN, its fluorescence intensity was increased. The fluorescence

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intensity of curcumin-WPN complexes was also significantly higher than the curcumin-WPI

324

complexes. These observations suggested that the curcumin was transferred from a polar

325

(hydrophilic) to a less polar (more hydrophobic) environment resulting from binding to the

326

hydrophobic patches of whey protein molecules (Li et al., 2013; Liu et al., 2016). The higher

327

intensity for curcumin-WPN compared to the curcumin-WPI counterpart also can be justified by

328

the higher surface hydrophobicity of whey protein nanofibrils than native WPI which increases

329

its capacity to form complexes with curcumin. Our findings are in accordance with previous

330

reports who studied the interaction between curcumin and different food proteins such as WPI

331

(Liu et al., 2016), β-lactoglobulin (Li et al., 2013), casein (Pan et al., 2013), and soy proteins

332

(Tapal & Tiku, 2012).

333

The fluorescence quenching of proteins at an excitation wavelength of 280 nm also was

334

investigated to estimate the accessibility of curcumin to fluorophore groups of WPI and WPN

335

and the resulting spectra are represented in Fig. 4B. Complexation of WPI and WPN with

336

curcumin decreased their fluorescence intensity; more decrease was observed for curcumin-WPN

337

complexes compared to the curcumin-WPI sample. The fluorescence emission of proteins at an

338

excitation wavelength of 280 nm is related to tryptophan and tyrosine residues (Li et al., 2013).

339

So, the quenching of WPI and WPN fluorescence upon the complexation with curcumin can be

340

due to the ability of curcumin to bind the tyrosine and tryptophan residues (Liu et al., 2016). The

341

more decrease in the fluorescence intensity in the case of curcumin-WPN compared to curcumin-

342

WPI complexes also suggested a higher curcumin binding capacity for whey protein nanofibrils

343

in comparison with the native WPI. In agreement with our findings, Tapal and Tiku (2012) also

344

reported that the intrinsic fluorescence of SPI was decreased upon binding to curcumin.

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The zeta-potential of different samples including WPI, WPN, and their complexes with curcumin

346

was determined to study the effect of curcumin binding on the stability of the delivery system

347

(Fig. 5). Fibrillation led to an increase in the positive charge of WPI which can be due to the

348

exposure of charged groups resulting from the hydrolysis and partial unfolding of whey protein

349

molecules during the formation of fibrillar aggregates (Mantovani et al., 2018). Complexation

350

with curcumin significantly (p < 0.05) increased the positive surface charge of WPI and WPN,

351

with a higher extent for curcumin-WPN nanocomplexes. In accordance, a greater zeta-potential

352

was also observed for curcumin-loaded caseinate/zein nanoparticles compared to curcumin-free

353

systems (Chang et al., 2017). The greater change in the surface charge density of whey protein

354

nanofibrils due to the complexation with curcumin compared to WPI-curcumin systems can be

355

attributed to the higher curcumin binding capacity of nanofibrils as a consequence of their higher

356

surface hydrophobicity. Chen et al. (2015) also reported a remarkable change in the net charge of

357

SPI at a so high load amount of curcumin due to the full coverage of protein surface hydrophobic

358

zones by the curcumin molecules. In general, the results of zeta-potential measurements showed

359

a good colloidal stability for curcumin-WPN complexes (with respect to their greater zeta-

360

potential) which makes them suitable candidates for using in functional acidic beverage systems.

361

FT-IR spectroscopy was employed to study the molecular attributes of different samples (Fig. 6).

362

Many characteristic peaks were observed for free curcumin related to its various functional

363

groups such as aromatic rings. However, these peaks were disappeared when curcumin was

364

complexed with WPI and WPN. This can be probably due to the limited stretching and bending

365

of the vibrations in curcumin when bound to the proteins. Xue et al. (2018) also reported that the

366

characteristic peaks of curcumin were diminished when it was encapsulated in zein-caseinate

367

composite nanoparticles. Amid I (1700-1600 cm-1, related to C=O stretching vibrations) and

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amid II (1575-1480 cm-1, related to 60% N-H bending and 40% C-H stretching vibrations)

369

regions of FT-IR spectra can be employed to study changes in the secondary structures of

370

proteins (Kong & Yu, 2007). FT-IR results indicated that the complexation of WPI and WPN did

371

not cause any noteworthy alteration in the position of these characteristic peaks associated with

372

the secondary structure of proteins. Therefore, it seems that the secondary structure of WPI and

373

WPN was not significantly influenced through the complexation with curcumin which also was

374

confirmed by CD spectroscopy. However, complexation with curcumin resulted in a peak

375

displacement from 2944 cm-1 to 2938 cm-1 for WPI and from 2964 cm-1 to 2925 cm-1 for whey

376

protein nanofibrils. These peaks in the spectrum of proteins are corresponded to the C-H

377

stretching vibrations of CH3 and CH2 functional groups (Kong & Yu, 2007). Hence, the above-

378

mentioned changes can be due to the formation of hydrogen bonds between the curcumin and

379

WPI/WPN which as a general rule lowers the frequency of stretching vibrations (Mohammadian

380

& Madadlou, 2016b). Accordingly, it was investigated that the binding of curcumin to proteins

381

such as β-lactoglobulin and zein-caseinate was occurred mainly through the hydrophobic

382

interactions and hydrogen bonding (Liu et al., 2017; Xue et al., 2018).

383

Circular dichroism spectroscopy in the far-UV region was employed to evaluate the influence of

384

curcumin binding on the secondary structures of WPI and WPN (Fig. 7). The secondary

385

structures contents of different specimens also were predicted using CDNN program and the

386

results are shown in Table 1. The CD spectrum of WPI showed two troughs at wavelengths of

387

around 218 and 210 nm which are characteristics of β-sheet and α-helical structures related to the

388

main fractions of whey proteins including β-lactoglobulin and α-lactalbumin (Liu et al., 2017;

389

Tomczynska-Mleko et al., 2014). Fibrillation significantly changed the CD spectrum of native

390

WPI which is in accordance with the observations of Mohammadian and Madadlou (2016a) and

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Gao et al. (2013) who reported that the heating of WPI at acidic conditions influenced its

392

secondary structure. As can be seen in Fig. 7 and Table 1, complexation of WPI and WPN with

393

curcumin did not significantly change their secondary structures. WPI/WPN-curcumin

394

nanocomplexes also showed similar CD spectra to those of non-complexed corresponding parent

395

proteins. In agreement with our findings, Liu et al. (2017) also investigated similar far-UV CD

396

spectra for native β-lactoglobulin and its complex with curcumin. This was attributed to the

397

relative flexibility of β-lactoglobulin loops which allows the protein to interact with ligands such

398

as curcumin without requiring extensive structural changes. Moreover, it was reported by Li et

399

al. (2013) that the change in the secondary structures of β-lactoglobulin resulting from the

400

binding to curcumin was dependent on the pH. They investigated no significant alteration in the

401

second structure of β-lactoglobulin upon interaction with curcumin at acidic conditions.

402

Whereas, binding of β-lactoglobulin to curcumin at pH value of 7.0 increased the contents of α-

403

helix, β-sheet, and β-turn.

404

The X-ray diffraction analysis was employed to examine the structural phase of different

405

samples (Fig. 8). The diffraction diagram of pure curcumin exhibited several characteristic peaks

406

at 2θ of 9.21°, 12.46°, 14.76°, 17.56°, 21.46°, 25.86°, 29.26°, etc. corresponding to its high

407

crystalline structure (Liu et al., 2016). Native WPI also showed an XRD pattern with no

408

significant peaks indicating its amorphous nature; whereas, fibrillation resulted in the formation

409

of a distinguishable peak at 2θ of 43.31° suggesting a partially crystalline structure for WPN.

410

This can be due to the hydrolysis of whey proteins during the fibrillation process which also was

411

confirmed by SDS-PAGE (Fig. 9). In this regard, it was reported that the hydrolysis of WPI at

412

pH 2.0 using pepsin caused the formation of a new peak at 45.7° in the XRD pattern of whey

413

protein hydrolysates (Nourbakhsh et al., 2016). The XRD analysis also indicated that the

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complexation of WPI and WPI resulted in the formation of new sharp peaks in their patterns.

415

These peaks were observed at 2θ of 43.16° for curcumin-WPI complexes and at 2θ of 41.81° and

416

48.71° for curcumin-WPN nanocomplexes. These peaks can be due to the presence of free

417

curcumin molecules which did not form complexes with proteins. Moreover, it was reported that

418

the binding of food protein molecules to ligands such as phenolic compounds affected their

419

regular arrangement (Malik, Sharma, & Saini, 2016). More changes in the XRD pattern which

420

was observed for whey protein nanofibrils resulting from the complexation with curcumin

421

compared to WPI can be justified by their higher capacity for binding to curcumin molecules.

422

However, other characteristic peaks related to curcumin were not observed in the XRD spectra of

423

C-WPI and C-WPN complexes suggesting the formation of amorphous curcumin (Patel et al.,

424

2010; Liu et al., 2016).

425

The SDS-PAGE profiles of different samples under reducing condition are represented in Fig. 9.

426

Characteristic peaks at 66 kDa, 18 kDa, and 14 kDa respectively related to the bovine serum

427

albumin (BSA), β-lactoglobulin (BLG), and α-lactalbumin (ALA) were observed for native WPI.

428

These peaks were disappeared or their intensity was decreased after fibrillation attributing to the

429

hydrolysis of whey proteins during the fibrillation process by acid and heat resulting in the

430

formation of peptides (Mohammadian and Madadlou, 2016a). These results are in accordance

431

with those of Bateman, Ye, and Singh (2010) who reported that peptides with molecular mass

432

lower than 10 kDa were formed after 20 h heating of β-lactoglobulin at 80°C and pH value of

433

2.0. However, it is noteworthy that any new band formation was not observed for whey protein

434

nanofibrils which can be due to the disassembly of nanofibrillar structures by SDS (Oboroceanu,

435

Wang, Brodkorb, Magner, & Auty, 2010). Moreover, the results showed that after the

436

complexation of WPI with curcumin, the SDS-PAGE pattern remained similar to that of native

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WPI. Whereas, the intensity of bands in the SDS-PAGE profile of curcumin-WPN complexes

438

were higher than those of curcumin-free sample. These results can be due to the significant

439

covering of WPN hydrophobic zones by curcumin molecules. In fact, in SDS-PAGE, the

440

molecules of SDS bind to hydrophobic patches of the polypeptide chain and break the

441

hydrophobic interactions (Guo & Chen, 1990). Therefore, it seems that in the case of whey

442

protein fibril-curcumin complexes, hydrophobic parts of proteins are less available for SDS,

443

resulting in the formation of bands with higher intensity compared to the curcumin-free

444

counterpart. However, to the best of our knowledge, there is no study on the effects of curcumin

445

binding on the molecular weight pattern of proteins; hence further studies are required to

446

investigate this interesting phenomenon.

447

3.5. Viscosity and surface tension

448

The apparent viscosity as a function of shear rate for different samples is shown in Fig. 10A. The

449

viscosity of WPI solution was increased by nanofibrillation process. The higher viscosity of

450

fibrillated WPI compared to native counterpart was also reported by other studies (Ng et al.,

451

2016; Akkermans et al., 2008) attributing to the formation of nanofibrillar structures with high

452

ability to entangle and form densely packed networks and also increasing of protein

453

hydrodynamic diameter due to the fibrillation process. Mohammadian and Madadlou (2016a)

454

also reported that the nanofibrillation of whey protein isolate for 5 h at 85°C increased its

455

consistency coefficient about 20 folds. Complexation with curcumin also increased the apparent

456

viscosity of WPI and WPN solutions. In agreement with our results, a higher viscosity was

457

reported for curcumin-treated collagen compared to the native counterpart due to the aggregation

458

of collagen in the presence of curcumin (Fathima, Devi, Rekha, & Dhathathreyan, 2009).

459

Hypothetically, it is possible that curcumin forms new inter- and intra-molecular linkages

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460

between proteins/fibrils through binding to their hydrophobic patches resulting in the formation

461

of associated particles increasing the flow resistance of the solutions and enhancing their

462

apparent viscosity. Among the samples, the highest apparent viscosity was observed for

463

curcumin-WPN complexes, so these nanocomplexes can be used as efficient bioactive thickening

464

and viscosity enhancer agents in the formulation of food products.

465

The surface tension of different samples as a function of time is displayed in Fig. 10B. All of the

466

samples including the WPI, WPN, and their complexes with curcumin had the ability to reduce

467

the surface tension at air/water interface which can be due to the presence of surface active

468

compounds. The surface tension of the samples decreased during the time and reached 46.25,

469

44.32, 44.77, and 42.66 mN/m for WPI, WPN, C-WPI, and C-WPN respectively. The lower

470

surface tension of whey protein nanofibrils compared to the native WPI can be explained by

471

increasing the surface hydrophobicity of proteins during the fibrillation process as well as the

472

presence of unconverted peptides and protein monomers in the fibrillated WPI system (Ruhs,

473

Scheuble, Windhab, & Fischer, 2013). In accordance with our results, Mantovani et al. (2018)

474

also investigated a higher surface activity for fibrillated WPI compared to the intact whey

475

proteins. Moreover, our results indicated that the complexation of WPI and WPN with curcumin

476

increased their surface activity. In agreement with our findings, it was reported that the surface

477

activity of collagen was improved through treating with curcumin (Fathima, Dhathathreyan, &

478

Ramasami, 2010). This was attributed to the exposure of more non-polar groups of protein

479

resulted from binding to curcumin which forces the protein to arrive at the air/water interface.

480

Furthermore, the hydrophobic nature of curcumin may involve in the changes of proteins surface

481

tension. A lower energy barrier for adsorption to the interfaces was reported for hydrophobic

482

compounds compared to the hydrophilic counterparts (Yang, Liu, Zeng, & Chen, 2018). It seems

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483

that the complexation of WPI and WPN with a hydrophobic bioactive molecule (curcumin) helps

484

them to diffuse to the air/water interface. According to the results of surface tension

485

measurement, it was indicated that the whey protein nanofibril-curcumin complexes with a high

486

affinity towards the interfaces consisted of hydrophilic and hydrophobic phases can be used to

487

design new engineered specialty food emulsions and foams.

488

3.6. Antioxidant characteristics

489

Reducing power assay and DPPH radical scavenging test were employed to determine the in

490

vitro antioxidant activity of different samples including WPI, WPN, and their complexes with

491

curcumin as well as the pure curcumin which was dispersed in distilled water (Fig. 11). The

492

results revealed a higher antioxidant activity for WPN compared to native WPI. This can be

493

justified by the conformational modifications and generation of peptides during the fibrillation

494

presses due to the acid and heat hydrolysis of the proteins. In accordance, Mohammadian and

495

Madadlou (2016a) and Feng et al. (2018) also reported a higher antioxidant capacity for

496

fibrillated WPI in comparison with non-fibrillated counterparts. The complexation of curcumin

497

with WPI and WPN also drastically improved its antioxidant capacity in both reducing power

498

and DPPH radical scavenging activity assays compared to the aqueous state. Li et al. (2013) also

499

reported a higher reduction capability for curcumin in the presence of β-lactoglobulin. This was

500

attributed to the promoting effect of protein for transferring of electron from curcumin to Fe3+

501

which improves the rate of sequential proton loss electron transfer (SPLET) process as the main

502

mechanism for antioxidant activity of curcumin. Additionally, it was investigated that the

503

increasing of curcumin water solubility resulted from encapsulation in pectin-coated casein/zein

504

nanoparticles improved its free radical scavenging activity in comparison with free curcumin

505

dissolved in ethanol or dispersed in water (Chang et al., 2017). Moreover, it was reported that the

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506

antioxidant activity (determined by reducing power and DPPH radical scavenging activity

507

measurements) of curcumin encapsulated within core-shell pectin-zein nanoparticle was even

508

higher than curcumin which was solubilized in ethanol (Huang et al., 2016). In fact, it is well-

509

known that curcumin forms large aggregates in water due to its poor solubility restricting the

510

amount of available curcumin for interacting with free radicals such as DPPH (Yi et al., 2016).

511

Moreover, our results indicated that the curcumin-WPN nanocomplexes had higher antioxidant

512

activity than curcumin-WPI counterparts, which can be due to the more curcumin binding

513

capacity of nanofibrils which was confirmed by fluorescence spectroscopy and solubility test.

514

Therefore, the whey protein nanofibrils-curcumin complexes could be utilized as antioxidant

515

agents to prevent oxidation in different food systems especially functional beverages thanks to

516

their high radical scavenging activity and reducing power in combination with good water

517

dispersibility. However, further studies are required to evaluate their in vivo antioxidant

518

properties and biological attributes.

519

3.7. In vitro release properties

520

The cumulative release profiles of curcumin from nanocomplexes based on WPI and whey

521

protein nanofibrils under simulated gastrointestinal conditions are shown in Fig. 12. The

522

cumulative release of curcumin from WPI and whey protein nanofibrils was found to be about

523

17% and 10% after 2 h in the simulated gastric fluid, respectively. This property of whey protein

524

nanofibril-based complexes is beneficial to deliver curcumin into the intestinal tract without

525

significant release in stomach condition. Moreover, the results showed that the percentage of

526

released curcumin from WPI and WPN was respectively about 41% and 19% after 6 h (2 h in

527

SGF plus 4 h in SIF) release experiment. Generally, the release of curcumin from whey protein

528

nanofibrils was significantly lower than the WPI counterpart over the entire period of release

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529

experiment in the simulated gastric and intestinal fluids. This can be due to the higher ability of

530

whey protein nanofibrils for binding to curcumin which was mentioned earlier. These results are

531

in a good accordance with those of Xue et al. (2018) who studied the release of curcumin from

532

complexes made of zein and caseinate under simulated gastric, intestinal, and colonic conditions.

533

They also reported a sustained release profile for curcumin without any burst release suggesting

534

the high complexation of curcumin with the proteins. In general, these observations suggested

535

that a controlled curcumin release can be achieved from the whey protein nanofibrils which can

536

expand their applications in the site-specific delivery of bioactive compounds and drugs.

537

4. Conclusion

538

In the present study, whey protein nanofibrils with nanometric diameter and high surface

539

hydrophobicity were formed by heating of WPI at pH 2.0 and then were employed as carriers for

540

enhancing the water dispersibility of curcumin as a water-insoluble bioactive compound. The

541

complexation of curcumin with nanofibrils at pH value of 3.2 drastically increased its aqueous

542

solubility and decreased its sedimentation during storage. FT-IR and fluorescence spectroscopy

543

results indicated that the non-covalent interactions such as hydrogen bonding and hydrophobic

544

interactions were mainly contributed to the formation of curcumin-whey protein fibril

545

nanocomplexes. The results of circular dichroism spectroscopy and atomic force microscopy also

546

showed that the secondary structures and morphology of WPN were not significantly affected by

547

binding to curcumin. The results of surface tension and viscosity measurements also indicated

548

that the whey protein nanofibrils-curcumin complexes with a high affinity towards the interfaces

549

and high viscosity can be used to design new engineered specialty functional food products.

550

These complexes also showed a high antioxidant activity which was determined by DPPH

551

radical scavenging test and reducing power assay. The evaluation of in vitro curcumin release

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552

from nanocomplexes under simulated gastrointestinal conditions also showed that the curcumin

553

was released slower from WPN-based complexes compared to the WPI counterpart. Generally, it

554

was concluded that the whey protein nanofibril can be considered as a potential multi-functional

555

carrier for incorporating of curcumin into the formulation of food beverages and drinks which

556

can improve their health-promoting attributes.

557

Acknowledgement

558

The support of University of Tehran is gratefully acknowledged.

559

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Yang, J., Liu, G., Zeng, H., & Chen, L. (2018). Effects of high pressure homogenization on faba

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bean protein aggregation in relation to solubility and interfacial properties. Food Hydrocolloids,

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83, 275−286.

681 682

Yi, J., Fan, Y., Zhang, Y., Wen, Z., Zhao, L., & Lu, Y. (2016). Glycosylated α-lactalbumin-

683

based nanocomplex for curcumin: physicochemical stability and DPPH-scavenging activity.

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Food Hydrocolloids, 61, 369−377.

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686

Fig. 1. Solubility of curcumin in different samples including water, whey protein isolate (WPI),

687

and whey protein nanofibrils (WPN). Means followed by different letters are significantly

688

different (p < 0.05).

689

Fig. 2. Visual appearance of different samples during one month of storage. In each image, vials

690

from left to right are representing the curcumin in water, curcumin-WPI complexes, and

691

curcumin-whey protein nanofibril complexes.

692

Fig. 3. AFM images of (A) WPI, (B) curcumin-WPI complexes, (C) WPN, (D) curcumin-WPN

693

complexes (pH 3.2), and (E) curcumin-WPN complexes (pH 7.0).

694

Fig. 4. Fluorescence emission spectra of different samples including free curcumin, WPI, WPN,

695

and their complexes with curcumin (C-WPI and C-WPN) at excitation wavelengths of (A) 420

696

nm and (B) 280 nm.

697

Fig. 5. Zeta-potential values for different samples including WPI, WPN and their complexes

698

with curcumin (C-WPI and C-WPN) at pH value of 3.2. Means followed by different letters are

699

significantly different (p < 0.05).

700

Fig. 6. FT-IR spectra of curcumin, WPI, WPN, and their complexes with curcumin (C-WPI and

701

C-WPN).

702

Fig. 7. Far-UV CD spectra of WPI, WPN, and their complexes with curcumin (C-WPI and C-

703

WPN).

704

Fig. 8. XRD patterns of curcumin, WPI, WPN, and their complexes with curcumin (C-WPI and

705

C-WPN).

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Fig. 9. SDS-PAGE patterns of different samples including WPI, WPN, and their complexes with

707

curcumin (C-WPI and C-WPN).

708

Fig. 10. Viscosity (A) and surface tension (B) for different sample solutions including WPI,

709

WPN, and their nanocomplexes with curcumin (C-WPI and C-WPN).

710

Fig. 11. DPPH radical scavenging activity (A) and reducing power (B) of different samples

711

including curcumin dispersed in distilled water (C-DW), WPI, WPN, and their complexes with

712

curcumin (C-WPI and C-WPN). Means followed by different letters are significantly different (p

713

< 0.05).

714

Fig. 12. Cumulative release profiles of curcumin from nanocomplexes based on WPI (C-WPI)

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and whey protein nanofibril (C-WPN) during the sequential simulated gastric (SGF) and

716

intestinal (SIF) digestion.

717 718 719

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Curcumin solubility (%)

70 a

60 50 40 30 20 10

b c

0 Curcumin-water Curcumin-WPI

Fig. 1

Curcumin-WPN

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Fig. 2

Fig. 3

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Fig. 4

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40

Zeta-potential (mV)

a 30 b

b

WPN

C-WPI

c 20

10

0 WPI

Fig. 5

C-WPN

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Fig. 6

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30 WPI

Ellipticity (mdeg)

20

WPN C-WPI

10

C-WPN

0 -10 -20 -30 190

210

230 Wavelength (nm)

Fig. 7

250

270

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Fig. 8

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Fig. 9

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Fig. 10

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Scavenging activity (%)

100

(A)

a

b

80 60 40

c d e

20 0 C-DW

WPI

WPN

C-WPI

0.250000004

a

(B)

b

0.200000003 Abs at 700 nm

C-WPN

0.150000002 c 0.100000001

d e

0.050000001 0 C-DW

WPI

Fig. 11

WPN

C-WPI

C-WPN

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Fig. 12

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Highlights 

Whey protein nanofibrils (WPN) with high surface hydrophobicity were produced.



Binding of curcumin to WPN drastically increased its aqueous solubility at pH 3.2.



Curcumin was bound to WPN mainly by hydrophobic interactions and hydrogen bonds.



Curcumin-WPN nanocomplexes showed a good antioxidant activity and high viscosity.

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Table 1 Estimations of secondary structures contents for different samples. Sample

α-helix (%)

β-sheet (%)

β-turn (%)

Random coils (%)

WPI

24.60

29.58

17.05

28.75

WPN

28.14

27.00

18.98

25.85

Curcumin-WPI

23.93

28.85

18.94

28.28

Curcumin-WPN

27.35

26.58

18.04

27.92