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Nanocomplexation between Curcumin and Soy Protein Isolate: Influence on Curcumin Stability/Bioaccessibility and in vitro Protein Digestibility Fei-Ping Chen, Bian-Sheng Li, and Chuan-He Tang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00448 • Publication Date (Web): 17 Mar 2015 Downloaded from http://pubs.acs.org on March 18, 2015

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Journal of Agricultural and Food Chemistry

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Nanocomplexation between Curcumin and Soy Protein Isolate:

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Influence on Curcumin Stability/Bioaccessibility and in vitro Protein

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Digestibility

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Fei-Ping Chen1, Bian-Sheng Li1*, Chuan-He Tang1,2,* 1

6 7 8 9

Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People’s Republic of China

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State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, People’s Republic of China

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ABSTRACT. The complexation of nanoparticles in unheated and heated (at 75-95º) soy protein

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isolate (SPI) with curcumin, as well as the effects on curcumin stability/bioaccessibility and in vitro

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protein digestibility were investigated. The nanoparticles did not display noticeable changes in size

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and morphology upon nanocomplexation with curcumin, except their surface hydrophobicity. The

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encapsulation efficiency of curcumin progressively decreased with increasing initial curcumin

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concentration in the dispersion, while the load amount linearly increased. The solubility of curcumin

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in water was enhanced by the complexation above 98,000-fold (vs free curcumin in water). The

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formation of the nanocomplexes considerably improved the storage stability of curcumin. In vitro

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simulated digestion experiments indicated that the complexation also improved the bioaccessibility of

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curcumin; the bioaccessibility was greatly impaired by hydrolysis-induced protein aggregation.

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Addtionally, the nanocomplexation significantly improved the in vitro protein digestibility of both

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unheated and heated SPI.

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KEYWORDS. Soy protein isolate (SPI); nano vehicle; curcumin; nano-encapsulation; protein

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digestibility


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INTRODUTION

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The complexation between proteins and phenolic compounds has attracted increasing attention, due to

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the potential of the formed complexes to perform as (nano)carriers for enhanced solubility, stability

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and even bioavailability of hydrophobic bioactives, especially those with poor water solubility and

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bioavailability, e.g., curcumin 1-3. Curcumin (diferuloylmethane) is a natural polyphenolic compound

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isolated from the spice turmeric, and has been confirmed to possess a number of biological activities,

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e.g., antioxidant, anti-inflammatory, anticancer or antitumor, anti-allergy and other disease-healing

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properties.4 However, a major limitation for curcumin to be applied in functional foods or medicinal

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formulations is its extremely low water solubility and poor bioavailability 5-7. The complexation with

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proteins, including bovine serum albumin (BSA) 8-10, αs1-casein 11, β-lactoglobulin nanoparticle 12, soy

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protein isolate (SPI) 13, and even zein colloidal particles14, has been confirmed to improve the stability

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and solubility (in aqueous phase) of curcumin. The complexation occurs occurs mainly via

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hydrophobic interactions 7, 12, 13, 15-20, though in some cases, hydrogen bonds may also be involved 21.

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In contrast with milk proteins, plant proteins (e.g. zein and soy proteins) seem to be more promising

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to perform as carriers for bioactives, due to their abundant renewable sources and less cost, and even

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potential health effects. However, to date, only a limited number of works are available addressing the

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potential of plant proteins in this aspect.

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The water solubility improvement of curcumin by the complexation with proteins is highly

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dependent on type and nature of the applied proteins and even the investigated conditions (e.g.,

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protein concentration), in a complex way. Esmaili et al. 15 reported that the water solubility of

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curcumin was increased at least 2500-fold by the complexation with beta casein-micelle, which is 3 - Environment ACS Paragon -Plus


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considerably higher than that (812-fold) observed for SPI-curcumin complex (1%, w/v; 13). It should

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be to note that in the latter case (SPI), the complexation with curcumin did not occur at optimal

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conditions, and furthermore, the importance of structural features and physicochemical properties

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(especially surface properties) of proteins to the complexation was not addressed. In fact, structural

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and physicochemical properties of soy proteins are highly dependent on a number of parameters,

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including composition and nature of proteins in starting materials, processing and/or preparation

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history, and environment conditions. Another noteworthy point is that most of the proteins in

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laboratory-prepared SPI are usually present in the nanoparticle form, and their surface hydrophobicity

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could be greatly increased by a heat treatment (e.g., 95ºC) 22-24. In the case of heat-induced β-

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lactoglobulin nanoparticles, it has been confirmed that the water solubility (at a concentration of 1%

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complex) of curcumin was increased by above 20,000 folds 12. Thus, a much higher potential for SPI

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to bind curcumin than that previously reported by Tapal & Tiku 13 can be reasonably expected.

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On the other hand, the in vitro digestibility of proteins is affected by the complexation with

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phenolic compounds. In this regard, there is generally a dispute about the influence of the

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complexation on the digestibility of food proteins 25-30. For example, Stojadinovic et al. 30 observed

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that the complexation with some dietary polyphenols impaired the digestibility of many proteins, e.g.,

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β-lactoglobulin. Using green tea catechins, Tantoush et al. 28 contrarily reported that the complexation

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could distinctly improve the pepsin digestibility of some food allergens. Venkatachalam and Sathe 29

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reported that the presence of some phenolic compounds including catechin and ellagic acid did not

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adversely affect the in vitro pepsin hydrolysis of phaseolin (a 7S globulin from kidney bean). With in

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vivo experiments, Carbonaro and others 31 also indicated that the binding of polyphenols produced an 4 - Environment ACS Paragon -Plus

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insignificant influence on the protein digestibility of proteins from several legumes. All these

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observations suggest that the influence of complexation with phenolic compounds on the protein

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digestibility is dependent on the type and nature of tested proteins and even phenolic compounds. For

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legume storage proteins, the structural characteristics seem to play a vital role in their digestibility 25,

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31

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would be a very interesting subject to characterize the influence of complexation with curcumin on

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the in vitro digestibility of soy proteins.

, which might be highly affected by some pretreatments, including heating. From this viewpoint, it

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The main objective of this study was systematically to investigate the complexation between

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nanoparticles of unheated and preheated (75-95ºC) SPI and curcumin. The correspondingly formed

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nanocomplexes were characterized in terms of surface characteristics, particle size and

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microstructure, as well as encapsulation efficiency of curcumin. Furthermore, the influence of the

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nanocomplexation on the storage stability and/or bioaccessibility of curcumin, as well as the in vitro

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digestibility of SPI was also characterized using a gastric-intestinal in vitro model. Our findings

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indicate that the nanocomplexation between SPI and curcumin not only provides a promising strategy

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to improve the stability and bioaccessibility of curcumin, but also can be applied to improve the

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nutritional value of soy proteins.

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MATERAILS AND METHODS

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Materials. Curcumin (~98% purity, from Curcuma longa) was purchased from Sigma-Aldrich

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Chemicals Co. (St. Louis, MO). Defatted soy flour with low protein denaturation was obtained from



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Shandong Yuwang Industrial and Commercial Co. Ltd. (Yucheng, Shandong province, China). All

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other chemicals were of analytical grade. Soy protein isolate (SPI) was prepared according to the

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conventional process as follows. In brief, the flour was dispersed in de-ionized water at a weight-to-

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volume ratio of 1:15, and the pH of the resultant slurry was adjusted to 8.0 using 2 M NaOH. The

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slurry was stirred for 2 h (if necessary, the pH was adjusted to 8.0 during the stirring), and then

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centrifuged at 8000 g for 20 min to remove the insoluble material. The resulting supernatant was

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adjusted to pH 4.5 with 2 M HCl, and the isoelectric-precipitated curd collected by centrifugation at

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5000 g for 20 min. The obtained precipitate was redispersed in de-ionized water at a weight-to-

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volume ratio of 1:5, and the pH of the dispersion was adjusted to 7.0 with 2 M NaOH. The final

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dispersion was lyophilized to produce the SPI. The protein content of this SPI was approximately

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91.5% (wet basis), as determined using a Dumas combustion method (Elemental Analyzer rapid N

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cube, Germany), with a nitrogen conversion factor of 6.25.

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Complexation of curcumin with SPI. The SPI stock solution (5%, w/v) at pH 7.0 was prepared

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by dispersing the freeze-dried SPI sample in water, under the stirring conditions, at least for 2 h. The

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heated SPI solutions were obtained by heating a same volume of the SPI stock solution in a water bath

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at 75, 85 and 95ºC for 15 min, and then immediately cooling to room temperature in ice bath. Stock

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curcumin solution of a concentration ([cur]) of 4.5 mg/mL was prepared in ethyl alcohol absolute (if

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necessary, the preparation was centrifuged to remove the insoluble). For the characterization of SPI-

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curcumin complexes, an amount of curcumin stock solution was dropwisely added at a volume ratio

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of 1:50 to 5% (w/v) unheated or heated SPI solutions, under magnetically-stirred conditions. The final

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[cur] in the mixtures was about 88 µg/mL. The mixtures were further kept for 8 h on a magnetic 6 - Environment ACS Paragon -Plus

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stirrer at room temperature. Last, the insoluble curcumin was removed by centrifugation at 8000 g for

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20 min. The resultant supernatants were subject to the particle property characterization, or freeze-

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dried to produce the SPI-curcumin complex powders.

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To investigate the influence of variation in [cur] on the encapsulation efficiency (EE) and load

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amount (LA) of curcumin, increasing amount of the curcumin stock solution was added to a specific

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volume of 5% (w/v) unheated SPI solution. If necessary, a certain volume of ethanol was added to

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make sure that in the final mixtures, the final ethanol and protein concentrations in the mixtures were

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constant, with 30% (v/v) and 3.5% (w/v), respectively.

Determination of surface hydrophobicity (Ho) and ζ-potential of proteins in solutions. Surface

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hydrophobicity (Ho) of proteins in unheated and heated SPIs, before or after complexation with

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curcumin, was determined using a fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid (ANS-)

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phosphate buffer (pH 7.0). Twenty microliters of ANS- stock solution was added to 4 mL of the

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buffer containing increasing amounts (0-50 µL) of 1.5% (w/v) SPI solutions. The relative

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fluorescence intensity (FI) was measured at 25°C, with excitation and emission wavelengths of 390

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and 470 nm, respectively. For each protein, the initial slope of FI versus protein concentration plot

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was calculated by linear regression analysis and used as an index of surface hydrophobicity (Ho).

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. In brief, stock solutions of 8×10-3 M ANS- and 1.5% (w/v) SPIs were prepared in 0.01 M

The ζ-potential of proteins in the unheated or heated SPI solutions at pH 7.0 was measured using a

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Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, Worcestershire, UK) in combination with a

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multipurpose autotitrator (model MPT-2, Malvern Instruments, Worcestershire, UK). Freshly 7 - Environment ACS Paragon -Plus


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prepared protein solutions were diluted to 0.1% (w/v) with the same applied buffer and filtered

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through a 0.45 µm HA Millipore membrane prior to analysis.

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Differential scanning calorimetry (DSC). The thermal transition of unheated and heated SPIs was

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examined using a TA Q100-DSC thermal analyzer (TA Instruments, New Castle, Delaware 19720

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USA), according to the process described in our previous work 33. All the unheated and heated SPI

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samples with or without complexation with curcumin for the DSC examination were prepared by

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freeze-drying the corresponding solutions (5%, w/v). Approximately 2.0 mg of the freeze-dried

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samples were weighed into aluminum liquid pans (Dupont), and 10 µL of 0.05 M phosphate buffer

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(pH 7.0) was added. The pans were hermetically sealed and heated from 20 to 110 °C at a rate of 5

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°C/min. A sealed empty pan was used as a reference. Denaturation temperature (Td) and enthalpy

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change of the endotherm (∆H) of β-conglycinin and glycinin components in the samples were

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computed from the thermograms by the Universal Analysis 2000, Version 4.1D (TA Instruments-

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Waters LLC). All experiments were conducted in triplicate. The sealed pans containing protein

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samples and buffers were equilibrated at 25 °C for 12 h.

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Particle size distribution and z-average diameter (Dz) measurement. The particle size

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distribution of particles in unheated and heated SPI solutions, before or after the complexation with

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curcumin, was evaluated using dynamic light scattering (DLS) technique. Each SPI solution was

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diluted to a protein concentration of about 0.1% (w/v) with 0.05 M phosphate buffer (pH 7.0), and

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filtered with a 0.22 µm filter. DLS analysis was performed at a ﬁxed angle of 173° using a Zetasizer

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Nano-ZS instrument (Malvern Instruments, Worcestershire, UK) equipped with a 4 mW He-Ne laser

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(633 nm wavelength) at 25 °C. The Dz of particles was calculated based on the Stokes-Einstein 8 - Environment ACS Paragon -Plus

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equation, assuming protein particles to be spherical. Each determination was performed in duplicates

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and the means used for statistical analyses.

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Atomic force microscopy (AFM). AFM images were acquired in tappng mode using a Dimension

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3000 microscope (Digital Instruments-Veeco, Santa Barbara, CA, USA), equipped with a “G”

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scanning head (maximum scan size 10um) and driven by a NanoscopeⅡIa controller. Various

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unheated or heated SPI solutions (5%, w/v), with or without complexation with curcumin, were

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diluted by 5,000 fold with de-ionized water. A droplet (2 µL) of each diluted sample was immediately

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spread on a freshly cleaved mica disc in air for 30 min at ambient temperature. For imaging under

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ambient condition, single-beam uncoated silicon cantilevers (type OMCL-AC, Olympus, and RTESP,

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Veeco) were used. The drive frequency was set at 300 kHz and the scan rate was 1.2 Hz. Images were

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processed by the Digital Nanoscope Software (Version 5.3 or 3, Digital Instruments). For each

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preparation condition, at least two samples were used for duplication. Approximately 10 images were

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obtained for each preparation.

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Determination of encapsulation efficiency (EE%) and load amount (LA) of curcumin. The

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encapsulation efficiency (EE%) of curcumin in the SPI-curcumin complexes was estimated as the

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percentage of curcumin encapsulated in the proteins, by the following equation (1):

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EE (%) = [1-

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where the amount of free curcumin is determined from the precipitate obtained by the centrifugation

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(see section 2.2). The precipitate was extracted in 5 mL of ethanol with mild stirring for 5 min, under

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magnetically stirred conditions, and then centrifuged at 9000 g for 15 min at 20°C to remove the

()

] × 100

()

(1),



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protein aggregates. The supernatant was subject to the spectrophotometric analysis at 426 nm with a

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UV754N UV-Vis spectrophotometer, and the curcumin concentration was determined using an

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established standard curve of curcumin (with R2 > 0.999).

172 173

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The LA of curcumin was calculated by dividing the amount of encapsulated curcumin by total protein amount in the system.

Stability measurement. The storage stability of free curcumin in water, or curcumin in the SPI-

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curcumin complexes in water (pH 7.0) was evaluated by monitoring the decrease kinetics of

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absorbance at 426 nm at 25 ºC or 95ºC. The SPI-curcumin complex solutions (1%, w/v) were

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prepared by dispersing the freeze-dried samples in water, under stirred conditions for more than 2 h.

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The free curcumin solution was prepared by dispersing 1.2 mL of the stock curcumin solution (4.5

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mg/mL in ethanol) into 40 mL water, under the same conditions as for the SPI-curcumin complex

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solution. Then, all the samples were centrifuged at 8000 g for 20 min and the supernatants were

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subjected to storage stability experiment. The initial absorbance for all the cases was set as 100%.

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Each experiment was performed in duplicates and means used for the statistical analyses.

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Sequential in vitro gastric and intestinal digestion. An in vitro model that simulated sequential

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gastric and intestinal digestion was applied to evaluate the influence of digestion on bioaccessibility

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of curcumin (free, or encapsulated in the nanocomplexes with SPI), and/or in vitro protein

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digestibility of unheated or heated (at 95ºC) SPI, according to the process described in our previous

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work 34, with a few modifications. In brief, 10 mL of SPI solutions, or solutions containing SPI-

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curcumin nanocomplexes at a protein concentration of 5.0 % (w/v), were well mixed with 40 mL of 10 - Environment ACS Paragon-Plus

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0.1 M HCl (pH 1.5), and preincubated in a shaker (at 37 ºC) at a rate of 95 rpm for around 10 min. If

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necessary, the pH of the mixtures was adjusted to 1.5 with 1.0 M HCl. The curcumin content in the

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nanocomplexes was about 871.5~892 µg curcumin per gram of protein. Then, 10 mg of pepsin

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powder was added and well mixed to start the simulated gastric digestion (0-60 min). After 60 min,

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the pH of the resultant pepsin-digests was immediately adjusted to 7.0 with 4 M NaOH, and 250 mg

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of bile extract powder was added and well dispersed in the shaker for 10 min. Last, 20 mg of

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pancreatin powder was added to start the intestinal digestion (60-180 min). For free curcumin

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(control), the curcumin dispersion was prepared by mixing 500 µL of curcumin ethanol solution (4.5

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g/L) with 25 mL deionized water, and then subject to the same digestion process as above. At the end

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of the whole digestion, 1 mL of digest dispersions was collected and centrifuged at 10,000 g for 30

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min, at 10 ºC. The amount of curcumin in the resultant supernatants, or the whole digests was

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extracted and determined (see the next section).

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Curcumin extraction and determination. Aliquots (200 µL) of the final digests (obtained after

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180 min of digestion) or their supernatants (after the centrifugation) were extracted two times with 2

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mL of ethyl acetate for 5 min, under vortexing conditions, and then centrifuged at 5,000 g for 5 min.

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The absorbance of the pooled ethyl acetate extracts was determined at 420 nm with a UV-Vis

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spectrophotometer, and the curcumin concentration in the extracts was calculated using an established

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standard curve of curcumin in ethyl acetate (with a coefficient factor > 0.999).

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Characterization of the digests of SPIs or their nanocomplexes with curcumin. The hydrolysis

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of proteins in unheated and heated SPIs, or their nancomplexes with curcumin, was characterized

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using sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) and release of 11 - Environment ACS Paragon-Plus


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trichloroacetic acid (TCA)-soluble nitrogen. For SDS-PAGE experiments, aliquots (50 µL) of the

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digest samples at specific period times of digestion (0-180 min) were taken, and immediately mixed

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with 300 µL of sample buffer (2-fold), namely, 60 mM Tris-HCl buffer (pH 8.0) containing 2.0%

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(w/v) SDS, 25% (v/v) glycerol, 0.1% bromophenol blue and 5% (v/v) β-mercaptoethanol. The

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electrophoresis was performed on a discontinuous buffered system using 12% separating gel and 4%

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stacking gel with some modification. The gel was stained with 0.1% Coomassie brilliant blue (R250)

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in a solution containing 45% methanol and 10% acetic acid for 40min, and further destained in a

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methanol/ehanol/water=1/1/8 solution for 24 h.

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To characterize the TCA-soluble nitrogen release, aliquots (1 mL) of the digests were taken after

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specific periods of digestion, and immediately mixed with 2 mL of 20% TCA solution to precipitate

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the proteins with large molecular weights. The mixtures were placed for 10 min and then centrifuged

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at 500 g for 5 min. The nitrogen content of the resultant supernatants was determined by micro

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Kjeldahl method. The percentage of TCA-soluble nitrogen released during the digestion was

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estimated from the following equation,

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% release of TCA-soluble nitrogen =100% −

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!"#$%$& '()& $ '()&

× 100%

(2)

The particle size distribution profiles of different digests were characterized using DLS technique,

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according to the same process as above. All the digests were diluted with the same background

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solvent to a concentration of about 0.05% (w/v), prior to the determination.

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Statistical analysis. An analysis of variance (ANOVA) of the data was performed, and a least significant difference (LSD) with a confidence interval of 95% was used to compare the means. 12 - Environment ACS Paragon-Plus

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RESULTS AND DISCUSSION

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Formation and characterization of SPI-curcumin nanocomplexes. Curcumin binds to hydrophobic

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clusters of proteins, mainly through hydrophobic interactions 7, 12, 15, 17, 19, 20, 35. Heating can result in

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structural unfolding and denaturation of proteins, thus increasing their surface hydrophobicity (Ho).

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The improvement of the Ho would be favorable for the binding of curcumin to the proteins. In the

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present work, the potential of both unheated and heated (at 75, 85 and 95°C for 15 min) SPIs to form

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complexes with curcumin was investigated. As expected, increasing the temperature from 75 to 95°C

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resulted in a progressive increase in protein denaturation of β-conglycinin and/or glycinin components

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in the SPIs, as well as Ho, e.g., the Ho considerably increased from 2248 to 5173 (Tables S1 and S2).

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The observations about the influence of heating on the protein denaturation of SPI are in accordance

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with those of our previous findings 33. In contrast, the ζ-potential (-13.4~-13.9 mV) of SPI at pH 7.0

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was almost unchanged by the heating (Table S2). Figure 1 A shows the typical size distribution

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profiles of particles in unheated and heated SPI dispersions, and the z-average diameter (Dz) data are

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summarized in Figure 1B. Most of particles in these unheated and heated SPI dispersions were

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present in the nanoparticle form, with Dz progressively but insignificantly increasing from 74 to 90

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nm with the temperature of heating (Figure 1B). The progressive increase in Dz was closely

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associated with the heating-induced denaturation and subsequent aggregation. The Dz of particles in

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unheated and heated SPIs is consistent with that of our previous findings 24. This can be further

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confirmed by the AFM observations (Figure 2 A-D), where most of particles in these SPIs were

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present in dissociated nanoparticles with heights of several nanometers. 13 - Environment ACS Paragon-Plus


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After mixing unheated or heated SPI (5%, w/v) with the curcumin stock solution (4.5 mg/mL; in

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100% ethanol) at a volume ratio of 50:1, all the resultant dispersions were transparent and yellow in

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appearance (Figure 3 A), indicating formation of curcumin-SPI complexes at the nanoscale. Under

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the conditions, the encapsulation efficiency (EE%) of curcumin for all the preparations was within the

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range of 96.8%-99.1%, and there was no significant difference in EE% between the different

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preparations (Table S1). The EE% is comparable to that (>96%) of curcumin encapsulated in β-

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lactoglobulin nanoparticles produced by desolvation 12, but considerably higher than that of curcumin

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encapsulated in casein nanocapsules by spray-drying (about 83.1%; 19), and in zein-based colloidal

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particles synthesized by an antisolvent precipitation (71%-87%; 14). The differences in EE% might be

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related to the differences in incorporated curcumin amount between these previous works and the

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present case. When all the SPI-curcumin nanocomplex preparations were freeze-dried and

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reconstituted, the transparency and yellow of the resultant reconstituted dispersions were the same as

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that of the original counterparts (Figure 3), indicating good dispersion and curcumin stability of these

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formed nanocomplexes. As expected, the curcumin load amount (LA) of different dried samples was

265

similar and within the range 1.743~1.784 µg/mg SPI (Table S1). The LA is much lower than that (19

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µg/mg casein) in the supernatant of curcumin encapsulated in casein nancapsules 19, but close 2 times

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that (0.892 µg/mg SPI) encapsulated in the same protein by spray-drying 14. If the dried SPI-curcumin

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nanocomplex preparation in the present work is prepared at a protein concentration (c) of 1.0% (w/v),

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the solubility of curcumin in water will reach 17.43~17.84 mg/L, which is more than 1600-folds

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higher than that (approximately 11 µg /L) for free curcumin in water 18.

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To further unravel the potential of SPI to bind curcumin, we evaluated the complexation of

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unheated SPI with increasing amount of incorporated curcumin (in ethanol). When the fraction of

273

curcumin-containing ethanol was above 0.3 (relative to total volume of mixed dispersions), the

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dispersions would exhibit a sol-gel state. Thus, the complexation of SPI with curcumin was performed

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at a final c value and ethanol volume fraction of 3.5% (w/v) and 0.3, respectively, with increasing

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[cur] from 0.0315 to 1.35 mg/mL dispersion. From Figure 4, we can interestingly see that as the [cur]

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increased, the EE% progressively decreased from 98% to 78%, almost in an exponentially decaying

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way, while the LA of curcumin linearly increased from about 0.9 to 31 µg/mg SPI. Similar changes in

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EE% and LA with the increase in [cur] in the water-ethanol phase have been observed for the zein-

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curcumin composite colloidal particles 14. At the highest [cur] (1.35 mg/mL), in the present work, the

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solubility of curcumin in water was enhanced by more than 98,000-folds, as compared with the 11

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µg/L of free curcumin in water 18. The solubility improvement was slightly lower than that (131,000

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times) observed for a synthesized diblock copolymer micelle 6, but considerably higher than those

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observed for hydrophobically modified starch and HP-γ-cyclodextrin (1670 and 4700 folds,

285

respectively) 36, 37, camel β-casein (at least 2500 fold) 15. The [cur] of soluble curcumin in the

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supernatant of the 3.5% SPI dispersion at [cur]=1.35 mg/mL was determined to be about 1.085

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mg/mL, which was several-fold higher than the 136.7 µg/mL for the supernatant obtained by

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centrifuging the reconstituted spray-dried curcumin in casein nanocapsules (at 7.2 mg/mL casein; 19).

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The influence of complexation with curcumin on DSC and surface characteristics, as well as

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particle size of unheated and heated SPIs was also investigated. The thermal denaturation of β-

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conglycinin and glycinin components in these SPIs was nearly unaffected by the complexation, but 15 - Environment ACS Paragon-Plus


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the complexation resulted in a considerable reduction in Ho, with the decreasing extent depending on

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the extent of heating (Tables S1 and S2). Highest reduction in Ho was observed for the SPI heated at

294

85 ºC among all the test preparations. Furthermore, the complexation also resulted in a slight but

295

insignificant decline in magnitude of ζ-potential in all the cases (Table S2). Curcumin molecules have

296

two phenolic OH groups with pKa values of 8.38 and 9.88 in aqueous solution, and are usually in the

297

neutral form at around pH 7.0 6. Thus, the decreased Ho might be largely due to the masking of

298

hydrophobic clusters of the proteins by binding with curcumin. Since the hydrophobic clusters are

299

usually even distributed over the whole protein molecules, the complexation with curcumin will also

300

mask some charged groups, thus causing the reduction in ζ-potential. The gradual decrease in ζ-

301

potential of the particles upon increasing the [cur] has also been observed for the complexation of zein

302

colloidal particles with curcumin, though the complexation was performed at acidic pHs (3.5-4.1)14.

303

When these SPI-curcumin nanocomplexes were freeze-dried and reconstituted, the Ho considerably

304

increased (relative to that before the freeze-drying), but the magnitude of ζ-potential remarkably

305

declined (Table S2), indicating that the additional freeze-drying markedly changed the surface

306

properties of the SPI-curcumin nanocomplexes.

307

For any test SPI solution, the complexation with curcumin did not significantly change the size of

308

particles in the aqueous solutions (Figure S1), as could be further confirmed by the AFM

309

observations of particles (Figure 2). However, when the solutions containing SPI-curcumin

310

complexes were freeze-dried and reconstituted, the particle size significantly increased relative to that

311

before the freeze-drying (Figure S1), suggesting that the noticeable changes in surface characteristics

312

of proteins after the freeze-drying led to protein aggregation, or formation of larger particles. On the 16 - Environment ACS Paragon-Plus

Page 17 of 43


313

other hand, the complexation with curcumin did not affect the secondary structure of the proteins in

314

unheated or heated SPIs, as evidenced by no discernible changes in fourier transform infrared (FTIR)

315

spectra (Figure S2). Previous works also indicated that no distinct conformational changes (at

316

secondary and tertiary levels) occur for many proteins, e.g., β-lactoglobulin and BSA, upon

317

complexation with curcumin or its derivatives 12, 17, 20.

318

Storage stability. Curcumin in aqueous solution is readily susceptible to hydrolysis or

319

degradation, even at physiological pH 8, 9. Wang et al. 9 indicated that curcumin at neutral and basic

320

pH values degraded by about 90%, within 30 min of storage at room temperature. This can be further

321

confirmed in our experiment that free curcumin degraded by about 70%, after storage of 30 min at 25

322

ºC (Figure 5 A). If the storage was further elongated up to 4 h, more than 80% of curcumin was

323

degraded. In contrast, almost 90% of curcumin in the nanocomplexes with SPIs remained stable after

324

storage up to 4 h (Figure 5 A), indicating remarkable improvement of degradation stability of

325

curcumin. There was no noticeable difference in the degradation kinetics of curcumin between the

326

nanocomplexes with unheated and heated (at 95ºC) SPIs (Figure 5 A). Tapal and Tiku 16 similarly

327

reported that more than 80% of curcumin in the SPI-curcumin complexes was stable upon storage up

328

to 12 h, in water or even in simulated gastric fluid and simulated intestinal fluid. A similar

329

improvement of storage stability has also been observed for curcumin in complexes with serum

330

albumins 8, 9, 38, and milk proteins including αs1-casein and β-lactoglobulin 11, 12.

331

As expected, the degradation of curcumin, free or protein-bound, was greatly accelerated by

332

increasing the temperature of storage to 95ºC (Figure 5 B). In this instance, the curcumin in all

333

preparations (free or protein-bound) dramatically decreased within 30 min, and the rate of degradation 17 - Environment ACS Paragon-Plus


Page 18 of 43

334

gradually slowed down upon further storage. However, at any specific time of storage, the amount of

335

remaining curcumin was significantly higher for the SPI-curcumin nanocomplexes than for free

336

curcumin (Figure 5 B). For example, approximately 25% of curcumin remained in the SPI-curcumin

337

nanocomplexes, after storage of 4 h at 95ºC, while it was about 12% for free curcumin. The

338

observations indicated that the nanocomplexation of curcumin with SPI greatly improved the heat

339

stability of curcumin.

340

Simulated gastric and intestinal digestion.

341

Bioaccessibility and stability of curcumin. During the digestion, the nanocomplexes of curcumin

342

with SPI may undergo dramatic changes in environmental conditions (pH and ionic strength), action

343

of proteases on the proteins, and even the presence of different active surfactants. After sequential

344

processes of in vitro gastric (60 min) and intestinal (120 min) digestion, in the absence or presence of

345

proteases, the bioaccessible amount of curcumin transferred to the aqueous phase of the digests for

346

free curcumin or its nanocomplexes with SPI is shown in Figure 6 A. It should be noted that the

347

bioaccessibility of curcumin might be dependent on its stability throughout the digestion process. If

348

curcumin encapsulated in the nanocomplexes is susceptible to the digestion process, the stability of

349

curcumin would be the prominent parameter affecting its bioaccessibility. Due to this consideration,

350

we also evaluated the stability (against degradation) of curcumin upon digestion, as displayed in

351

Figure 6 B.

352 353

Interestingly, it can be observed that the presence of proteases during digestion greatly improved the stability of curcumin, but the improvement was closely dependent on the present state of 18 - Environment ACS Paragon-Plus

Page 19 of 43


354

curcumin, free or bound (Figure 6 B). For free curcumin, about 88% of curcumin degraded after the

355

digestion of 120 min without addition of proteases, whereas in the bound form case, it was only 30%.

356

This observation is consistent with the observation of storage stability at ambient temperature (Figure

357

5 A), indicating strong stabilization of complexation with SPI towards degradation for curcumin. The

358

presence of proteases considerably increased the amount of free curcumin that survived the whole

359

digestion from 12% to 80%; for the curcumin in the nanocomplexes, there was no significant loss

360

throughout the digestion, in the presence of enzymes. The improvement of curcumin stability by the

361

presence of enzymes during digestion might be largely attributed to the curcumin-protein interaction

362

or complexation, since the enzymes are proteins themselves.

363

On the other hand, the bioaccessible amount of curcumin also highly depended on the present

364

state of curcumin, as well as whether the presence of enzymes or not (Figure 6 A). Without the

365

enzymes during the digestion, only less than 1% of curcumin was transferred to the aqueous phase for

366

free curcumin, indicating extremely low bioaccessibility. In contrast, the bioaccessibility of curcumin

367

in the nanocomplexes with unheated or heated SPI reached approximately 60% (Figure 6 A). If the

368

degradation of curcumin during the digestion is considered into account, the ideal bioaccessibility of

369

curcumin in the nanocomplexes with SPI, in this case, may reach about 85%. In the presence of

370

enzymes during the digestion, the bioaccessibility of free curcumin reached about 20% (Figure 6 A).

371

Relative to the amount of undegraded curcumin, the ideal bioaccessiblity for free curcumin would be

372

around 25%. The presence of enzymes also significantly improved the bioaccessibility of curcumin in

373

the nanocomplex with unheated SPI (from 60% to 90%), but did not significantly affect that with



374

heated SPI (Figure 6 A). The difference in the improvement between the unheated and heated SPI

375

might be due to the differences in their protein hydrolysis and protein aggregation.

376

Page 20 of 43

Protein in vitro digestibility. The bioaccessibility of curcumin in the nanocomplexes with proteins

377

may be associated with protease hydrolysis of the proteins during digestion. Furthermore, the

378

complexation with curcumin also produces an influence on the digestibility of the proteins. During the

379

simulated SGF and SIF digestion, the protease-induced hydrolysis of proteins in unheated or heated

380

SPI, with or without complexation with curcumin, was characterized using SDS-PAGE and TCA-

381

soluble nitrogen analyses, as displayed in Figures 7 and 8. As expected, the digestion resulted in a

382

progressive hydrolysis of protein polypeptides in SPI, with dramatic changes observed in the SGF

383

digestion, as evidenced by SDS-PAGE and TCA method (Figures 7 and 8). By comparison between

384

the unheated and heated SPI (without complexation with curcumin), it can be generally observed that

385

1) all the protein components in SPI were more susceptible to the gastric protease (pepsin) hydrolysis

386

relative to the SIF hydrolysis; 2) the polypeptides of glycinin were much easily digested by the

387

protease hydrolysis than those from β-conglycinin (Figures 7 A, B and 8). The protease hydrolysis

388

and TCA-soluble nitrogen release of unheated SPI during digestion are similar to those observed in

389

our previous work 34. Furthermore, the heat pretreatment could increase the susceptibility of β-

390

conglycinin polypeptides to the pepsin hydrolysis, but it to a certain extent impaired the TCA-soluble

391

nitrogen release during the SIF digestion (Figures 7 A, B and 8). The observations confirm the

392

previous viewpoint that the protease susceptibility and/or in vitro digestibility of β-conglycinin or

393

other vicilins is affected by the heat-induced protein denaturation (positive), as well as subsequent

394

protein aggregation (negative) 25, 26, 29. The improvement of the susceptibility to pepsin hydrolysis by 20 - Environment ACS Paragon-Plus

Page 21 of 43


395

the heating could be thus largely attributed to the thermal denaturation of the protein, as similarly

396

observed for phaseolin (a 7S globulin from kidney bean; 25, 29). However, the influence of heating

397

on the digestibility of these proteins is also dependent on the nature of their conformations and

398

whether or not the presence of other components (e.g., in the protein isolate or flour form). For

399

example, Deshphande and Damodaran 25 also pointed out that the vicilin from green peas with a

400

flexible conformation underwent an undesirable change in conformation upon heating, and as a

401

consequence, the heated vicilin exhibited more resistance to proteolysis than the native one. In fact,

402

the undesirable change in conformation in this case can also be explained as the occurrence of high

403

extent of heat-induced protein aggregation. The importance of structural characteristics to the

404

digestibility of legume proteins has been also confirmed using in vivo experiments with growing rats

405

26

406

.

On the other hand, in both unheated and heated SPI cases, the proteins in the nanocomplexes with

407

curcumin were much more susceptible to the protease hydrolysis, especially during the gastric

408

digestion (Figure 7). For example, the α´-subunit of β-conglycinin in the unheated SPI remained

409

almost intact after 60 min of the SGF digestion, while that in the its nanocomplex counterpart had

410

been completely degraded even after 30 min of digestion. The improvement of protein hydrolysis by

411

the nanocomplexation can be further confirmed by the TCA-soluble nitrogen release observations

412

(Figure 8), wherein it can be observed that the release of TCA-soluble nitrogen during the SGF

413

digestion was significantly higher in the nanocomplex case than in the controls. There was no

414

significant difference in the TCA-soluble nitrogen release between the nanocomplexes with unheated

415

and heated SPI (Figure 8). Since curcumin is a phenolic compound, the observations thus indicated 21 - Environment ACS Paragon-Plus


Page 22 of 43

416

that the nanocomplexation with curcumin greatly improved the protein digestibility of SPI,

417

irrespective of heat pretreatment or not. A similar improvement of pepsin digestibility has been

418

reported for some major food allergens when they interacted with green tea catechins 28. Tagliazucchi

419

and others 27 even observed that the complexation with some phenolic compounds distinctly improved

420

the pepsin activity during simulated gastric digestion. However, some contrasting observations have

421

been available that the complexation with phenolic compounds may also produce an adverse or

422

insignificant influence on the digestibility of many proteins, e.g., β-lactoglobulin 30, and legume

423

proteins 29, 31. Thus, the influence of complexation with phenolic compounds on the digestibility of

424

proteins is a complex process related to the nature of both the phenolic compounds and proteins.

425

Hydrolysis-induced aggregation. The protease hydrolysis of proteins during digestion may lead to

426

exposure of their hydrophobic clusters to aqueous phase, or release of hydrophobic peptides, thus

427

resulting in protein aggregation 39. Figure 9 shows changes in particle size distribution profiles of

428

unheated or heated SPI, or their nancomplexes with curcumin, upon increasing digestion time (0-180

429

min), in sequential in vitro SGF and SIF digestion. As expected, unheated SPI consisted of

430

approximately two protein fractions with sizes centered at 17 (major) and 100 (minor) nm,

431

respectively (Figure 9 A). The heat pretreatment led to a remarkable reduction of the 17 nm protein

432

fraction, and concomitantly, a distinct increase of the fraction with large size (~100 nm) (Figure 9 A,

433

B), indicating occurrence of heat-induced aggregation of proteins. When both unheated and heated

434

SPI were subject to the SGF digestion, it was observed that a new particle distribution peak occurred

435

at a much larger size (> 1000 nm), at the expense of all the initially present protein fractions (centered

436

at 17 and 100 nm) (Figure 9 A, B). The observations clearly indicated that the pepsin hydrolysis 22 - Environment ACS Paragon-Plus

Page 23 of 43


437

resulted in aggregation of all the proteins. By comparison, we can approximately see that the extent of

438

pepsin-induced aggregation was higher in the heated SPI case than in the unheated counterpart, and it

439

also increased with increasing the digestion time (0-60 min).

440

When the pepsin-treated digests of both unheated and heated SPI were further subject to the SIF

441

digestion for more than 30 min (and accordingly, the pH also changed from 1.2 to 6.8), the size of the

442

formed aggregates (at 60 min) considerably decreased to much less values (Figure 9 A, B), indicating

443

disruption of the initially formed aggregates. However, the changing pattern of particle sizes was

444

dependent on the type of proteins (unheated or heated) and the time of SIF digestion. In the unheated

445

SPI case, minimal particle sizes (larger than that of control) were observed at 120 min of digestion,

446

while in the heated case, the digestion with increasing time up to 180 min resulted in a progressive

447

decrease in particle size (Figure 9 A, B). The observations suggest that although the heat pretreatment

448

greatly increased the pepsin-induced aggregation of proteins, it facilitated the SIF digestion.

449

The particle size distribution of unheated or heated SPI was nearly unchanged by the

450

nanocomplexation with curcumin, but the changing pattern of their particle size distributions upon

451

protease hydrolysis, especially during the SIF digestion, was remarkably affected (Figure 9). In the

452

unheated SPI case, the nanocomplexation with curcumin greatly favored the formation of smaller

453

sizes of particles during the SIF digestion, as compared with the control (Figure 9 A, C). In this case,

454

the particle sizes of the final digests seemed to be slightly dependent on the digestion time. In

455

contrast, the situation is different in the heated SPI case, and in the latter case, it can be observed that

456

increasing the digestion time during the SIF digestion resulted in a progressive increase in particle

457

size (Figure 9 C, D). The observations indicated that upon the sequential SGF and SIF digestion, the 23 - Environment ACS Paragon-Plus


458

nanocomplexation of unheated SPI with curcumin greatly lessened the extent of protein aggregation

459

occurring during the digestion, while the situation for the heated SPI was the reversal. Thus, the

460

remarkably lower bioaccessibility of curcumin in the nanocomplex with heated SPI (relative to that

461

with unheated SPI; Figure 6 A) could be largely attributed to the occurrence of protease-induced

462

protein aggregation, especially in the SIF digestion.

Page 24 of 43

463

In summary, we reported that SPI can easily form nanocomplexes with curcumin, thus remarkably

464

improving its solubility (in aqueous phase), stability and even bioaccessibility. The complexation with

465

curcumin did not distinctly change the size and morphology of nanoparticles in SPI solutions, though

466

the surface hydrophobicity of the proteins considerably decreased upon the binding. Under the

467

investigated conditions, the amount of loaded curcumin at a given c (e.g., 3.5%, w/v) was mainly

468

dependent on the initial [cur] in the dispersion. Although the heated SPI exhibited a higher capacity to

469

bind curcumin than the unheated one, the bioaccessibility was significantly lower. The lower

470

bioaccessibility of curcumin in the nanocomplexes with heated SPI relative to that with the unheated

471

one was largely attributed to the protein aggregation occuring during the SIF digestion. The present

472

work reveals that SPI is an excellent nano vechicle for the delivery of water-insoluble bioactive

473

ingredients, and the nanocomplexation exhibit a great potential to be applied in functional food

474

formulations. However, the denatured or aggregated state of the proteins in SPI might produce a

475

significant influence on the bioavailability of encapsulated bioactive ingredients. Further work is

476

necessary to testify the effectiveness of curcumin in the nanocomplexes of SPI using in vivo digestion

477

models.


Page 25 of 43


478

AUTHOR INFORMATION

479

Corresponding Author

480

(C.H.T.) Fax (086)20-87114263. E-mail: [email protected].

481

Notes

482

The authors declare no competing financial interest.

483

ACKNOWLEDGEMENTS

484

This work was supported by the NNSF of China (serial numbers: 31471695, 31171632 and 31130042).

485

SUPPORTING INFORMATION FOR PUBLICATION PARAGRAPH.

486

Additional tables (Tables S1 and S2) and figures (Figures S1 and S2). This material is available free of

487

charge via the Internet at http://pubs.acs.org.

488



489

490 491 492 493 494 495 496 497 498 499

Page 26 of 43

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582 583


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584

Figure captions.

585

Figure 1. A) Typical size distribution profiles of particles for unheated and heated SPI solutions; B) Z-

586

average diameter of particles in unheated and heated (at 75, 85 and 95°C for 15 min) SPI solutions or

587

dispersions, before or after nanocomplexation with curcumin. Different characters (a-b) on the top of

588

columns represent significant difference at p < 0.05, between the different treatments.

589

Figure 2. Typical AFM height images of particles in unheated and heated SPIs (A-D), or SPI-curcumin

590

complexes (A´-D´). Panels A-D indicate the images of unheated SPI, or SPIs heated at 75, 85 and 95ºC,

591

respectively; Panels A´-D´ indicate the images of the SPI-curcumin complexes from unheated SPI, or

592

SPIs heated at 75, 85 and 95ºC, respectively.

593

Figure 3. Visual observations of original (A) and reconstituted (B) SPI-curcumin nanocomplexes in

594

water. The unheated or heated SPI (5%, w/v) mixed with the curcumin stock solution (4.5 mg/mL; in

595

100% ethanol) at a volume ratio of 50:1. The samples from left to right represent untreated SPI

596

(control), and SPI preheated at 75, 85 and 95°C, respectively.

597

Figure 4. Influence of curcumin concentration (in the mixed dispersions) on the encapsulation

598

efficiency (EE) and load amount (LA) of curcumin in unheated SPI-curcumin nanocomplexes. The final

599

c value and curcumin-containing ethanol volume fraction were kept constant, with 3.5% (w/v) and 0.3,

600

respectively. Each data is the means and standard deviations of triplicate measurements performed on

601

separate samples.



Page 32 of 43

602

Figure 5. Degradation kinetics of free curcumin, or curcumin in the nanocomplexes with unheated and

603

heated (95ºC) SPIs in water at pH 7.0, upon storage up to 4 h, at 25ºC (A) and 95ºC (B), respectively.

604

Data are presented as means and standard deviations of triplicate measurements.

605

Figure 6. A) Percentage of curcumin remained in the aqueous phase for free curcumin and curcumin-

606

nanocomplexes with unheated and heated (at 100 ºC) SPI, after the whole simulated digestion of 120

607

min, in the absence or presence of proteases. B) Percentage of curcumin in the whole digests of free

608

curcumin, or curcumin-nanocomplexes with unheated and heated SPI, after the digestion of 120 min, in

609

the absence or presence of proteases. Data are presented as triplicate measurements on separate samples.

610

Error bars represent standard deviations. Different letters (a-b) on the top of columns represent

611

significant difference at p < 0.05 among curcumin in free or complexed form, due to the application of

612

proteases or not. Different letters (e-g) on the top of columns represent significant difference among the

613

same set of samples without or with enzyme, due to the different forms of curcumin (free, or

614

nanocomplexes with unheated or heated SPI).

615

Figure 7. Changes in SDS-PAGE patterns of unheated (A, C) and heated (B, D) SPI, during the

616

digestion in SGF (0-60 min) and SIF (60-180 min), as a function of digestion time. Panels A and B:

617

controls (without complexation with curcumin); Panels C and D: nanocomplexes with curcumin. The

618

symbols (α´, α and β) within the figures represent the major α´-, α- and β-subunits of β-conglycinin,

619

while AS and BS represent the acidic and basic subunits (or polypeptides) of glycinin, respectively.

32 Environment ACS Paragon Plus

Page 33 of 43


620

Figure 8. Release kinetics of TCA-soluble nitrogen during the sequential SGF and SIF digestion of

621

unheated and heated SPI, or their nanocomplexes with curcumin. Each datum is the means and standard

622

deviation of triplicate measurements on separate samples.

623

Figure 9. Typical particle size distribution profiles of unheated (A, C) or heated (B, D) SPI, without (A,

624

B) or with complexation with curcumin (C, D), as a function of digestion time (0-180 min). The

625

numbers (1-5) within figures correspond to the digestion times of 30, 60, 90, 120 and 180 min,

626

respectively. The digestion consisted of sequential SGF (0-60 min) and SIF (60-180 min) processes,

627

respectively.



628

Fig. 1. (Chen et al.)

A

20 unheated SPI heated SPI (75oC) heated SPI (85oC) heated SPI (95oC)

Volume (%)

16 12 8 4 0 1

10

100

1000

10000

Size (µ µm)

629

B

120

SPI

SPI-cur

b

SPI-cur (reconstituted)

b

Z-average diameter (nm)

100

630

80

a a

a

a,b

b

a

a

Unheated

75

b

b

b

60 40 20 0 85

95

Heat pretreatment (oC)


Page 34 of 43

Page 35 of 43

631



632

A)

A´)

633

B)

B´)

634

C)

C´)



635 636

D)

D´)


637


Page 36 of 43

Page 37 of 43


100

35

EE LA

95

30

EE (%)

25 90

20 15

85 10 80

5 0

75 0.0

0.3

0.6

0.9

1.2

Curcumin concentration (mg/mL)

639 640


1.5

LA (ug/mg SPI)

638



641


A

1.2

Relative Intensity (A.U.)

1.0 0.8 Nanocomplex (Unheated SPI) Nanocomplex (Heated SPI) Free curcumin

0.6 0.4 0.2 0.0 0

1

2

3

4

Storage time (h)

642

Relative Intensity (A.U.)

B

1.0

Nanocomplex (Unheated SPI) Nanocomplex (Heated SPI) Free curcumin

0.8 0.6 0.4 0.2 0.0 0

1

2

3

Storage time (h)

643 644


4

Page 38 of 43

Page 39 of 43


% curcumin in aqueous phase

A

646

100

B

b, g

Free curcumin Nanocomplex (unheated SPI) Nanocomplex (heated SPI)

80 a, f

a, f

a, f

60

40 b, e

20 a, e

0

without enzyme

with enzyme

% curcumin in the whole digests

645


100

Free curcumin Nanocomplex (unheated SPI) Nanocomplex (heated SPI)

80

a, f

b, f

b, f

b, e

a, f

60

40

20

a, e

0

without enzyme

647 648


with enzyme


649


650

651 652


Page 40 of 43

Page 41 of 43


TCA-soluble Nitrogen (%)

653


100 pepsin digestion

pancreatin digestion

80

60

40 Unheated SPI Heated SPI Unheated SPI-curcumin Heated SPI-curcumin

20

0 0

30

60

90

120

150

Digestion time (min) 654 655


180


656


A

35

35

B

30

4

30

2

4

20

Volume (%)

Volume (%)

20

1

3

15 5

10

15 10 control (0 min)

5

5

0

0 1

10

100

1000

1

10000

10

100

657 35 4

30

D

5 1

3

25

35 4

10000

Volume (%)

20 15 10 control (0 min)

1

2

30 3

25

2

5

1000

Size (nm)

Size (nm)

Volume (%)

2

25 control (0 min)

C

1

3

5

25

20

5

control (0 min)

15 10 5

0

0 1

658

Page 42 of 43

10

100

1000

10000

1

Size (nm)

10

100

Size (nm)

659 660


1000

10000

Page 43 of 43

661


TOC Graphic

662 663


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