CoreâShell Soy ProteinâSoy Polysaccharide Complex (Nano)particles

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Core-shell Soy Protein-Soy Polysaccharide Complex (Nano)particles as Carriers for Improved Stability and Sustained-Release of Curcumin Fei-Ping Chen, Shiyi Ou, and Chuan-He Tang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01176 • Publication Date (Web): 31 May 2016 Downloaded from http://pubs.acs.org on June 3, 2016

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Journal of Agricultural and Food Chemistry

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Core-shell Soy Protein-Soy Polysaccharide Complex (Nano)particles as

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Carriers for Improved Stability and Sustained-Release of Curcumin

4 Fei-Ping Chen1, Shi-Yi Ou2, Chuan-He Tang1,3,*

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1

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

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Republic of China 2

Department of Food Science and Engineering, Jinan University, Guangzhou 510632, 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. Using soy protein isolate (SPI) and soy polysaccharides (SSPS) as polymer matrixes,

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this study reported a novel process to fabricate unique core-shell complex (nano)particles to perform

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as carriers for curcumin (a typical poorly soluble bioactive). In the process, curcumin-SPI

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nanocomplexes were first formed at pH 7.0, and then coated by SSPS. At this pH, the core-shell

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complex was formed in a way the SPI nanoparticles might be incorporated into the interior of SSPS

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molecules without distinctly affecting the size and morphology of particles. The core-shell structure

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was distinctly changed by adjusting pH from 7.0 to 4.0. At pH 4.0, SSPS was strongly bound to the

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surface of highly aggregated SPI nanoparticles, and as a consequence, much larger complexes were

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formed. The bioaccessibility of curcumin in the SPI-curcumin complexes was unaffected by the SSPS

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coating. However, the core-shell complex formation greatly improved the thermal stability and

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controlled release properties of encapsulated curcumin. The improvement was much better at pH 4.0

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than that at pH 7.0. All the freeze-dried core-shell complex preparations exhibited a good redispersion

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behavior. The findings provide a simple approach to fabricate food-grade delivery systems for

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improved water dispersion, heat stability and even controlled release of poorly soluble bioactives.

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KEYWORDS. Curcumin; soy protein; soy polysaccharide; carrier; core-shell particles

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INTRODUTION

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Food proteins as natural vehicles for bioactive have recently attracted increasing interest in the food

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science and medicine fields, since the complexation with proteins can greatly improve water

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dispersibility, stability and bioaccessibility of poorly soluble bioactives 1, 2. Hydrophobic bioactives

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are generally bound to proteins mainly through hydrophobic interactions, though in some cases, van

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der Waals attraction and hydrogen bonds may be also involved. For a given bioactive, the

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effectiveness of proteins to perform as carriers is largely determined by the nature of proteins. Due to

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the ability to self-assemble into micellar nanostructures, casein or its caseinates has been recognized

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to be an excellent carrier system for hydrophobic bioactives such as curcumin 3. For caseins,

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hydrophobic bioactives are usually encapsulated within the interior of their self-assembled micelles.

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By contrast, the complexation between these bioactives and globular proteins, e.g., whey proteins or

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soy proteins, usually occurs on their surface, wherein the nature and number of hydrophobic clusters

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seem to play an important role in the complexation 4. Heat-induced denaturation and subsequent

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formation of aggregated particles has been confirmed to increase the effectiveness of β-lactoglobulin

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or soy protein isolate (SPI) to act as carriers for curcumin 4, 5. The increased effectiveness is largely

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due to the enhanced surface hydrophobicity of heat-treated proteins. However, one point is

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noteworthy that the enhanced surface hydrophobicity might be unfavorable for the colloidal stability

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of the protein systems. Furthermore, if the loaded amount of hydrophobic bioactives on the proteins is

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high, the bound bioactives may act as ‘bridges’, thus further favoring protein-protein or particle-

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particle interactions.



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On the other hand,(nano)complexes or complex coacervates arising from protein-polysaccharide

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electrostatic interaction seem to provide a solution to inhibit protein-protein aggregation, thus

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exhibiting a great potential to perform as carriers for bioactives 6, 7. The complexation of proteins with

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anionic polysaccharides (e.g., pectin, alginate, and soy polysaccharides) not only provides a steric

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stabilization of proteins, but also may improve the stabilization of bioactives encapsulated in their

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corresponding complex coacervates. For the protein-polysaccharide coacervates as the carriers,

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bioactives are usually bound to proteins prior to the complexation between proteins and

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polysaccharides 6. The effectiveness of these complex coacervates to perform as the carriers for

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bioactives seems to be highly related to the nature of proteins and the applied pH (at which the

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complexation occurs). For β-lactoglobulin-sodium alginate systems, Hosseini et al. 6 demonstrated

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that their soluble nanocomplexes could be formed at both pH 7.0 and 4.25, and the solubility in water

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of many nutraceutical agents, including β-carotene, folic acid, curcumin and ergocalciferol was

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greatly improved. In this case, the nanocomplexes at pH 7.0 seemed to be a better carrier than at pH

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4.25, due to higher molar binding ratios of the former. Santipanichwong and others 8 reported that the

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electrostatic deposition of an anionic polysaccharide (beet pectin) onto heat-denatured β-lactoglobulin

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aggregates at pH 7.0 resulted in formation of core-shell biopolymer nanoparticles. Similar complex

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nanoparticles from heated β-lactoglobulin and pectin have been confirmed to act as carriers for

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enhanced stability of linoleic acid 9. Ding & Yao 10 proposed an assembly approach to produce a folic

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acid-loaded complex between soy protein and soy polysaccharide at pH 4.0. If a heat treatment was

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performed on this complex, a kind of nanogels that were dispersible in acidic conditions could be

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fabricated. Although the protein-polysaccharide complexes exhibit a great potential to perform as 4 - Environment ACS Paragon -Plus

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carriers for bioactives, very few information has been available addressing pH-dependent formation

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mechanism of the complexes, stability, release behavior and bioaccessibility of encapsulated

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bioactives.

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The main objective of the current work was to systematically investigate the formation of SPI-soy

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soluble polysaccharide (SSPS) complex coacervates at acidic (pH 4.0) or neutral (pH 7.0) pHs and

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their potential as carriers for curcumin, a low-molecular-weight, natural polyphenolic compound

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found in the rhizome of turmeric (Curcuma longa). Most of the proteins in SPI are usually present in

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the nanoparticle aggregated state 11, and thus can perform as nanocarriers for curcumin4, 12, 13. SSPS is

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an anionic and pectin-like polysaccharide, containing a covalently bound protein (with molecular

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weight of ~50 kDa) 14. To form the core-shell particles, curcumin was first bound with SPI, and the

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resultant curcumin-SPI complexes were further coated with SSPS. The core-shell (nano)particles have

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been confirmed to act as nutraceutical carriers15.

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MATERAILS AND METHODS

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Materials. Soy protein isolate (SPI) was prepared in the laboratory from defatted soy flour according

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to the same process as described in our previous work 4, with a protein content of about 91.5% (dry

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basis; as determined using a Dumas combustion method, Elemental Analyzer rapid N cube, Germany,

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with a nitrogen conversion factor of 6.25). Soybean soluble polysaccharides (SSPS) (purity > 95%)

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was purchased from Gen-view Scientific Inc. (China). Porcine bile extract (B8631), pancreatin (from

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porcine pancreas, P1750, 4×USP), pepsin (from porcine gastric mucosal, P7000, 975 units/mg of 5 - Environment ACS Paragon -Plus


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protein) and curcumin (with a purity > 98%) were purchased from Sigma Chemical Co. (St. Louis,

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MO, USA). All other chemicals were of analytical grade.

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Formation of core-shell SPI-curcumin-SSPS complexes. The stock solutions of SPI and SSPS

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were prepared at 2.0% and 1.0% (w/v) in distilled water respectively, with the help of a magnetic

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stirrer for 3h, and then placed overnight for complete hydration. The pH of both solutions was

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precisely adjusted to 7.0 with 0.2 M HCl. One milliliter of curcumin solution (5 mg/mL in absolute

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ethyl alcohol) was first added dropwise to 20 mL of the SPI solution, and the mixture was mixed

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under magnetic stirring conditions for 60 min. After that, 20 mL of SSPS solution was further added

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dropwise to the curcumin-SPI mixture. The resultant SPI-curcumin complex was denoted as the

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complex-I, while the complex-I together with SSPS was denoted as the complex-II. If the pH of the

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dispersion containing the complex-II was further adjusted to 4.0 using 1 M HCl, the final resultant

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complex (at pH 4.0) was denoted as the complex-III.

To evaluate the redispersion behavior, the dispersions containing different complexes (I-III) were freeze-dried to produce the corresponding powders.

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Encapsulation efficiency (EE) and retention ratio (RR) of curcumin. For the determination, all

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the dispersions containing the complexes prior to the freeze-drying were centrifuged at 8000 g for 20

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min to remove any insoluble curcumin crystal. The curcumin content in the resultant supernatants

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(after the centrifugation) was determined as in the following section. The EE of curcumin in the

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complexes was determined by the relative ratio of the curcumin amount in the supernatants to the

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initial total curcumin amount (added to the system). 6 - Environment ACS Paragon -Plus

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The RR of curcumin was determined on the freeze-dried complexes. The curcumin amount in the

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freeze-dried complexes was also determined as the following section. The percentage of RR (RR%)

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of curcumin was determined by the relative ratio of curcumin amount in the freeze-dried complexes to

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the curcumin amount in their corresponding supernatants (after the centrifugation).

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Curcumin extraction and determination. Aliquots (100 µL) of the aqueous samples containing

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curcumin were diluted with 900 µL of deionized water, or about 10 mg of freeze-dried complex

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powders were well dispersed in 1 mL of deionized water, and then 400 µL of the diluted samples

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were extracted in 3 mL of extracting agent, a mixture of ethyl acetate and absolute ethyl alcohol at a

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volume ratio of 10:1, under magnetic stirring conditions for 10 s. After that, the mixtures were placed

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quiescently for 20 min to allow lamination. The curcumin content in the resultant supernatants was

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determined at 420 nm with a UV754N UV-Vis spectrophotometer (Precision & Scientific Instrument,

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Shanghai, China), according to an established standard curve of curcumin (with R2> 0.999).

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Particle size and ξ-potential. The particle size distribution and z-average diameter (Dz) of particles

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in SPI, SPI-curcumin complex or core-shell SSPS-SPI complex dispersions, at pH 7.0 and 4.0, were

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evaluated using dynamic light scattering (DLS). Each solution was diluted to a protein concentration

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of about 0.1% (w/v) with 5 mM phosphate buffer at pH 7.0 or 4.0 (PBS; filtered with a 0.22 µm HA

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Millipore membrane). DLS analysis was performed at a ﬁxed angle of 173° using a Zetasizer Nano-

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ZS instrument (Malvern Instruments, Worcestershire, UK) equipped with a 4 mW He-Ne laser (633

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nm wavelength) at 25 °C. Droplet sizing was performed at 10-s intervals in a particle-sizing cell using

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backscattering technology. The Dz of particles was calculated based on the Stokes-Einstein equation,



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

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separate samples.

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The ζ-potential of particles in all the aforementioned dispersions, or of SSPS, at pH 7.0 and 4.0 was

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measured using a Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, Worcestershire, UK) in

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combination with a multipurpose autotitrator (model MPT-2, Malvern Instruments, Worcestershire,

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UK). Freshly prepared solutions were diluted to a concentration of protein or polysaccharide to 0.1%

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(w/v) with the same pH of PBS prior to analysis.

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Atomic force microscopy (AFM). Morphological observations of SSPS, SPI-curcumin complexes,

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or core-shell SPI-curcumin-SSPS complexes at pH 7.0 and 4.0 were performed using atomic force

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microscopy (AFM, a Dimension 3000 microscope, Digital Instruments-Veeco, Santa Barbara, CA,

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USA), according to the same processes using the same equipment as described in our previous work

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Fourier transform infrared spectroscopy (FTIR). The structural characteristics of SPI, SSPS,

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curcumin, SPI-curcumin complexes or core-shell SSPS-SPI complexes, formed at pH 4.0 or 7.0, were

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monitored by FTIR with a Vector33 model Fourier transform infrared instrument (Bruker Co.,

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Germany) at 25°C. All the samples were first freeze-dried and then mixed with KBr, and pressed into

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a pellet. The spectra were acquired in the range 400~4000 cm-1 at a resolution of 4 cm-1.

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Sequential in vitro gastrointestinal digestion. The bioaccessibility of curcumin in different

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complexes with SPI alone, or with both SPI and SSPS, at pH 7.0 and 4.0,were evaluated using a

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simulated gastrointestinal digestion model, according to the process as described in our previous work 8 - Environment ACS Paragon -Plus

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concentration of 1.0% (w/v) was adjusted to pH 2.0 with 2 M HCl and then preincubated in a shaker

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(37°C, 95 rpm) for 10 min. Four micrograms of pepsin powder was added and well mixed to start the

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simulated gastric digestion (0~60min). After 60 min of digestion at 37°C, the pH of the pepsin-digests

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was adjusted to 7.0 with 4.0 M NaOH, and then100 mg of bile extract powder was added and well

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dispersed in the shaker for 10 min. Last, 8 mg of pancreatin powder was added to start the simulated

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intestinal digestion (60~120 min). At the end of the whole digestion, 1 mL of the digest samples was

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collected and centrifuged at 10000 g for 30 min, then the curcumin in the resultant supernatants was

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extracted and determined as described above. The bioaccessibility was defined as percent of curcumin

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amount remaining in the aqueous phase (after the centrifugation), after the whole gastric and intestinal

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digestion (180min).

, with a few modifications. In brief, 20 mL of freshly prepared complex dispersions at a protein

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The degradation kinetics of curcumin encapsulated in these complexes during the digestion was

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monitored by detecting the relative decrease in curcumin amount of the whole digests (as a function

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of digestion time).

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In vitro release of encapsulated curcumin in PBS. The in vitro release behavior of curcumin from

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different complexes formed at pH 7.0 or 4.0 was evaluated in pH 7.0 PBS, according to the method of

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Teng and others 16, with a few modifications. All the PBS buffers applied in this section contained 5

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mg/mL Tween 20. Four milliliters of different complexes (at a protein concentration of 1.0%, w/v)

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were filled into dialysis bags (MW cutoff 6~8 kDa). Each dialysis bags was then immersed in a flask

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containing 100 mL of PBS (preheated at 37°C). The flask was incubated in a reciprocal shaking bath

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(AoHua Ltd, Changzhou, China) at 37°C under constant shaking conditions at a rate of 120 rpm. At 9 - Environment ACS Paragon -Plus


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required incubation time intervals, aliquots (2 mL) of samples were taken from the flask, and diluted

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with an appropriate amount of PBS. The concentration of released curcumin in the resultant

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dispersions was determined at 426 nm using a UV754N UV-Vis spectrophotometer, according to an

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established standard curve of curcumin (with R2 > 0.999). After each sampling, 2 mL of fresh PBS

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was added to the flask to maintain the total volume of samples constant. The release kinetics of

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curcumin was plotted as a function of time.

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Thermal stability. The thermal stability of curcumin encapsulated in different complexes at pH 7.0

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or 4.0 was evaluated by monitoring the degradation kinetics of curcumin, upon heating at 80°C up to

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3 h. In brief, 20 mL of the complexes freshly prepared, or free curcumin dispersion (prepared by

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adding 0.5 mL of curcumin solution (in absolute ethyl alcohol; at 5 mg/mL, into 20 mL of water at pH

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7.0) was transferred to a clean amber bottle and incubated at 80°C in a water bath. At predetermined

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time intervals, aliquots (100 uL) of the heated samples were taken out. The curcumin remaining in

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these samples was extracted and determined as described aforementioned.

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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.

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RESULTS AND DISCUSSION

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Characterization of curcumin-loaded core-shell particles. Encapsulation efficiency (EE) and

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retention ratio (RR) of curcumin. The dispersion containing SPI and curcumin at pH 7.0 was

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transparent, but orange-yellow in color (Figure 1 A), indicating the formation of SPI-curcumin 10 - Environment ACS Paragon-Plus

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complex at nanoscale (complex-I). The visual observation did not distinctly change upon the addition

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of SSPS (Figure 1 A), suggesting good compatibility between SPI and SSPS at pH 7.0 (due to the

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same charge on the surface). When the pH of the dispersion containing the complex-I in the presence

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of SSPS (complex-II) was adjusted from 7.0 to 4.0, the dispersion became turbid, but still kinetically

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stable (even after a storage up to 3 days; Figure 1 A). In contrast, if the pH of the complex-I

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dispersion was directly adjusted to 4.0, severe aggregation and phase separation would occur within a

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short period of storage (data not shown), clearly as a result of remarkably decreased electrostatic

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repulsion. Considering the fact that soy proteins at pH 4.0 is positively charged, while the charge of

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SSPS is negative, it can be reasonably hypothesized that the coating with negatively charged SSPS on

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the surface of the complex-II might increase the inter-particle electrostatic repulsion, thus improving

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the colloidal stability of the dispersion.

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The EE% of curcumin in the curcumin-loaded complexes I, II and III was determined to be 89.12%,

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93.1% and 96.0%, respectively (data not shown), indicating that although the complex-I (using SPI

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alone as the vehicle) exhibited high encapsulation efficiency, the formation of core-shell structure

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could further improve the encapsulation efficiency. Interestingly, in the complex-III case, the EE%

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reached 96.0%, implying that almost all of the curcumin molecules could be transformed into the

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complex. We also evaluated the influence of a freeze-drying on the redispersion behavior of the

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curcumin-loaded complexes (I-III), and found that all the curcumin-loaded preparations exhibited a

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good redispersion behavior, as evidenced by no distinct changes in visual observation between the

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original and freeze-dried/reconstituted dispersions (Figure 1 A). Figure 1 B shows the retention ratio

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of curcumin in the freeze-dried curcumin-loaded complex preparations (I-III). For the curcumin in the 11 - Environment ACS Paragon-Plus


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complex-I, almost 30% of curcumin was degraded after the freeze-drying, while in the case of the

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complex-II, the loss was only 5.5% (Figure 1 B), indicating that the presence of SSPS considerably

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improved the stability of curcumin against the freeze-drying. The loss of curcumin for the complex-III

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was similar to that for the complex-II (Figure 1 B). The observations clearly showed that the

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formation of core-shell complex nanoparticles remarkably improved the encapsulation efficiency and

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retention ratio of curcumin.

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Particle size and ξ-potential. Figure 2 shows the z-average diameter (Dz) and ξ-potential of

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particles in dispersions containing curcumin-loaded SPI, or SPI-SSPS complexes, at pH 7.0 and 4.0.

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At pH 7.0, the Dz of particles in the SPI dispersion was about 82 nm, which is in accordance with our

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previous work 17. At this pH, the Dz did not suffer a significant change after complexation with

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curcumin, but significantly increased to about 300 nm when SSPS was further introduced (Figure 2

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A). The increase in particle size upon the addition of SSPS might reflect the coating of negatively

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charged polysaccharide on the surface of the SPI-curcumin complex, resulting in formation of a core-

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shell structure. The coating may be attributed to electrostatic, hydrophobic interactions and hydrogen

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bonding between the proteins and the polysaccharide 15. The pH adjustment (from 7.0 to 4.0) further

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significantly increased the Dz of particles (Figure 2 A). At pH 4.0, the protein and SSPS were

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contrarily charged, and as a consequence, their electrostatic interaction would be strengthened. On the

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other hand, the inter-particle attractive interactions of the proteins themselves at pH 4.0 (close to their

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isoelectric point) also increased as compared to that at pH 7.0. Thus, the distinct increase in particle

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size for the core-shell complex upon the pH decreasing might be a result of re-arrangement of core-

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shell structure, and subsequently, formation of more compact and stable ‘core-shell’ structure. 12 - Environment ACS Paragon-Plus

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Another point is noteworthy that if the complex-I was not coated by the SSPS, the pH adjustment

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(from 7.0 to 4.0) would lead to formation of much larger particles as compared to the complex-II (6.0

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µm vs 0.8 µm; data not shown). This observation suggests that the SSPS coating did produce a de-

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aggregation effect on the protein particles at acidic pH conditions. The protein de-aggregation in

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acidic SPI dispersions by the SSPS addition has been similarly observed in previous works 14, 18,

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wherein the increased stability against aggregation was largely attributed to the steric repulsion.

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As expected, the magnitude of ξ-potential for the SPI particles at pH 7.0 was greater than that at pH

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4.0 (-27 mV vs 13 mV; Figure 2 B). The binding of curcumin at pH 7.0 did not significantly affect

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the surface charge of the particles in the dispersions. However, remarkable changes in ξ-potential of

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particles (from -27 mV to -43 mV) were observed after further addition of SSPS, which is clearly as a

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result of the formation between protein particles and negatively changed polysaccharides.

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Interestingly, the magnitude of ξ-potential of core-shell particles at pH 7.0 was close to that of SSPS

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(Figure 2 B), suggesting that the surface charge of core-shell particles be largely dominated by SSPS.

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This is an indirect evidence to indicate that the surface of the curcumin-loaded SPI nanoparticles at

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pH 7.0 was coated by the SSPS. As the pH was adjusted from 7.0 to 4.0, the magnitude of ξ-potential

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of particles to a certain extent decreased, which is consistent with the decrease in ξ-potential of SSPS

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alone (Figure 2 B), further confirming the dominant role of the SSPS on the colloidal stability of the

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dispersions.

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Particle morphology and intra-particle interactions. The microstructural morphology of particles

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for curcumin-loaded SPI or SPI-SSPS complexes at pH 7.0 or 4.0 was evaluated using AFM, with the

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3D AFM images displayed in Figure 3. For the curcumin-loaded SPI complexes at pH 7.0, most of 13 - Environment ACS Paragon-Plus


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particles were present in a dissociated state with heights of several nanometers (Figure 3 A). The

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particle morphology of this complex is similar to that for the SPI alone (data not shown), which is in

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agreement with the DLS data (Figure 2 A), confirming that the complexation with curcumin did not

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distinctly affect the particle size. When the curcumin-loaded SPI complexes were further coated with

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SSPS, it can be observed that the heights for a large portion of particles were unaffected by the

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coating with SSPS, while the fraction of particles with large sizes increased (Figure 3 A, B). On the

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other hand, the SSPS molecules alone could not be observed in the AFM images (Figure 3 D),

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possibly due to their linear and flexible structure in nature. Taking the DLS data (Figure 2 A) into

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account together, it can be reasonably hypothesized that the increased Dz by the SSPS coating might

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be largely due to increased hydration layer of the SPI nanoparticles, as a result of the SSPS coating on

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their surface. In other words, the observations suggest that the core-shell structure formed at pH 7.0

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might be relatively loose, and the curcumin-loaded SPI nanoparticles be entrapped within the flexible

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chain of SSPS.

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Compared with the core-shell complexes at pH 7.0, the particles in the core-shell SPI-SSPS

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complexes at pH 4.0 had much greater heights and contour sizes (Figure 3 B, C). In this core-shell

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complex, the width sizes ranged about 100 nm to 500 nm, which is basically consistent with the

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particle size distribution profile as observed by DLS (data not shown). In addition, it can be observed

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that the surface of the particles for the core-shell complexes at pH 4.0 was more smooth and regular,

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implying stronger intra-particle interactions. The observations clearly indicated that the pH

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adjustment (from 7.0 to 4.0) might be favorable for the formation of more compact and ordered core-

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shell complexes. The proposed mechanism for the formation of core-shell SPI-curcumin-SSPS

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complexes at pH 7.0 and 4.0 can be illustrated in Figure 5.

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To further unravel the intra-particle interactive force pattern, we evaluated the structural

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characteristics of freeze-dried SPI, SSPS or their complexes using FTIR technique, and their typical

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FTIR profiles are shown in Figure 4. The major characteristic peaks of SPI were 1650cm-1(amide Ⅰ,

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C=O stretching) and 1541cm-1(amide II, N-H bending), which are well consistent with previous

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works 19, 20. SSPS has a pectin-like structure, which is composed of rhamnogalacturonan as the

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backbone. The featured IR bands of SSPS were 3432 cm-1 (N-H bending), 1630 cm-1 (COO-

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stretching) and 1057 cm-1 (rhamnogalacturonan moiety) 20. The characteristic FTIR profiles of SPI

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were nearly unaffected by the complexation with curcumin (Figure 4), indicating that the

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complexation did not involve the peptide amides of the proteins. This indirectly supported our

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previous argument that the complexation with curcumin occurred on the surface hydrophobic domains

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of the proteins 4. At pH 7.0, distinct blue shifts for the amide I peaks (2-4 nm) were observed after the

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curcumin-loaded SPI complexes were further coated by the SSPS (Figure 4). A similar phenomenon

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has been observed in the formation of α-tocopherol-loaded zein-chitosan complexes 21, wherein it was

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largely attributed to the occurrence of inter-molecular electrostatic interactions between proteins and

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polysaccharides. In a review, Ye 7 confirmed that the electrostatic interactions between anionic

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polysaccharides and positively charged part of proteins can occur at neutral pH.

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Besides the band shift, the magnitude of the amide I and II bands for the proteins was distinctly

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decreased by the SSPS coating (Figure 4). In contrast, the magnitude of the amide I and II bands at

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pH 4.0 was much less affected by the SSPS (Figure 4). Considering the fact that the particles in SPI 15 - Environment ACS Paragon-Plus


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highly varied in size between at pH 7.0 and 4.0 (72 nm vs 6.2 µm; Figure 2 A), the observations thus

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strongly suggest that the intra-particle interactions of SPI particles at pH 7.0 were distinctly weakened

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by the SSPS coating, while that at pH 4.0 was slightly affected. On the other hand, the bands assigned

297

to carboxylate in SSPS at around 1630 cm-1 was not detectable in the SSPS-SPI complexes (Figure

298

4), further indicating interactions between the protein and the polysaccharide.

299

Bioaccessibility and stability of curcumin during in vitro simulated digestion. The

300

bioaccessibility of curcumin in different complexes (I-III), defined as percent of curcumin remaining

301

in the supernatants after sequential simulated gastric (1 h) and intestinal (2 h) digestion (relative to the

302

initial total curcumin), was evaluated using an in vitro gastrointestinal digestion model. All the test

303

curcumin-loaded complexes have a similar bioaccessibility of around 60% (Figure 6 A), indicating

304

that the SSPS coating did not distinctly affect the bioaccessibility of curcumin in the complexes with

305

SPI particles. The bioaccessibility of curcumin in the SPI complex at pH 7.0 is much less than that

306

reported in our previous work 12. The lower curcumin bioaccessibility in the current work might be

307

related to the much higher loading amount of curcumin in the complexes (1.25% vs ~0.18% in the

308

previous work).

309

On the other hand, the degradation of curucmin occurring during the digestion was also evaluated,

310

as displayed in Figure 6 B. As expected, the curcumin in these complexes suffered a gradual

311

degradation as the digestion proceeded, indicating its instability in an aqueous environment in nature.

312

There was no significant difference in stability of curcumin during the gastric digestion between

313

different complexes. During the intestinal digestion, the curcumin encapsulated in the core-shell

314

complex at pH 7.0 was degraded at a significantly higher rate than that in the complexes with SPI 16 - Environment ACS Paragon-Plus

Page 17 of 33


315

alone at the same pH, or in the core-shell preparation at pH 4.0 (Figure 6 B). The lower stability of

316

curcumin in the complex II (relative to that in the complexes I or III) might be related to the

317

differences in polarity of microenvironment of encapsulated curcumin in the complexes. At pH 7.0,

318

the coating of SSPS on the surface of SPI-curcumin complexes seemed to increase the polarity of

319

microenvironment of encapsulated curcumin, due to the formation of loose hydration layer.

320

In vitro release behavior of curcumin in complexes. The release kinetics of curucmin encapsulated

321

in different complexes (I-III) was evaluated using a pH7.0 PBS/Tween 20 system as adopted by Teng

322

et al. 16, and the results are shown in Figure 7. All the curcumin-loaded complexes exhibited a release

323

behavior that can be well described using a first-order kinetic model, suggesting that the release of

324

curcumin from these complexes was mainly dominated by the concentration-dependent diffusion

325

from the polymer matrix. The release behavior is similar to that observed for curcumin released from

326

β-lactoglobulin-based nanoparticles that were fabricated by glutaraldehyde crosslinking 16. By

327

contrast, it can be observed that irrespectively of the applied pH, the SSPS coating distinctly slowed

328

down the release rate of curucmin from the SPI complexes (Figure 7). The released amount of

329

curcumin at 20 h (56-60%) from the core-shell complexes at pH 4.0 or 7.0 was considerably lower

330

than that (~90%) from the complex with SPI alone. The delayed release was clearly associated with

331

the formation of core-shell structure that could to a great extent limit the diffusion of encapsulated

332

curucmin molecules from SPI particles.

333

Thermal stability of curcumin. Curcumin is very unstable in aqueous environment, especially at

334

high temperatures. About 70% of total amount for free curcumin in water was degraded after a

335

storage of 30 min at 25 °C; the complexation with SPI could greatly increase the stability of curucmin 17 - Environment ACS Paragon-Plus


Page 18 of 33

336

against degradation during storage at room or high temperatures 12. In the current work, we evaluated

337

the degradation kinetics of curcumin encapsulated in different complexes (I-III), at 80°C, as displayed

338

in Figure 8. As expected, all the curcumin-loaded complex preparations exhibited an exponential

339

decay behavior of encapsulated curcumin, as the time of heating proceeded (Figure 8). For the

340

curcumin encapsulated in the complex-I, almost 65% was degraded after the heating up to 3 h. In

341

contrast, the loss in the core-shell complexes after the same heating was only 44% (complex-II) and

342

19% (complex-III) (Figure 8), indicating improvement of thermal stability of curucmin by the

343

formation of a core-shell complex structure. The formation of more compacted ‘core-shell’ SPI-SSPS

344

complex structure could exhibit an extraordinary stabilization for the encapsulated curcumin against

345

heating-induced degradation.

346

In summary, the present work reported the elaboration of core-shell SPI-SSPS complexes as

347

carriers for improved water dispersibility, stability and sustained-release of curcumin. The curcumin-

348

loaded core-shell complexes were formed via two steps: a nanocomplex between curcumin and SPI

349

nanoparticles was formed at pH 7.0 by molecular complexation, and the SSPS was then introduced to

350

coat the surface of the initially curcumin-loaded SPI nanocomplexes. In the core-shell structure, the

351

SPI nanoparticles might be incorporated within the interior of flexible SSPS molecules. The

352

bioaccessibility of encapsulated curcumin in the complexes was unaffected by the coating of SSPS.

353

However, the core-shell complex formation greatly improved the thermal stability and controlled-

354

release behavior of encapsulated curcumin. When the pH of the SPI-curcumin-SSPS complex

355

dispersion was adjusted from 7.0 to 4.0, the core-shell structure distinctly changed accordingly. At pH

356

4.0, the SSPS would be strongly bound to the surface of the aggregated SPI nanoparticles, and as a 18 - Environment ACS Paragon-Plus

Page 19 of 33


357

result, much compact and large core-shell structure was formed. As compared with that at pH 7.0, the

358

pH adjustment (from 7.0 to 4.0) further improved the thermal stability of encapsulated curcumin. The

359

findings would be of great importance for providing a strategy to develop a kind of water dispersible

360

and heat-stable curcumin ingredients suitable for curcumin-enriched functional food formulations.

361

362

AUTHOR INFORMATION

363

Corresponding Author

364

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

365

Notes

366

The authors declare no competing financial interest.

367

ACKNOWLEDGEMENTS

368

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

369



370

371 372

373 374 375

Page 20 of 33

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Livney, Y. D. (2010). Milk proteins as vehicles for bioactives. Curr. Opin. Colloid Interface

Sci. 2010, 15, 73-83.

(2)

McClements, D. J. Nanoscale nutrient delivery systems for food applications: Improving

bioactive dipersibility, stability, and bioavailability. J. Food Sci. 2015, 80, N1602-N1611. (3)

Esmaili, M.; Ghaffari, M.; Moosavi-Movahedi, Z.; Atri, M. S.; Sharifizadeh, A.; Farhadi, M.;

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Yousefi, R.; Chobert, J. M.; Haertlé, T.; Moosavi-Movahedi, A. A. Beta casein-micelle as a nano

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vehicle for solubility enhancement of curcumin; food industry application. LWT 2011, 44, 2166-2172

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(2011).

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(4)

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Chen, F. P.; Li, B. S.; Tang, C. H. Nanocomplexation of soy protein isolate with curcumin:

Influence of ultrasonic treatment. Food Res. Int. 2015, 75, 157-165. (5)

Sneharani, A. H.; Karakkat, J. V.; Singh, S. A.; Rao, A. G. A. Interaction of curcumin with β-

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lactoglobulin -Stability, spectroscopic analysis, and molecular modeling of the complex. J. Agric.

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Food Chem. 2010, 58, 11130-11139.

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(6)

Hoseeini, S. M. H.; Emam-Djomeh, Z.; Sabatino, P.; Van der Meeren, P. Nanocomplexes

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arising from protein-polysaccharide electrostatic interaction as a promising carrier for nutraceutical

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compounds. Food Hydrocolloids 2015, 50, 16-26.

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(7)

Ye, A. Complexation between milk proteins and polysaccharides via electrostatic interaction:

principles and applications -a review. Int. J. Food Sci. Technol. 2008, 43, 406-415.


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Santipanichwong, R.; Suphantharika, M.; Weiss, J.; McClements, D. J. Core-shell biopolymer

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nanoparticles produced by electrostatic deposition of beet pectin onto heat-denatured beta-

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lactoglobulin aggregates. J. Food Sci. 2008, 73, N23-30.

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(9)

Perez, A.; Sponton, O. E.; Andermattern, R. B.; Rubiolo, A.; Santiago, L. G. Biopolymer

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nanoparticles designed for polyunsaturated fatty acid vehiculizaition: Protein-polysaccharide ratio

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study. Food Chem. 2015, 188, 543-550.

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(10) Ding, X.; Yao, P. Soy protein/soy polysaccharide complex nanogels: Folic acid loading, protection, and controlled delivery. Langmuir 2013, 29, 8636-8644. (11)

Tang, C. H. Emulsifying properties of soy proteins: A critical review with emphasis on the

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role of conformational flexibility. Cri. Rev. Food Sci. Nutr. 2015, DOI:

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10.1080/10408398.2015.1067594.

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(12)

Chen, F. P.; Li, B. S.; Tang, C. H. Nanocomplexation between curcumin and soy protein

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isolate: Influence on curcumin stability/ bioaccessibility and in vitro protein digestibility. J. Agric.

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Food Chem. 2015, 63, 3559-3569.

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(13)

Tapal, A.; Tiku, P. K. Complexation of curcumin with soy protein isolate and its implications

on solubility and stability of curcumin. Food Chem. 2012, 130, 960-965. (14)

Ray, M.; Rousseau, D. Stabilization of oil -in -water emulsions using mixtures of denatured

soy whey proteins and soluble soybean polysaccharides. Food Res. Int. 2013, 52, 298-307. (15)

Chen, L.Y.; Subirade, M. Chitosan/β-lactoglobulin core-shell nanoparticles as nutraceutical

carriers. Biomaterials 2005, 26, 6041-6053. 21 - Environment ACS Paragon-Plus


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Teng, Z.; Li, Y.; Wang, Q. Insight into curcumin-loaded β-lactoglobulin nanoparticles:

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Incorporation, particle disintegration, and releasing profiles. J. Agric. Food Chem. 2014, 62, 8837-

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8847.

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Chen, F. P.; Zhang, N.; Tang, C. H. Enhanced water solubility and stability of coenzyme Q10

by complexation with food protein. LWT 2015, submitted for publication. (18)

Tran, T.; Rousseau, D. Stabilization of acidic soy protein-based dispersions and emulsions by

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Liu, Y. Y.; Zeng, X. A.; Deng, Z.; Yu, S.J.; Yamasaki, S. Effect of pulsed electric field on

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the secondary structure and thermal properties of soy protein isolate. Eur. Food Res. Technol. 2011,

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Teng, Z.; Luo, Y. C.; Wang, Q. Nanoparticles synthesized from soy protein: preparation,

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characterization, and application for nutraceutical encapsulation. J. Agric. Food Chem. 2012, 60,

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Luo, Y. C.; Zhang, B. C.; Whent, M.; Yu, L. L.; Wang, Q. Preparation and characterization

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of zein/chitosan complex for encapsulation of α-tocopherol, and its in vitro controlled release study.

424

Colloids Surfaces B 2011, 85, 145-152.

425


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426

Figure captions.

427

Figure 1. A) Visual observations of dispersions of original and freeze-dried/reconstituted SPI-curcumin

428

or SPI-curcumin-SSPS complexes (I-III). Complex-I: SPI-curcumin complex at pH 7.0; Complex-II:

429

SPI-curcumin-SSPS complex at pH 7.0; Complex-III: SPI-curcumin-SSPS complex at pH 4.0. B)

430

Retention ratio (RR) of curcumin in these freeze-dried complexes (I-III). Each data are represented as

431

means and standard deviations (n=3). Different letters (a-b) on the top of columns represent significant

432

difference at p < 0.05 level.

433

Figure 2. Z-average diameter (A) and ξ-potential (B) of particles in dispersions containing SPI alone, or

434

SPI-curcumin complexes (with or without soy soluble polysaccharide), at pH 7.0 and 4.0. The

435

complexes (I-III) are the same as in caption of Figure 1. Each datum is the means and standard

436

deviations (n = 3).

437

Figure 3. AFM observations of SSPS and curcumin-loaded complex samples, A: complex-I; B:

438

complex-II; C: complex-III; D: SSPS. The complexes (I-III) are the same as in caption of Figure 1.

439

Figure 4. FTIR spectra of single ingredients and their complex samples, A: curcumin; B: SSPS; C: SPI;

440

D: complex-I; E: complex-II; F: complex-III. The complexes (I-III) are the same as in caption of Figure

441

1.

442

Figure 5. Schematic illustration for the formation of ‘core-shell’ SPI-curcumin-SSPS complexes at pH

443

7.0 and 4.0.

444

Figure 6. A) Bioaccessibility of curcumin in the different complexes with SPI and/or soy soluble

445

polysaccharide after the whole in vitro simulated gastric and intestinal digestion; B) Degradation kinetic 23 - Environment ACS Paragon-Plus


Page 24 of 33

446

of curcuminin the complexes during the simulated gastric and intestinal digestion. The complexes (I-III)

447

are the same as in caption of Figure 1. Each datum is the means and standard deviations (n = 3). The

448

same letter (a) on the top of columns represent insignificant difference at p < 0.05 level between

449

different complexes.

450

Figure 7. Kinetic release profiles of curcumin from SPI-curcumin complexes and SPI-curcumin-SSPS

451

complexes in 5 mM PBS (pH 7.0; containing 5 mg/mL Tween 20). The complexes (I-III) are the same

452

as in caption of Figure 1. Each datum is the means and standard deviations (n = 3).

453

Figure 8. Degradation kinetic of curcumin encapsulated in the SPI complexes or in core-shell SPI-SSPS

454

complexes in water upon storage for up to 3 h at 80°C. The complexes (I-III) are the same as in caption

455

of Figure 1. Each data is the means and standard deviations (n = 3).

24 Environment ACS Paragon Plus

Page 25 of 33

456


Fig. 1. (Chen et al.)

R etention R atio (% )

B)

100 80

b

b

a

60 40 20 0

Complex I

Complex II

457 458


Complex III



Z -average diam ter (nm )

A)

1000 c

800 600 400

b

200 a

B

20 SPI SPI-cur complex SPI-cur-SSPS complex SSPS

10

ξ-potential (mV)

459

Page 26 of 33

0

-10 -20 -30

b

b

c c a

-50

SPI

Complex-I Complex-II Complex-III

d

0

-40

a

d

pH 7.0

460 461


a

pH 4.0

Page 27 of 33

462



A

C

463


B

D


464

Page 28 of 33


F

1062

1546

1645

1063

E

1546 1642

D 1650

1543

C 1541

1650

B

1057

1630

A

2000

1205

1601

1800

1600

1400

1200 -1

Wavenumbers(cm )

465 466


1000

Page 29 of 33

467



468 469 470




Bioaceessibility of curcumin (%)

A

70

a

a a

60

B Curcumin retention (%)

471

Page 30 of 33

50 40 30 20 10 0

Complex-I

Complex-II

Complex-III

Complex-I Complex-II Complex-III

100

90

80

70

60 0.0

pepsin digestion 0.5

472 473


pancreatin digestion

1.0

1.5

2.0

Digestion Time (h)

2.5

3.0

Page 31 of 33

474



Curcumin release (%)

100 Complex-I Complex-II Complex-III

80 60 40 20 0 0

475

5

10

15

20

Time (h)

476



477


Curcumin Retention (%)

100

-0.067x y=98.02e

80 -0.186x y=98.621e

60 -0.353x y=91.403e

complex-I complex-II complex-III free curcumin

40 20 0 0.0

0.5

1.0

1.5

2.0

2.5

Time (h)

478 479


3.0

Page 32 of 33

Page 33 of 33

480


TOC Graphic

481

482


CoreâShell Soy ProteinâSoy Polysaccharide Complex (Nano)particles

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