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

Accepted Manuscript Nanocomplexes arising from protein-polysaccharide electrostatic interaction as a promising carrier for nutraceutical compounds Seyed Mohammad Hashem Hosseini, Zahra Emam-Djomeh, Paolo Sabatino, Paul Van der Meeren PII:

S0268-005X(15)00156-3

DOI:

10.1016/j.foodhyd.2015.04.006

Reference:

FOOHYD 2949

To appear in:

Food Hydrocolloids

Received Date: 10 October 2014 Revised Date:

3 March 2015

Accepted Date: 5 April 2015

Please cite this article as: Hosseini, S.M.H., Emam-Djomeh, Z., Sabatino, P., Van der Meeren, P., Nanocomplexes arising from protein-polysaccharide electrostatic interaction as a promising carrier for nutraceutical compounds, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.04.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Graphical abstract: Schematic representation of the nutraceuticals entrapment within electrostatically stable nanocomplexes arising from β-lactoglobulin-sodium alginate interaction Title: Nanocomplexes arising from protein-polysaccharide electrostatic interaction as a promising carrier for nutraceutical compounds

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Seyed Mohammad Hashem Hosseinia,*, Zahra Emam-Djomehb, Paolo Sabatinoc, Paul Van der Meerenc a

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Department of Food Science and Technology, College of Agriculture, Shiraz University, 71441-65186 Shiraz, Iran b Department of Food Science, Technology and Engineering, Faculty of Agricultural Engineering and Technology, Agricultural Campus of the University of Tehran, 31587-11167 Karadj, Iran, P. O. Box: 4111 c Particle and Interfacial Technology Group, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Gent, Belgium * Corresponding author. Tel.: +98 71 32286110; fax: +98 71 32286110 E-mail address: [email protected] (S. M. H. Hosseini).

Nutraceutical

Anionic polysaccharide

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β-lactoglobulin– nutraceutical complex

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β-lactoglobulin

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Soluble nanocomplexes

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Nanocomplexes arising from protein-polysaccharide electrostatic interaction

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as a promising carrier for nutraceutical compounds

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Seyed Mohammad Hashem Hosseinia,b,*, Zahra Emam-Djomehb, Paolo Sabatinoc,

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Paul Van der Meerenc

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University, 71441-65186 Shiraz, Iran

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b

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Department of Food Science and Technology, College of Agriculture, Shiraz

Department of Food Science, Technology and Engineering, Faculty of Agricultural

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Engineering and Technology, Agricultural Campus of the University of Tehran,

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31587-11167 Karadj, Iran, P. O. Box: 4111

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c

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Ghent University, Coupure Links 653, B-9000 Gent, Belgium

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Particle and Interfacial Technology Group, Faculty of Bioscience Engineering,

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* Corresponding author. Tel.: +98 71 32286110; fax: +98 71 32286110

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E-mail address: [email protected] (S. M. H. Hosseini).

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ACCEPTED MANUSCRIPT ABSTRACT

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The main purpose of the current work was exploring the potential application of the

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protein–polysaccharide soluble nanocomplexes as delivery systems for

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nutraceuticals in liquid foods. In this study, the intrinsic transporting property of β-

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lactoglobulin (BLG) was utilized to develop nanoscale green delivery systems. The

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binding analysis using fluorescence spectroscopy suggested that the complexation

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between BLG and four nutraceutical models including β-carotene, folic acid,

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curcumin and ergocalciferol occurred under all conditions but varied as a function of

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pH and nutraceutical type. The 1H-NMR study of hydrophilic ligands binding to BLG

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provided complementary information on the interactions between protein and water

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soluble ligands. These findings resulted in designing nanoscopic delivery systems for

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encapsulation of both hydrophobic and hydrophilic bioactives in clear liquid food

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products of acidic pH. The stability experiments demonstrated that the nutraceuticals

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of low solubility in water were successfully entrapped within electrostatically stable

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nanocomplexes arising from BLG-sodium alginate interactions. The electrophoretic

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mobility analysis showed that soluble nanocomplexes had good stability against

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aggregation.

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Keywords: Complex coacervation; Beta-lactoglobulin; Nanoparticle; Delivery system;

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Fluorescence spectroscopy; 1H-NMR

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Chemical compounds studied in this article 41

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Beta-carotene (PubChem CID: 5280489); Curcumin (PubChem CID: 969516); Ergocalciferol (PubChem CID: 5280793); Folic acid (PubChem CID: 6037)

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1. Introduction Recently, some classes of the chemical compounds such as minerals (Fe+2, Mg+2), antioxidants (tocopherols, flavonoids, phenolic compounds), carotenoids (β-

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carotene, lycopene, lutein, zeaxanthin), vitamins (D, B1, B2), fatty acids (omega 3,

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conjugated linoleic acid), phytosterols (stigmasterol, β-sitosterol, campesterol), fibers

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(inulin) and prebiotics have been the focus of research. After isolation from a food

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matrix such compounds are called nutraceuticals: ‘the link between nutrients and

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pharmaceuticals’, a term coined by Stephen DeFelice in 1979 and defined as ‘food

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or parts of food that provide medical or health benefits, including the prevention and

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treatment of disease’ (DeFelice, 1995). The value of these traditional supplements

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led to their application for food enrichment and fortification and ultimately to develop

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new functional foods (McClements, Decker, Park, & Weiss, 2009; Livney, 2010).

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Most of the nutraceuticals show instability against chemical or physical

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degradation and tend to degrade during storage when incorporated into foods.

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Encapsulation systems, also known as ‘delivery systems’, are typically used to

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incorporate them into the foods (Shimoni, 2009). So, the development of structured

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delivery systems for the encapsulation of bioactives is an important area of research

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for the food industry in order to improve the quality of foods and beverages

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(Matalanis, Jones, & McClements, 2011). Due to the hydrophobic nature of the most

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functional compounds, their incorporation into non-fat aqueous foods and beverages

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(especially clear ones) is challenging (Sagalowicz, & Leser, 2010; Matalanis et al.,

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2011). In this case, the encapsulated bioactive ingredients must be stabilized in a

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liquid environment, which is completely different from stabilizing the bioactives in a

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solid environment (Sagalowicz et al., 2010).

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Different types of delivery systems have been used to introduce bioactives into foods including oil in water ordinary and multilayered emulsions (Saberi, Fang, &

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McClements, 2014; Yoksan, Jirawutthiwongchai, & Arpo, 2010), multiple emulsions

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(Jiménez-Colmenero, 2013; Santos, Bozza, Thomazini, & Favaro-Trindade, 2015),

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microemulsions and nanoemulsions (Qian, Decker, Xiao, & McClements, 2012;

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Rao, & McClements, 2012, Davidov-Pardo, & McClements, 2015), solid lipid

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nanoparticles (SLNs) (Pandita, Kumar, Poonia, & Lather, 2014), cyclodextrins (Choi,

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Ruktanonchai, Min, Chun, & Soottitantawat, 2010), amylose (Zabar, Lesmes, Katz,

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Shimoni, & Bianco-Peled, 2010), liposomes (Takahashi, Inafuku, Miyagi, Oku, Wada,

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Imura, & Kitamoto, 2007), and micelles (Zimet, Rosenberg, & Livney, 2011).

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It seems that the main attention in the research field of aqueous delivery systems is to try to physically or chemically ‘complex’ or ‘bind’ the active ingredients

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(specially hydrophobic ones) to a molecular or supramolecular structure in order to

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protect it in this way from chemical or physical deterioration (Sagalowicz et al.,

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2010). Many types of biopolymers are capable of binding lipophilic and hydrophilic

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molecules and forming molecular complexes. The bioactive molecules may be

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bound to individual biopolymer molecules (such as globular proteins, amylose and

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starch derivatives) at one or more active binding sites by either specific or non-

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specific interactions with different molecular origins (hydrophobic or electrostatic)

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(Zabar et al., 2010; Liang, & Subirade, 2010), or they may be incorporated within

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clusters formed by a single type (such as casein micelles) (Shapira, Assaraf, &

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Livney, 2010; Zimet et al., 2011) or mixed types (such as molecular clusters arising

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from electrostatic attraction between proteins and ionic polysaccharides) of

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biopolymers (Zimet, & Livney, 2009; Ron, Zimet, Bargarum, & Livney, 2010).

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The formation of non-covalent electrostatic complexes between proteins and polysaccharides can potentially lead to different functional properties, compared to

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the two biopolymers taken individually (Schmitt, Sanchez, Desobry-Banon, & Hardy,

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1998; McClements, 2006). This is generally due to a synergistic combination of the

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functional features of both the protein and the polysaccharide (Schmitt, Aberkane, &

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Sanchez, 2009). Mixing proteins and polysaccharides in an aqueous medium leads

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to two different types of phase separation phenomena including thermodynamic

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incompatibility (repulsive) and thermodynamic compatibility (attractive) depending on

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the charged properties of both biopolymers, and hence on the factors influencing

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them, such as the pH and the ionic strength. Other important parameters influencing

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the complex coacervation between biopolymers include total biopolymer

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concentration, protein to polysaccharide mixing ratio, molecular weight, charge

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density, molecular conformation, charge distribution, processing factors such as

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temperature, pressure, shearing and acidification method (Schmitt et al., 2009;

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Turgeon, & Laneuville, 2009). During attractive interactions, protein and

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polysaccharide in the biopolymer rich phase are held together mainly by electrostatic

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forces and can appear as coacervates, complexes (soluble or insoluble) and gels

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(Turgeon et al., 2009). Among the formed structures, coacervates can be used as

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delivery systems for encapsulation purposes. However, interaction between

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coacervates leads to coalescence and formation of transient multivesicular

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structures that can coalesce further and eventually completely separate into a dense

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coacervated phase (Sanchez, Mekhloufi, Schmitt, Renard, Robert, Lehr, Lamprecht,

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& Hardy, 2002), which limits its application as a delivery system in fortification of

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clear liquid foods where a uniform structure is a concern. During recent years, some

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researchers have examined the potential applications of soluble and/or insoluble

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complexes arising from protein-polysaccharide interactions in order to encapsulate

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hydrophilic and lipophilic molecules (Bedie, Turgeon, & Makhlouf, 2008; Zimet et al.,

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2009; Ron et al., 2010). BLG as a small globular protein contains 162 amino acid residues with one free

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thiol group and two disulfide bonds and has a molecular weight of 18.4 kDa (Fox,

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2009). It is a member of the lipocalin family of proteins because of its ability to bind

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small hydrophobic molecules. The quaternary structure (association properties) of

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the protein varies among monomers, dimers or oligomers depending on the pH,

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temperature, concentration and ionic strength as a result of a delicate balance

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among hydrophobic, electrostatic and hydrogen-bond interactions (Sakurai, & Goto,

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2002; Gottschalk, Nilsson, Roos, & Halle, 2003). At pH 5–8, BLG exists as a dimer,

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at pH 3–5 the dimers associate to form octamers, and at extreme pH values (<2 or

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>8) most protein exists as monomers. At pH>9, the molecule is irreversibly

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denatured (Harnsilawat, Pongsawatmanit, & McClements, 2006).

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BLG folds up into an 8-stranded (A-H) antiparallel β-barrel (Fig. 1). A ninth βstrand (I) flanks the first strand. It is this strand that forms a significant part of the

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dimer interface in the bovine protein. The conical central cavity (the calyx or β-barrel)

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which provides the main ligand-binding site is made of β-strands A-D forming one

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sheet, and β-strands E-H forming a second sheet. It seems that the β-barrel can

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accommodate linear molecules like palmitic acid and also retinol with the β-ionone

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ring system inside (Kontopidis, Holt, & Sawyer, 2004; Edwards, Creamer, &

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Jameson, 2009). There is a 3-turn α-helix on the outer surface of the β-barrel

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between strands G and H. The loops that connect the β-strands at the closed end of

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the β-barrel (BC, DE, and FG) are generally quite short, whereas those at the open

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end are significantly longer and more flexible (Kontopidis et al., 2004; Edwards et al.,

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ACCEPTED MANUSCRIPT 2009). In particular, the EF loop (residues 85-90) acts as a gate over the binding site.

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The latch for this gate is the side chain of the Glu89 (Qin, Bewley, Creamer, Baker,

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Baker, & Jameson, 1998). Indeed, the pH-dependent conformational change of this

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loop is regulated by the protonation of Glu89 (Ragona, Fogolari, Catalano, Ugolini,

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Zetta, & Molinari, 2003). At low pH, the gate is in closed position, and binding is

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inhibited or impossible, whereas at high pH it is open, allowing ligands to penetrate

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into the calyx (Kontopidis et al., 2004). Computational studies have shown that three

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potential binding sites are possible for ligand binding to BLG: the calyx, the surface

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pocket with a hydrophobic lining in a groove between the α-helix and the β-barrel

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and the outer surface near Trp19-Arg124 (Liang, Tajmir-Riahi, & Subirade, 2008).

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Among the large spectrum of protein-polysaccharide pairs which have been

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studied under the effects of different parameters, biopolymer complexes formation

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between sodium alginate (Na-ALG) and milk proteins was extensively investigated.

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Harnsilawat et al. (2006) investigated the characteristics of Na-ALG, β-lactoglobulin

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(BLG) and their mixtures in aqueous solutions under the effect of pH (3-7) using

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isothermal titration calorimetry (ITC), dynamic light scattering (DLS), turbidity, zeta-

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potential and soluble protein measurements. At pH 5, BLG and Na-ALG formed fairly

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soluble complexes due to the electrostatic attraction between negatively charged

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carboxyl groups (-COO-) of Na-ALG and positively charged surface patches (resulted

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from basic amino acids) of BLG. Zhao, Li, Carvajal, and Harris (2009) used both the

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native bovine serum albumin (BSA) and the heat-denatured BSA to study the effect

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of protein conformational changes during protein-alginate complex formation. In this

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work a comparison was performed between the mixtures of Na-ALG with either

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native BSA or the heat-denatured BSA using zeta-potential analyzer, DLS and

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turbidimetric analysis in combination with protein conformational studying tools,

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ACCEPTED MANUSCRIPT Fourier transform infrared spectroscopy (FT-IR) and size exclusion chromatography.

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Peinado, Lesmes, Andrés, and McClements (2010) fabricated sub-micrometer

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biopolymer particles by electrostatic complexation of heat-denatured lactoferrin

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particles with Na-ALG. Biopolymer particles were formed by mixing the protein

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particles with Na-ALG at pH 8 and then lowering the pH to promote electrostatic

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deposition of polysaccharides onto the protein particle surfaces. The effects of pH (2-

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11) and ionic strength (0-200 mM NaCl) on the properties and the stability of the

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complexes were studied using turbidity, DLS and electrophoresis measurements.

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Hosseini et al. (2013a, b), studied the interaction of BLG with either Na-ALG or κ-

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carrageenan (before and after an ultrasound treatment) using ITC, streaming current

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detector (SCD), turbidity, dynamic light scattering and electrophoretic mobility

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measurements. The results showed that the sonication decreased the interaction

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strength between BLG and treated anionic polysaccharide. Fioramonti, Perez,

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Aríngoli, Rubiolo, and Santiago (2014) designed and characterized soluble

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biopolymer complexes formed by self-assembly of Na-ALG and whey protein isolate

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(WPI). Resultant self-assembled biopolymer complexes were then characterized by

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the same set of spectroscopic techniques. Higher WPI:Na-ALG ratios produced

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soluble self-assembled biopolymer particles, where hydrophobic patches of protein

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aggregates would be more occluded ensuring the protection of potential lipophilic

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bioactive agents attached inside. It has also been reported that BLG can form water-

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soluble complexes with docosahexaenoic acid (DHA) and ergocalciferol and protect

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these lipophilic compounds from degradation by heat and oxidation (Zimet et al.,

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2009; Ron et al., 2010). Liang et al. (2008) and Forrest, Yada, and Rousseau (2005)

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concluded that interaction with BLG may strongly influence the stability of phenolic

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compounds and vitamin D3, respectively and hence their bioavailability in processed

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foods. It has been shown that BLG has some antioxidant activity, apparently due to

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its free thiol group (Liu, Chen, & Mao, 2007). Therefore, BLG can be used as a

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versatile carrier of bioactive molecules in controlled delivery applications. The hypothesis of the current study is that the binding properties of BLG (a

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member of the lipocalin protein family) towards some bioactive compounds can be

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used to produce green delivery systems. To form a core-shell structure, the

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produced nanoscale delivery system can be overprotected by deposition of an

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anionic polysaccharide as a secondary layer (or shell) around the protein core in the

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pH range between the pKa of the polysaccharide and the isoelectric point of BLG.

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The first objective of the current study was to determine the binding properties of four

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nutraceutical models including curcumin (CUR), folic acid (FA), β-carotene (βC) and

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vitamin D2 (VD) to β-lactoglobulin (BLG) under the effect of pH using

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spectrofluorometry technique. The second objective was to study the binding

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between BLG and hydrophilic ligands using 1H-NMR. The third objective was to

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assess the stabilization efficiencies of nutraceutical compounds in an acidic (pH

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4.25) clear beverage model using nanoparticles (soluble complexes) produced via

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electrostatic interactions between BLG and Na-ALG.

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2. Materials and methods

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2.1. Materials

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Guluronate-rich sodium alginate (ALG, composition: 66.26% (w/w) ALG,

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14.19% (w/w) moisture and 9.55% (w/w) ash) from Laminaria hyperborean was

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purchased from BDH Co. (Poole, UK). The molecular weight of sodium alginate was

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200 kDa and the uronate compositions were 37.5% mannuronate and 62.5%

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guluronate. β-lactoglobulin isolate (BLG, 18.4 kDa, minimum purity 90% (w/w)) from 9

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USA). Sodium azide (as a preservative), ergocalciferol (vitamin D2) (VD), β-carotene

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(βC), curcumin (CUR), folic acid (FA) and N-acetyl-L-tryptophanamide (NATA) were

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obtained from Sigma Chemical Co. (St. Louis, MO, USA). Deuterium oxide (D2O,

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99.8% D) was obtained from Armar Chemicals (Döttingen, Switzerland). Glacial

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acetic acid, analytical grade hydrochloric acid, sodium hydroxide, monosodium

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dihydrogen phosphate, 8-anilinonaphthalene-1-sulfonic acid ammonium salt (ANS)

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and absolute ethanol were purchased from Acros Organics (Geel, Belgium).

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Deionized water (18.2 MΩ cm resistivity) from a MilliQ water system

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(Millipore Corporation, MA, USA) was used for the preparation of all solutions. In this

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study, all materials were used directly from the sample containers without additional

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purification taking into account their purity.

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2.2. Preparation of solutions

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ALG (0.8% (w/w)) and BLG (0.4% (w/w)) stock solutions were prepared by

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dispersing in deionized water containing 0.03% (w/w) sodium azide. The solutions

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were then stirred at 250 rpm at ambient temperature for 12 h to ensure complete

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hydration of the biopolymers in order to use on the following day.

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2.3. Bioactives binding to BLG

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Fluorescence spectroscopy was used to study the binding of bioactives to BLG

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by measuring the binding-induced quenching of the intrinsic fluorescence of BLG

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tryptophanyl residue (Trp19), which is particularly sensitive to changes of its

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microenvironment (Wang, Allen, & Swaisgood, 1998). For this experiment, BLG

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stock solutions were made fresh daily by dissolving in either 10 mM phosphate buffer

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at pH 7 or 10 mM acetate buffer at pH 4.25 (to determine the effect of pH on the

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binding properties of ligands). After filtration through a 0.22-µm syringe filter to obtain 10

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determined by UV spectroscopy using an Ultrospec 1000 UV-visible

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spectrophotometer (Pharmacia-Biotech, Biochrom Ltd., Cambridge, England). The

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samples were measured in a 1 cm quartz cell at 278 nm against buffer solutions as

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the reference. BLG concentrations were calculated using an extinction coefficient (ε)

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of 17600 M-1 cm-1 (Liang et al., 2008) and amounted to 2.90 and 3.52 µM at pH 7

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and 4.25, respectively. The low concentration of the BLG solutions avoided inner

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filter effects which could occur during the experiment. To facilitate the binding of

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nutraceutical components to BLG, they were prepared daily by dissolving in absolute

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ethanol, except with FA which was dissolved in phosphate buffer (pH 7). To prevent

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degradation, nutraceutical stock solutions of the appropriate concentrations (βC:

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0.130 and 0.104 mM, FA: 0.213 and 0.199 mM, CUR: 0.201 and 0.255 mM, VD:

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0.393 and 0.509 mM, at pH 7 and 4.25, respectively) were purged with nitrogen gas,

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and stored in the dark at 4 ºC. Samples were prepared at room temperature in 2.5 ml

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plastic tubes covered with aluminum foil, by mixing BLG (2 ml) and different amounts

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of nutraceutical (0, 2, 5, 9, 14, 20, 27, 35, 44, 54 µl) stock solutions. The protein-

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nutraceutical solutions were vortexed for 30 s and allowed to equilibrate for 10 min

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prior to fluorescence measurement. As the ethanol dissipates in the water, most of

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the nutraceutical components bind to the protein’s binding site(s). The highest

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resulting ethanol concentration was about 2.7% (v/v), which had no appreciable

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effect on protein structure. Fluorescence spectra were recorded at room temperature

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on a Cary Eclipse Fluorescence spectrophotometer (Varian Inc., Walnut Creek, CA,

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USA) using an excitation wavelength of 287 nm and an emission wavelength of 332

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nm (Cogan, Kopelman, Mokady, & Shinitzky, 1976). The band slit (spectral

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resolution) was 5 nm for both excitation and emission. To eliminate the effects of

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ACCEPTED MANUSCRIPT protein dilution by the added ligand solution and the possible tryptophan

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fluorescence changes induced by ethanol, for each sample, a blank BLG solution

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containing an identical concentration of ethanol (and/or buffer for FA) was prepared

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and treated in the same manner as the sample. A second blank containing NATA

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was also prepared in a manner similar to all samples. NATA is unable to interact with

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nutraceutical models; however it displays a fluorescence spectrum typical of

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tryptophan. The decrease in fluorescence intensity of blanks containing NATA is not

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due to the interaction but it results from the inner filter effect as a consequence of

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ligand absorbance at 287 nm. NATA was also used to exclude the possibility of

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unspecific interactions of the nutraceutical model with the protein’s tryptophan

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indoles, where at increasing nutraceutical concentrations the nutraceutical may

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absorb light, which would otherwise excite the indole groups, and thus fluorescence

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would decrease for this reason (Dufour, & Haertlé, 1990) not for binding. The

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concentration of NATA (0.228 mg/150 ml buffer and 0.265 mg/150 ml buffer, at pH 7

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and 4.25, respectively) had been selected in the way that it had the same emission

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at 332 nm as the studied BLG solution. The fluorescence intensity changes of the

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blanks were subtracted from fluorescence intensity measurements of the

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ligand/protein complexes for every considered sample. In these experiments, before

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correction for the blanks, the fluorescence intensity at 332 nm of the original BLG

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solution was normalized to 1. All experiments were run in duplicate samples put in

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quartz cuvettes of 1 cm optical path length. After correcting for the blanks, the

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differences in fluorescence intensity at 332 nm between complex and free protein

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were used to measure the apparent dissociation constant (K'd) and the apparent

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mole ratio of ligand to protein at saturation (n). The direct linear plotting method of

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Eisenthal and Cornish-Bowden (1974), where the corrected fluorescence is plotted

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ACCEPTED MANUSCRIPT directly against ligand concentrations, was used to obtain K'd directly from the

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median of intersecting regression lines representing individual observations on the

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abscissa. The n values were obtained directly from the fluorescence titration curve

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plotted against nutraceutical/protein molar ratio correlating to the saturation point.

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2.4. NMR study of hydrophilic ligands binding to BLG

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Interactions between BLG and FA and/or ANS were investigated by proton

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nuclear magnetic resonance (1H-NMR) spectroscopy. Stock solution of ligand of

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appropriate concentration was prepared by dissolving in deuterium oxide (D2O). To

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determine the effect of protein on ligand binding, BLG was added to ligand stock

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solution at different concentrations. All NMR experiments were performed on a

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BrukerAvance II spectrometer operating at a 1H- frequency of 700.13 MHz. A 5 mm

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TXI gradient probe with a maximum gradient strength of 57.5 G·cm-1 was used

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throughout. Temperature was controlled to within ±0.1 °C with a Eurotherm 2000VT

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controller. Diffusion coefficients were measured by PFG-NMR with a convection

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compensated double-stimulated-echo experiment (Jerschow, & Muller, 1997) using

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monopolar smoothened square shaped gradient pulses and a modified phase cycle

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(Connell, Bowyer, Bone, Davis, Swanson, Nilsson, & Morris, 2009).

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The echo-decay of the resonance intensity obtained with the double stimulated

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echo sequence obeys equation (1), from which it is clear that the diffusion coefficient

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(D) is derived from the echo-decay as a function of the parameter k

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[

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I = I 0 exp − D ( γ G δ s ) 2 ∆ '

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I = I 0 exp[− D ⋅ k ]

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(1)

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ACCEPTED MANUSCRIPT where I is the echo intensity with gradient; I0 is the echo intensity at zero gradient; γ

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is gyromagnetic ratio; G the maximum gradient amplitude; δ the duration of the

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gradient pulse and ∆' is the diffusion delay corrected for the finite gradient pulse

315

duration ∆' = ∆ − 0.6021 ⋅ δ . The gradient shape factor s was set to 0.9, to account for

316

the smoothed rectangular gradient shape used in the experiments.

317

2.5. Nanoencapsulation of nutraceutical models

After finding nutraceutical binding characteristics to BLG, a series of solutions

SC

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312

containing constant final BLG concentration (0.1% w/w), and constant final

320

nutraceutical concentration at a molar ratio of 1:1 were prepared by adding

321

nutraceutical compound dissolved in absolute ethanol (except for FA which was

322

dissolved in deionized water) to the protein solutions. The protein solution (at neutral

323

pH) was then stirred for half an hour. Polysaccharide stock solution was added to the

324

BLG-nutraceutical solution at the desired quantity (0.75% w/w to obtain a transparent

325

system containing nanoparticles) (Hosseini et al., 2013) and deionized water was

326

added to obtain a final constant volume as well as constant protein and

327

polysaccharide concentrations. Then the pH was adjusted while stirring to 4.25 using

328

0.4, 0.1 and/or 0.01 M HCl solutions (the post-blending acidification method), and the

329

samples were stirred further for half an hour for equilibration. For each sample, a

330

blank consisting of deionized water which contained an identical concentration of

331

nutraceutical compound dissolved in ethanol (and/or deionized water for folic acid)

332

was prepared and treated in the same manner as the sample. A second blank

333

containing BLG and nutraceutical compound without sodium alginate was also

334

prepared in a manner similar to all samples.

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ACCEPTED MANUSCRIPT 335 336

2.6. Particle size and electrophoretic mobility analyses Measurements of particle size distribution were carried out using a dynamic light scattering (DLS) instrument (90Plus, Brookhaven Instruments Corp., Vienna,

338

Austria). Analyses were carried out at a scattering angle of 90° at 25 °C. The effective

339

diameter (also called Z-average mean diameter) was obtained by cumulant analysis.

340

The electrophoretic mobility was determined by laser Doppler anemometry with

341

palladium electrodes using a ZetaPals instrument (Brookhaven Instruments Corp.,

342

Vienna, Austria) at fixed light scattering angle of 90° at 25 °C. During both dynamic

343

light scattering and electrophoretic light scattering measurements, the viscosity of

344

the continuous phase was assumed to correspond to pure water.

345

2.7. Statistical analysis

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Measurements were performed two or three times using freshly prepared samples and analyzed by ANOVA using the MSTATC program (version 2.10, East

348

Lansing, MI, USA). Results were reported as means and standard deviations.

349

Comparison of means was carried out using Duncan’s multiple range tests at a

350

confidence level of 0.05.

351

3. Results and discussion

352

3.1. Binding properties of the nutraceutical compounds to BLG

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353

The raw data was analyzed to measure the apparent dissociation constant (K'd)

354

and the apparent mole ratio of ligand to protein at saturation (n) which are presented

355

in Tables 1 and 2, respectively. A low K'd value indicates high binding affinity. The

356

analysis suggested that binding occurred under all conditions but varied as a

357

function of pH and nutraceutical model. From these observations (n>1 as well as

358

binding dependence on pH and nutraceutical type), it can be concluded that the four

15

ACCEPTED MANUSCRIPT nutraceutical models investigated are bound on proteins on different binding sites

360

and/or by different binding mechanisms. An opposite trend was observed for the two

361

estimated parameters at both pH values, where the weaker observed affinity was

362

associated with the higher n value.

363

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Among the four nutraceutical models investigated, β-carotene had the highest binding affinity and the lowest binding stoichiometry at both pH values. The more

365

hydrophobic character of β-carotene may explain the higher affinity of this molecule

366

for the hydrophobic binding sites of BLG, whereas its large size may be responsible

367

for the lower binding stoichiometry. Frapin, Dufour and Haertlé (1993) reported that

368

the affinity of BLG for saturated fatty acids increases gradually from lauric acid to

369

palmitic acid (as a longer aliphatic chain) due to an increase in the hydrophobic

370

nature of the ligand. The β-carotene-BLG molar binding ratio at pH 7 was 0.48± 0.04

371

which is in good agreement with the results (0.49 ± 0.03) reported by Dufour and

372

Haertlé (1991). The solvent exposure of a part of the retinol isoprenoid chain, when

373

bound to BLG could explain the 1:2 stoichiometry of β-carotene-BLG complexes.

374

This tetraterpen has two β-ionone rings, joined by an isoprenoid chain, which may

375

interact distally with two BLG molecules (Dufour et al., 1991). Another possibility is

376

that this ligand can bind at the dimer interface. According to Wang, Allen and

377

Swaisgood (1998, 1999) the site found at the dimer interface is the highest affinity

378

site as observed in the current study.

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379

Folic acid is less hydrophobic than curcumin and ergocalciferol but is bound to

380

a higher extent. This suggests possible interactions that are not only hydrophobic in

381

nature, and/or binding to the other binding sites than the calyx. Liang et al. (2010)

382

proposed that the binding site of resveratrol may be on the outer surface near Trp19-

383

Arg124, while folic acid binds to the surface hydrophobic pocket in a groove between 16

ACCEPTED MANUSCRIPT the α-helix and the β-barrel. The n value of folic acid-BLG complex at pH 7 was

385

1.25± 0.05. This value is in very good agreement with those (1.30 ± 0.03 and 1.17 ±

386

0.04) obtained by Liang et al. (2010) during excitation at 280 and 295 nm,

387

respectively.

388

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The curcumin-BLG ratio at pH 7 was 0.95± 0.06 which is in good agreement with the results (1 and 0.85) reported by Sneharani, Karakkat, Singh and Rao (2010)

390

and by Mohammadi, Bordbar, Divsalar, Mohammadi and Saboury (2009) at pH 7

391

and 6.4, respectively. Based on the obtained results (no significant change in binding

392

stoichiometry) at the lower pH level (4.25) and on those reported in literature

393

(Riihimäki, Vainio, Heikura, Valkonen, Virtanen, & Vuorela, 2008), it seems that

394

curcumin (as a phenolic compound) binds to a site other than the calyx.

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The ergocalciferol-BLG molar ratio at pH 7 was 1.31± 0.12. Wang, Allen, and Swaisgood (1997) reported the value of 2.00 ± 0.16 at pH 7. It seems that the

397

increased stoichiometry (binding to the external hydrophobic surface patch) is

398

accompanied by relatively loose binding as evidenced by the decreased observed

399

affinity. According to Forrest et al., (2005), the weakest affinity site is found at the

400

hydrophobic surface patch. A higher than 1 stoichiometry indicated that the other

401

binding sites (crevice next to the alpha helix and the dimer interface) of higher or

402

equal affinity are involved in the binding to BLG. These binding sites may be

403

saturated prior to, or simultaneously with the main binding site. Zimet et al. (2009)

404

reported that 2.67 ± 1.26 moles of DHA were bound per mole of BLG.

405

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The binding capacities of BLG mediated by pH were evident in the present

406

study. Upon lowering the pH, the binding constants changed depending on the

407

nutraceutical nature. Since the EF loop, that acts as a gate in the central cavity, is in

17

ACCEPTED MANUSCRIPT a closed conformation at acidic pH (Kontopidis et al., 2004), the binding

409

stoichiometry of β-carotene was decreased with decreasing pH. Previous NMR

410

studies have shown that palmitic acid starts to be released at a pH lower than 6, and

411

80% of the palmitic acid has already been released at pH 2 (Ragona, Fogolari, Zetta,

412

Perez, Puyol, De Kruif, Lohr, Ruterjans, & Molinari, 2000). The binding affinity of β-

413

carotene at pH 4.25 was increased because hydrophobic interactions are enhanced

414

at pH values around the isoelectric point of BLG (~4.7). Since the EF loop is blocking

415

ligand access, another possibility is that a single β-carotene molecule was tightly

416

bound between the monomers (at the interfaces) found within the octamers as

417

evidenced by the strongest observed affinity.

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The decrease in the binding stoichiometry of folic acid can be attributed to its lower solubility at pH 4.25 than at pH 7 (~1 and ~5 mg/L, respectively) (Younis,

420

Stamatakis, Callery, & Meyer-Stouta, 2009).

421

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The binding stoichiometry of curcumin was not significantly (p<0.05) different at pH 4.25 as compared to pH 7.00. Riihimäki et al. (2008) reported that, contrary to

423

retinol, the release of phenolic compounds was not observed at acidic pH, which

424

suggests that phenolic compounds and their derivatives do not bind to the central

425

calyx. Liang et al. (2008) studied the binding of the natural polyphenolic compound

426

resveratrol to bovine BLG. The observed blue shift of the fluorescence emission

427

maxima and the increase in the emission intensity implied that the environment of

428

the polyphenol bound to BLG is not as hydrophobic as the cavity of BLG, suggesting

429

binding on the surface of the protein. Moreover, the qualitative docking results

430

performed by Riihimäki et al. (2008), showed that phenolic compounds and their

431

derivatives would not bind to the central calyx, supporting the results of this study for

432

the curcumin-BLG complex.

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18

ACCEPTED MANUSCRIPT 433

The binding stoichiometry of ergocalciferol was significantly decreased by decreasing pH. At pH 4.25, approximately 0.85 molecule of vitamin D2 were bound

435

per BLG monomer with a relatively weak affinity. Since the EF loop is in a closed

436

position at this pH, the results indicate that loose binding occurred at the external

437

hydrophobic surface patch. Since protein association is increased after lowering the

438

pH to 4.25 (formation of the octamers), it seems that the decreased available surface

439

area inhibited greater ligand access at external hydrophobic surface patches and

440

interfaces. The binding to the external hydrophobic surface patches and the

441

interfaces is accompanied by relatively loose and tight affinity, respectively. The

442

absence of a significant change in binding affinity of ergocalciferol to BLG with

443

decreasing pH is likely due to a combination of both a decrease in binding affinity

444

arising from the contribution of the hydrophobic surface patches in binding and an

445

increase in binding affinity caused by the binding to interfaces resulting in an overall

446

unchanged binding affinity.

447

3.2. NMR results

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NMR spectroscopy is a useful technique to study molecular interactions. NMR

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provides different measurable parameters that depend on the amount and the

450

strength of the interaction. The former may be quantified by analysis of the diffusion

451

behavior of ligand and protein both separately and upon mixing. The latter gives rise

452

to important changes in the chemical environment and in the rotational as well as

453

translational mobility of the ligand, which is reflected in a change in the chemical

454

shift, the line width and relaxation rate, as well as the molecular diffusion rate

455

constants of the ligand. Indeed, binding of ligands to hydrophobic sites may cause a

456

change in chemical shift to lower values. In addition, bound ligand molecules will

457

have a reduced mobility which provokes a peak broadening. Considering the

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19

ACCEPTED MANUSCRIPT relatively limited binding of hydrophobic ligands (ranging from 1/2 to 2/1 molar ratio),

459

the large difference in molar mass and the absence of highly intense peaks in the

460

NMR spectrum of the ligands (as compared to the intense CH2-signal in typical

461

surfactant-like chemicals), NMR detection of the ligand in the presence of a large

462

amount of protein is hampered. To overcome this problem, initial measurements

463

were performed using Anilino Naphthalene Sulphonate (ANS, data not shown). This

464

fluorescent dye is widely used as a surface hydrophobicity probe, based on the fact

465

that its fluorescence intensity is largely increased when present in a rather

466

hydrophobic environment. ANS is known to bind to proteins. In addition, its aromatic

467

nature is responsible for the fact that most ligands NMR peaks are resolved from the

468

protein peaks. 1D proton NMR is a quick method to achieve qualitative information

469

about the degree of interaction. In this type of experiment the variation in chemical

470

shift of the ligand resonances upon sorption as compared to the dissolved molecules

471

can be used to roughly estimate the partitioning coefficient as well as the type of

472

interactions. A more accurate quantification of the interaction of the ligands with BLG

473

is obtained by using Diffusion Ordered SpectroscopY (DOSY-NMR) measurements.

474

Hereby, the observed diffusion coefficient of the ligand in the presence of proteins

475

(Dobs) is the weighted average of non-bound molecules (with diffusion coefficient

476

Dfree) and protein bound molecules (with the same diffusion coefficient as the protein

477

Dpro). If the bound fraction is represented by P, the weighted average may be

478

calculated according to the following equation:

479

480

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Dobs = PD sorb + (1 − P ) D free

(2)

from which

20

ACCEPTED MANUSCRIPT 481

P=

D free − Dobs

(3)

D free − Dsorb

Hence, the bound protein fraction follows from experimental values of the diffusion

483

coefficient of the ligands in the absence (Dfree) and presence (Dobs) of protein, as well

484

as from the diffusion coefficient of the protein (Dpro).

RI PT

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Fig. 2 shows the chemical structure of folic acid. In this case a complete

486

assignment is possible as summarized in Fig. 3. From Fig. 4, it is clear that the

487

addition of protein only slightly influences the NMR contribution of the folic acid. The

488

resonances show little chemical shift variation and there are no signs of line

489

broadening. However, the diffusion measurements show that the diffusion behavior

490

of folic acid changes (slower diffusion) upon addition of protein, as visible in Fig. 5.

491

The sorbed amount of folic acid can be estimated according to the previously

492

mentioned equation, where Dfree is (3.19 ± 0.04)10-10 m2s-1, Dobs is (2.96 ± 0.05)10-10

493

m2s-1 and Dpro is (0.75 ± 0.01)10-10 m2s-1. The solubilized fraction is 9.4%, which

494

corresponds on average to 1.1 folic acid molecules per BLG monomer. This result is

495

in good agreement with the results of fluorimetry. The same series of experiments

496

were repeated for the same system but at different pH conditions (pH ≈ 4.25).

497

Unfortunately, in these conditions, the NMR spectrum resulted in a complete loss of

498

the folic acid resonances. As a further consequence, the diffusion experiments were

499

not possible. The very poor aqueous solubility at the acidic pH is the main reason of

500

the loss of folic acid resonance at lower pH conditions.

501

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485

A more accurate analysis of the chemical shift variations is also required to

502

reveal which ligand functional group is more involved in contact with protein and as

503

consequence to establish the nature of the interactions. However, this technique can

504

only be used when the ligand has a sufficiently large solubility (i.e. at least some 21

ACCEPTED MANUSCRIPT 505

mM) in (heavy) water. Hence, NMR could not be used to study the binding of β-

506

carotene, curcumin or ergocalciferol.

507

509

3.3. Nanoencapsulation and colloidal stability of nutraceutical models

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The effects of BLG and Na-ALG on the colloidal stability of nanoencapsulated curcumin (as hydrophobic model) and folic acid (as hydrophilic model with low

511

solubility at acidic pH) at pH 4.25 are shown in Figs. 6 and 7, respectively. Except for

512

the blank sample of curcumin (which was first dissolved in ethanol before being

513

added to the aqueous phase) in deionized water, there was not any significant

514

difference between the samples just after production (Figs. 6.A and 7.A). After 2

515

hours of production, some differences were observed. The curcumin and folic acid,

516

incorporated into deionized water, started to precipitate and separated almost

517

completely after 24 hours of production. The samples containing BLG and BLG+ALG

518

did not show any precipitation after 24 hours (Figs. 6.B and 7.B). However, the

519

turbidity of both samples was slightly different. 48 hours after production, the

520

differences between these two samples were more obvious (Figs. 6.C and 7.C): the

521

blank sample containing the nutraceutical models and BLG showed some

522

precipitation, while the main sample, which contained the nutraceutical models, BLG

523

and ALG, remained completely transparent without any sign of precipitation. This

524

clearly demonstrated the efficacy of soluble nanocomplexes arising from protein-

525

polysaccharide interactions on nanoencapsulation and colloidal stability of

526

nutraceuticals of low solubility in water even 30 days after production without storing

527

in the dark (Fig. 6.D and 7.D). The low solubility of curcumin in aqueous solution

528

significantly limits its application. Recently, it has been shown that polyelectrolyte-

529

coated curcumin nanoparticles (obtained through a layer by layer shell assembly)

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510

22

ACCEPTED MANUSCRIPT are hydrosoluble (Zheng, Zhang, Carbo, Clark, Nathan, & Lvov, 2010). In our study,

531

curcumin binding to BLG increased its colloidal stability in water. The stability of the

532

main sample containing folic acid was lower than those containing curcumin. The

533

nutraceutical models were incorporated in a 1:1 molar ratio into the protein solution.

534

The apparent mole ratio of folic acid to BLG at pH 4.25 was determined to be 0.39 ±

535

0.04 (Table 2). Hence, it is possible that after decreasing the pH to 4.25, the BLG

536

became overloaded and some precipitation occurred. As the apparent mole ratio of

537

curcumin to BLG at pH 4.25 was found to be 0.82 ± 0.08 (Table 2), BLG overloading

538

was relatively prevented in this case. The particle size and electrophoretic mobility

539

(EM) analyses of the samples containing BLG (0.1% w/w) and ALG (0.075 % w/w)

540

with and without nutraceutical model compounds showed that curcumin addition into

541

the soluble complexes of BLG-ALG, slightly increased the particle size from 269 nm

542

to 278 nm and also slightly decreased the EM from -4.52 to -4.30 (10-8 m2/Vs). Folic

543

acid incorporation into soluble complexes resulted from BLG and Na-ALG

544

interactions, increased the particle size from 269 nm to values higher than 1µm

545

(maybe due to the low solubility of folic acid at pH 4.25) and also decreased the EM

546

from -4.52 to -3.95 (10-8 m2/Vs). These experimental values are in good agreement

547

with the qualitative visual observations, which indicated an increased turbidity in the

548

presence of folic acid. A similar decrease in the absolute value of the EM was

549

reported by Zimet et al. (2009) upon DHA addition into BLG-pectin soluble

550

complexes. One should keep in mind that the measured results are intensity-

551

weighted, which means that the larger particles have the larger contribution. If

552

volume- or number- weighted distributions are considered, much smaller average

553

diameters are obtained. For any delivery system, it is essential that the system

554

remains stable throughout the entire life cycle of the product. Furthermore, the

AC C

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530

23

ACCEPTED MANUSCRIPT biopolymer nanoparticles should not adversely impact the normal shelf-life of the

556

product itself (Matalanis et al., 2011). EM can be obtained from the perturbations of

557

Brownian diffusivity under a pulsating electrical field and is a crucial parameter for

558

predicting the stability of colloidal delivery systems (Cooper, Dubin, Kayitmazer, &

559

Turksen, 2005; Matalanis et al., 2011).

560

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Nanoparticle formation from the protein – polysaccharide electrostatic

interaction could be understood as the shrinkage of the protein-polysaccharide

562

complexes which occurs at low ionic strength. This shrinkage is a result of a

563

decrease in the intramolecular repulsion of like-charged groups of polysaccharide

564

induced by the interaction of the BLG molecules with the carboxyl groups of the Na-

565

ALG led to a decrease in the backbone chain rigidity (Fig. 8) (Tolstoguzov, 2003).

566

This compaction phenomenon was well predicted by Monte Carlo simulations which

567

showed that at low ionic strengths, a polyelectrolyte chain would wrap around an

568

oppositely charged spherical particle (Girard, Turgeon, & Gauthier, 2003). Indeed,

569

mutual neutralization decreases the net charge, hydrophilicity and chain rigidity of

570

the junction zones resulting in a compact conformation of the complex with the

571

hidden junction zones (Tolstoguzov, 2003).

M AN U

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Another important attribute of a suitable delivery system is its stability against

AC C

572

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561

573

processing conditions (such as thermal processing and pressure) which can result in

574

protein denaturation and hence affecting its transporting properties. The precise

575

denaturation process is complex and is influenced by factors such as pH, protein

576

concentration, ionic environment, genetic variant and presence of ligands. Both

577

lowering the pH (Relkin, Eynard, & Launay, 1992) and adding calyx-bound ligands

578

(Busti, Gatti, & Delorenzi, 2006) make the protein more resistant to thermal

579

unfolding. Enzymatic proteolysis observations indicate that BLG is less susceptible 24

ACCEPTED MANUSCRIPT 580

to pressure-induced changes at acidic pH than at neutral or basic pH (Edwards et

581

al., 2009).

582

The targeted delivery and controlled release are also very important. Active ingredients release from whey protein gels was investigated by Tomczy ska-Mleko,

584

& Mleko (2014). As an example, it is beneficial for the encapsulated folic acid to be

585

released in the small intestine where most of the absorption of vitamins takes place.

586

The jejunum is the site of maximum absorption of free folates, where absorption

587

occurs by a pH dependent, carrier-mediated system (Kailasapathy, 2008). Therefore,

588

the biopolymers used for encapsulation should be able to protect the folate in the

589

upper gastrointestinal tract (acidic stomach conditions) and release the folate in the

590

alkaline conditions of the small intestine. The BLG structure is relatively compact and

591

stable in aqueous solutions at acidic pH, as demonstrated by its resistivity to

592

proteolysis by pepsin (Mohan Reddy, Kella, & Kinsella, 1988). The compact structure

593

of BLG at acidic pH can be overprotected by using an anionic polysaccharide due to

594

the electrostatic interactions. The BLG-anionic polysaccharide complexes will be

595

dissociated at alkaline pH of the small intestine (due to the charge similarity of both

596

biopolymers). Hydrophobic interactions are mainly responsible for the binding of

597

nutraceuticals to BLG. Since those interactions are entropy driven and depend on

598

the presence of the native structure in BLG, the release process will occur when the

599

protein loses its native structure during digestion in small intestine.

600

4. Conclusion

AC C

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601

In this study, the intrinsic transporting properties of BLG were utilized to

602

develop nano-sized green delivery systems. The binding analysis suggested that the

603

binding occurred under all conditions but varied as a function of pH and nutraceutical 25

ACCEPTED MANUSCRIPT model compound. From those observations, it could be concluded that the four

605

nutraceutical models investigated are bound to proteins on different binding sites

606

and/or by different binding mechanisms. The binding capacities of BLG mediated by

607

pH were evident in the present study. Upon lowering the pH, the binding constants

608

changed depending on the nutraceutical nature. Whereas NMR enables a more

609

direct determination of the binding to proteins, this technique suffers from the fact

610

that it can only be applied for ligands with a sufficiently large solubility (i.e. in the mM

611

range) in the aqueous phase. These findings resulted in designing nanoscopic

612

delivery systems for encapsulation of both hydrophilic and hydrophobic bioactives in

613

liquid food products. The preliminary stability experiments demonstrated the efficacy

614

of soluble complexes arising from protein-polysaccharide interactions on

615

nanoencapsulation and colloidal stability of nutraceuticals of low solubility in water.

616

Nevertheless, further work is required in the future to determine the bioprotection

617

efficiency, morphology of the stable biopolymer nanocomplexes, stability toward

618

processing conditions and controlled release properties.

619

Acknowledgment

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The authors are thankful to University of Tehran, Iranian Nanotechnology Initiative Council and Ghent University for financial support.

622

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623

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isolate-low methoxyl pectin complexes as a matrix for hydro-soluble food

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Busti, P., Gatti, C. A., & Delorenzi, N. J. (2006). Binding of alkylsulfonate ligands to

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whey proteins. In A. Thompson, M. Boland, & H. Singh (Eds.).Milk proteins:

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Fioramonti, S. A., Perez, A. A., Aríngoli, E. E., Rubiolo, A. C., & Santiago, L. G. (2014). Design and characterization of soluble biopolymer complexes produced

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Table captions

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Table 1: Apparent dissociation constant (K'd, expressed in nM) of BLG and

841

nutraceutical model compounds at pH 7.00 and 4.25.

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Table 2: Apparent molar binding ratios (n, expressed in mol ligand per mol of protein

844

monomer) of nutraceutical model compounds to BLG at pH 7.00 and 4.25.

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Table 1

846 K'd (nM) β-carotene

Folic acid

Curcumin

Ergocalciferol

7.00

21 ± 3Ba

34 ± 27Aa

201 ± 72Bb

144 ± 28Ab

4.25

15 ± 2Aa

27 ± 4Ab

51 ± 17Ac

847

173 ± 16Ad

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pH

The superscript letters (A, B) mean that the results within the same

850

column without a common letter are significantly different (p< 0.05); the

851

subscript letters (a, b, c, d) mean that the results within the same row

852

without a common letter are significantly different (p< 0.05).

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Table 2

855 N β-carotene

Folic acid

Curcumin

Ergocalciferol

7.00

0.48 ± 0.04Ba

1.25 ± 0.05Bc

0.95 ± 0.06Ab

1.31 ± 0.12Bc

4.25

0.23 ± 0.03Aa

0.39 ± 0.04Ab

0.82 ± 0.08Ac

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pH

856 857

0.85 ± 0.06Ac

The superscript letters (A, B) mean that the results within the same

859

column without a common letter are significantly different (p< 0.05); the

860

subscript letters (a, b, c) mean that the results within the same row without

861

a common letter are significantly different (p< 0.05).

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Figure Captions

864

Fig. 1: 3D illustration of asymmetric dimer of BLG showing the hydrophobic

866

binding sites

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Fig. 2: Folic acid chemical structure

868

Fig. 3: NMR spectrum of 3 mM folic acid in D2O at 25 °C with relative peak

869

assignment

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Fig. 4: NMR spectra of 3 mM folic acid in D2O at 25 °C: alone (blue), in

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presence of 0.25 mM BLG (red) and in presence of BLG at pH 4.25 (green)

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Fig. 5: DOSY spectra of 3 mM folic acid in D2O at 25 °C: alone (blue), in presence of

873

0.25 mM BLG (red)

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Fig. 6: Effects of curcumin nanoencapsulation on its colloidal stability at pH 4.25 (I:

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dissolved curcumin in ethanol added to deionized water; II: dissolved curcumin in

876

ethanol added to BLG dispersion (0.1% w/w); III: dissolved curcumin in ethanol

877

added to BLG-Na-ALG soluble complexes (Na-ALG/BLG weight ratio of 0.75).

878

Fig. 7: Effects of folic acid nanoencapsulation on its colloidal stability at pH 4.25

879

(I: dissolved folic acid in deionized water added to deionized water; II: dissolved

880

folic acid in deionized water added to BLG dispersion (0.1% w/w); III: dissolved

881

folic acid in deionized water added to BLG-Na-ALG soluble complexes (Na-

882

ALG/BLG weight ratio of 0.75).

883

Fig. 8: Increasing in chain flexibility as a result of mutual neutralization

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890 891

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Fig. 1

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4 H N

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H

O

H

H

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8 H

6

H

5

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Fig. 2

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H 3

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7

6

5

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2(3)-3(2)

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Fig. 3

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1

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910 911 912

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Fig. 4

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Fig. 5

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A III

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II

I

III

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III

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Junction zone

-

-

+

- + +

-

-

-

-

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Anionic polysaccharide

+ -

Globular protein

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-

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Chain segment of increased hydrophobicity and flexibility as a result of mutual neutralization

-

Inflexible hydrophilic chain segment

941 942

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Fig. 8

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Utilizing intrinsic transporting property of BLG to develop green delivery system BLG-bioactives complexation varied as a function of pH and

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nutraceutical type.

Successful entrapment of bioactives within electrostatically stable

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nanocomplexes

1