Efficient delivery of quercetin after binding to beta-lactoglobulin followed by formation soft-condensed core-shell nanostructures

Highlights

• Sodium alginate/β-lactoglobulin interaction resulted in core-shell nanocarriers.

• Quercetin was able to bind to β-lactoglobulin even at acidic conditions.

• Enhanced protection of quercetin by electrostatic deposition of an alginate shell.

• No release in simulated gastric and sustained-release in intestinal conditions.

Abstract

Nature-made inherent transporting property of beta-lactoglobulin (BLG) was exploited to develop delivery systems for quercetin. After binding to BLG, quercetin was nanoencapsulated within soft-condensed nanostructures of BLG and sodium alginate (ALG). Fluorimetry results revealed that quercetin could bind to BLG even at acidic conditions. The amounts of stoichiometry binding (n) were 1.24 and 1.62 at pH values of 4 and 7, respectively. Formation of core-shell type nanostructures was confirmed by transmission electron microscopy. Quercetin was efficiently entrapped (>93%). The ejection from the carrier was very limited over time (<1% during 1 month). The protection of nanoencapsulated quercetin was at least 3 times better than that of free quercetin. Quercetin was not released (<3.5% during 6 h) in simulated gastric fluids (pH 1.2 and 4); while, a sustained release (77% during 12 h) was observed in simulated intestinal fluid (pH 7.4).

Introduction

Among various nutraceuticals, natural antioxidants have gained a great deal of attention due to a large number of health claims ascribed to them (Giampieri, Alvarez-Suarez, & Battino, 2014). Flavonoids as natural antioxidants have six subdivisions including flavones, flavonols, flavanones, flavan-3ols, anthocyanidins and isoflavones (Ross & Kasum, 2002). Quercetin, as a flavonol, is naturally present in vegetables (e.g., onions), fruits (e.g., apples), soft drinks (e.g., tea), alcoholic beverages (e.g., red wine) and medicinal plants (e.g., yellow sweet clover). Similar to other flavonoids, quercetin possess several potential health benefits such as antioxidant and antiviral activities (Zhang, Yang, Tang, Hu, & Zou, 2008) and may contribute to lowering the risk of cardiovascular disease, cancer, diabetes and obesity (Gutierrez, Prater, & Holladay, 2014). Most of quercetin present in plants is in glycosidic form; however, it can be also found as free aglycone.

Because of crystallinity and low water-solubility, quercetin has low bioavailability after oral administration (Aceituno-Medina, Mendoza, Rodríguez, Lagaron, & López-Rubio, 2015). Moreover, chemical instability, under the influence of oxygen, light and high temperature, makes the incorporation of quercetin into liquid functional foods very difficult.

Many efforts have been made to develop quercetin delivery systems using amorphous solid dispersions based on cellulose derivatives (Gilley et al., 2017), electrospun hybrid fibers synthesized from a mixture of amaranth protein isolate and pullulan (Aceituno-Medina et al., 2015), nanoparticles of chitosan oligosaccharide and β-lactoglobulin (Ha, Kim, Lee, & Lee, 2013), self-assembled lecithin and chitosan nanoparticles (Souza et al., 2013), poly-d, l-lactic acid (Kumari, Yadav, Pakade, Singh, & Yadav, 2010) and ionically cross-linked chitosan nanoparticles (Zhang et al., 2008).

Encapsulation systems based on protein-polysaccharide complexes offer many advantages such as increasing bioactivity, masking unpleasant tastes, targeted delivery and controlled release (Arroyo-Maya & McClements, 2015). Electrostatic complexes are formed at pH conditions between the isoelectric point (pI) of protein and the pK_a of ionic polysaccharide (Perez et al., 2015, Sanchez et al., 2002). Among various structures which can be formed in the mixture of oppositely charged biopolymers, spherical coacervates are the best choice for the encapsulation. However, the phase separation resulting from the coalescence of coacervates (Sanchez et al., 2002) limits their application as delivery system in clear beverages.

In recent years, the potential application of the soluble complexes in the encapsulation of bioactives has been extensively investigated (Arroyo-Maya and McClements, 2015, Hosseini et al., 2015, Ilyasoglu and El, 2014, Perez et al., 2015, Zimet and Livney, 2009). To our knowledge, binding of quercetin to beta-lactoglobulin (BLG) and then its delivery by means of mixed biopolymer nanocomplexes have not been studied yet. The hypotheses of this study were i) BLG (as a member of lipocalin protein family) can be used as a carrier for quercetin. However, its transporting properties may be influenced by pH; ii) the loaded quercetin may be better protected by deposition of a shell around the protein core via complex coacervation between BLG and an anionic polysaccharide; iii) the characteristics of the nanocarrier may be influenced by the ratio of both biopolymers; iv) the heat stability of mixed biopolymer nanocomplexes may be superior to individual BLG molecules.

Therefore, the objectives of the current work were i) the assessment of quercetin binding to BLG at various pH levels; ii) evaluating the protection conferred to quercetin after binding to BLG and then deposition of a sodium alginate (ALG) shell around the quercetin-loaded BLG (as the core); iii) evaluating the efficacy of the resultant delivery system (prepared at different biopolymer mixing ratios) in various aspects of entrapment, delivery and controlled release of quercetin; and iv) monitoring the effect of heat processing on the characteristics of the delivery system.