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Mixed biopolymer nanocomplexes conferred physicochemical stability and sustained release behavior to introduced curcumin
Food Hydrocolloids 71 (2017) 216e224
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Mixed biopolymer nanocomplexes conferred physicochemical stability and sustained release behavior to introduced curcumin Seyedeh Fatemeh Mirpoor a, Seyed Mohammad Hashem Hosseini a, *, Gholam Hossein Yousefi b, c a b c
Department of Food Science and Technology, School of Agriculture, Shiraz University, Shiraz, Iran Department of Pharmaceutics, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Sciences, Shiraz, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2017 Received in revised form 17 April 2017 Accepted 17 May 2017 Available online 20 May 2017
Beta-lactoglobulin (BLG)-sodium alginate (ALG) electrostatic nanocomplexes were utilized to encapsulate and deliver curcumin in clear acidic media. Relative viscosity measurements confirmed that ALG wrapping around the globular BLG molecules occurred in the aqueous biopolymer mixture upon acidification. Curcumin was efficiently entrapped within developed green delivery systems. The encapsulation efficiency (EE) of curcumin was around 98%. The carriers were efficiently able to retain the curcumin inside over time. The physical stability of hydrophobic curcumin toward precipitation in aqueous solution was significantly improved after introduction into nanostructures. Moreover, nanocomplexes conferred considerable protection to curcumin against heat-induced degradation. At the end of 15-day storage at 45 C, the amounts of curcumin loaded in nanocomplexes were significantly (p < 0.05) larger than those loaded in blank samples. Non-significant changes in the particle size after high temperature short time (HTST) heat treatment indicated that the presence of polysaccharide shell around the protein core increased the thermal stability of BLG at acidic conditions. No release occurred in simulated fasting and non-fasting gastric conditions. However, a sustained release behavior (78.5% during 12 h) was observed in simulated intestinal fluid. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Complex coacervation Wrapping phenomenon Curcumin Nanoparticles Encapsulation Controlled release
1. Introduction Curcumin is a low molecular weight polyphenol present in yellow rhizome of Curcuma longa (Prasad, Gupta, Tyagi, & Aggarwal, 2014). This bright orange-yellow pigment has been used for centuries as a condiment and a medicinal plant (Shaikh, Ankola, Beniwal, Singh, & Ravi Kumar, 2009). A wide range of pharmacological activities such as anticancer, antioxidant, antiinflammatory, antibacterial, wound healing and anti-amyloid properties have been ascribed to this lipophilic fluorescent molecule (Maheshwari, Singh, Gaddipati, & Srimal, 2006; Kumar, Dorga, & Prakash, 2009). Despite multiple medicinal benefits, curcumin has very low bioavailability upon oral administration and hence low clinical efficacy. Curcumin suffers from low water-solubility and poor chemical stability toward light (Jourghanian, Ghaffari,
* Corresponding author. E-mail address: [email protected] (S.M.H. Hosseini). http://dx.doi.org/10.1016/j.foodhyd.2017.05.021 0268-005X/© 2017 Elsevier Ltd. All rights reserved.
Ardjmand, Haghighat, & Mohammadnejad, 2016). Moreover, its conjugated diene structure is sensitive to degradation at neutral to basic pH conditions; therefore, curcumin stability increases by decreasing pH (Shaikh et al., 2009). Bioactive-loaded delivery systems can be used for fortification purposes of various types (e.g., solid, semi-solid and liquid) of food products and supplements. Fortification of low fat or fat-free aqueous-based liquid food products (particularly clear beverages) using hydrophobic bioactives is more challenging than the other fields of fortification (Sagalowicz & Leser, 2010; Matalanis, Jones, & McClements, 2011); The designed carrier system must be incorporated in sufficient amount into liquid food without losing the protective effects conferred to the core. Moreover, the carrier should not adversely affect the optical properties of final product. Sub-micrometer (and preferably nano) scale delivery systems are the best candidates for this purpose, provided having the ability to keep the wall integrity after incorporating into aqueous system. Some encapsulation systems such as those prepared by spray drying technique usually lose the wall integrity just after contact
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with water. In-situ formation of carriers in large amounts of water may help to keep the wall integrity. Therefore, emulsion- and surfactant-based delivery systems such as nanoemulsion, microemulsion and nanoliposome can be as carriers in the fortification of liquid systems. However, they generally suffer from low loading capacity, the presence of organic solvents and inappropriate taste characteristics as a result of large amounts of surfactants (Sagalowicz & Leser, 2010). Colloidal biopolymer structures, assembled from individual or mixed biopolymer molecules, can offer many advantages including protection from the loss of bioactivity under the effect of harsh environmental factors, masking unpleasant tastes and targeted delivery (Arroyo-Maya & McClements, 2015; Perez, Sponton, Andermatten, Rubiolo, & Santiago, 2015). Complex coacervation (thermodynamic compatibility) is a catch-all term for the spontaneous association of oppositely charged biopolymers (usually proteins and polysaccharide). This phenomenon occurs at low concentrations (<3e4.5%) and at pH values between protein's isoelectric point (pI) and the pKa of polysaccharide (Ganzevles, Stuart, van Vliet, & de Jongh, 2006). During acidification of a mixture of protein/anionic polysaccharide, a soluble complex at a molecular level is firstly formed, followed by formation of coacervates and/or fractal aggregates (depending on the involved contributors) upon further reduction in pH (Paraskevopoulou & Kiosseoglou, 2013). When polysaccharide is present in excess amount, soluble nanocomplexes may be developed even at low pH conditions. These nanocomplexes may have potential applications in the fortification purposes of clear liquid food products (using hydrophobic bioactives) and also in the lipophilic drug delivery systems. Recently, the application of biopolymers for the encapsulation of curcumin has been extensively studied. Sarika and Nirmala (2016) and Sarika, James, and Raj (2016) studied the application of oxidized gum Arabic and alginate-gelatin conjugate for the delivery of curcumin, respectively. In spite of promising results, utilization of toxic organic solvents limits their application in food products. Self-assembly of k-carrageenan and lysozyme was used to encapsulate curcumin (Xu et al., 2014). Large amount of encapsulation efficiency (EE) and increased stability against heat and UV radiation were reported. However, the strong electrostatic interaction between anionic k-carrageenan and cationic lysozyme at neutral pH may prevent the efficient release of curcumin in its site of action (i.e., intestine). Kafirin and kafirin/carboxymethyl-chitosan nanoparticles were successfully used to enhance the cellular uptake of curcumin (Xiao, Nian, & Huang, 2015). Hu et al. (2015) reported that curcumin is in an amorphous rather than crystalline form after nanoencapsulation within coreeshell nanoparticles fabricated from zein and pectin. To our knowledge, the application of stable nanocomplexes arising from beta-lactoglobulin/sodium alginate interaction in the nanoencapsulation of curcumin has not been studied yet. Stable nanocomplexes can be defined as very small complex coacervates with a slow rate of coalescence and macroscopic separation (Hosseini et al., 2016). Therefore, this study was conducted to evaluate the efficacy of soluble nanostructures, developed from electrostatic soft-condensation of beta-lactoglobulin (BLG) and sodium alginate (ALG), in various aspects of encapsulation, protection, delivery, heat stability and controlled release of curcumin as a hydrophobic nutraceutical model. Our hypothesis was that the intrinsic transporting properties of beta-lactoglobulin as a member of lipocalin protein family may be used for developing protein-curcumin complexes (cores); which could then be overprotected by deposition of an anionic polysaccharide shell at acidic pH levels.
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2. Materials and methods 2.1. Materials Sodium alginate (ALG, MW: 200 kDa) with a mannuronate:guluronate (M:G) ratio of 0.6 was obtained from BDH Co. (Poole, UK). The amounts of carbohydrate, moisture and total ash were 66.3, 14.2 and 9.5 (%w/w), respectively. Beta-lactoglobulin (BLG, MW: 18.4 kDa, composition (%w/w): 93% BLG, 5.4% moisture and 1.6% ash), pepsin (extracted from porcine gastric mucosa, activity: 600e1800 unit/mg protein), pancreatin extracted from porcine pancreas (>3x USP specifications), glucono-d-lactone (GDL) and curcumin were obtained from Sigma Chemical co. (St. Louis, MO, USA). Citric acid, analytical grade hydrochloric acid, sodium azide and absolute ethanol were purchased from Merck Co. (Darmstadt, Germany). In this study, double distilled water (DDW) was used to prepare all dispersions. 2.2. Dispersion preparation Biopolymers including ALG (0.2 %w/w) and BLG (0.1 %w/w) were separately dissolved in DDW already containing 0.03 %w/w sodium azide. The dispersions were stirred at 200e250 rpm at room temperature for 4e5 h and then stored at 4 C overnight to ensure complete hydration. 2.3. Changes in the absorbance as a function of pH Formation of complexes was studied by turbidimetric analysis. Biopolymer mixtures were prepared by mixing the required amounts of stock biopolymer dispersions so that to obtain a 2:1 protein to polysaccharide mixing ratio (MR) and total biopolymer concentration (TBC) of 0.075 %w/w. The mixture was then gradually titrated by HCl; while, gentle magnetic stirring was carried out for 2 min at each pH level before reduction to the next pH (of about 0.1 unit difference). Titrations were done at room temperature and dilution effects were considered to be negligible. Changes in the absorbance were monitored using a UV/visible light spectrophotometer at 600 nm (UNICO UV-2100, USA). Distilled water was used as blank. 2.4. Changes in the relative viscosity during complexation We assumed that changes in the flexibility and hence the conformation of biopolymers during acidification and subsequent complexation might influence the viscosity of the system. Therefore, the relative viscosity of ALG/BLG mixture (TBC 0.075 %w/w and BLG:ALG MR 2:1) was studied as a function of pH using the method described by Weinbreck, Nieuwenhuijse, Robijn, and de Kruif (2003) with a slight modification. To measure the relative viscosity, a U-tube capillary viscometer (No. 518 10, Schott Ger€ ate, Germany) was used at 25 ± 0.1 C. In-situ acidification was carried out using GDL (0.1 %w/w). Relative viscosity (t/t0) was plotted against the pH of the sample; where t and t0 are the flow times (second) of sample and pure solvent (water), respectively. BLG and ALG blank dispersions were also studied at their relative concentrations (0.05 and 0.025 %w/w, respectively). The results were then indirectly related to the complexation phenomenon. 2.5. Curcumin nanoencapsulation A fresh alcoholic solution of curcumin was gradually added into BLG dispersion (0.025 %w/w) so that to obtain a curcumin:BLG molar ratio of 1:1. This molar ratio was selected based on our previously published fluorimetry results regarding the interaction
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between BLG and curcumin (Hosseini, Emam-Djomeh, Sabatino, & Van der Meeren, 2015). An increase in the molar ratio may lead to protein overloading and hence ejection of curcumin from the inside of the delivery system over time. After stirring for 30 min, different amounts of ALG dispersion were added into curcumin/BLG complexes in order to reach two different TBCs, namely 0.075 and 0.044 %w/w (corresponding to MRs of 0.5 and 1.33, respectively). Constant final volumes were obtained by DDW addition. Post blending acidification method was used to complete the nanoencapsulation process through adjusting the pH to 4 and then stirring for additional 30 min. Nanoencapsulated curcumin dispersion was equilibrated overnight at room temperature before analysis. For each sample, three blanks including a DDW (biopolymer-free) sample containing equal concentration of curcumin, ALG-free curcumin/ BLG dispersion and curcumin-free mixed biopolymer dispersion were also prepared and treated in a similar manner. 2.6. Measurement of encapsulation parameters The efficiency of curcumin entrapment within nanocomplexes was determined by separating free (unloaded) curcumin from curcumin-loaded nanocomplexes using Amicon Ultra centrifugal filter units (MW cut off 10 kDa, Merck Millipore Ltd., Ireland). After centrifugation (4000 g, 30 min, 25 C) of nanocomplexes (4 ml), the concentration of free curcumin in permeate was determined from a standard curve. To develop the standard curve, the standard solutions of curcumin were prepared by curcumin dissolution in 50:50 water:ethanol mixture and then measuring the absorbance at 468 nm. To determine the curcumin concentration in permeate, absolute ethanol was added into the permeate so that to obtain a water:ethanol ratio of 50:50 followed by measuring the absorbance. The encapsulation (entrapment) efficiency (EE) was calculated according to the following equation (1).
EE% ¼ Mic Mpc
Mic 100
(1)
where, Mic is the initial mass of curcumin present in the sample (before centrifugation); Mpc is the mass of free curcumin determined in permeate (after centrifugation). Loading capacity (LC) was calculated according to equation (2).
LC% ¼ ðmass of curcumin=mass of BLG þ ALGÞ 100
water ratio of 4:1 (80:20) resulted in the complete extraction of curcumin from the nanocarriers. Curcumin was not detected in the precipitates after centrifugation and subsequent washing with ethanol. Therefore, after sampling (4 ml), absolute ethanol was added (16 ml) and mixed thoroughly. After curcumin extraction, the biopolymer precipitates were settled by centrifugation at 1400 rpm and 25 C for 20 min. Distilled water was added to the supernatant to adjust the ethanol content to 50:50. After that, curcumin was quantified using the standard curve (section 2.6). 2.9. HTST heat processing effects on particle size and surface potential of loaded and un-loaded nanocomplexes To determine the heat processing effects, thin layers of different liquid samples were provided in glass vials and then subjected to heating in water bath pre-set at 75 C for 30 s. After cooling, the particle size and the surface potential was measured. Volumeweighted mean diameter was determined using a dynamic light scattering (DLS) instrument (Nanotrac Wave, Microtrac, USA) at a scattering angle of 90 and 25 C. The amounts of the surface potential (mV) of complexes were determined using a Microtrac Zeta Check (Microtrac, Germany) at 25 C. 2.10. In-vitro release of nanoencapsulated quercetin In-vitro release studies were performed according to the method described by Chen and Subirade (2006). Simulation of fasting and non-fasting gastric conditions was performed by mixing equal volumes of sample and an HCl solution (containing pepsin at 0.1 %w/w concentration) at pH values of 1.2 and 4, respectively. The mixture was incubated in a shaker at 37 C for 6 h in dark conditions. To simulate intestinal conditions, a mixture of equal volumes of sample and phosphate-buffered solution (PBS, 10 mM, pH 7, containing 1 %w/w pancreatin) was prepared and then incubated at 37 C for 12 h in dark conditions. Sampling (4 ml) was performed during 1-h intervals. The released curcumin was then separated from the mixture using Amicon filter units centrifuged at 6000 g and 4 C for 30 min. Low temperature was used to stop the digestion process. Absolute ethanol was added into the permeate so that to reach a 50:50 water:ethanol ratio. Curcumin concentration was determined from the standard curve after measuring the absorbance at 468 nm (section 2.6).
(2)
The ability of biopolymer nanocomplexes to retain the curcumin inside over time (loading or delivery efficiency, DE) was determined by measuring the EE during 1-month storage. 2.7. Physical stability measurement Physical stability of curcumin-loaded nanostructures against precipitation was evaluated by measuring the absorbance at 600 nm using a spectrophotometer during 1-month storage at 25 C. The samples were stored in dark to prevent from lightinduced degradation. 2.8. Curcumin protection An enhanced shelf-life stress test described by Zimet and Livney (2009) was used with a slight modification to determine the protective effects conferred by nanocomplexes to curcumin. After preparation at room temperature, samples containing nanoencapsulated curcumin dispersion and their respective blanks were placed in dark glass containers and shaken in a water bath at 45 C for 15 days. Our preliminary experiments showed that an ethanol to
2.11. Statistical analysis The results obtained in this work were reported as mean ± standard deviation. Duncan's multiple range tests at a probability of 0.95 were used to compare the means using the SPSS software (version 21, IBM Corp. USA). 3. Results and discussion 3.1. Changes in the absorbance and relative viscosity during complexation process The ionization degree of functional side groups of biopolymers is influenced by pH (Ye, 2008). Fig. 1a depicts the phase diagram and critical pH values upon acidification. At pH values more than 5.2e5.3, both biopolymers had negative charges; therefore, no significant increase in the absorbance of mixture was observed due to the strong electrostatic repulsion. Formation of soluble complexes at pHc (~5.0e5.2) is the first experimentally detectable noncovalent interaction in BLG/ALG mixture. The formation of soluble complexes occurs at molecular levels and hence the value of pHc is independent from TBC and MR but not ionic strength (Hadian et al.,
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decreasing the effective volume. The relative viscosity of the blank BLG dispersion remained unchanged, which was due to the globular structure of protein molecules (Kontopidis, Holt, & Sawyer, 2004). The viscosity of biopolymer mixture was lower than that of blank ALG dispersion. A possible explanation is that ALG molecules in biopolymer mixture had less effective volumes (or less stretched conformation) in the presence of protein molecules even in the non-complexed conditions. The difference in the effective volume could be resulted from the competition for the solvent molecules or the predominance of thermodynamic incompatibility (i.e., during segregative phase separation polysaccharide molecules may acquire a different conformation after surrounding by molecules of their own type). The viscosity of the ALG/BLG mixture decreased as a function of pH. The sharp decrease (breaking point) in Fig. 1b coincided with the initiation of the interactions at molecular levels (i.e., formation of soluble complexes) in Fig. 1a. The release of counterions upon complexation would cause a slight increase in the ionic strength (de Kruif, Weinbreck, & de Vries, 2004). An increase in the ionic strength increases the chain flexibility (i.e., less viscosity). Another explanation is that mutual neutralization decreases the net charge, hydrophilicity and chain stiffness of the junction zones and resulted in ALG bending around BLG molecules and developing complexes with a compact structure (Tolstoguzov, 2003). It should be pointed out that the alginate type used in this study was rich in guluronate (G) residues, which are responsible for the stiff nature of the alginate chain (Draget, Moe, Skjåk-Bræk, & Smidsrød, 2006). The decrease in the relative viscosity was a proof for the wrapping phenomenon in biopolymer mixture during acidification and formation of soft-condensed coreshell type structures. Further reduction in pH was not possible due to the complex growth and U-tube fouling. Fig. 1. Changes in the (a) absorbance and (b) relative viscosity values of mixed biopolymer dispersion as a function of pH measured at 25 C.
2016; Turgeon & Laneuville, 2009). Formation of soluble complexes at pHc values higher than the pI of BLG (z4.9e5, where the protein net charge is negative) could be ascribed either to the presence of localized basic (positive) peptides (Turgeon & Laneuville, 2009; Weinbreck et al., 2003) or to the inherent electrical capacitance of globular proteins leading to regulate the charge under the effect of polysaccharide's negative charge (Dickinson, 2008). Further decrease in pH resulted in phase separation and formation of interpolymeric (insoluble) complexes at pH41 and detected by an abrupt increase in the absorbance. At this pH, the solution changed from transparent to cloudy. Cloudiness was due to the nucleation and growth of sufficiently large biopolymer complexes capable of light scattering (Bengoechea, Jones, Guerrero, & McClements, 2011). Absorbance increased steadily (due to further complex growth) and reached to a maximum at pHopt (2.2e1.8) corresponding to an electrical equivalence. Dissociation of complexes at pH values below pHopt led to formation of another critical point denoted as pH42. Mixed dispersion became fully transparent close to pH 0.5, indicating complete dissociation as a result of deionization of alginate's carboxyl groups (eCOO- þ Hþ 4 eCOOH). Size and conformation of biomacromolecules are crucial to the viscosity of dilute biopolymer dispersions (Schmitt, Sanchez, DesobryBanon, & Hardy, 1998). Fig. 1b represents the changes in the relative viscosity of ALG/BLG mixture (TBC 0.075 %w/w, BLG:ALG MR 2:1) and the respective blanks as a function of pH. The relative viscosity of the blank dispersion of polysaccharide decreased during pH reduction. The decreased charge density of ALG reduced the intramolecular repulsion between similarly charged monomers. This phenomenon resulted in backbone bending and hence
3.2. Encapsulation parameters Low water solubility of curcumin limits its bioavailability and hence its incorporation as a nutraceutical into functional foods. Curcumin solubilization and vehiculization using different encapsulation systems is an appropriate solution for this challenge. To determine the appropriate delivery system, the entrapment characteristics are of paramount importance. Encapsulation efficiency (EE) provides an idea about the fraction of bioactive successfully entrapped within nanovehicles. The amounts of EEs were around 98% for both MRs of 0.5 and 1.33. These large amounts of EEs revealed high affinity of curcumin toward mixed biopolymer nanocomplexes. Since curcumin was added into BLG dispersion in equimolar concentration, the amounts of EEs obtained in this work were significantly larger than those reported in previous studies for curcumin and other nutraceutical compounds: 71± 0.02% for curcumin encapsulation within sodium k-carrageenan/lysozyme complexes (Xu et al., 2014); 85% for 10% sweet orange oil loaded microcapsules prepared by complex coacervation of soybean protein isolate/gum Arabic (Jun-xia, Hai-yan, & Jian, 2011); 70% for tea polyphenol encapsulation within complex coacervate core micelles (Zhou et al., 2012); 64% for DHA entrapment within BLG/pectin nanocomplexes (Zimet & Livney, 2009) and 83.7% and 93.6% for ferulic acid and quercetin entrapment within pullulan electrospun fibers, respectively (Aceituno-Medina, Mendoza, Rodríguez, pez-Rubio, 2015). Huang et al. (2016) reported that Lagaron, & Lo curcumin encapsulation within core-shell nanostructures developed from a mixture of pectin (70%) and alginate (30%) (as the shell) and zein nanoparticles (as the core) resulted in higher antioxidant and free radical scavenging activities than curcumin dissolution in ethanol. Loading capacity (LC) gives an idea about the nutraceutical content of delivery system. As mentioned already the amounts of total biopolymers were 0.044 and 0.075 g per 100 g of
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Fig. 2. Encapsulation efficiency (EE) profiles of curcumin-loaded nanocomplexes as a function of time obtained at two different MRs.
dispersion. Therefore, LCs were 1.14% and 0.67% at TBCs of 0.044 and 0.075%, respectively (taking into account the mass of the added curcumin: 0.0005 g/100 g of dispersion). The LC was lower than normal capacity ranges (5e10%); which should be addressed in future studies. The results of loading (delivery) efficiency (DE), indicating the ability of nanocomplexes to retain the curcumin inside over time, are shown in Fig. 2. The protective effects of delivery systems would be lost upon nutraceutical ejection from the carrier. A very slight decrease in EE was observed for both MRs during a 31day storage period at room temperature. The decreasing trend was attributed to the removing of loosely bound curcumin from biopolymer nanocomplexes likely due to unknown re-structuration phenomena. 3.3. Physical stability of nanoencapsulated curcumin dispersion over time The effects of BLG/ALG self assembly on the physical stability of nanoencapsulated curcumin dispersion are shown in Fig. 3 and Fig. 4. In Fig. 3, lower physical stability could be detected by a rapid decrease in the absorbance. The highest physical stability was observed in the curcumin-free biopolymer mixtures. Large electrostatic repulsion between nanoparticles (discussed later in section 3.5) prevented them from aggregation and hence sedimentation. The stability profiles exhibited that curcumin encapsulation within BLG/ALG nanocomplexes led to higher
Fig. 3. Physical stability of various delivery systems and their respective blanks as a function of time.
physical stability than its entrapment within single BLG molecules (Figs. 3 and 4). The instability of curcumin-loaded BLG sample (denoted as sample III in Fig. 4) could be attributed to the proteinpolyphenol interactions and also to the proximity of the sample pH to the pI of the protein. Polyphenol-induced cross-linking of separate protein molecules resulted in the formation of curcuminBLG large complexes and hence rapid precipitation (Figs. 3 and 4). This interaction is related to the polyphenols molecular size and number of binding locations available for association with proteins (Howell, 2006). Staszewski, Jagus, and Pilosof (2011) concluded that the initial binding of polyphenols to whey proteins is essentially a non-selective hydrophobically-driven interaction; but the formation of insoluble complexes is likely determined by surface charge effects. The electrostatic repulsion conferred by ALG shell to the curcumin-loaded BLG core resulted in higher physical stability of curcumin-loaded ALG/BLG nanostructures than curcumin-loaded BLG (Figs. 3 and 4). The physical stability of curcumin-loaded nanocomplexes obtained at MR of 1:2 (0.5) was relatively higher than that obtained at MR of 4:3 (Fig. 3). Higher stability at lower BLG:ALG MR was attributed to the presence of ALG in sufficiently large (excess) amount leading to effective coating of curcuminloaded protein core. As shown in Fig. 4, no precipitation could be observed in the sample denoted as V (the alcoholic solution of curcumin in water). It should be noted that the absence of precipitation in this sample was not a proof for the system stability. As discussed later, this sample had the lowest chemical stability. Moreover, most of the curcumin present in this sample adhered to the inside wall of the cell. 3.4. Curcumin protection Due to having active phenolic hydroxyl groups in the molecular structure, polyphenols stability is influenced by a variety of factors such as pH, temperature, light and UV radiation (Lee, Choi, Kim, & Hong, 2013). Fig. 5 shows the amounts of curcumin (%) remained in different samples during storage for 15 days at 45 C. The amount of the remained curcumin in the sample denoted as Blank 1 decreased rapidly and reached to 34.5% of initial curcumin content (i.e., 65.5% loss) after 4 days of production. This blank was prepared by dissipating the alcoholic solution of curcumin in water. Curcumin loss was continued but at a decreased degradation rate and reached to 21% of initial curcumin content. Changes in the rate of curcumin loss during storage could be attributed to the protection conferred by the self-association of hydrophobic curcumin in aqueous solution. In aqueous environments, lipophilic compounds such as carotenoids tend to form aggregates or adhere to the surfaces (Wilska-Jeszka, 2007). A similar pattern in curcumin degradation was observed after entrapment by protein particles. After 4 days of storage, the curcumin loss was 43% and reached to 74% at the end of storage. The BLG-induced protection of curcumin against heat was lower than the protection of other nutraceuticals reported already (Ron, Zimet, Bargarum, & Livney, 2010; Zimet & Livney, 2009). Different bioactives have different binding locations in the BLG molecule. It has been reported that the binding location of phenolic compounds is the hydrophobic surface patches but not the central €ki et al., 2008). calyx (the main binding site of BLG) (Riihima Different binding locations may have different protection effects. ALG/BLG core-shell nanostructures could effectively prevent curcumin loss during storage. No significant difference was observed between two MRs. The amount of curcumin loss at the end of storage was about 40%; which means the stability of curcumin loaded in mixed biopolymer nanostructures was improved about 4 fold and 2.3 fold as compared with the stability provided by curcumin self-association (in Blank 1) and by individual BLG molecules (in Blank 2), respectively. These results clearly indicated that the
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Fig. 4. Visual observation of the physical stability of various delivery systems and their respective blanks toward precipitation at different storage times; I1:2 and I4:3 indicate curcumin-free mixed biopolymer nanocomplexes fabricated at BLG:ALG mixing ratios of 1:2 and 4:3, respectively; II1:2 and II4:3 indicate curcumin-loaded mixed biopolymer nanocomplexes fabricated at BLG:ALG mixing ratios of 1:2 and 4:3, respectively; III indicates curcumin loaded BLG (Blank 2); V indicates Blank 1 (alcoholic solution of curcumin in water).
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Fig. 5. Curcumin bioprotection in nanostructures and in respective blanks as a function of time.
Table 1 Changes in the surface potential and particle size of ALG/BLG nanostructures under the effects of curcumin loading and HTST heat processing. ALG þ BLG mixture type
A: surface potential (mV) TBC 0.044 %w/w, MR 4:3 TBC 0.075 %w/w, MR 1:2 B: particle size (nm) TBC 0.044 %w/w, MR 4:3 TBC 0.075 %w/w, MR 1:2
Control sample
Curcumin-loaded sample HTST ()
HTST (þ)
98.9Bb 191.8Aa
109.9Ab 193.8Aa
108.4Ab 169.3Ba
81.1Cb 188.9Aa
109.2Bb 139.5Ba
114.5Ab 141.4Ba
In each row, different superscript capital letters indicate significant differences (p < 0.05). In each column, different subscript small letters indicate significant differences (p < 0.05). HTST () and HTST (þ) indicate samples without and with heat treatment, respectively.
core (curcumin-loaded BLG molecules) could be efficiently overprotected after electrostatic deposition of ALG molecules as a secondary layer (shell). 3.5. HTST heat processing effects on surface potential and particle size of delivery systems In this study, surface potential (mV) (Table 1a) and particle size (nm) (Table 1b) of curcumin-free and curcumin-loaded mixed biopolymer particles (containing fixed BLG concentration) were measured to provide an insight into the stability of delivery system under the effects of curcumin loading and heat processing. The
effects of curcumin loading and heat treatment on the amounts of surface potential and particle size were dependent on the ALG concentration which might lead to different conformations. As reported in Table 1a, decreasing BLG:ALG MR from 1.33 to 0.5 (or increasing the concentration of negatively charged ALG) led to an increase in the negative charge carried by unloaded nanocomplexes. Curcumin encapsulation increased the surface potential value of ALG/BLG nanostructures at MR of 1.33. This phenomenon could be attributed to the changes in conformation of nanoparticles (adopting structures with higher charge density) upon curcumin loading. Curcumin was expected to interact with BLG through hydrophobic interactions. However, electrostatic interactions between curcumin and ionic BLG may also play role in binding mode and location particularly at acidic pH levels (Hosseini et al., 2015). The possible electrostatic attractive interactions between curcumin and cationic BLG could decrease the number of BLG cationic functional groups available for complexation with anionic ALG. Therefore, the fraction of free carboxyl groups of ALG and hence the absolute surface potential of nanoparticles increased after curcumin loading. Changes in the surface potential after curcumin loading were MR-dependent. The increase in the surface potential of BLG/ALG nanostructures at MR of 1.33 was higher than that of mixed biopolymers obtained at MR of 0.5, likely due to the presence of sufficiently large amount of anionic carboxyl groups at lower MR and hence decreasing the effect of curcumin loading on the amount of charge carried by complexes. Similarly, the effect of heat processing on the surface potential absolute values of curcumin-loaded nanocomplexes was MR dependent. We could not find any reasonable explanation for the observed changes. Changes in the particle size under the effects of curcumin loading and heat treatment are shown in Table 1b. A decrease in the MR (i.e., an increase in the weight ratio of ALG to BLG) increased the particle size. Zimet and Livney (2009) concluded that the increased microviscosity as a result of increasing polysaccharide concentration decreases the Brownian motion of particles and results in detecting an apparently increased particle size by DLS. Formation of more opened structures as a result of increasing intramolecular repulsions along ALG backbone (arising from the decreased ratio of BLG molecules attached per ALG chain) are likely another plausible explanation for the increased particle diameter by decreasing MR (Hosseini et al., 2013). Curcumin loading increased the particle size of nanocomplexes developed at MR of 1.33. However, a decrease in the particle size (shrinkage phenomenon) was observed in the MR of 0.5 upon curcumin loading. It seems that the interaction of ALG with curcumin-loaded BLG is different from its interaction with
Fig. 6. Gastric and intestinal release profiles of curcumin from ALG/BLG core-shell nanostructures. The inset shows the gastric release in fasting and non-fasting conditions.
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curcumin-free BLG which should be addressed in future studies. Curcumin might favor the electrostatic interactions between BLG and ALG, likely via the higher accessibility of charges carried by curcumin-BLG complex. Aberkane et al. (2012) reported that the interaction of BLG and total acacia gum increases in the presence of quercetin. In spite of the heat instability of BLG, particle size of curcumin-loaded nanoparticles was not significantly influenced by HTST heat treatment indicating that the ALG shell could effectively protect the curcumin-loaded protein core against heat-induced aggregation. 3.6. Release kinetics of curcumin during in-vitro digestion For each delivery system, the release profile of the encapsulated compound is very important. The designed carrier should be finely tuned so that to release its content at the desired site of gastrointestinal tract (GIT). Prasad et al. (2014) reported that different organs of body have different concentrations of curcumin after its uptake. Most of curcumin are accumulated in liver and intestine. A small quantity could be detected in the other organs (Prasad et al., 2014). To our knowledge, the main absorption site of orally administered curcumin is not precisely detected yet. However, a degree of similarity between the absorption mechanisms of curcumin and other polyphenols (mainly via small intestine) is not beyond the mind. Therefore, the encapsulating system should limit the curcumin release in gastric conditions. To have higher bioavailability, curcumin should be released slowly in the alkaline conditions of intestine. Fig. 6 shows the gastric and intestinal release profiles of quercetin. The inset shows the gastric release profiles at fasting and non-fasting (corresponding to pH values of 1.2 and 4, respectively) conditions. After 6 h, only 3e4% of curcumin was released in simulated gastric fluid. The relatively compact structure of BLG is stable in aqueous acidic solutions. Moreover, BLG is resistant to pepsin proteolysis (Reddy, Kella, & Kinsella, 1998). A relatively higher release could be observed at pH 1.2. Therefore, it can be concluded that the deposition of ALG shell around curcumin-loaded BLG core may also decrease the release of curcumin from its carriers. The relatively increased release of curcumin at fasting gastric conditions (pH 1.2) could be attributed to the dissociation of mixed complexes at very low pH values (ALG unwrapping) (Fig. 1a). Under these conditions, the negative charge of ALG becomes zero as a result of extensive protonation of carboxyl groups. Therefore, biopolymer complexes dissociated after weakening the electrostatic interaction. Curcumin sustained-release (78.5% of total curcumin during 12 h) was occurred in intestinal condition. The dissociation of BLG/ALG complexes at alkaline pH (as a result of electrostatic repulsion) facilitated the pancreatic hydrolysis of BLG and hence controlled release of curcumin. The release profile of curcumin entrapped within dextran sulphate/ chitosan complexes was investigated by Anitha et al. (2011). 70% of curcumin released during 1 week; however, the maximum rate of release was at the first 3 h (Anitha et al., 2011). Kumar Das, Kasoju, and Bora (2010) reported that curcumin release from the alginate/ chitosan/pluronic was about 36% at the first 12 h and reached to 75% after 96 h. 4. Conclusion In conclusion, curcumin-loaded core-shell type nanostructures were developed from ALG/BLG soft nanocomplexes at different MRs and TBCs. Encapsulation of curcumin significantly enhanced its water dispersibility and delivery. The application of a relatively simple and green process using food-grade ingredients, significant bioprotection, physical stability toward heat processing and controlled release characteristics make these soluble
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nanostructures a suitable candidate for the application in the encapsulation technology. However, finding appropriate strategies to increase the loading capacity of nanocomplexes should be addressed in future studies. Acknowledgment This work was financially supported by Shiraz University (Grant number 93GCU3M194065). References Aberkane, L., Jasniewski, J., Gaiani, C., Hussain, R., Scher, J., & Sanchez, C. (2012). Structuration mechanism of b-lactoglobulin e acacia gum assemblies in presence of quercetin. Food Hydrocolloids, 29, 9e20. pez-Rubio, A. Aceituno-Medina, M., Mendoza, S., Rodríguez, B. A., Lagaron, J. M., & Lo (2015). Improved antioxidant capacity of quercetin and ferulic acid during invitro digestion through encapsulation within food-grade electrospun fibers. Journal of Functional Foods, 12, 332e341. Anitha, A., Deepagan, V. G., Divya Rani, V. V., Menon, D., Nair, S. V., & Jayakumar, R. (2011). Preparation, characterization, in vitro drug release and biological studies of curcumin loaded dextran sulphateechitosan nanoparticles. Carbohydrate Polymers, 48, 1158e1164. Arroyo-Maya, I. J., & McClements, D. J. (2015). Biopolymer nanoparticles as potential delivery systems for anthocyanins: Fabrication and properties. Food Research International, 69, 1e8. Bengoechea, C., Jones, O. G., Guerrero, A., & McClements, D. J. (2011). Formation and characterization of lactoferrin/pectin electrostatic complexes: Impact of composition, pH and thermal treatment. Food Hydrocolloids, 25(5), 1227e1232. Chen, L., & Subirade, M. (2006). Alginateewhey protein granular microspheres as oral delivery vehicles for bioactive compounds. Biomaterials, 27(26), 4646e4654. Dickinson, E. (2008). Interfacial structure and stability of food emulsions as affected by proteinepolysaccharide interactions. Soft Matter, 4(5), 932e942. Draget, K. I., Moe, S. T., Skjåk-Bræk, G., & Smidsrød, O. (2006). Alginates. In A. M. Stephen, G. O. Phillips, & P. A. Williams (Eds.), Food polysaccharides and their applications (2nd ed., pp. 289e334). Florida: CRC Press. Ganzevles, R. A., Stuart, M. A. C., van Vliet, T., & de Jongh, H. H. (2006). Use of polysaccharides to control protein adsorption to the airewater interface. Food Hydrocolloids, 20(6), 872e878. Hadian, M., Hosseini, S. M. H., Farahnaky, A., Mesbahi, G. R., Yousefi, G. H., & Saboury, A. A. (2016). Isothermal titration calorimetric and spectroscopic studies of b-lactoglobulin-water-soluble fraction of Persian gum interaction in aqueous solution. Food Hydrocolloids, 55, 108e118. Hosseini, S. M. H., Emam-Djomeh, Z., Negahdarifar, M., Sepeidnameh, M., Razavi, S. H., & Van der Meeren, P. (2016). Polysaccharide type and concentration affect nanocomplex formation in associative mixture with b-lactoglobulin. International Journal of Biological Macromolecules, 93, 724e730. Hosseini, S. M. H., Emam-Djomeh, Z., Razavi, S. H., Moosavi-Movahedi, A. A., Saboury, A. A., Atri, M. S., et al. (2013). b-Lactoglobulinesodium alginate interaction as affected by polysaccharide depolymerization using high intensity ultrasound. Food Hydrocolloids, 32(2), 235e244. Hosseini, S. M. H., Emam-Djomeh, Z., Sabatino, P., & Van der Meeren, P. (2015). Nanocomplexes arising from protein-polysaccharide electrostatic interaction as a promising carrier for nutraceutical compounds. Food Hydrocolloids, 50, 16e26. Howell, N. K. (2006). Interaction of proteins with selected small molecules. In A. Gaonkar, & A. McPherson (Eds.), Ingredient interactions: Effects on food quality (2nd ed., pp. 309e341). Florida: CRC Press. Huang, X., Huang, X., Gong, Y., Xiao, H., McClements, D. J., & Hu, K. (2016). Enhancement of curcumin water dispersibility and antioxidant activity using coreeshell proteinepolysaccharide nanoparticles. Food Research International, 87, 1e9. Hu, K., Huang, X., Gao, Y., Huang, X., Xiao, H., & McClements, D. J. (2015). Coreeshell biopolymer nanoparticle delivery systems: Synthesis and characterization of curcumin fortified zeinepectin nanoparticles. Food Chemistry, 182, 275e281. Jourghanian, P., Ghaffari, S., Ardjmand, M., Haghighat, S., & Mohammadnejad, M. (2016). Sustained release curcumin loaded solid lipid nanoparticles. Advanced Pharmaceutical Bulletin, 6(1), 17e21. Jun-xia, X., Hai-yan, Y., & Jian, Y. (2011). Microencapsulation of sweet orange oil by complex coacervation with soybean protein isolate gum Arabic. Food Chemistry, 125, 1267e1272. Kontopidis, G., Holt, C., & Sawyer, L. (2004). Invited review: b-lactoglobulin: Binding properties, structure, and function. Journal of Dairy Science, 87(4), 785e796. de Kruif, C. G., Weinbreck, F., & de Vries, R. (2004). Complex coacervation of proteins and anionic polysaccharides. Current Opinion in Colloid & Interface Science, 9(5), 340e349. Kumar Das, R., Kasoju, N., & Bora, U. (2010). Encapsulation of curcumin in alginatechitosan-pluronic composite nanoparticles for delivery to cancer cells. Nanomedicine: Nanotechnology, Biology, and Medicine, 6, 153e160. Kumar, A., Dorga, S., & Prakash, A. (2009). Protective effect of curcumin (Curcuma longa), against aluminium toxicity: Possible behavioral and biochemical
224
S.F. Mirpoor et al. / Food Hydrocolloids 71 (2017) 216e224
alterations in rats. Behavioural Brain Research, 205(2), 384e390. Lee, B. H., Choi, H. A., Kim, M. R., & Hong, J. (2013). Changes in chemical stability and bioactivities of curcumin by ultraviolet radiation. Food Science and Biotechnology, 22(1), 279e282. Maheshwari, R. K., Singh, A. K., Gaddipati, J., & Srimal, R. C. (2006). Multiple biological activities of curcumin: A short review. Life Sciences, 78(18), 2081e2087. Matalanis, A., Jones, O. G., & McClements, D. J. (2011). Structured biopolymer-based delivery systems for encapsulation, protection, and release of lipophilic compounds. Food Hydrocolloids, 25(8), 1865e1880. Paraskevopoulou, A., & Kiosseoglou, V. (2013). Interfacial properties of biopolymers, emulsions, and emulsifiers. In C. Chassenieux, D. Durand, P. Jyotishkumar, & S. Thomas (Eds.), Handbook of biopolymer-based Materials: From blends and composites to gels and complex networks (pp. 717e740). Weinheim: Wiley. Perez, A. A., Sponton, O. E., Andermatten, R. B., Rubiolo, A. C., & Santiago, L. G. (2015). Biopolymer nanoparticles designed for polyunsaturated fatty acid vehiculization: Proteinepolysaccharide ratio study. Food Chemistry, 188, 543e550. Prasad, S., Gupta, S. C., Tyagi, A. K., & Aggarwal, B. B. (2014). Curcumin, a component of golden spice: From bedside to bench and back. Biotechnology Advances, 32(6), 1053e1064. Reddy, I. M., Kella, N. K. D., & Kinsella, J. E. (1998). Structural and conformational basis of the resistance of b-lactoglobulin to peptic and chymotryptic digestion. Journal of Agricultural and Food Chemistry, 36, 737e741. Riihim€ aki, L. H., Vainio, M. J., Heikura, J. M., Valkonen, K. H., Virtanen, V. T., & Vuorela, P. M. (2008). Binding of phenolic compounds and their derivatives to bovine and reindeer b-lactoglobulin. Journal of Agricultural and Food Chemistry, 56(17), 7721e7729. Ron, N., Zimet, P., Bargarum, J., & Livney, Y. D. (2010). Beta-lactoglobulinepolysaccharide complexes as nanovehicles for hydrophobic nutraceuticals in non-fat foods and clear beverages. International Dairy Journal, 20(10), 686e693. Sagalowicz, L., & Leser, M. E. (2010). Delivery systems for liquid food products. Current Opinion in Colloid & Interface Science, 15(1), 61e72. Sarika, P. R., James, N. R., & Raj, D. K. (2016). Preparation, characterization and biological evaluation of curcumin loaded alginate aldehydeegelatin nanogels. Materials Science and Engineering: C, 68, 251e257. Sarika, P. R., & Nirmala, R. J. (2016). Curcumin loaded gum Arabic aldehyde-gelatin nanogels for breast cancer therapy. Materials Science and Engineering C, 65,
331e337. Schmitt, C., Sanchez, C., Desobry-Banon, S., & Hardy, J. (1998). Structure and technofunctional properties of protein-polysaccharide complexes: A review. Critical Reviews in Food Science and Nutrition, 38(8), 689e753. Shaikh, J., Ankola, D. D., Beniwal, V., Singh, D., & Ravi Kumar, M. N. V. (2009). Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when compared to curcumin administered with piperine as absorption enhancer. European Journal of Pharmaceutical Sciences, 37, 223e230. Staszewski, M. V., Jagus, R. J., & Pilosof, A. M. R. (2011). Characterization of whey protein-polyphenol interactions by dynamic light scattering. In Presented in part at 11th international congress on engineering and food. Athens, Greece, May. Tolstoguzov, V. (2003). Some thermodynamic considerations in food formulation. Food Hydrocolloids, 17(1), 1e23. Turgeon, S. L., & Laneuville, S. I. (2009). Protein þ polysaccharide coacervates and complexes: From scientific background to their application as functional ingredients in food products. In S. Kasapis, I. T. Norton, & J. B. Ubbink (Eds.), Modern biopolymer science (pp. 327e363). New York: Elsevier. Weinbreck, F., Nieuwenhuijse, H., Robijn, G. W., & de Kruif, C. G. (2003). Complex formation of whey proteins: Exocellular polysaccharide EPS B40. Langmuir, 19(22), 9404e9410. Wilska-Jeszka, J. (2007). Food colorants. In Z. E. Sikorski (Ed.), Chemical and functional properties of food components (3rd ed., pp. 245e274). Florida: CRC Press. Xiao, J., Nian, S., & Huang, Q. (2015). Assembly of kafirin/carboxymethyl chitosan nanoparticles to enhance the cellular uptake of curcumin. Food Hydrocolloids, 51, 166e175. Xu, W., Jin, W., Zhang, C., Li, Z., Lin, L., Huang, Q., et al. (2014). Curcumin loaded and protective system based on complex of k-carrageenan and lysozyme. Food Research International, 59, 61e66. Ye, A. (2008). Complexation between milk proteins and polysaccharides via electrostatic interaction: Principles and applicationsea review. International Journal of Food Science & Technology, 43(3), 406e415. Zhou, H., Sun, X., Zhang, L., Zhang, P., Li, J., & Liu, Y. N. (2012). Fabrication of biopolymeric complex coacervation core micelles for efficient tea polyphenol delivery via a green process. Langmuir, 28(41), 14553e14561. Zimet, P., & Livney, Y. D. (2009). Beta-lactoglobulin and its nanocomplexes with pectin as vehicles for u-3 polyunsaturated fatty acids. Food Hydrocolloids, 23(4), 1120e1126.
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Mixed biopolymer nanocomplexes conferred physicochemical stability and sustained release behavior to introduced curcumin Seyedeh Fatemeh Mirpoor a, Seyed Mohammad Hashem Hosseini a, *, Gholam Hossein Yousefi b, c a b c
Department of Food Science and Technology, School of Agriculture, Shiraz University, Shiraz, Iran Department of Pharmaceutics, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Sciences, Shiraz, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2017 Received in revised form 17 April 2017 Accepted 17 May 2017 Available online 20 May 2017
Beta-lactoglobulin (BLG)-sodium alginate (ALG) electrostatic nanocomplexes were utilized to encapsulate and deliver curcumin in clear acidic media. Relative viscosity measurements confirmed that ALG wrapping around the globular BLG molecules occurred in the aqueous biopolymer mixture upon acidification. Curcumin was efficiently entrapped within developed green delivery systems. The encapsulation efficiency (EE) of curcumin was around 98%. The carriers were efficiently able to retain the curcumin inside over time. The physical stability of hydrophobic curcumin toward precipitation in aqueous solution was significantly improved after introduction into nanostructures. Moreover, nanocomplexes conferred considerable protection to curcumin against heat-induced degradation. At the end of 15-day storage at 45 C, the amounts of curcumin loaded in nanocomplexes were significantly (p < 0.05) larger than those loaded in blank samples. Non-significant changes in the particle size after high temperature short time (HTST) heat treatment indicated that the presence of polysaccharide shell around the protein core increased the thermal stability of BLG at acidic conditions. No release occurred in simulated fasting and non-fasting gastric conditions. However, a sustained release behavior (78.5% during 12 h) was observed in simulated intestinal fluid. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Complex coacervation Wrapping phenomenon Curcumin Nanoparticles Encapsulation Controlled release
1. Introduction Curcumin is a low molecular weight polyphenol present in yellow rhizome of Curcuma longa (Prasad, Gupta, Tyagi, & Aggarwal, 2014). This bright orange-yellow pigment has been used for centuries as a condiment and a medicinal plant (Shaikh, Ankola, Beniwal, Singh, & Ravi Kumar, 2009). A wide range of pharmacological activities such as anticancer, antioxidant, antiinflammatory, antibacterial, wound healing and anti-amyloid properties have been ascribed to this lipophilic fluorescent molecule (Maheshwari, Singh, Gaddipati, & Srimal, 2006; Kumar, Dorga, & Prakash, 2009). Despite multiple medicinal benefits, curcumin has very low bioavailability upon oral administration and hence low clinical efficacy. Curcumin suffers from low water-solubility and poor chemical stability toward light (Jourghanian, Ghaffari,
* Corresponding author. E-mail address: [email protected] (S.M.H. Hosseini). http://dx.doi.org/10.1016/j.foodhyd.2017.05.021 0268-005X/© 2017 Elsevier Ltd. All rights reserved.
Ardjmand, Haghighat, & Mohammadnejad, 2016). Moreover, its conjugated diene structure is sensitive to degradation at neutral to basic pH conditions; therefore, curcumin stability increases by decreasing pH (Shaikh et al., 2009). Bioactive-loaded delivery systems can be used for fortification purposes of various types (e.g., solid, semi-solid and liquid) of food products and supplements. Fortification of low fat or fat-free aqueous-based liquid food products (particularly clear beverages) using hydrophobic bioactives is more challenging than the other fields of fortification (Sagalowicz & Leser, 2010; Matalanis, Jones, & McClements, 2011); The designed carrier system must be incorporated in sufficient amount into liquid food without losing the protective effects conferred to the core. Moreover, the carrier should not adversely affect the optical properties of final product. Sub-micrometer (and preferably nano) scale delivery systems are the best candidates for this purpose, provided having the ability to keep the wall integrity after incorporating into aqueous system. Some encapsulation systems such as those prepared by spray drying technique usually lose the wall integrity just after contact
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with water. In-situ formation of carriers in large amounts of water may help to keep the wall integrity. Therefore, emulsion- and surfactant-based delivery systems such as nanoemulsion, microemulsion and nanoliposome can be as carriers in the fortification of liquid systems. However, they generally suffer from low loading capacity, the presence of organic solvents and inappropriate taste characteristics as a result of large amounts of surfactants (Sagalowicz & Leser, 2010). Colloidal biopolymer structures, assembled from individual or mixed biopolymer molecules, can offer many advantages including protection from the loss of bioactivity under the effect of harsh environmental factors, masking unpleasant tastes and targeted delivery (Arroyo-Maya & McClements, 2015; Perez, Sponton, Andermatten, Rubiolo, & Santiago, 2015). Complex coacervation (thermodynamic compatibility) is a catch-all term for the spontaneous association of oppositely charged biopolymers (usually proteins and polysaccharide). This phenomenon occurs at low concentrations (<3e4.5%) and at pH values between protein's isoelectric point (pI) and the pKa of polysaccharide (Ganzevles, Stuart, van Vliet, & de Jongh, 2006). During acidification of a mixture of protein/anionic polysaccharide, a soluble complex at a molecular level is firstly formed, followed by formation of coacervates and/or fractal aggregates (depending on the involved contributors) upon further reduction in pH (Paraskevopoulou & Kiosseoglou, 2013). When polysaccharide is present in excess amount, soluble nanocomplexes may be developed even at low pH conditions. These nanocomplexes may have potential applications in the fortification purposes of clear liquid food products (using hydrophobic bioactives) and also in the lipophilic drug delivery systems. Recently, the application of biopolymers for the encapsulation of curcumin has been extensively studied. Sarika and Nirmala (2016) and Sarika, James, and Raj (2016) studied the application of oxidized gum Arabic and alginate-gelatin conjugate for the delivery of curcumin, respectively. In spite of promising results, utilization of toxic organic solvents limits their application in food products. Self-assembly of k-carrageenan and lysozyme was used to encapsulate curcumin (Xu et al., 2014). Large amount of encapsulation efficiency (EE) and increased stability against heat and UV radiation were reported. However, the strong electrostatic interaction between anionic k-carrageenan and cationic lysozyme at neutral pH may prevent the efficient release of curcumin in its site of action (i.e., intestine). Kafirin and kafirin/carboxymethyl-chitosan nanoparticles were successfully used to enhance the cellular uptake of curcumin (Xiao, Nian, & Huang, 2015). Hu et al. (2015) reported that curcumin is in an amorphous rather than crystalline form after nanoencapsulation within coreeshell nanoparticles fabricated from zein and pectin. To our knowledge, the application of stable nanocomplexes arising from beta-lactoglobulin/sodium alginate interaction in the nanoencapsulation of curcumin has not been studied yet. Stable nanocomplexes can be defined as very small complex coacervates with a slow rate of coalescence and macroscopic separation (Hosseini et al., 2016). Therefore, this study was conducted to evaluate the efficacy of soluble nanostructures, developed from electrostatic soft-condensation of beta-lactoglobulin (BLG) and sodium alginate (ALG), in various aspects of encapsulation, protection, delivery, heat stability and controlled release of curcumin as a hydrophobic nutraceutical model. Our hypothesis was that the intrinsic transporting properties of beta-lactoglobulin as a member of lipocalin protein family may be used for developing protein-curcumin complexes (cores); which could then be overprotected by deposition of an anionic polysaccharide shell at acidic pH levels.
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2. Materials and methods 2.1. Materials Sodium alginate (ALG, MW: 200 kDa) with a mannuronate:guluronate (M:G) ratio of 0.6 was obtained from BDH Co. (Poole, UK). The amounts of carbohydrate, moisture and total ash were 66.3, 14.2 and 9.5 (%w/w), respectively. Beta-lactoglobulin (BLG, MW: 18.4 kDa, composition (%w/w): 93% BLG, 5.4% moisture and 1.6% ash), pepsin (extracted from porcine gastric mucosa, activity: 600e1800 unit/mg protein), pancreatin extracted from porcine pancreas (>3x USP specifications), glucono-d-lactone (GDL) and curcumin were obtained from Sigma Chemical co. (St. Louis, MO, USA). Citric acid, analytical grade hydrochloric acid, sodium azide and absolute ethanol were purchased from Merck Co. (Darmstadt, Germany). In this study, double distilled water (DDW) was used to prepare all dispersions. 2.2. Dispersion preparation Biopolymers including ALG (0.2 %w/w) and BLG (0.1 %w/w) were separately dissolved in DDW already containing 0.03 %w/w sodium azide. The dispersions were stirred at 200e250 rpm at room temperature for 4e5 h and then stored at 4 C overnight to ensure complete hydration. 2.3. Changes in the absorbance as a function of pH Formation of complexes was studied by turbidimetric analysis. Biopolymer mixtures were prepared by mixing the required amounts of stock biopolymer dispersions so that to obtain a 2:1 protein to polysaccharide mixing ratio (MR) and total biopolymer concentration (TBC) of 0.075 %w/w. The mixture was then gradually titrated by HCl; while, gentle magnetic stirring was carried out for 2 min at each pH level before reduction to the next pH (of about 0.1 unit difference). Titrations were done at room temperature and dilution effects were considered to be negligible. Changes in the absorbance were monitored using a UV/visible light spectrophotometer at 600 nm (UNICO UV-2100, USA). Distilled water was used as blank. 2.4. Changes in the relative viscosity during complexation We assumed that changes in the flexibility and hence the conformation of biopolymers during acidification and subsequent complexation might influence the viscosity of the system. Therefore, the relative viscosity of ALG/BLG mixture (TBC 0.075 %w/w and BLG:ALG MR 2:1) was studied as a function of pH using the method described by Weinbreck, Nieuwenhuijse, Robijn, and de Kruif (2003) with a slight modification. To measure the relative viscosity, a U-tube capillary viscometer (No. 518 10, Schott Ger€ ate, Germany) was used at 25 ± 0.1 C. In-situ acidification was carried out using GDL (0.1 %w/w). Relative viscosity (t/t0) was plotted against the pH of the sample; where t and t0 are the flow times (second) of sample and pure solvent (water), respectively. BLG and ALG blank dispersions were also studied at their relative concentrations (0.05 and 0.025 %w/w, respectively). The results were then indirectly related to the complexation phenomenon. 2.5. Curcumin nanoencapsulation A fresh alcoholic solution of curcumin was gradually added into BLG dispersion (0.025 %w/w) so that to obtain a curcumin:BLG molar ratio of 1:1. This molar ratio was selected based on our previously published fluorimetry results regarding the interaction
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between BLG and curcumin (Hosseini, Emam-Djomeh, Sabatino, & Van der Meeren, 2015). An increase in the molar ratio may lead to protein overloading and hence ejection of curcumin from the inside of the delivery system over time. After stirring for 30 min, different amounts of ALG dispersion were added into curcumin/BLG complexes in order to reach two different TBCs, namely 0.075 and 0.044 %w/w (corresponding to MRs of 0.5 and 1.33, respectively). Constant final volumes were obtained by DDW addition. Post blending acidification method was used to complete the nanoencapsulation process through adjusting the pH to 4 and then stirring for additional 30 min. Nanoencapsulated curcumin dispersion was equilibrated overnight at room temperature before analysis. For each sample, three blanks including a DDW (biopolymer-free) sample containing equal concentration of curcumin, ALG-free curcumin/ BLG dispersion and curcumin-free mixed biopolymer dispersion were also prepared and treated in a similar manner. 2.6. Measurement of encapsulation parameters The efficiency of curcumin entrapment within nanocomplexes was determined by separating free (unloaded) curcumin from curcumin-loaded nanocomplexes using Amicon Ultra centrifugal filter units (MW cut off 10 kDa, Merck Millipore Ltd., Ireland). After centrifugation (4000 g, 30 min, 25 C) of nanocomplexes (4 ml), the concentration of free curcumin in permeate was determined from a standard curve. To develop the standard curve, the standard solutions of curcumin were prepared by curcumin dissolution in 50:50 water:ethanol mixture and then measuring the absorbance at 468 nm. To determine the curcumin concentration in permeate, absolute ethanol was added into the permeate so that to obtain a water:ethanol ratio of 50:50 followed by measuring the absorbance. The encapsulation (entrapment) efficiency (EE) was calculated according to the following equation (1).
EE% ¼ Mic Mpc
Mic 100
(1)
where, Mic is the initial mass of curcumin present in the sample (before centrifugation); Mpc is the mass of free curcumin determined in permeate (after centrifugation). Loading capacity (LC) was calculated according to equation (2).
LC% ¼ ðmass of curcumin=mass of BLG þ ALGÞ 100
water ratio of 4:1 (80:20) resulted in the complete extraction of curcumin from the nanocarriers. Curcumin was not detected in the precipitates after centrifugation and subsequent washing with ethanol. Therefore, after sampling (4 ml), absolute ethanol was added (16 ml) and mixed thoroughly. After curcumin extraction, the biopolymer precipitates were settled by centrifugation at 1400 rpm and 25 C for 20 min. Distilled water was added to the supernatant to adjust the ethanol content to 50:50. After that, curcumin was quantified using the standard curve (section 2.6). 2.9. HTST heat processing effects on particle size and surface potential of loaded and un-loaded nanocomplexes To determine the heat processing effects, thin layers of different liquid samples were provided in glass vials and then subjected to heating in water bath pre-set at 75 C for 30 s. After cooling, the particle size and the surface potential was measured. Volumeweighted mean diameter was determined using a dynamic light scattering (DLS) instrument (Nanotrac Wave, Microtrac, USA) at a scattering angle of 90 and 25 C. The amounts of the surface potential (mV) of complexes were determined using a Microtrac Zeta Check (Microtrac, Germany) at 25 C. 2.10. In-vitro release of nanoencapsulated quercetin In-vitro release studies were performed according to the method described by Chen and Subirade (2006). Simulation of fasting and non-fasting gastric conditions was performed by mixing equal volumes of sample and an HCl solution (containing pepsin at 0.1 %w/w concentration) at pH values of 1.2 and 4, respectively. The mixture was incubated in a shaker at 37 C for 6 h in dark conditions. To simulate intestinal conditions, a mixture of equal volumes of sample and phosphate-buffered solution (PBS, 10 mM, pH 7, containing 1 %w/w pancreatin) was prepared and then incubated at 37 C for 12 h in dark conditions. Sampling (4 ml) was performed during 1-h intervals. The released curcumin was then separated from the mixture using Amicon filter units centrifuged at 6000 g and 4 C for 30 min. Low temperature was used to stop the digestion process. Absolute ethanol was added into the permeate so that to reach a 50:50 water:ethanol ratio. Curcumin concentration was determined from the standard curve after measuring the absorbance at 468 nm (section 2.6).
(2)
The ability of biopolymer nanocomplexes to retain the curcumin inside over time (loading or delivery efficiency, DE) was determined by measuring the EE during 1-month storage. 2.7. Physical stability measurement Physical stability of curcumin-loaded nanostructures against precipitation was evaluated by measuring the absorbance at 600 nm using a spectrophotometer during 1-month storage at 25 C. The samples were stored in dark to prevent from lightinduced degradation. 2.8. Curcumin protection An enhanced shelf-life stress test described by Zimet and Livney (2009) was used with a slight modification to determine the protective effects conferred by nanocomplexes to curcumin. After preparation at room temperature, samples containing nanoencapsulated curcumin dispersion and their respective blanks were placed in dark glass containers and shaken in a water bath at 45 C for 15 days. Our preliminary experiments showed that an ethanol to
2.11. Statistical analysis The results obtained in this work were reported as mean ± standard deviation. Duncan's multiple range tests at a probability of 0.95 were used to compare the means using the SPSS software (version 21, IBM Corp. USA). 3. Results and discussion 3.1. Changes in the absorbance and relative viscosity during complexation process The ionization degree of functional side groups of biopolymers is influenced by pH (Ye, 2008). Fig. 1a depicts the phase diagram and critical pH values upon acidification. At pH values more than 5.2e5.3, both biopolymers had negative charges; therefore, no significant increase in the absorbance of mixture was observed due to the strong electrostatic repulsion. Formation of soluble complexes at pHc (~5.0e5.2) is the first experimentally detectable noncovalent interaction in BLG/ALG mixture. The formation of soluble complexes occurs at molecular levels and hence the value of pHc is independent from TBC and MR but not ionic strength (Hadian et al.,
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decreasing the effective volume. The relative viscosity of the blank BLG dispersion remained unchanged, which was due to the globular structure of protein molecules (Kontopidis, Holt, & Sawyer, 2004). The viscosity of biopolymer mixture was lower than that of blank ALG dispersion. A possible explanation is that ALG molecules in biopolymer mixture had less effective volumes (or less stretched conformation) in the presence of protein molecules even in the non-complexed conditions. The difference in the effective volume could be resulted from the competition for the solvent molecules or the predominance of thermodynamic incompatibility (i.e., during segregative phase separation polysaccharide molecules may acquire a different conformation after surrounding by molecules of their own type). The viscosity of the ALG/BLG mixture decreased as a function of pH. The sharp decrease (breaking point) in Fig. 1b coincided with the initiation of the interactions at molecular levels (i.e., formation of soluble complexes) in Fig. 1a. The release of counterions upon complexation would cause a slight increase in the ionic strength (de Kruif, Weinbreck, & de Vries, 2004). An increase in the ionic strength increases the chain flexibility (i.e., less viscosity). Another explanation is that mutual neutralization decreases the net charge, hydrophilicity and chain stiffness of the junction zones and resulted in ALG bending around BLG molecules and developing complexes with a compact structure (Tolstoguzov, 2003). It should be pointed out that the alginate type used in this study was rich in guluronate (G) residues, which are responsible for the stiff nature of the alginate chain (Draget, Moe, Skjåk-Bræk, & Smidsrød, 2006). The decrease in the relative viscosity was a proof for the wrapping phenomenon in biopolymer mixture during acidification and formation of soft-condensed coreshell type structures. Further reduction in pH was not possible due to the complex growth and U-tube fouling. Fig. 1. Changes in the (a) absorbance and (b) relative viscosity values of mixed biopolymer dispersion as a function of pH measured at 25 C.
2016; Turgeon & Laneuville, 2009). Formation of soluble complexes at pHc values higher than the pI of BLG (z4.9e5, where the protein net charge is negative) could be ascribed either to the presence of localized basic (positive) peptides (Turgeon & Laneuville, 2009; Weinbreck et al., 2003) or to the inherent electrical capacitance of globular proteins leading to regulate the charge under the effect of polysaccharide's negative charge (Dickinson, 2008). Further decrease in pH resulted in phase separation and formation of interpolymeric (insoluble) complexes at pH41 and detected by an abrupt increase in the absorbance. At this pH, the solution changed from transparent to cloudy. Cloudiness was due to the nucleation and growth of sufficiently large biopolymer complexes capable of light scattering (Bengoechea, Jones, Guerrero, & McClements, 2011). Absorbance increased steadily (due to further complex growth) and reached to a maximum at pHopt (2.2e1.8) corresponding to an electrical equivalence. Dissociation of complexes at pH values below pHopt led to formation of another critical point denoted as pH42. Mixed dispersion became fully transparent close to pH 0.5, indicating complete dissociation as a result of deionization of alginate's carboxyl groups (eCOO- þ Hþ 4 eCOOH). Size and conformation of biomacromolecules are crucial to the viscosity of dilute biopolymer dispersions (Schmitt, Sanchez, DesobryBanon, & Hardy, 1998). Fig. 1b represents the changes in the relative viscosity of ALG/BLG mixture (TBC 0.075 %w/w, BLG:ALG MR 2:1) and the respective blanks as a function of pH. The relative viscosity of the blank dispersion of polysaccharide decreased during pH reduction. The decreased charge density of ALG reduced the intramolecular repulsion between similarly charged monomers. This phenomenon resulted in backbone bending and hence
3.2. Encapsulation parameters Low water solubility of curcumin limits its bioavailability and hence its incorporation as a nutraceutical into functional foods. Curcumin solubilization and vehiculization using different encapsulation systems is an appropriate solution for this challenge. To determine the appropriate delivery system, the entrapment characteristics are of paramount importance. Encapsulation efficiency (EE) provides an idea about the fraction of bioactive successfully entrapped within nanovehicles. The amounts of EEs were around 98% for both MRs of 0.5 and 1.33. These large amounts of EEs revealed high affinity of curcumin toward mixed biopolymer nanocomplexes. Since curcumin was added into BLG dispersion in equimolar concentration, the amounts of EEs obtained in this work were significantly larger than those reported in previous studies for curcumin and other nutraceutical compounds: 71± 0.02% for curcumin encapsulation within sodium k-carrageenan/lysozyme complexes (Xu et al., 2014); 85% for 10% sweet orange oil loaded microcapsules prepared by complex coacervation of soybean protein isolate/gum Arabic (Jun-xia, Hai-yan, & Jian, 2011); 70% for tea polyphenol encapsulation within complex coacervate core micelles (Zhou et al., 2012); 64% for DHA entrapment within BLG/pectin nanocomplexes (Zimet & Livney, 2009) and 83.7% and 93.6% for ferulic acid and quercetin entrapment within pullulan electrospun fibers, respectively (Aceituno-Medina, Mendoza, Rodríguez, pez-Rubio, 2015). Huang et al. (2016) reported that Lagaron, & Lo curcumin encapsulation within core-shell nanostructures developed from a mixture of pectin (70%) and alginate (30%) (as the shell) and zein nanoparticles (as the core) resulted in higher antioxidant and free radical scavenging activities than curcumin dissolution in ethanol. Loading capacity (LC) gives an idea about the nutraceutical content of delivery system. As mentioned already the amounts of total biopolymers were 0.044 and 0.075 g per 100 g of
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Fig. 2. Encapsulation efficiency (EE) profiles of curcumin-loaded nanocomplexes as a function of time obtained at two different MRs.
dispersion. Therefore, LCs were 1.14% and 0.67% at TBCs of 0.044 and 0.075%, respectively (taking into account the mass of the added curcumin: 0.0005 g/100 g of dispersion). The LC was lower than normal capacity ranges (5e10%); which should be addressed in future studies. The results of loading (delivery) efficiency (DE), indicating the ability of nanocomplexes to retain the curcumin inside over time, are shown in Fig. 2. The protective effects of delivery systems would be lost upon nutraceutical ejection from the carrier. A very slight decrease in EE was observed for both MRs during a 31day storage period at room temperature. The decreasing trend was attributed to the removing of loosely bound curcumin from biopolymer nanocomplexes likely due to unknown re-structuration phenomena. 3.3. Physical stability of nanoencapsulated curcumin dispersion over time The effects of BLG/ALG self assembly on the physical stability of nanoencapsulated curcumin dispersion are shown in Fig. 3 and Fig. 4. In Fig. 3, lower physical stability could be detected by a rapid decrease in the absorbance. The highest physical stability was observed in the curcumin-free biopolymer mixtures. Large electrostatic repulsion between nanoparticles (discussed later in section 3.5) prevented them from aggregation and hence sedimentation. The stability profiles exhibited that curcumin encapsulation within BLG/ALG nanocomplexes led to higher
Fig. 3. Physical stability of various delivery systems and their respective blanks as a function of time.
physical stability than its entrapment within single BLG molecules (Figs. 3 and 4). The instability of curcumin-loaded BLG sample (denoted as sample III in Fig. 4) could be attributed to the proteinpolyphenol interactions and also to the proximity of the sample pH to the pI of the protein. Polyphenol-induced cross-linking of separate protein molecules resulted in the formation of curcuminBLG large complexes and hence rapid precipitation (Figs. 3 and 4). This interaction is related to the polyphenols molecular size and number of binding locations available for association with proteins (Howell, 2006). Staszewski, Jagus, and Pilosof (2011) concluded that the initial binding of polyphenols to whey proteins is essentially a non-selective hydrophobically-driven interaction; but the formation of insoluble complexes is likely determined by surface charge effects. The electrostatic repulsion conferred by ALG shell to the curcumin-loaded BLG core resulted in higher physical stability of curcumin-loaded ALG/BLG nanostructures than curcumin-loaded BLG (Figs. 3 and 4). The physical stability of curcumin-loaded nanocomplexes obtained at MR of 1:2 (0.5) was relatively higher than that obtained at MR of 4:3 (Fig. 3). Higher stability at lower BLG:ALG MR was attributed to the presence of ALG in sufficiently large (excess) amount leading to effective coating of curcuminloaded protein core. As shown in Fig. 4, no precipitation could be observed in the sample denoted as V (the alcoholic solution of curcumin in water). It should be noted that the absence of precipitation in this sample was not a proof for the system stability. As discussed later, this sample had the lowest chemical stability. Moreover, most of the curcumin present in this sample adhered to the inside wall of the cell. 3.4. Curcumin protection Due to having active phenolic hydroxyl groups in the molecular structure, polyphenols stability is influenced by a variety of factors such as pH, temperature, light and UV radiation (Lee, Choi, Kim, & Hong, 2013). Fig. 5 shows the amounts of curcumin (%) remained in different samples during storage for 15 days at 45 C. The amount of the remained curcumin in the sample denoted as Blank 1 decreased rapidly and reached to 34.5% of initial curcumin content (i.e., 65.5% loss) after 4 days of production. This blank was prepared by dissipating the alcoholic solution of curcumin in water. Curcumin loss was continued but at a decreased degradation rate and reached to 21% of initial curcumin content. Changes in the rate of curcumin loss during storage could be attributed to the protection conferred by the self-association of hydrophobic curcumin in aqueous solution. In aqueous environments, lipophilic compounds such as carotenoids tend to form aggregates or adhere to the surfaces (Wilska-Jeszka, 2007). A similar pattern in curcumin degradation was observed after entrapment by protein particles. After 4 days of storage, the curcumin loss was 43% and reached to 74% at the end of storage. The BLG-induced protection of curcumin against heat was lower than the protection of other nutraceuticals reported already (Ron, Zimet, Bargarum, & Livney, 2010; Zimet & Livney, 2009). Different bioactives have different binding locations in the BLG molecule. It has been reported that the binding location of phenolic compounds is the hydrophobic surface patches but not the central €ki et al., 2008). calyx (the main binding site of BLG) (Riihima Different binding locations may have different protection effects. ALG/BLG core-shell nanostructures could effectively prevent curcumin loss during storage. No significant difference was observed between two MRs. The amount of curcumin loss at the end of storage was about 40%; which means the stability of curcumin loaded in mixed biopolymer nanostructures was improved about 4 fold and 2.3 fold as compared with the stability provided by curcumin self-association (in Blank 1) and by individual BLG molecules (in Blank 2), respectively. These results clearly indicated that the
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Fig. 4. Visual observation of the physical stability of various delivery systems and their respective blanks toward precipitation at different storage times; I1:2 and I4:3 indicate curcumin-free mixed biopolymer nanocomplexes fabricated at BLG:ALG mixing ratios of 1:2 and 4:3, respectively; II1:2 and II4:3 indicate curcumin-loaded mixed biopolymer nanocomplexes fabricated at BLG:ALG mixing ratios of 1:2 and 4:3, respectively; III indicates curcumin loaded BLG (Blank 2); V indicates Blank 1 (alcoholic solution of curcumin in water).
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Fig. 5. Curcumin bioprotection in nanostructures and in respective blanks as a function of time.
Table 1 Changes in the surface potential and particle size of ALG/BLG nanostructures under the effects of curcumin loading and HTST heat processing. ALG þ BLG mixture type
A: surface potential (mV) TBC 0.044 %w/w, MR 4:3 TBC 0.075 %w/w, MR 1:2 B: particle size (nm) TBC 0.044 %w/w, MR 4:3 TBC 0.075 %w/w, MR 1:2
Control sample
Curcumin-loaded sample HTST ()
HTST (þ)
98.9Bb 191.8Aa
109.9Ab 193.8Aa
108.4Ab 169.3Ba
81.1Cb 188.9Aa
109.2Bb 139.5Ba
114.5Ab 141.4Ba
In each row, different superscript capital letters indicate significant differences (p < 0.05). In each column, different subscript small letters indicate significant differences (p < 0.05). HTST () and HTST (þ) indicate samples without and with heat treatment, respectively.
core (curcumin-loaded BLG molecules) could be efficiently overprotected after electrostatic deposition of ALG molecules as a secondary layer (shell). 3.5. HTST heat processing effects on surface potential and particle size of delivery systems In this study, surface potential (mV) (Table 1a) and particle size (nm) (Table 1b) of curcumin-free and curcumin-loaded mixed biopolymer particles (containing fixed BLG concentration) were measured to provide an insight into the stability of delivery system under the effects of curcumin loading and heat processing. The
effects of curcumin loading and heat treatment on the amounts of surface potential and particle size were dependent on the ALG concentration which might lead to different conformations. As reported in Table 1a, decreasing BLG:ALG MR from 1.33 to 0.5 (or increasing the concentration of negatively charged ALG) led to an increase in the negative charge carried by unloaded nanocomplexes. Curcumin encapsulation increased the surface potential value of ALG/BLG nanostructures at MR of 1.33. This phenomenon could be attributed to the changes in conformation of nanoparticles (adopting structures with higher charge density) upon curcumin loading. Curcumin was expected to interact with BLG through hydrophobic interactions. However, electrostatic interactions between curcumin and ionic BLG may also play role in binding mode and location particularly at acidic pH levels (Hosseini et al., 2015). The possible electrostatic attractive interactions between curcumin and cationic BLG could decrease the number of BLG cationic functional groups available for complexation with anionic ALG. Therefore, the fraction of free carboxyl groups of ALG and hence the absolute surface potential of nanoparticles increased after curcumin loading. Changes in the surface potential after curcumin loading were MR-dependent. The increase in the surface potential of BLG/ALG nanostructures at MR of 1.33 was higher than that of mixed biopolymers obtained at MR of 0.5, likely due to the presence of sufficiently large amount of anionic carboxyl groups at lower MR and hence decreasing the effect of curcumin loading on the amount of charge carried by complexes. Similarly, the effect of heat processing on the surface potential absolute values of curcumin-loaded nanocomplexes was MR dependent. We could not find any reasonable explanation for the observed changes. Changes in the particle size under the effects of curcumin loading and heat treatment are shown in Table 1b. A decrease in the MR (i.e., an increase in the weight ratio of ALG to BLG) increased the particle size. Zimet and Livney (2009) concluded that the increased microviscosity as a result of increasing polysaccharide concentration decreases the Brownian motion of particles and results in detecting an apparently increased particle size by DLS. Formation of more opened structures as a result of increasing intramolecular repulsions along ALG backbone (arising from the decreased ratio of BLG molecules attached per ALG chain) are likely another plausible explanation for the increased particle diameter by decreasing MR (Hosseini et al., 2013). Curcumin loading increased the particle size of nanocomplexes developed at MR of 1.33. However, a decrease in the particle size (shrinkage phenomenon) was observed in the MR of 0.5 upon curcumin loading. It seems that the interaction of ALG with curcumin-loaded BLG is different from its interaction with
Fig. 6. Gastric and intestinal release profiles of curcumin from ALG/BLG core-shell nanostructures. The inset shows the gastric release in fasting and non-fasting conditions.
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curcumin-free BLG which should be addressed in future studies. Curcumin might favor the electrostatic interactions between BLG and ALG, likely via the higher accessibility of charges carried by curcumin-BLG complex. Aberkane et al. (2012) reported that the interaction of BLG and total acacia gum increases in the presence of quercetin. In spite of the heat instability of BLG, particle size of curcumin-loaded nanoparticles was not significantly influenced by HTST heat treatment indicating that the ALG shell could effectively protect the curcumin-loaded protein core against heat-induced aggregation. 3.6. Release kinetics of curcumin during in-vitro digestion For each delivery system, the release profile of the encapsulated compound is very important. The designed carrier should be finely tuned so that to release its content at the desired site of gastrointestinal tract (GIT). Prasad et al. (2014) reported that different organs of body have different concentrations of curcumin after its uptake. Most of curcumin are accumulated in liver and intestine. A small quantity could be detected in the other organs (Prasad et al., 2014). To our knowledge, the main absorption site of orally administered curcumin is not precisely detected yet. However, a degree of similarity between the absorption mechanisms of curcumin and other polyphenols (mainly via small intestine) is not beyond the mind. Therefore, the encapsulating system should limit the curcumin release in gastric conditions. To have higher bioavailability, curcumin should be released slowly in the alkaline conditions of intestine. Fig. 6 shows the gastric and intestinal release profiles of quercetin. The inset shows the gastric release profiles at fasting and non-fasting (corresponding to pH values of 1.2 and 4, respectively) conditions. After 6 h, only 3e4% of curcumin was released in simulated gastric fluid. The relatively compact structure of BLG is stable in aqueous acidic solutions. Moreover, BLG is resistant to pepsin proteolysis (Reddy, Kella, & Kinsella, 1998). A relatively higher release could be observed at pH 1.2. Therefore, it can be concluded that the deposition of ALG shell around curcumin-loaded BLG core may also decrease the release of curcumin from its carriers. The relatively increased release of curcumin at fasting gastric conditions (pH 1.2) could be attributed to the dissociation of mixed complexes at very low pH values (ALG unwrapping) (Fig. 1a). Under these conditions, the negative charge of ALG becomes zero as a result of extensive protonation of carboxyl groups. Therefore, biopolymer complexes dissociated after weakening the electrostatic interaction. Curcumin sustained-release (78.5% of total curcumin during 12 h) was occurred in intestinal condition. The dissociation of BLG/ALG complexes at alkaline pH (as a result of electrostatic repulsion) facilitated the pancreatic hydrolysis of BLG and hence controlled release of curcumin. The release profile of curcumin entrapped within dextran sulphate/ chitosan complexes was investigated by Anitha et al. (2011). 70% of curcumin released during 1 week; however, the maximum rate of release was at the first 3 h (Anitha et al., 2011). Kumar Das, Kasoju, and Bora (2010) reported that curcumin release from the alginate/ chitosan/pluronic was about 36% at the first 12 h and reached to 75% after 96 h. 4. Conclusion In conclusion, curcumin-loaded core-shell type nanostructures were developed from ALG/BLG soft nanocomplexes at different MRs and TBCs. Encapsulation of curcumin significantly enhanced its water dispersibility and delivery. The application of a relatively simple and green process using food-grade ingredients, significant bioprotection, physical stability toward heat processing and controlled release characteristics make these soluble
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nanostructures a suitable candidate for the application in the encapsulation technology. However, finding appropriate strategies to increase the loading capacity of nanocomplexes should be addressed in future studies. Acknowledgment This work was financially supported by Shiraz University (Grant number 93GCU3M194065). References Aberkane, L., Jasniewski, J., Gaiani, C., Hussain, R., Scher, J., & Sanchez, C. (2012). Structuration mechanism of b-lactoglobulin e acacia gum assemblies in presence of quercetin. Food Hydrocolloids, 29, 9e20. pez-Rubio, A. Aceituno-Medina, M., Mendoza, S., Rodríguez, B. A., Lagaron, J. M., & Lo (2015). Improved antioxidant capacity of quercetin and ferulic acid during invitro digestion through encapsulation within food-grade electrospun fibers. Journal of Functional Foods, 12, 332e341. Anitha, A., Deepagan, V. G., Divya Rani, V. V., Menon, D., Nair, S. V., & Jayakumar, R. (2011). Preparation, characterization, in vitro drug release and biological studies of curcumin loaded dextran sulphateechitosan nanoparticles. Carbohydrate Polymers, 48, 1158e1164. Arroyo-Maya, I. J., & McClements, D. J. (2015). Biopolymer nanoparticles as potential delivery systems for anthocyanins: Fabrication and properties. Food Research International, 69, 1e8. Bengoechea, C., Jones, O. G., Guerrero, A., & McClements, D. J. (2011). Formation and characterization of lactoferrin/pectin electrostatic complexes: Impact of composition, pH and thermal treatment. Food Hydrocolloids, 25(5), 1227e1232. Chen, L., & Subirade, M. (2006). Alginateewhey protein granular microspheres as oral delivery vehicles for bioactive compounds. Biomaterials, 27(26), 4646e4654. Dickinson, E. (2008). Interfacial structure and stability of food emulsions as affected by proteinepolysaccharide interactions. Soft Matter, 4(5), 932e942. Draget, K. I., Moe, S. T., Skjåk-Bræk, G., & Smidsrød, O. (2006). Alginates. In A. M. Stephen, G. O. Phillips, & P. A. Williams (Eds.), Food polysaccharides and their applications (2nd ed., pp. 289e334). Florida: CRC Press. Ganzevles, R. A., Stuart, M. A. C., van Vliet, T., & de Jongh, H. H. (2006). Use of polysaccharides to control protein adsorption to the airewater interface. Food Hydrocolloids, 20(6), 872e878. Hadian, M., Hosseini, S. M. H., Farahnaky, A., Mesbahi, G. R., Yousefi, G. H., & Saboury, A. A. (2016). Isothermal titration calorimetric and spectroscopic studies of b-lactoglobulin-water-soluble fraction of Persian gum interaction in aqueous solution. Food Hydrocolloids, 55, 108e118. Hosseini, S. M. H., Emam-Djomeh, Z., Negahdarifar, M., Sepeidnameh, M., Razavi, S. H., & Van der Meeren, P. (2016). Polysaccharide type and concentration affect nanocomplex formation in associative mixture with b-lactoglobulin. International Journal of Biological Macromolecules, 93, 724e730. Hosseini, S. M. H., Emam-Djomeh, Z., Razavi, S. H., Moosavi-Movahedi, A. A., Saboury, A. A., Atri, M. S., et al. (2013). b-Lactoglobulinesodium alginate interaction as affected by polysaccharide depolymerization using high intensity ultrasound. Food Hydrocolloids, 32(2), 235e244. Hosseini, S. M. H., Emam-Djomeh, Z., Sabatino, P., & Van der Meeren, P. (2015). Nanocomplexes arising from protein-polysaccharide electrostatic interaction as a promising carrier for nutraceutical compounds. Food Hydrocolloids, 50, 16e26. Howell, N. K. (2006). Interaction of proteins with selected small molecules. In A. Gaonkar, & A. McPherson (Eds.), Ingredient interactions: Effects on food quality (2nd ed., pp. 309e341). Florida: CRC Press. Huang, X., Huang, X., Gong, Y., Xiao, H., McClements, D. J., & Hu, K. (2016). Enhancement of curcumin water dispersibility and antioxidant activity using coreeshell proteinepolysaccharide nanoparticles. Food Research International, 87, 1e9. Hu, K., Huang, X., Gao, Y., Huang, X., Xiao, H., & McClements, D. J. (2015). Coreeshell biopolymer nanoparticle delivery systems: Synthesis and characterization of curcumin fortified zeinepectin nanoparticles. Food Chemistry, 182, 275e281. Jourghanian, P., Ghaffari, S., Ardjmand, M., Haghighat, S., & Mohammadnejad, M. (2016). Sustained release curcumin loaded solid lipid nanoparticles. Advanced Pharmaceutical Bulletin, 6(1), 17e21. Jun-xia, X., Hai-yan, Y., & Jian, Y. (2011). Microencapsulation of sweet orange oil by complex coacervation with soybean protein isolate gum Arabic. Food Chemistry, 125, 1267e1272. Kontopidis, G., Holt, C., & Sawyer, L. (2004). Invited review: b-lactoglobulin: Binding properties, structure, and function. Journal of Dairy Science, 87(4), 785e796. de Kruif, C. G., Weinbreck, F., & de Vries, R. (2004). Complex coacervation of proteins and anionic polysaccharides. Current Opinion in Colloid & Interface Science, 9(5), 340e349. Kumar Das, R., Kasoju, N., & Bora, U. (2010). Encapsulation of curcumin in alginatechitosan-pluronic composite nanoparticles for delivery to cancer cells. Nanomedicine: Nanotechnology, Biology, and Medicine, 6, 153e160. Kumar, A., Dorga, S., & Prakash, A. (2009). Protective effect of curcumin (Curcuma longa), against aluminium toxicity: Possible behavioral and biochemical
224
S.F. Mirpoor et al. / Food Hydrocolloids 71 (2017) 216e224
alterations in rats. Behavioural Brain Research, 205(2), 384e390. Lee, B. H., Choi, H. A., Kim, M. R., & Hong, J. (2013). Changes in chemical stability and bioactivities of curcumin by ultraviolet radiation. Food Science and Biotechnology, 22(1), 279e282. Maheshwari, R. K., Singh, A. K., Gaddipati, J., & Srimal, R. C. (2006). Multiple biological activities of curcumin: A short review. Life Sciences, 78(18), 2081e2087. Matalanis, A., Jones, O. G., & McClements, D. J. (2011). Structured biopolymer-based delivery systems for encapsulation, protection, and release of lipophilic compounds. Food Hydrocolloids, 25(8), 1865e1880. Paraskevopoulou, A., & Kiosseoglou, V. (2013). Interfacial properties of biopolymers, emulsions, and emulsifiers. In C. Chassenieux, D. Durand, P. Jyotishkumar, & S. Thomas (Eds.), Handbook of biopolymer-based Materials: From blends and composites to gels and complex networks (pp. 717e740). Weinheim: Wiley. Perez, A. A., Sponton, O. E., Andermatten, R. B., Rubiolo, A. C., & Santiago, L. G. (2015). Biopolymer nanoparticles designed for polyunsaturated fatty acid vehiculization: Proteinepolysaccharide ratio study. Food Chemistry, 188, 543e550. Prasad, S., Gupta, S. C., Tyagi, A. K., & Aggarwal, B. B. (2014). Curcumin, a component of golden spice: From bedside to bench and back. Biotechnology Advances, 32(6), 1053e1064. Reddy, I. M., Kella, N. K. D., & Kinsella, J. E. (1998). Structural and conformational basis of the resistance of b-lactoglobulin to peptic and chymotryptic digestion. Journal of Agricultural and Food Chemistry, 36, 737e741. Riihim€ aki, L. H., Vainio, M. J., Heikura, J. M., Valkonen, K. H., Virtanen, V. T., & Vuorela, P. M. (2008). Binding of phenolic compounds and their derivatives to bovine and reindeer b-lactoglobulin. Journal of Agricultural and Food Chemistry, 56(17), 7721e7729. Ron, N., Zimet, P., Bargarum, J., & Livney, Y. D. (2010). Beta-lactoglobulinepolysaccharide complexes as nanovehicles for hydrophobic nutraceuticals in non-fat foods and clear beverages. International Dairy Journal, 20(10), 686e693. Sagalowicz, L., & Leser, M. E. (2010). Delivery systems for liquid food products. Current Opinion in Colloid & Interface Science, 15(1), 61e72. Sarika, P. R., James, N. R., & Raj, D. K. (2016). Preparation, characterization and biological evaluation of curcumin loaded alginate aldehydeegelatin nanogels. Materials Science and Engineering: C, 68, 251e257. Sarika, P. R., & Nirmala, R. J. (2016). Curcumin loaded gum Arabic aldehyde-gelatin nanogels for breast cancer therapy. Materials Science and Engineering C, 65,
331e337. Schmitt, C., Sanchez, C., Desobry-Banon, S., & Hardy, J. (1998). Structure and technofunctional properties of protein-polysaccharide complexes: A review. Critical Reviews in Food Science and Nutrition, 38(8), 689e753. Shaikh, J., Ankola, D. D., Beniwal, V., Singh, D., & Ravi Kumar, M. N. V. (2009). Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when compared to curcumin administered with piperine as absorption enhancer. European Journal of Pharmaceutical Sciences, 37, 223e230. Staszewski, M. V., Jagus, R. J., & Pilosof, A. M. R. (2011). Characterization of whey protein-polyphenol interactions by dynamic light scattering. In Presented in part at 11th international congress on engineering and food. Athens, Greece, May. Tolstoguzov, V. (2003). Some thermodynamic considerations in food formulation. Food Hydrocolloids, 17(1), 1e23. Turgeon, S. L., & Laneuville, S. I. (2009). Protein þ polysaccharide coacervates and complexes: From scientific background to their application as functional ingredients in food products. In S. Kasapis, I. T. Norton, & J. B. Ubbink (Eds.), Modern biopolymer science (pp. 327e363). New York: Elsevier. Weinbreck, F., Nieuwenhuijse, H., Robijn, G. W., & de Kruif, C. G. (2003). Complex formation of whey proteins: Exocellular polysaccharide EPS B40. Langmuir, 19(22), 9404e9410. Wilska-Jeszka, J. (2007). Food colorants. In Z. E. Sikorski (Ed.), Chemical and functional properties of food components (3rd ed., pp. 245e274). Florida: CRC Press. Xiao, J., Nian, S., & Huang, Q. (2015). Assembly of kafirin/carboxymethyl chitosan nanoparticles to enhance the cellular uptake of curcumin. Food Hydrocolloids, 51, 166e175. Xu, W., Jin, W., Zhang, C., Li, Z., Lin, L., Huang, Q., et al. (2014). Curcumin loaded and protective system based on complex of k-carrageenan and lysozyme. Food Research International, 59, 61e66. Ye, A. (2008). Complexation between milk proteins and polysaccharides via electrostatic interaction: Principles and applicationsea review. International Journal of Food Science & Technology, 43(3), 406e415. Zhou, H., Sun, X., Zhang, L., Zhang, P., Li, J., & Liu, Y. N. (2012). Fabrication of biopolymeric complex coacervation core micelles for efficient tea polyphenol delivery via a green process. Langmuir, 28(41), 14553e14561. Zimet, P., & Livney, Y. D. (2009). Beta-lactoglobulin and its nanocomplexes with pectin as vehicles for u-3 polyunsaturated fatty acids. Food Hydrocolloids, 23(4), 1120e1126.