Article Cite This: J. Agric. Food Chem. 2018, 66, 6717−6726
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Fabrication and Characterization of β‑Lactoglobulin-Based Nanocomplexes Composed of Chitosan Oligosaccharides as Vehicles for Delivery of Astaxanthin Chengzhen Liu,† Zhuzhu Liu,† Xun Sun,† Shuaizhong Zhang,† Shuhui Wang,‡ Fuxian Feng,§ Dongfeng Wang,† and Ying Xu*,†
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College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Shinan, Qingdao, Shandong 266003, People’s Republic of China ‡ Qingdao Municipal Center for Disease Control and Prevention, 175 Shandong Road, Shibei District, Qingdao, Shandong Province 266033, China § Dalian Bangchuidao Seafood Company, Limited, 987 Wuyi Road, Jinzhou, Dalian, Liaoning 116100, People’s Republic of China ABSTRACT: Astaxanthin (Ax), a type of carotenoid, has limited use as a result of its poor water solubility, low bioavailability, and decomposition under harsh conditions. This study reports a delivery system for Ax through a simple affinity binding with βlactoglobulin and then coated with chitosan oligosaccharides. Ax-loaded β-lactoglobulin nanocomplexes and chitosan oligosaccharide-coated nanocomplexes were successfully prepared. The nanocomplexes exhibited a smooth spherical shape with diameters of about 40 and 60 nm measured by transmission electron microscopy. Spectroscopic techniques (ultraviolet−visible, fluorescence, and Fourier transform infrared spectroscopy) combined with molecular docking were used to determine the binding mechanism of Ax and β-lactoglobulin. In comparison to native Ax, the nanocomplexes maintain the hydroxyl radical scavenging activity of Ax under the treatment of acid, high temperature, and ultraviolet radiation. The release experiment of nanocomplexes revealed that the encapsulation could provide prolonged release of Ax in simulated gastrointestinal juices. This study aimed to fabricate and characterize Ax−β-lactoglobulin nanocomplexes, which can improve the Ax stability and slow release. KEYWORDS: carotenoid, affinity binding, milk protein, encapsulation
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and microencapsulation.8,8b The encapsulation could both protect Ax from the harm of harsh processing conditions and control its release. In addition, their unique physicochemical properties entrust them with the cellular uptake features compared to bulk materials.9,10 It reported that the nanoparticles with a diameter below 500 nm remarkably enhanced the bioavailability of substances that were encapsulated into nanoparticles.11 When the size is around 200 nm, the nanoparticles can stabilize the clathrin-coated pits, and thus, they are easily internalized into cells via clathrin-mediated endocytosis. These direct cellular-delivered substrates could help to keep bioactivity of Ax. Until now, there have been many studies showing successful encapsulation of Ax in nanocarriers. Tachaprutinun et al. prepared an Ax nanosystem by encapsulating Ax into a chitosan derivative.12 Khalid et al. reported the formulation of Ax-loaded nanoemulsions via a high-pressure homogenization.13 Tamjidi et al. have successfully fabricated nanostructured lipid carriers stabilized with lecithin and emulsifier and investigated the effect of different environmental stresses on the stability of Ax-nanostructured lipid carriers.14 Nonetheless, these encapsulation methods could protect Ax from various adverse conditions. They hinge
INTRODUCTION Astaxanthin (Ax) is a type of carotenoid found in marine animals, such as salmon, shrimp, and lobster,1 which is red in color. However, when the native Ax combines with proteins (carotenoproteins) or lipoproteins (carotenolipoproteins), it produces characteristic blue or purple tones, but upon denaturation, the complex turns red.2 Ax has a range of health benefits, such as anticancer, antiobesity, and antidiabetic activities and positive effects against inflammation and cardiovascular diseases,3 which are at least partially due to its potent antioxidant activity. Previous research has indicated that Ax is 10−500 times greater than the widely recognized antioxidants, such as β-carotene and α-tocopherol.4 Ax and related carotenoids are highly unsaturated3 and prone to degrading in harsh environmental conditions, such as high temperature, light, and oxygen, during food processing.5 Ax degradation can be observed with the distinctive red color fading and directly correlated with the loss of functional attributes. Another factor affecting the applications of Ax is its water insolubility, like most other carotenoids, which results in its low solubility as well as low bioavailability in aqueous matrices, especially in aqueous-based foods. The bioavailability of Ax with an occasional solubility is improved by increasing its solubility. In recent years, nanoencapsulation has been interested in increasing the stability and bioavailability of Ax, including nanoemulsions,6 nanodispersions,7 liposomic encapsulation, © 2018 American Chemical Society
Received: Revised: Accepted: Published: 6717
February 20, 2018 May 6, 2018 June 8, 2018 June 8, 2018 DOI: 10.1021/acs.jafc.8b00834 J. Agric. Food Chem. 2018, 66, 6717−6726
Article
Journal of Agricultural and Food Chemistry
Scheme 1. Schematic Diagram of the Formation of Ax−β-lg and Ax−β-lg@COS Nanocomplexes via Simple Affinity Binding
Development Co., Ltd. (Tianjin, China). 1,10-Phenanthroline was supplied by Solarbio Science & Technology Co., Ltd. (Beijing, China). All other reagents used were of analytical grade. Synthesis of Nanocomplexes. The schematic diagram of the formation process of astaxanthin−β-lactoglobulin (Ax−β-lg) nanocomplexes and chitosan oligosaccharide-coated nanocomplexes (Ax−β-lg@COS nanocomplexes), illustrated in Scheme 1, were prepared via spontaneous self-assembly. First, 4.5 mg of β-lg was fully dissolved in 50 mL of distilled water and incubated under magnetic agitation at 50 °C for 10 min. A specified amount of Ax was dissolved in 3 mL of ethanol to obtain various concentrations. Ax solution of different concentrations was added dropwise to aliquots of β-lg (at the molar ratios of β-lg/Ax of 1:0, 1:1, 1:2, and 1:3). After vortex blending for 5 min for the self-assembly to occur, Ax−β-lg nanocomplexes were obtained. Subsequently, the mixtures were cooled to room temperature, and the pH of reaction mixtures was adjusted to 7. COS solution (0.5%, w/v) was filtered using 0.22 μm syringe filters and added to the Ax−β-lg nanocomplex dispersion dropwise. The mixtures were stirred mildly for 1 h, and then the free unreacted COS and ethanol in reaction solution were removed by dialysis against ultrapure water to obtain Ax−β-lg@COS nanocomplexes. Finally, two freshly prepared nanocomplex dispersions were used for the measurement of various characteristics, and then the nanoparticles were lyophilized at −80 °C for 48 h to obtain dry particles. Measurement of Nanocomplex Properties. Transmission Electron Microscopy (TEM) Analysis. The morphology and size of Ax−β-lg and Ax−β-lg@COS nanocomplexes were measured with a Hitachi 7700 transmission electron microscope (Tokyo, Japan) at an acceleration voltage of 80 kV. About 5 μL of nanocomplex dispersion (1‰, w/v) was dropped onto a carbon-coated copper grid (400 mesh) and freeze-dried for 6 h. Size, ζ Potential, and Polydispersity Index (PDI). The measurements of the mean particle size, ζ potential, and PDI were performed using a Zetasizer Nano ZS90 (Malvern Instruments, Ltd., Malvern, U.K.). The two nanocomplex dispersions were equilibrated at 25 °C prior to measurement. Encapsulation Efficiency and Loading Capacity. Accurately weighed amounts (0.05 g) of two kinds of nanocomplexes were dispersed completely in methanol and then sonicated for 30 min. The concentration of Ax in all of the samples was measured through highperformance liquid chromatography (HPLC). The encapsulation efficiency (EE) and loading capacity (LC) of the nanocomplexes were determined using the following equations:
on the emulsifier selection, system composition, and end usage, with polydispersity that can have low emulsion stability. In addition, using organic solvents in these methods is still a big challenge for food application. β-Lactoglobulin (β-lg), with a molecular mass of 18.5 kDa and isoelectric point (pI) of around 5.0, is a very ample protein (58%, w/w) in milk.15 As a ligand-binding protein, it is biodegradable and has a tendence of binding various small hydrophobic molecules, for instance, carotenoids, fatty acids, and vitamin D, within its central calyx.16 It has reported that there are a few high-affinity active sites for polyphenol compounds on β-lg.17,18 β-lg has been extensively designed in delivery systems for its ability to resist pepsin degradation at low pH, bind various compounds, cost effectiveness, and availability.10,19 Chitosan oligosaccharide (COS) is a cationic polymer of a low molecular weight (≤10 kDa), obtained via the decomposition of chitosan. It is generally regarded as watersoluble, bioactive, degradable, and biocompatible and widely used as the material of active substance delivery carriers. As a result of its cationic properties, COS can be adsorbed on the surface of protein-based nanoparticles (NPs) to form the coating via electrostatic interactions, and COS-coated proteinbased NPs exhibit better oral delivery performance for Ax than the other Ax encapsulation systems. In this study, we report the synthesis of nanocomplexes of Ax, β-lg, and COS using spontaneously self-assembles via affinity binding and electrostatic interaction in suitable environmental conditions. The encapsulation and characteristics of Ax-encapsulated nanocomplexes are investigated. We also evaluate the chemical stability and release properties of Ax in buffer solution (pH 2 and 7). This process provides valuable information for carotenoids used in designing nutritional products and opens the possibility of the application of whey protein as nanovehicles to produce fortified products.
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MATERIALS AND METHODS
Materials. Ax (>97%) was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, U.S.A.) and used as a standard. β-lg (≥95.0% purity, 18.3 kDa) from bovine milk was also purchased from SigmaAldrich Co., Ltd. (St. Louis, MO, U.S.A.). COS with a low molecular weight (
