Fabrication and characterization of Î²-lactoglobulin-based

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Bioactive Constituents, Metabolites, and Functions

Fabrication and characterization of #-lactoglobulin-based nanocomplexes composed of chitosan oligosaccharides as vehicles for delivery of astaxanthin Chengzhen Liu, Zhuzhu Liu, Xun Sun, Shuaizhong Zhang, Shuhui Wang, Fungxian Feng, Dongfeng Wang, and Ying Xu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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Journal of Agricultural and Food Chemistry

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Fabrication and characterization of β-lactoglobulin-based nanocomplexes

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composed of chitosan oligosaccharides as vehicles for delivery of astaxanthin

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Chengzhen Liu†, Zhuzhu Liu†, Xun Sun†, Shuaizhong Zhang†, Shuhui Wang‡, Fungxian Feng§, Dongfeng Wang†, Ying Xu*†

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† College of Food Science and Engineering, Ocean University of China (5

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Yushan Road, Shinan District, Qingdao, Shandong Province, 266003, China) ‡ Qingdao Municipal Center for Disease & Prevention (175 Shandong Road, Shinan District, Qingdao, Shandong Province, 266033, China) § Dalian Bangchuidao Seafood Co., Ltd. (987 Wuyi Road, Jinzhou District,

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Dalian, Liaoning Province, 116100, China)

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ABSTRACT

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Astaxanthin (Ax), a type of carotenoid, is limited used due to its poor

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water-solubility, low bioavailability, and the decomposition under harsh condition.

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This study reports a delivery system for Ax through a simple affinity binding with

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β-lactoglobulin and then coated with chitosan oligosaccharides. The Ax-loaded

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β-lactoglobulin nanocomplexes and chitosan oligosaccharides-coated nanocomplexes

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were successfully prepared. The nanocomplexes exhibited a smooth spherical shape

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with diameters of about 40 and 60 nm measured by transmission electron microscope.

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Spectroscopic techniques (UV–vis, Fluorescence, and Fourier transform infrared

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spectroscopy) combined with molecular docking were used to determine the binding

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mechanism of the Ax and β-lactoglobulin. Compared with native Ax, the

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nanocomplexes maintain the hydroxyl radicals scavenging activity of Ax under the

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treatment of acid, high temperature, and ultraviolet radiation. The release experiment

ACS Paragon Plus Environment


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of nanocomplexes revealed that the encapsulating could provide prolonged release of

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Ax in simulated gastrointestinal juices. This study aimed to fabricate and characterize

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Ax-β-lactoglobulin nanocomplexes which can improve the Ax stability and

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slow-released.

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KEYWORDS: Carotenoid; affinity binding; milk protein; Encapsulation

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INTRODUCTION

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Astaxanthin (Ax) is a type of carotenoids found in marine animals such as salmon,

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shrimp and lobster,1 which is red in color. However, when the native Ax combines

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with proteins (carotenoproteins) or lipoproteins (carotenolipoproteins), it produces a

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characteristic blue or purple tones, but upon denaturation the complex turns red.2 Ax

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has a range of health benefits such as anticancer, antiobesity, antidiabetic activities,

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and positive effects against inflammation and cardiovascular diseases,3 which are at

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least partially due to its potent antioxidant activity. Previous research has indicated

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that Ax is 10–500 times greater than the widely recognized antioxidants, such as

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β-carotene and α-tocopherol.4 Ax and related carotenoids are highly unsaturated3 and

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prone to degrading in harsh environmental conditions like high temperature, light, and

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oxygen during food processing.5 Ax degradation can be observed with the distinctive

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red color fading and directly correlated with loss of functional attributes. Another

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factor affecting the applications of Ax is its water insolubility like most other

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carotenoids, which results in their low solubility as well as low bioavailability in

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aqueous matrices, especially in aqueous-based foods. The bioavailability of Ax with

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an occasional solubility is improved by increasing its solubility.


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Recent years, nano-encapsulation has been interested in increasing the stability

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and bioavailability of Ax, including nanoemulsions,6 nanodispersions,7 liposomic

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encapsulation and microencapsulation.8,8b The encapsulation could both protect Ax

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from hurt of harsh processing conditions and control its release. In addition, their

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unique physicochemical properties entrust them the cellular uptake features compared

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to bulk materials.9,10 It reported that the nanoparticles with diameter below 500 nm

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remarkably enhanced the bioavailability of substance which were encapsulated into

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nanoparticles.11 When the size is around 200 nm, the nanoparticles can stabilize the

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clathrin-coated pits and thus, they are easily internalized into cells via

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clathrin-mediated endocytosis. These direct cellular delivered substrates could help to

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keep bioactivity of Ax. Until now, there have been many studies showing successful

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encapsulation of Ax in nanocarriers. Tachaprutinun et al. (2009) prepared Ax

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nanosystem by encapsulating Ax into a chitosan derivative.12 Khalid et al. (2017)

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reported the formulation of Ax-loaded nanoemulsions via a high-pressure

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homogenization.13 Tamjidi et al. (2017) have successful fabricated nanostructured

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lipid carriers stabilized with lecithin and emulsifier, and investigate the effect of

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different environmental stresses on the stability of Ax-nanostructured lipid carriers.14

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Nonetheless, these encapsulation methods could protect Ax from various adverse

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conditions. They hinges on the emulsifier selection, system composition, and end

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usage with polydispersity that can have low emulsion stability. In addition, using

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organic solvents in these methods is still a big challenge for food application.

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β-lactoglobulin (β-lg), with a molecular mass of 18.5 kDa and isoelectric point



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(pI) around 5.0, is the very ample protein (58%, w/w) in milk, 15 As a ligand-binding

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protein, it is biodegradable and has a tendence of binding various small hydrophobic

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molecules for instance carotenoids, fatty acids, and vitamin D, within their central

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calyx16. It has reported that there are a few high affinity active sites for polyphenols

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compounds on β-lg.17,18 β-lg has been extensively designed into delivery systems for

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its ability to resist to pepsin degradation at low pH, bind various compounds, cost

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effectiveness and availability.10,19

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Chitosan Oligosaccharides (COS) are cationic polymers of low molecular weight

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(≤10 kDa) which obtained via the decomposition of chitosan. It is generally regarded

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as water-soluble, bioactive, degradable and biocompatible, and widely used as the

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materials of active substance delivery carriers. Due to its cationic properties, COS can

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be adsorbed on the surface of protein-based NPs to form the coating via electrostatic

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interactions, and COS-coated protein-based NPs exhibit better oral delivery

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performance for Ax than the other Ax encapsulation system.

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In this study, we report the synthesis of nanocomplexes of Ax, β-lg, and COS

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using spontaneously self-assembles via affinity binding and electrostatic interaction in

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suitable environmental conditions. The encapsulation and characteristics of

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Ax-encapsulated nanocomplexes are investigated. We also evaluate the chemical

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stability and release properties of the Ax in buffer solution (pH 2 and pH 7). This

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process provides the valuable information for carotenoids used in designing

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nutritional products and opens the possibility of the application of whey protein as

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nanovehicles to produce fortified products.


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MATERIAL AND METHODS Materials

Ax (>97%) was purchased from Sigma-Aldrich Co., Ltd. (St. Louis,

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MO, USA) and used as standard. β-lg (≥ 95.0% purity, 18.3kDa) from bovine milk,

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was also purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). COS with

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low-molecular weight (˂ 3kDa) was obtained from Tianjin Hiromi Biotechnology

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Development Co., Ltd. (Tianjin, China). 1, 10 - phenanthroline was supplied by

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Solarbio Science & Technology Co., Ltd. (Beijing, China). All other reagents used

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were of analytical grade.

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Synthesis of nanocomplexes The schematic diagram of the formation process of

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astaxanthin-β-lactoglobulin (Ax-β-lg) nanocomplexes and chitosan oligosaccharides

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coated nanocomplexes (Ax-β-lg@COS nanocomplexes) illustrated in Scheme 1, were

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prepared via the spontaneously self-assembles. Firstly, 4.5 mg β-lg fully dissolved in

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50 mL distilled water and incubated under magnetic agitation at 50 °C for 10 min. A

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specified amount of Ax was dissolved in 3 mL ethanol to obtain various

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concentrations. The Ax solution of different concentrations were added dropwise into

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aliquots of the β-lg (at the molar ratios of β-lg to Ax, 1 : 0, 1 : 1, 1 : 2, and 1 : 3,

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respectively). After vortex blending for 5 min for the self-assembly to occur, the

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Ax-β-lg nanocomplexes were obtained. Subsequently, the mixtures were cooled to

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room temperature and the pH of reaction mixtures were adjusted to 7. COS solution

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(0.5%, w/v) was filtered using a 0.22 µm syringe filters and added to the Ax-β-lg

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nanocomplexes dispersion dropwise. The mixtures were stirred mildly for 1 h and

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then the free unreacted COS and ethanol in reaction solution was removed by dialysis



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against ultrapure water to obtain Ax-β-lg@COS nanocomplexes. Finally, two freshly

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prepared nanocomplexes dispersions were used for the measurement of various

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characteristics and then the nanoparticles were lyophilized at -80 °C for 48 h to obtain

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dry particles.

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Measurement of nanocomplex properties

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Transmission electron microscopy analysis The morphology and size of the

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Ax-β-lg nanocomplexes and Ax-β-lg@COS nanocomplexes were measured with a

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Hitachi 7700 transmission electron microscope (TEM) (Tokyo, Japan) at an

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acceleration voltage of 80 kV. About 5 µL of nanocomplex dispersion (1 ‰, w/v) was

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drop onto a carbon-coated copper grid (400 meshes) and freeze-dried for 6 h.

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Size, ζ-potential, and polydispersity index The measurement of mean particle

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size ζ-potential, and polydispersibility index (PDI) was using a Zetasizer Nano ZS90

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(Malvern Instruments Ltd., Worcestershire, U.K.). The two nanocomplex dispersions

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were equilibrated at 25 °C prior to measurement.

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Encapsulation efficiency and loading capacity Accurately weighed amounts

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(0.05 g) of two kinds of nanocomplexes were dispersed completely in methanol and

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then sonicated for 30 min. The concentration of Ax in all the samples was measured

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through the HPLC. The encapsulation efficiency (EE) and loading capacity (LC) of

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the nanocomplexes was determined using the equation:

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EE (%) = content of Ax in nanocomplexes/total content of Ax×100

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LC (%) = content of Ax in nanocomplexes/total weight of dry nanocomplexes×100

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(2)


(1)

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HPLC analysis was performed using an Agilent 1260 liquid chromatogram

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system (Agilent Technologies Inc., USA), equipped with a SB-C18 Waters HPLC

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column (4.6×250 mm), using an isocratic mobile phase consisting of 85% v/v

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methanol, 5% v/v dichloromethane, 5% v/v acetonitrile and 5% v/v water with a flow

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rate of 1 mL/min. The detection was performed at 474 nm. The injection volume was

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20 µL. The calibration of peak area versus Ax content was linear in the range of

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determined concentrations.

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Ultraviolet−visible absorbance spectrum The ultraviolet-visible (UV-vis)

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absorbance spectrum of pure Ax, β-lg, COS and the nanocomplex suspensions were

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measured separately with an UV-vis spectrophotometer (UV-2800, Unico Instrument

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Co., Ltd., Shanghai, China) from 200-600 nm with a quartz cell (1 cm path length).

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The concentration of Ax, β-lg, COS was kept 150 µM, 50 µM and 0.5%, respectively.

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The absorbance of Ax and β-lg nanocomplexes with β-lg to Ax molar ratio from 1:0 to

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1:3 was recorded, respectively.

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Fluorescence spectroscopy The fluorescence spectrum of pure Ax, β-lg, COS

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and nanocomplex suspensions was measured by a fluorescence spectrophotometer

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(F-7000, Hitachi, Japan) in a 1.0 cm path length quartz cell. An aliquot of 50 µM of

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β-lg solution interacted with various concentrations of Ax (0, 50, 100, 150 µM). After

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vortex blending for 5 min, 3 mL of the mixture were added into a quartz cell.

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The emission spectra were recorded from 290 to 460 nm with an excitation

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wavelength of 280 nm. The excitation wavelength was 295 nm (to excite Trp) and the

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scan an emission wavelength ranged between 290 and 550 nm to follow protein



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folding state. The slit width of excitation and emission was set at 10 nm.

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Fourier transform infrared spectroscopy The Ax, β-lg, COS and the

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nanocomplex powders were compressed into thin potassium bromide disks. The

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pellets were determined by a Fourier transform infrared (FTIR) spectrometer (Tensor

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27, Bruker, Germany) with an MCT-A detector. The spectrum in the range of 400 to

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4000 cm-1 was obtained. For analyzing the secondary structure of β-lg, Ax-β-lg

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nanocomplexes, the IR spectra (1700 to 1600 cm-1) were further subjected to smooth

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and Fourier self-deconvolute using OMNIC software. The FTIR spectrum was

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analyzed using Peakfit 4.10 software.20

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Antiradical activity stability assessment of nanocomplexes

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Acid treatment The pH of nanocomplex was adjusted to acidity (about pH 2)

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with HCl solution (0.5 M). Then, the samples were transferred into sample tubes and

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stored for 1 week in the dark place.

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Thermal treatment The nanocomplex dispersions (about 5 mL) were pipetted

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into a 10 mL sample tube and incubated in a water bath (90 °C) for 1 h and then

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cooled to room temperature.

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Ultraviolet (UV) inactivation treatment The nanocomplex dispersions were

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subjected to ultraviolet irradiation (25 w) for 7 days. After different treatment the

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samples were to measure the antiradical activity.

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Antiradical activity The hydroxyl radicals (OH) scavenging activity of pure Ax,

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COS and nanocomplexes was measured after treatment of acid, high temperature, and

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UV light with Fenton reaction according to the literature.21 Reaction mixture


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contained 1 mL of FeSO4 (0.75 mM), 1 mL of 1, 10 –phenanthroline (0.75 mM), 2 mL

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of phosphate buffer (0.2 M, pH 7.4), 1 mL of H2O2 (0.01%), and 1 mL of sample

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solution. Adding H2O2 started the reaction. After incubation at 25 °C for 30 min, the

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absorbance of the mixtures at 510 nm was determined with UV-vis spectrophotometer.

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Pure Ax solution was used as control for all the treatments. The OH scavenging

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activity was calculated as shown in Eq. 3: OH scavenging (%) =

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( ) ()

× 100

(3)

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in which As is the absorbance of the test sample. A1 represents the absorbance of

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the control (PBS mixed with the 1, 10 -phenanthroline, FeSO4 and H2O2) and A0 is

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the absorbance of the blank sample (PBS mixed with the 1,10 -phenanthroline and

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FeSO4).

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Molecular docking analysis The molecular docking calculation was performed

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using AutoDock software based on previous literatures.1 The three dimensional (3D)

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structure

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(http://www.rcsb.org). The Ax crystal structure was obtained from the PubChem

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website (https://www.ncbi.nlm.nih.gov/). AutoDock Tools (ADT) version 1.5.6 was

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used to design the Ax binding to β-lg. The whole protein was selected as potential

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binding site for Ax and the procedure was performed in a blind manner. The

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conformation was ranked using the scoring function (Ascore), which estimates the

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free binding energy (∆G). The conformer with the lowest binding free energy was

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used.

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of

β-lg

was

available

from

the

RCSB

Protein

Data

Bank

In vitro release The controlled release of Ax from the Ax-β-lg nanocomplexes



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and Ax-β-lg@COS nanocomplexes was achieved in phosphate buffered saline (pH 2

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and 7).The dialysis bag (Mw = 3.0 kDa) was placed in 50 mL phosphate buffered

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saline (pH 2 and pH 7), and incubated (37 ± 0.5 °C, 120 rpm) for 24 h in a water bath

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with magnetic stirring. The total quantity of Ax released from the nanocomplexes was

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determined using HPLC over different time intervals.

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Statistical analysis All the experiments were conducted in triplicate. The

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experimental data was subjected to statistical analysis with SPSS 19.0 software (SPSS

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Inc., Chicago, USA). Duncan's multiple range tests were also applied to determine the

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difference of means from the ANOVA, using a significance test level of 5% (P

Fabrication and characterization of Î²-lactoglobulin-based

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