Physicochemical Properties of Whey-Protein-Stabilized Astaxanthin

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Physicochemical properties of whey protein-stabilized astaxanthin nanodispersion and its transport via Caco-2 monolayer Xue Shen, Changhui Zhao, Jing Lu, and Mingruo Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05284 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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Journal of Agricultural and Food Chemistry

Physicochemical properties of whey protein-stabilized astaxanthin nanodispersion and its transport via Caco-2 monolayer

Xue Shena, Changhui Zhaoa, Jing Lua, Mingruo Guo*a b c a College of Food Science and Engineering, Jilin University, Changchun, 130062, China b Department of Food Science, Northeast Agricultural University, Harbin, 150030, China c Department of Nutrition and Food Sciences, College of Agriculture and Life Sciences, University of Vermont, Burlington, Vermont, 05405, USA

* Corresponding author: Mingruo Guo E-mail: [email protected] Mailing address: University of Vermont, 109 Carrigan Drive, 351 Carrigan Wing, Burlington, VT 05405, USA Tel: (802) 656-8168 Fax: 802-656-0001

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ABSTRACT

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Astaxanthin nanodispersion was prepared using whey protein isolate (WPI) and

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polymerized whey protein (PWP) through an emulsification–evaporation technique.

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The physicochemical properties of the astaxanthin nanodispersion were evaluated and

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the transport of astaxanthin was assessed using a Caco-2 cell monolayer model. The

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astaxanthin nanodispersions stabilized by WPI and PWP (2.5%, w/w) had a small

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particle size (121 ± 4.9 and 80.4 ± 5.9 nm), negative zeta potential (-19.3 ± 1.5 and

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-35.0 ± 2.2 mV), and high encapsulation efficiency (92.1 ± 2.9% and 93.5 ± 2.4%).

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Differential scanning calorimetry (DSC) curves indicated that amorphous astaxanthin

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existed in both astaxanthin nanodispersions. Whey protein-stabilized astaxanthin

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nanodispersion showed resistance to pepsin digestion but readily released astaxanthin

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after trypsin digestion. The nanodispersions showed no cytotoxicity to Caco-2 cells at

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a protein concentration below 10 mg/mL. WPI and PWP stabilized nanodispersions

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improved the apparent permeability coefficient (Papp) of Caco-2 cells to astaxanthin

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by 10.3- and 16.1- fold, respectively. The results indicated that whey

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protein-stabilized nanodispersion is a good vehicle to deliver lipophilic bioactive

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compounds like astaxanthin and to improve their bioavailability.

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Keywords: Astaxanthin;

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Bioavailability

Whey

protein;

Nanodispersion;

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Caco-2

cells;

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Introduction

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Astaxanthin is a xanthophyll carotenoid that has antioxidant activity (1) and can

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be used to reduce the risk of cancer (2), cardiovascular diseases (3), Helicobacter

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pylori infections (4), and age-related diseases (5-8). Astaxanthin cannot be

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synthesized de novo by mammals and thus must be acquired from the diet (4).

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Utilization of carotenoids as nutraceutical ingredients for foods is therefore

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recommended (8). Unfortunately, the applications of astaxanthin in different food

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formulations are currently limited because of its poor water solubility, high melting

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point, low bioavailability, and susceptibility to chemical degradation under certain

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conditions, such as an acidic environment, heating, and exposure to light and oxygen

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(9).

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To use astaxanthin in functional foods, different strategies have been developed to

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improve its stability and bioavailability in foodstuffs, including microencapsulation

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(10, 11), liposomes (12), emulsions (13), and nanodispersions (14). The

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nanodispersion system has attracted much attention for increasing the bioavailability

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of lipophilic bioactive agents in water-based food products due to its special features,

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including very small particle size (20 to 200 nm), relatively high physical stability,

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enhanced chemical stability and improved water solubility. Nanodispersions also have

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thermodynamic stability and optical transparency, which is particularly useful for

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transparent beverages (15, 16). Stabilizers can affect the physicochemical and

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bioavailability properties of nanodispersions depending on the type and nature of the

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stabilizers (17). Various stabilizing agents have been used to prepare astaxanthin 3



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nanodispersions including gelatin (18), polysaccharides (19), Tween 20 (16), sodium

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caseinate (20, 21), and mixture stabilizers (22-24). Anarjan et al. reported that

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astaxanthin was more bioavailable when administered with protein or dietary fat

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ingredients such as milk (14). Therefore, milk protein should be one of the best

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candidates to stabilize astaxanthin nanodispersion.

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Whey protein is a valuable byproduct of cheese making. In addition to their

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desirable nutritional quality, whey proteins are amphiphilic molecules with high

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surface activity and emulsifying properties. Whey proteins can form a protective layer

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around fat droplets and protect them from aggregation or coalescence due to a

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combination of electrostatic effects and hydrophobic interactions. Another advantage

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of whey protein as an emulsifier or stabilizer is its antioxidant activity (25). The

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emulsifying properties of WPI can be increased by polymerized whey protein soluble

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aggregates also called PWP (26). However, information about WPI and PWP used as

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emulsifiers to stabilize astaxanthin nanodispersions is very limited.

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Therefore, the objective of this study was to prepare astaxanthin

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nanodispersions using an emulsification–evaporation method with WPI and/or PWP

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as stabilizers. The astaxanthin nanodispersions were characterized by their particle

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size distribution, zeta potential, encapsulation efficiency, flow behavior, viscosity, and

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thermal properties. Caco-2 cells were used to evaluate the cytotoxicity and

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bioavailability of the whey protein-astaxanthin nanodispersions.

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Materials and methods

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Chemicals 4


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WPI, containing 93.1% protein, 0.36% fat, 4.79% moisture, 1.60% ash and

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0.70% lactose, was purchased from Fonterra (Auckland, New Zealand).

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Dimethylsulfoxide (DMSO) was purchased from Beijing Solarbio Science and

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Technology Co., (China). Astaxanthin and its standard were purchased from Sigma

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(St. Louis, MO, USA). Methanol, methyl tert-butyl ether (MTBE), acetone, and

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dichloromethane were HPLC grade and purchased from Thermo Fisher Scientific Co.

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(Waltham, MA, USA). High-glucose and L-glutamine Dulbecco's Modified Eagle

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Medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher

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Scientific Co. (Waltham, MA, USA). All other chemicals used were of reagent grade

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and purchased through Sigma (St. Louis, MO, USA). Purified water used in this study

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was filtered using a Millipore Milli-Q™ water purification system (Millipore Corp.,

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Milford, MA, USA).

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Preparation of astaxanthin nanodispersions

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Astaxanthin (25 mg) was dissolved in 100 mL acetone/ dichloromethane (3:1, v/v)

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at room temperature as an organic phase. As previously reported, 0.10, 0.25, 0.50, 1.0,

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2.5, and 5.0% (w/w) WPI solution and PWP solution (85°C for 20 min) was prepared

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(26). Sodium azide (0.022%, w/w) was added to the aqueous phase to inhibit the

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growth of microorganisms. The organic phase and aqueous phase were mixed at ratio

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of 1:9 (v/v), and a coarse emulsion was formed using an Ultra-Turrax T25 high-speed

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blender (IKA, Staufen, Germany) at 12,000 rpm for 2 min. The coarse emulsion was

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then homogenized through an ultrasonic processor (VCX800, Vibra-Cell, Sonics,

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USA) with a 13-mm high grade titanium alloy probe in an ice bath at 40% amplitude 5



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for 2 min (10 s:5 s work/rest cycles). The solvent was subsequently removed from the

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emulsion by rotary evaporation (EYELA N-1100，Tokyo, Japan) at 37°C and 100 rpm

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(27). The container was wrapped in foil to keep astaxanthin protected from light

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during the treatment.

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Determination of particle size and zeta potential

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The mean particle size and zeta-potential (ζ) of nanodispersions were determined

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by dynamic light scattering (DLS) and electrophoretic mobility (UE) using laser

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Doppler velocimetry and phase analysis light scattering by a Zetasizer Nano ZS 90

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(Malvern Instruments, UK). The samples were diluted 100-fold using deionized water.

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Then 1 mL of diluted samples was transferred into the measuring cell. The refractive

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index (RI) values for oil droplets and water were 1.45 and 1.33, respectively. Particle

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size values were reported as Z-average (Dz), which is the intensity-weighted mean

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hydrodynamic size of the particles. ζ was calculated based on the Henry equation. All

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measurements were conducted at 25°C.

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Determination of rheological property

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Rheological property analyses were performed using a rheometer (DHR-1, TA

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Instrument, USA). A steel parallel plate geometry (diameter = 40 mm, sample,

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thickness = 1 mm) was used. The sample temperature was controlled by a Peltier unit

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attached to a water circulation system.

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Flow ramp analyses were performed for shear rate from 0.1 to 1000 s-1 at 25°C. Apparent viscosity was recorded as a function of shear rate. Peak hold analyses were performed at 200 s-1 for 60 s at 25°C. 6


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Differential Scanning Calorimetry

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The thermal properties of the samples were evaluated using a Differential

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Scanning Calorimeter (Q2000, TA Instrument, USA). Approximately 5 mg of

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freeze-dried samples were placed in aluminum pans and weighed accurately, and then

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the pans were sealed. The temperature was programmed to rise from 25 to 250°C at

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5°C /min. An empty pan was used as a reference. The TA Universal Analysis 2000

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was used to determine the melting temperature, peak temperature, and enthalpy (∆H).

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Release of astaxanthin in vitro

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Freeze-dried samples (2 mg/ml) were added to simulated gastric fluid (SGF, 3.2

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mg/ml pepsin in 0.035 M NaCl, 0.084 N HCl, pH 1.2) and simulated intestinal fluid

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(SIF, 1.0 mg/ml trypsin in 0.1 M PBS, pH 7.0), respectively. This mixture was

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incubated in a glass tube at 37oC in a shaking water bath. An aliquot of reactant was

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removed, and the hydrolysis was terminated by adjusting the pH at set intervals

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during 120 min of digestion time. The astaxanthin content was determined by the

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HPLC method described below.

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Extraction and determination of astaxanthin in nanodispersions

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Astaxanthin was extracted from nanodispersions using methanol and

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dichloromethane as described (14) with some modifications. Briefly, 0.5 mL of

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sample was mixed with 2 mL methanol/dichloromethane (1:1, v/v), vortexed for 10

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min, and then centrifuged at 1000 g for 10 min. The upper supernatant containing

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astaxanthin was transferred to a 10-mL brown volumetric flask. The extraction was

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repeated with 1 mL dichloromethane three times until the aqueous layer was clear. 7



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The extracts were brought up to 10 mL. The encapsulation efficiency of astaxanthin

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was calculated by comparing the astaxanthin content encapsulated in nanodispersions

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with that of the total content added.

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High Performance Liquid Chromatography (HPLC)

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Astaxanthin analysis was performed using an UHPLC system (Nexera UHPLC

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LC-30A, Shimadzu, Japan) with a PDA UV-vis absorption detector and a C30

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reverse-phase analytical column (250 × 4.6 mm i.d., 5µm, YMC, Co., LTD). The flow

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rate was set at 1.0 mL/min at room temperature. The injection volume was 20 µL, and

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the detection wavelength was 474 nm. The chromatography conditions were as

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follows: solvent A, methanol; solvent B, MTBE; solvent C, phosphoric acid/H2O

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(1:99, v/v). The solvent gradient program was 81% A/15% B/4% C at 0 min and was

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changed linearly to 66% A/30% B/4% C at 5 min, maintained for 10 min and was

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subsequently changed linearly to 16% A/80% B/4% C at 16-23 min, maintained for 4

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min, and followed by a linear return to 81% A/15% B/4% C at 28-30 min, maintained

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for 5 min. The calibration of peak area versus the astaxanthin standard concentration

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was linear in the measured concentration ranging from 0.1 to 50 µg/mL for all-trans

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astaxanthin (R2 > 0.99, n=3).

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Cytotoxicity of astaxanthin nanodispersions on Caco-2 Cells

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To determine the cell viability after incubation with astaxanthin nanodispersions,

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a methyl thiazolyl tetrazolium (MTT) assay was performed using a Caco-2 cell line

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(passages 30-40). Caco-2 cells were incubated in high-glucose and L-glutamine

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DMEM medium supplemented with 1% (v/v) penicillin/streptomycin and 10% (v/v) 8


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FBS. The cells were seeded at a density of 2.5 × 104 cells/well in 96-well plates and

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incubated at 37°C and 5% CO2 for 72 h. Culture medium was removed, and

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astaxanthin nanodispersions diluted with cell culture medium at different protein

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concentrations (1.0 - 10.0 mg/mL) were added to the wells. DMEM was used as a

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control. After 24 h of incubation, the cells were washed with 200 µL PBS/well three

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times. Then, 200 µL MTT containing medium (5 mg/mL MTT in DMEM) was added

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to each well. After 4 h of incubation, the medium was removed and 150 µL DMSO

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was added to dissolve the formed formazan crystals. Absorbance measurement was

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performed at 570 nm using a microplate reader (Synergy HT, BioTek, USA). Relative

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cell viability (%) was calculated by comparing the absorbance of nanoparticle cells

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with that of control cells (28).

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Caco-2 cell monolayer incubation

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Caco-2 cells were seeded at a density of 1.5 × 106 cells/well onto polyethylene

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terephthalate (PET) filters with a 0.4-µm pore size (Corning 6-well transwell, Corning,

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Massachusetts, USA) for 21 days to achieve a differentiated intestinal cell monolayer.

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Both the transepithelial electrical resistance (TEER) value and the phenol red flux (29,

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30) were measured to ensure the monolayer integrity.

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Astaxanthin transport via Caco-2 monolayers

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The differentiated Caco-2 monolayers were washed with Hanks buffer three

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times. Both astaxanthin nanodispersions and free astaxanthin (in DMSO) were diluted

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with DMEM to a final concentration of 12.5 µg/mL and added to the chambers. The

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transportation experiments were performed at 37°C. Next, 1 mL of sample was added 9



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to the apical side and 2.5 mL DMEM was added to basal side, which was sampled at

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intervals (0.5, 1, 1.5, 2, 3, and 4 h), extracted and analyzed for astaxanthin content by

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HPLC. The TEER (0, 0.5, 1, 2, 3, and 4 h) and phenol red flux (0, 4 h) were also

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measured to evaluate the integrity of Caco-2 monolayers. The apparent permeability

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coefficient (Papp, cm s−1) was calculated as follows (31): Papp =

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dQ 1 · dt AC0

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where dQ/dt = the permeability rate (µg s−1), A= the surface area of the filter (4.67

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cm-2), C0= the initial concentration in the chamber (µg mL−1).

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Statistical analyses

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All experiments were performed in triplicate. Statistical analyses were performed

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using the statistical program SPSS Version 17.0 SPSS (SPSS Inc. Chicago, IL, USA).

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Comparisons among data of different groups were performed with one-way ANOVA,

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where the LSD method and Dunnet’s C were used on the basis of the homogeneity

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test. The results were presented as the mean ± standard deviation (SD) and considered

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significantly different when P

Physicochemical Properties of Whey-Protein-Stabilized Astaxanthin

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