Construction of Fucoxanthin Vector Based on Binding of Whey Protein

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Food and Beverage Chemistry/Biochemistry

Construction of Fucoxanthin Vector Based on Binding of Whey Protein Isolate and Its Subsequent Complex Coacervation with Lysozyme Junxiang Zhu, Hao Li, Ying Xu, and Dongfeng Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06679 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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Journal of Agricultural and Food Chemistry

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Construction of Fucoxanthin Vector Based on Binding of Whey Protein Isolate

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and Its Subsequent Complex Coacervation with Lysozyme

3

Junxiang Zhu†,‡,§, Hao Li†, Ying Xu†, Dongfeng Wang*, †

4

†

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Republic of China

6

‡

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Republic of China

8

§

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Zhejiang, People's Republic of China

College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, the

Marine Fisheries Research Institute of Zhejiang, Zhoushan, 316021, Zhejiang, People's

Marine and Fisheries Research Institute, Zhejiang Ocean University, Zhoushan, 316021,

10 11

Corresponding author: Dongfeng Wang

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Email: [email protected]

ACS Paragon Plus Environment


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Abstract

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In this study, a novel vector for fucoxanthin was constructed using the ligand-binding property

15

of whey protein isolate and its subsequent heteroprotein complex coacervation with lysozyme.

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The results showed that fucoxanthin could quench the intrinsic fluorescence of whey protein

17

isolate by a static mechanism, indicating that they could spontaneously form nanocomplex

18

through non-covalent interactions. Moreover, the structural and electrostatic properties of whey

19

protein were different from before with the binding of fucoxanthin, and this reason could be well

20

explained by molecular dynamic simulation. The size and ζ-potential tests showed that when the

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whey protein isolate was combined with fucoxanthin and then coacervated with lysozyme, the

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heteroprotein ratio and pH that affected the coacervation process also changed compared to free

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whey protein isolate. The FT-IR results showed that fucoxanthin was successfully encapsulated

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into complex coacervates. In addition, the heteroprotein system exhibited a higher loading

25

efficiency and also provided a better protection for fucoxanthin in heating, storage and simulated

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gastrointestinal environments.

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Keywords: fucoxanthin, whey protein isolate, lysozyme, ligand binding, heteroprotein complex

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coacervation, molecular dynamics


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INTRODUCTION

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FX is a marine xanthophyll commonly found in macroalgae such as Undaria pinnatifida,

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Laminaria japonica, and Ecklonia cava.1 It is also the most abundant carotenoid in nature and

32

contributes to more than 10% of the total natural carotenoid products.2 Although FX is not a

33

provitamin A, its special structure endows a variety of biological activities that are beneficial to

34

human health, including hypolipidemic, anti-obesity, antidiabetic, and anticarcinogenic effects.3

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Among them, the anti-obesity property of FX attracts attention and has received support from

36

many in vitro and in vivo studies.4 A recent study also reported the effectiveness of FX reducing

37

the formation of β-Amyloid fibrils and attenuating β-Amyloid neurotoxicity in the early stage of

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Alzheimer’s disease.5 These findings indicate that FX can be developed as a nutritional

39

supplement or used to produce functional foods.

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However, similar to other carotenoids, FX is sensitive to degradation under ambient stresses (light,

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temperature, oxygen) and gastrointestinal chemistry. To improve its poor stability and low

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bioavailability, several delivery systems have been designed for FX. For instance, Salvia Trujillo

43

et al. investigated the bioavailability of FX in nanoemulsions and compared three different oils

44

used in carriers, which showed that oil type has a conspicuous impact on the bioavailability of

45

FX.6 Dai et al. constructed FX-loaded O/W microemulsions using medium chain triglycerides as

46

an oil phase, tween 80 as a surfactant, and polyethylene glycol 400 as a co-surfactant.7 The results

47

showed that about 95% of FX in microemulsions was retained for 4 weeks. These studies either

48

use an emulsification process that requires high energy, which reduces FX and is costly for the

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food industry or uses synthetic surfactants that may be toxic.8,9

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Whey proteins are GRAS materials according to the Food and Drug Administration of the United



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States.10 In addition to providing nutrition, they also have physicochemical properties suitable for

52

the preparation of delivery systems, including binding ions or small molecules, self-assembly,

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gelation, emulsification, and complex coacervation.11 Whey proteins have hydrophobic cavities

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that can bind ligands of different sizes by non-covalent interactions, which are usually located in

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the surface pocket or internal cavity of protein.12 Recently, some researchers have utilized this

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binding property to construct the heteroprotein complex coacervates to transport the ligand bound

57

by whey proteins. Chapeau et al. prepared complex coacervates of lactoferrin and β-Lg, which

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could load hydrophilic vitamin B9 at a content of 10 mg/g protein,13 mainly due to the binding

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properties of β-Lg.14 This approach was later successfully expanded to the pilot scale.15 However,

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lactoferrin is relatively expensive and may be replaced with other food protein such as Lyz, which

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can be easily extracted from egg whites.16 The hydrophobic vitamin D3 was loaded in complex

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coacervates of β-Lg and Lyz with a high encapsulation efficiency of 90.8 ± 4.8%.17 This study

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also utilized the fact that β-Lg can bind vitamin D3 to form a complex.18 In their subsequent study,

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this complex coacervate was shown to be effective in protecting vitamin D3 against UV-light

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irradiation and increased bioavailability in vivo.19 Our previous study showed that β-Lg, BSA,

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and α-La can form nanocomplexes with FX,20 suggesting we can try to construct FX vector based

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on complex coacervates using heteroprotein from whey and other sources. However, FX has a

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larger molecular weight and is not as heat resistant as vitamin D3.21 Another thing to note is that

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the presence of ligand can affect the heteroprotein complex coacervation.22 When a protein forms

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a complex with a ligand, the protonation of ionizable groups and the conformation of the

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hydrophobic region sometimes change.23 These phenomena can affect the electrostatic and

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hydrophobic forces between the two proteins, ultimately determining the coacervate size or


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product formulation.

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Therefore, The objective of this study was to construct a FX delivery system based on the binding

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property of whey protein and heteroprotein complex coacervation. Food proteins WPI and Lyz

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were selected in this study owing to their low cost and commercial availability, which might

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increase the scalability of food applications. WPI was initially used to bind FX and then interact

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with Lyz. In this process, the effect of FX on complex coacervation of WPI and Lyz was studied

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by theoretical simulation and experiment. Finally, the protective and release properties of FX in

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this heteroprotein complex coacervates were evaluated.

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MATERIALS AND METHODS

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Materials and Chemicals. WPI (HilmarTM-9410, protein 92.9% dry basis) was obtained from

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Hilmar Cheese Company, Inc. (Hilmar, CA, USA). Lyz from hen egg white (protein ≥ 95%, > 40000

84

units/mg protein), all-trans FX standard, and ANS-Na (≥ 97.0%, for fluorescence) were purchased

85

from Sigma Chemical Company (St. Louis, MO, USA). FX used in this study was isolated from

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Undaria pinnatifida and quantified by 1260 HPLC (Agilent Technologies Inc., Santa Clara, CA,

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USA).24 Solvents for HPLC including acetonitrile and MTBE were procured from Merck & Co.,

88

Inc. (Darmstadt, Germany). Pepsin from porcine gastric mucosa (10000 units/mg protein), trypsin

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from porcine pancreas (250 units/mg proteins), and porcine bile extracts were purchased from

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Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemicals were of

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analytical grade and procured from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

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Preparation of Protein Samples. WPI powder was dissolved at 0.2% (w/v) in deionized water

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under a continuous mild stirring for 2 h at 25°C to allow complete rehydration,25 followed by

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filtration through a 0.22 μm MCE syringe membrane filter to remove undissolved particulates. A



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Lyz solution was prepared at 0.6% (w/v) in deionized water. These stock solutions were kept at 4 °C

96

until diluted to a final concentration and then used within two days.

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Preparation of WPI–FX Complex. Nanocomplex comprising WPI and FX was prepared based on

98

our previous study.20 FX was pre-dissolved in ethanol at 0.5 mM, and various volumes of the FX

99

solution was respectively dropwise added to 3.0 mL of WPI (0.1%, w/v) followed by the vortex for

100

5 min, corresponding to a final FX concentration of 5.0 μM, 7.5 μM, 10.0 μM, 12.5 μM, and 15.0

101

μM. After standing for different time (0 min, 15 min, 60 min, and 120 min), the samples were

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measured for fluorescence at three temperatures (300 K, 305 K, 310 K), size and ζ-potential at

103

ambient.

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Steady-state Fluorescence. Intrinsic fluorescence of sample (with or without FX) was recorded by

105

a F-7000 fluorescence spectrophotometer (Hitachi Ltd., Tokyo, Japan). The excitation wavelength

106

was set at 280 nm, and the emission spectra were recorded in the range of 300–450 nm at room

107

temperature. The excitation and emission slit widths were fixed at 10 nm. To explore the

108

fluorescence quenching mechanism, the Stern−Volmer equation was used: 𝐹0

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𝐹

= 1 + 𝐾SV[𝑄] = 1 + 𝑘q𝜏0[𝑄]

(1)

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Where F0 and F represent the steady-state fluorescence intensities in the absence and presence of

111

quencher, respectively. KSV is the Stern–Volmer quenching constant (M−1). [Q] is the concentration

112

of the quencher (mol). kq is the bimolecular quenching constant (M−1 s−1), and τ0 is the average

113

lifetime of the molecule without any quencher (s), and the fluorescence lifetime of the biopolymer

114

is 10–8 s.26

115

When small molecules bind independently to a set of equivalent sites on a macromolecule, the

116

equilibrium between the free and bound molecules is given by the following equation,27


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log

𝐹0 ― 𝐹 𝐹

(2)

= log𝐾𝑎 +𝑛log[𝑄]

118

Where Ka and n are the binding constant (M−1) and the number of binding sites, respectively.

119

For studying thermodynamics, the van’t Hoff equation is used for determining the enthalpy change

120

(ΔH°, kJ mol−1) and entropy change (ΔS°, J mol−1 K−1):

121

ln𝐾a = ―

∆𝐻○ 𝑅𝑇

+

∆𝑆○ 𝑅

（3）

122

where R is the gas constant and T is the temperature (K). Then, the free energy change (ΔG°, kJ

123

mol−1) is calculated according to the following equation:

124

∆𝐺○ = ∆𝐻○ ― 𝑇∆𝑆○

（4）

125

Extrinsic Fluorescence Probe. The PSH was assessed by fluorescence spectroscopy with an ANS

126

probe based on the method.28 The WPI solution (0.1%, w/v) with or without FX was diluted to

127

0.05%, 0.0375%, 0.025%, 0.0125% (w/v). Then, 20 μL of ANS-Na (8 mM) was added to 1.6 mL

128

of each sample, vortexed for 15 s, and kept in the dark for 5 min. The same concentration of protein

129

solution without ANS was determined as the control, and the free ANS was used as the blank. For

130

fluorescence measurement, the excitation wavelength was fixed at 390 nm, and the emission spectra

131

were recorded in the range of 400–600 nm. The excitation and emission slit widths were 5 and 10

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nm, respectively. The scan speed was 1500 nm/min. The index of PSH was determined from the

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initial slope of the plot of fluorescence intensity versus protein concentration. Within the protein

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concentration range used in these experiments, linear relationships were obtained (R2 > 0.99).

135

Molecular Simulation. The molecular simulation was a complementary way to understand the

136

conformation transition and interaction of proteins and its ligand. Three major proteins in WPI,

137

including BSA, β-Lg, and α-La, were selected as model proteins for simulation. Initial

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conformations of whey proteins with FX were generated by Autodock 4.2 based on our previous



139

study,20 and then simulated by MD using the Gromacs 5.1.2 package.29 The topological file of FX

140

with a force field of GROMOS96 54a7 was prepared by the ATB online server.30 Then, a cube box

141

containing each whey protein with its FX ligand was constructed, in which SPC-type water

142

molecules and counterions were filled. After constructing the box, the energy was optimized by the

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steepest descent method. The maximum number of steps was 50000, and the optimization was to be

144

completed when the maximum force Fmax < 10.0 kJ mol−1. Then, the box was equilibrated under the

145

canonical and isothermal-isobaric ensembles. Upon completion of the two equilibration phases, the

146

system could release the position restraints and run MD with a time length of 20 ns for data

147

collection. Finally, the visualization of three-dimensional structure and electrostatic potential of

148

whey proteins before and after binding FX were carried out by PyMOL. The changes in the

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secondary structure of the protein are calculated by do_dssp. The titration curve and pI were

150

computed using APBS,31 and H++ web server.32

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Preparation of WPI–FX–Lyz Coacervates. To study the mass ratio on complex coacervation at

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25°C, the WPI–FX (0.1%, w/v) solution was mixed with the solution of 0.025%, 0.05%, 0.075%,

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0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5% (w/v) lysozyme at equal volumes. The solutions were

154

adjusted to pH 6.5 with NaOH or HCl (0.1 M). Then, to study the effect of pH on complex

155

coacervation, the pH 6.5 solutions were also adjusted to pH 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,

156

10.5, and 11 with NaOH and HCl (0.1 M). After pH adjustment and standing without vortex for 15

157

min, the samples were measured for size and ζ-potential. A mixed solution containing WPI and Lyz,

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but not including FX, was used as a control.

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Particles Size and ζ-Potential Measurement. The particle size and ζ-potential of one or two

160

proteins and those with FX were measured with a Zetasizer Nano ZS90 instrument (Malvern


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Instruments Ltd., Malvern, Worcestershire, U.K.) assuming the scattering particles to be spherical,

162

their particles size was calculated from the diffusion parameters using Stokes-Einstein equation.

163

The electrophoretic mobility of each sample was determined, then calculating the ζ-potential using

164

the Smoluchowski approximation.33

165

FT-IR. Infrared spectra were obtained from the free FX, WPI and Lyz samples, lyophilized WPI-

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FX, WPI-Lyz (1:2, w/w), and WPI-Lyz-FX (1:2, w/w). The analyses were performed with an FT-

167

IR spectrometer (Nicolet iS10, Thermo Scientific) using KBr (potassium bromide) and read in the

168

range of 4000–400 cm−1

169

Loading Efficiency of FX in WPI–FX and WPI–FX–Lyz. To determine the loading efficiency of

170

FX in nanocomplex and coacervates, the WPI–FX–Lyz solution was centrifuged at 12000 g and

171

4 °C for 15 min. The precipitate was collected and mixed with an equal volume of acetone/n-hexane

172

(1:1, v/v) solvent. For WPI–FX complex, the solution was centrifuged in Millipore (30 kDa, MWCO)

173

filters at 4000 g for 20 min and washed with deionized water 2–3 times to remove free FX. Then,

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the concentrate was diluted to 3 mL and also mixed with an equal volume of acetone/n-hexane (1:1,

175

v/v) solvent. After vortex for 20 s, the upper organic phase was collected, concentrated under a

176

stream of nitrogen, and dissolved in acetonitrile for HPLC analysis.24 The molar amounts of FX in

177

the precipitate or concentrate (M, μmol) and that used in sample preparation (M0, μmol) were used

178

to calculate the loading efficiency as follows:

179

Loading efficiency % =

𝑀0 𝑀

× 100%

(5)

180

Stability of FX in WPI–FX and WPI–FX–Lyz. Samples with FX were contained in amber vials

181

and kept at 25 °C for up to 21 d or 75 °C in a water bath for up to 9 h in the dark. Samples were

182

taken periodically and mixed with an equal volume of acetone/n-hexane (1:1, v/v) solvent to extract



183

FX for calculation of residual amount. After vortex for 3 min, the upper organic phase was collected,

184

concentrated under a stream of nitrogen, and dissolved in acetonitrile for HPLC analysis. The

185

degradation of FX was evaluated for the zero, first, and second-order kinetic models using Equations

186

(4), (5), and (6), respectively.34

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𝐹 = 𝐹0 ― 𝑘1𝑡

(6)

188

𝐹 = 𝐹0e ― 𝑘2𝑡

(7)

189

𝐹 = 1 + 𝑘3𝐹0𝑡

𝐹0

(8)

190

where F is the residual amount of FX (μmol) after a treatment time of t (d or h). F0 is the total

191

amount of FX (μmol) before incubation. k1, k2, and k3 are the respective rate constant at the

192

corresponding reaction order, d−1 or h−1.

193

Release Properties of FX after Simulated Digestions. To evaluate in vitro digestion of FX in

194

WPI–FX and WPI–FX–Lyz, the protocol was used to formulate simulated digestive juices and

195

digestion conditions with some modifications.1 A 4 mL of sample solutions containing a certain

196

amount of FX (F0, μmol) was first diluted with 10 mL of an electrolyte solution (pH 5.5) containing

197

120 mM NaCl, 5 mM KCl, and 6 mM CaCl2. The simulated gastric digestion was conducted at

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37 °C for 2 h in a shaking water bath after mixing the diluted FX sample with 0.5 mL of pepsin

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solution (0.075 g/mL dissolved in 0.1 M of HCl) and adjusting to pH 2.2 using HCl (10 mM). When

200

the stomach digestion was completed, FX in WPI–FX and WPI–FX–Lyz was extracted by

201

acetone/n-hexane (1:1, v/v). Its amount (F1, μmol) was determined using HPLC. The rate of FX

202

release (Rel) in the stomach was calculated as follows:

203 204

𝐹1

𝑅el(%) = (1 ― 𝐹0) × 100

(9)

The sample after the simulated gastric digestion was treated sequentially to simulate digestions at


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three intestinal phases (duodenum, jejunum, and ileum) in the shaking water bath. To simulate the

206

duodenum phase, 14.5 mL of the sample after the stomach phase treatment was mixed with 250 mg

207

of porcine bile extract, 0.5 mL of pancreatic lipase (0.01 g/mL), and 0.5 mL of trypsin (0.08 g/mL).

208

The mixture was adjusted to pH 6.5 using NaHCO3 (10 mM) and incubated at 37 °C for 30 min. To

209

mimic the jejunum phase, the mixtures after the duodenum phase digestion was changed to pH 6.8

210

using NaHCO3 (10 mM), followed by incubation at 37 °C for 90 min. Subsequently, the mixture

211

was adjusted to pH 7.2 using NaHCO3 (10 mM) and incubated at 37 °C for 5 h to simulate the ileum

212

phase of digestion.

213

After each phase of the simulated intestinal digestion, the mixture was centrifuged at 12000 g for

214

20 min at 4 °C to collect the supernatant to determine the amount of released FX (F2, μmol) using

215

the HPLC. The percentage of FX released (Rel) was then calculated with respect to the total amount

216

used in the simulated digestion, which was consistent with the stomach digestion. 𝐹2

217

𝑅el(%) = 𝐹0 × 100

(10)

218

Statistical Analysis. Data were presented as the mean ± standard deviation of three separate

219

experiments. All statistical analysis was conducted using SPSS software (version 19.0, SPSS Inc.,

220

Chicago, IL, USA). The Shapiro–Wilk test was used to assess the normality of data and the Levene

221

test was used to check the homoscedasticity. The difference between samples was evaluated using

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the one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons. All statements of

223

significance were based on the 0.05 probability level.

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RESULTS AND DISCUSSION

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Fluorescence of WPI quenched by FX. Fluorescence spectroscopy is currently one of the most

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reliable and convenient methods for studying the interaction between a protein and a ligand, wildly



227

applicated in food science, biophysics, and pharmacy. Commonly, the intrinsic fluorescence of a

228

protein is mainly derived from hydrophobic amino acid residues inside the protein molecule. The

229

fluorescence intensity of WPI in Figure S1 exhibited the maximum emission peaks at 335 nm,

230

mainly due to their Tyr and Trp residues.26 As the FX concentration increased, the fluorescence

231

intensity of WPI showed a progressive decrease at three temperatures, indicating that FX quenched

232

the intrinsic fluorescence of WPI mostly owing to molecular interaction resulting in a decrease in

233

the quantum yield of the fluorophore.20 Further, the quenching of WPI fluorescence was studied

234

using the Stern−Volmer model. The plot of F0/F versus FX concentration in Figure 1A showed good

235

linear relationships (R2 > 0.98) and the slope (KSV) decreased with an increasing temperature,

236

indicating that the fluorescence quenching of FX on WPI was static, which also meant that a stable

237

non-covalent complex was formed between WPI and FX because a high temperature usually leading

238

to dissociation of the unstable adduct.26 The bimolecular quenching constant kq calculated in Table

239

S1 also supported static quenching mechanism of WPI by FX, because the kq value far exceeded the

240

diffusion-controlled rate constant of various quenchers with a biopolymer (2.0 × 1010 M−1 s−1).35

241

Binding and thermodynamic behaviors of WPI with FX. After clarifying the static quenching

242

mechanism of WPI fluorescence by FX, the log[(F0–F)/F] versus log[Q] was plotted in Figure 1B

243

to evaluate the binding ability of WPI with FX. Obviously, three curves of binding affinity also

244

showed good linear relationships (R2 > 0.98). The binding constant Ka (300 K) of WPI to FX listed

245

in Table S1 was 3.38 × 104 M−1. This result had the same order (104 M−1) as the norbixin and bixin

246

that were bound to WPI,35,36 and higher than β-carotene (103 M−1)37, showing that WPI has the

247

stronger binding ability to FX than β-carotene. Further, the thermodynamic parameters calculated

248

in Table S1 demonstrated the binding process of FX to WPI was spontaneous owing to the negative


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values of ΔG° (−26.2 kJ mol−1 at 300 K). This result was also higher than β-carotene (−22.2 kJ

250

mol−1 at 298 K), well agreeing with the binding constant result.37 The negative values of ΔH° and

251

ΔS° indicated the major forces of WPI–FX interaction were van der Waals force and hydrogen

252

bond,38 which were different from hydrophobic interactions driving the formation of a complex

253

between WPI and β-carotene because both the ΔH° and ΔS° were positive.38 This result might be

254

due to the fact that the hydroxyl groups of FX had the ability to form hydrogen bonds with the side

255

chain of WPI, but not for β-carotene.

256

Surface hydrophobicity of WPI after binding FX. As discussed above, WPI and FX can form a

257

complex by non-covalent interaction forces. Here, the surface hydrophobicity of WPI with or

258

without FX was further measured by ANS probe. In aqueous solution, ANS fluoresces very weakly,

259

but its quantum yield increases significantly upon binding to some hydrophobic regions of proteins,

260

so it is widely used to characterize surface exposure of hydrophobic patches.39 The results in Figure

261

1C indicate that the PSH of WPI increases with an increasing FX concentration, indicating a tighter

262

binding of ANS to WPI in the presence of FX. One possible reason is the binding of FX exposed

263

some of the previously buried hydrophilic regions of WPI, generating new high-affinity ANS

264

binding sites and increasing the number of bound ANS per each protein. This was similar to Shahlaei

265

et al. and Jia et al. who studied the binding of sertraline with human serum albumin and ferulic acid

266

with β-Lg, respectively.39,40 It was worth noting that if the surface hydrophobicity of protein was

267

increased, the internal hydrophobic pocket would be exposed, enhancing the hydrophobic

268

interaction between the protein molecules.20 This result might promote the self-aggregation of WPI,

269

and affect the complex coacervation with Lyz. This inference was further studied in the following

270

experiments.



271

Size and ζ-Potential Change Upon Forming WPI–FX Complex. As described above, the PSH of

272

WPI increased after binding FX, meaning some hydrophobic areas of WPI had been exposed.

273

Besides, the previous research reported some ionizable residues of protein may change their charge

274

state during the binding process.23 For complex coacervation of two oppositely charged polymers,

275

this phenomenon can affect the electrostatic interaction. So, this section investigated the effects of

276

FX concentration and standing time on the size and ζ-potential of WPI. The particle size in Figure

277

2A indicated that the native WPI (183 ± 3 nm) became larger (> 200 nm) as FX concentration

278

enhanced. In Figure 2B, as the preparation time prolonged, the size of WPI was further increased

279

and eventually stabilize at about 220 nm. The above results found WPI aggregated after binding

280

FX, supporting the inference from surface hydrophobicity change. On the other hand, the data in

281

Figure 2C showed the ζ-potential of natural WPI was −18.83 ± 0.39 mV, which did not change

282

significantly after adding ethanol. This was because the ethanol volume was controlled within 3%

283

of the total volume of the WPI solution, which substantially preserved the original conformation of

284

the protein.41 After adding 5 μM, 10 μM, and 15 μM of FX, the ζ-potential significantly decreased

285

to −23.90 ± 0.57, −25.87 ± 0.33 and −25.40 ± 0.94 mV, respectively, suggesting the change in

286

surface potential of WPI after binding FX. In Figure 2D, the ζ-potential of the WPI–FX was further

287

reduced after sample solution was allowed to stand for different time, and then became gentler

288

similar to the size change, indicating that the binding process was gradually stabilized.

289

MD simulation. As previously mentioned, the PSH, size, and ζ-potential of WPI changed when

290

interacting with FX. To further explore its mechanism in detail, the complex conformation obtained

291

by docking three main proteins (β-Lg, α-La, and BSA) of WPI with FX was used as the initial

292

conformation of the MD simulation, and the simulation time was 20 ns. Both pure whey proteins


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293

and the protein–FX complexes were simulated in the meantime, which could display the dynamic

294

alterations of three proteins after the molecular recognition of FX. The variations of the RMSD

295

regarding β-Lg, α-La and BSA with or without FX were shown in Figure 3. Commonly, if the

296

fluctuations of RMSD value for a typical dynamic system was kept within 0.1 nm, the system could

297

be considered to reach a stable state of dynamic equilibrium.42 Obviously, the three non-covalent

298

whey protein–FX complexes became stable from 12 ns. Comparatively, the pure protein systems

299

were equilibrated with some mild fluctuations over a time period of 20 ns. When the MD simulations

300

were completed, equilibrium conformation of three complexes was also obtained in Figure 3. It was

301

noteworthy that hydrogen bonds between Leu87 in β-Lg, Ser286 in BSA and FX were formed, well

302

agreeing with the thermodynamic measurement as discussed earlier. Other amino acid residues with

303

hydrophobic side chain, e.g., Leu39, Val41, Ile71, Ala86, Met107 in β-Lg, Leu259, Ala290, Val432

304

in BSA, Phe31, Ala40, Ile41, Val42 in α-La, constituted the hydrophobic pockets to envelop the FX

305

molecule.

306

Then, in order to verify the hydrophobic region exposure of the WPI after binding FX, the changes

307

in the radius of gyration (Rg) and SASA during the MD simulation were calculated and shown in

308

Figure 4. The Rg is a physical quantity that describes the tightness of the protein structure. The larger

309

its value, the lower the protein density, also indicating that the protein has expanded.43 The data in

310

Figure 4A-4C showed the average Rg of complex systems was higher than the free whey proteins,

311

revealing the protein tightness was reduced and the structure transformed into a loose state caused

312

by FX binding. To further realize the variation of the radius of gyration, the SASA values of free

313

and FX-binding whey proteins were determined. Normally, during protein folding, the hydrophobic

314

amino acid residues tend to be buried inside the protein molecule to minimize the accessible surface



315

of their non-polar groups. Therefore, the surface hydrophobicity of proteins can be studied by

316

calculating their SASA, and there is a certain correlation between them.44 The data in Figure 4D-4F

317

showed the average SASA of systems containing FX was higher than that of the pure whey proteins,

318

indicating that the structure became loose after the protein was combined with FX, causing an

319

increase in SASA. Both SASA and Rg indicated that the structures of whey proteins were folded in

320

the presence of FX, exposing more hydrophobic areas.

321

Furthermore, time-dependent secondary structure fluctuation analysis provided some additional

322

information about protein structure upon binding a ligand. The secondary structures of β-Lg, BSA,

323

and α-La before and after binding FX were calculated and shown in Figure 4G-4I, respectively. In

324

the initial conformation of BSA, the main secondary structure was α-helix, accounting for 72.6%,

325

decreased to 69.5% after simulation, while the contents of random coil and β-turn increased from

326

20.9% to 23.2%. For β-Lg, the content of main β-sheet structure decreased from 40.7% to 34.0%.

327

Other structures, such as α-helix and random coil, accounted for 6.8% and 19.1%, respectively.

328

After MD, they were increased to 13.6% and 25.9%, respectively. Similar to BSA, the main

329

secondary structure of α-helix was also α-helix, whose content was reduced from 33.3% to 26.0%,

330

while the random coil increased from 18.7% to 25.2%. The above results showed that the major

331

secondary structure components of the whey proteins after binding FX were reduced, indicating the

332

presence of FX caused the conspicuous loosening and unfolding of polypeptide chain backbone

333

structure in whey proteins, resulting in exposure of the hydrophobic region. This also confirmed the

334

enhancement of PSH and particle size after spontaneous assembly of WPI and FX.

335

Further, to investigate the ζ-potential alteration of WPI, the electrostatic potential of whey proteins

336

with and without FX in Figure 5A, 5B, 5D, 5E, 5G, and 5H was calculated by APBS. The results


Page 16 of 40

Page 17 of 40


337

showed that the surface potential of the three whey proteins had undergone some changes when

338

interacting with the FX molecule, among which β-Lg and α-La were more obvious than BSA. In

339

addition, calculations of titration curves by H++ server in Figure 5C, 5F, and 5I showed the pI of β-

340

Lg and α-La reduced obviously after binding FX, while the change of BSA was minimal, indicating

341

the negative charges of β-Lg and α-La with FX were more than the parent proteins above a certain

342

pH of the isoelectric point. This result supported the ζ-potential change after adding FX to WPI and

343

revealed this change was mainly derived from β-Lg and α-La. Aguilar et al. also reported that

344

proteins often adjusted their conformation when they bound ligand, modifying the

345

microenvironment of the amino acids and affecting their pK values.45 In more detail, the electrically

346

charged amino acid residues of three whey proteins involved in interaction with FX molecule

347

(Figure 3) were selected to estimate their pKa change by pK_(1/2) calculation. In Figure S2, the

348

protonation probability of these residues against pH was plotted. The pK_(1/2) was the pH at which

349

the protonation probability was 0.5. In most cases, the protonation probability versus pH was

350

algebraically equivalent to the Henderson-Hasselbalch equation, in which case pK_(1/2) = pKa.46

351

The results showed that the pKa of the five charged residues from β-Lg (Lys69, Lys70, Asp85) and

352

α-La (His32, Asp37) participating in stabilizing FX were reduced, however, there was no change

353

for BSA (data now shown). This was also in line with changes in the isoelectric point of each whey

354

protein (Figure 5), indicating that the introduction of FX decreased the pKa of some amino acid

355

residues that interacted with it, leading to the shift of pI of the entire proteins.

356

Formation of complex coacervates between WPI–FX and Lyz. Based on the successful

357

formation of nanocomplex between WPI and FX, this study then constructed a FX-loaded vector

358

formed by WPI–FX and Lyz. These two proteins are commonly used proteins for complex



359

coacervation, one of which had an isoelectric point of 4.6 and the other had an isoelectric point

360

of about 11.0.46,47 As discussed above, WPI underwent a series of changes in its properties after

361

binding FX, such as increased particle size and reduced ζ-potential, which would inevitably affect

362

its complex coacervation with other proteins. Therefore, this experiment studied the effects of

363

WPI/Lyz mass ratio and pH on the heteroprotein complex coacervation process in the presence

364

and absence of FX. As shown in Figure 6A, at a fixed pH 6.5, the particle size of WPI–Lyz

365

continued to increase at WPI/Lyz ratios of 4:1–2:3 (w/w), then reached a plateau at ratios of 2:3–

366

1:5 (w/w) where the complex solutions have the highest size. This result indicated that complex

367

coacervation could occur between WPI and Lyz drove by electrostatic interaction at a suitable

368

mass ratio because pH 6.5 was between the pI of whey protein and lysozyme. After loading FX,

369

the size of WPI–FX–Lyz did not change significantly and also had the largest size at WPI/Lyz

370

ratios of 2:3–1:5 (w/w). The data of ζ-potential in Figure 6A showed something different. As the

371

WPI/Lyz ratio increased, the ζ-potentials of WPI–Lyz and WPI–FX–Lyz exhibited a progressive

372

rise and were close to 0 mV at the ratio from 2:3 (w/w) to 1:2 (w/w). However, after the addition

373

of FX, the negative charge carried by WPI increased, resulting in a lower potential of WPI–FX–

374

Lyz than WPI–Lyz at the same WPI/Lyz ratio. For example, under the WPI/Lyz condition of 1:2

375

(w/w), the ζ-potentials of WPI–Lyz and WPI–FX–Lyz were 1.24 ± 0.94 mV and 0.69 ± 0.05 mV,

376

respectively.

377

As previously mentioned, the pI of the WPI sample was 4.6, while the pI of Lyz sample was 11.0.

378

Thus, to study the effect of pH on the formation of the WPI–Lyz co-precipitates, all the pH values

379

were selected within the range of 5.0–11 to avoid the self-aggregation of a single protein and

380

ensure the opposite charge state of WPI and Lyz. The dynamic light scattering measurement in


Page 18 of 40

Page 19 of 40


381

Figure 6B showed the solutions of WPI–Lyz and WPI–FX–Lyz between pH 6.5 to 10.0 were

382

multiple dispersion systems and large aggregates existed. The particles of both systems exhibited

383

the micron levels, and there was no significant difference between them. Besides, the ζ-potential

384

of WPI–Lyz in Figure 6B showed close to zero at pH 8.0, which confirmed the larger size

385

particles produced by complex coacervation. However, after adding FX, there was a significant

386

change in the ζ-potential of complex coacervates formed by WPI–FX and Lyz. The pH of ζ-

387

potential closest to 0 mV had shifted to 7.5, which was due to the increased negative charge of

388

WPI. This was somewhat similar to the case where the Lyz concentration was fixed and the WPI

389

concentration was increased, resulting in a decrease in the pI of the complex coacervates.49

390

Combined with the results of the previous sections, this study indicated that the non-covalent

391

binding of FX to WPI altered the size and charge of whey proteins, which had no obvious effect

392

on the particle size of the complex coacervates subsequently formed with Lyz, but altered the

393

optimal pH of complex coacervation process.

394

FT-IR analysis was performed to evaluate the interaction between proteins and the encapsulation of

395

FX in complex coacervates. Infrared spectra of the single protein and the complex at the ratio of 1:2

396

were shown in Figure 7. The major FTIR spectra of WPI and Lyz were between the bands 1300

397

cm−1 and 1700 cm−1, corresponding to amides I, II, and III. In the FTIR spectrum of WPI–Lyz, the

398

similar structures were observed but the intensities were low. The reduction in the band (1300 cm−1

399

to 1700 cm−1) corresponding to amides was caused by the electrostatic interaction between the –

400

COO− (C=O) cluster of one protein and the –NH3+ (NH) cluster of the other protein.49 In addition,

401

the stretching of the N–H and O–H groups could be identified by the band near the 3300 cm−1. A

402

decrease in the strength of this band was still observed in the WPI–Lyz composite, indicating that



403

the formation of the complex coacervates not only involved electrostatic interaction but also

404

hydrogen bonding. In the spectrum of FX, the band at 1923 cm−1 was assigned as an allenic bond

405

(C=C=C), which was considered to be a representative group of FX. The bands at 3373 cm−1 and

406

1737 cm−1 were respectively identified as hydrogen bonded O−H stretching vibrations and the

407

ketones with −C=O bonds. Bands between 2800 and 3100 cm−1 showed the presence of alkanes

408

with C−H bonds. In addition, the bands at 970 cm−1 was the external torsional pendulum vibration

409

of the C−H bond in the double bond, which was the characteristic absorption band of the trans

410

substituted ethylene. In the spectrum of WPI–FX, the bands at 1637 and 1539 cm−1 was

411

corresponding to the carbonyl groups (C=O). However, the major bands in FX were not found in

412

this spectrum, indicating that the FX was well bound and encapsulated within WPI. Similarly, in

413

the spectrum of WPI–FX–Lyz, and the absorption peaks of FX could not be found. Taking all of

414

the results obtained by FT-IR together, it was concluded that FX had been successfully encapsulated

415

in heteroprotein complex coacervates.

416

Ability to deliver FX by WPI–FX–Lyz, and WPI–FX. Based on the above results, the

417

heteroprotein complex coacervates formed by WPI–FX and Lyz as a carrier for FX were constructed.

418

Then, the delivering ability of WPI–FX–Lyz (WPI/Lyz = 1:2, pH = 7.5), including loading

419

efficiency, chemical stability, and digestion in vitro, were investigated in the following experiments.

420

For comparison, WPI–FX was also used for the study. As shown in Figure 8A, when the amount of

421

added FX was 0.045 μmol, the loading efficiency of WPI–FX–Lyz was 82.36 ± 3.85%, which was

422

significantly higher than that of WPI–FX, corresponding to 55.60 ± 2.25%. This result indicated

423

that the spontaneous co-assembly of FX, WPI, and Lyz into coacervates could provide a higher FX-

424

loading capacity than WPI itself.


Page 20 of 40

Page 21 of 40


425

Subsequently, the chemical stabilities of WPI–FX–Lyz and WPI–FX were evaluated by the

426

degradation of FX under the heating and storage conditions. As shown in Figure 8B, for the thermal

427

stability test at 75 ℃, FX in the control group degraded 91.2% after 2 h of heating, while the WPI–

428

FX–Lyz and WPI–FX groups retained 74.4% and 20.9%, respectively. For storage stability test at

429

25 ℃ in Figure 8C, WPI–FX–Lyz group could still retain 63.4% of the total FX after storage for 2

430

d, while the WPI–FX group had degraded the vast majority, only 27.8% of the initial content was

431

left. These results indicated that WPI had a better protective effect on FX after self-assembled with

432

Lyz. Furthermore, the degradation dynamics illustrated that the degradation of FX in all samples

433

followed the second-order kinetic model (n = 2, R2> 0.96). The degradation rates k2 of FX in WPI–

434

FX–Lyz and WPI–FX were less than that of FX with no encapsulation, also indicating the excellent

435

protection property of WPI or WPI–Lyz carriers in the high-temperature or ambient environment.

436

Additionally, it could be found that the delivery system prepared by heteroprotein complex

437

coacervation had a better ability to prevent the degradation of FX than the carrier prepared by a

438

simple protein binding.

439

Finally, the release properties of WPI–FX–Lyz and WPI–FX in the stomach and various intestinal

440

segments were evaluated using the in vitro simulated digestion method. The digestion of FX-loaded

441

vector in the gastrointestinal tract was a complex process, which played a major role in the uptake,

442

distribution as well as metabolism of FX. The results in Figure 8D indicated that the encapsulated

443

FX was very stable and rarely released from the WPI–FX–Lyz and WPI–FX by the action of pepsin.

444

Nearly 90% of the total FX was still retained in the heteroprotein vehicle. The resistance of this

445

carrier toward pepsin digestion might be attributed to the presence of β-Lg above 50% of WPI. It

446

was well documented that the native β-Lg was resistant to hydrolysis in the gastric compartment



447

following simulated digestion owing to its compact globular structure.50 In addition, Lyz was

448

particularly stable to changes in pH, and there was no significant conformational transition in a

449

moderately dilute solution within the pH range of 1.2–11.3.51 In the digestion of small intestinal

450

phase, the duodenal incubation resulted in destabilization of the WPI–FX–Lyz and 62.56 ± 2.50%

451

of the encapsulated FX was released. However, after jejunal and ileal incubation, the release rate

452

increased to 66.76 ± 4.00% and 70.82 ± 2.83%, respectively. Yonekura and Nagao revealed that the

453

carotenoids in the food were mainly combined with proteins, which were released from the protein

454

complexes through the catalysis of the digestive enzyme in an animal, transformed into the

455

chylomicrons with the other lipids in the duodenum, and then absorbed by the cholesterol transfer

456

carrier or passive diffusion pathway.52 As an oxygenated carotenoid, the main release of FX

457

occurred in the small intestinal phase, which might be beneficial to the absorption process. Besides,

458

Fu et al. reported that β-Lg could be almost completely digested by pancreatic enzymes,53 which

459

might indicate that the heteroprotein complex coacervates formed by WPI and Lyz was unstable to

460

trypsin. All of these the clues helped explain the effectively cumulative release of FX in the small

461

intestine. It should be noted that the release rate of FX from WPI–FX in the intestine was

462

significantly lower than that from WPI–FX–Lyz, reconfirming the advantages and potential of

463

heteroprotein complex coacervation as the vector to deliver FX.

464 465

ABBREVIATIONS USED

466

α-La, α-lactalbumin; β-Lg, β-lactoglobulin; ANS-Na, 8-anilino-1-naphthalenesulfonic acid

467

ammonium salt; BSA, bovine serum albumin; FT-IR, Fourier transform infrared spectrometry; FX,

468

fucoxanthin; GRAS, generally recognized as safe; Lyz, lysozyme; MD, molecular dynamics;


Page 22 of 40

Page 23 of 40


469

MTBE, methyl tert-butyl ether; PSH, protein surface hydrophobicity; RMSD, Root-mean-square

470

deviation; SASA, solvent accessible surface area; WPI, whey protein isolate; WPI–FX, FX-

471

binding WPI; WPI–FX–Lyz, complex coacervates of FX-binding WPI and Lyz; WPI–Lyz,

472

complex coacervates of WPI and Lyz

473 474

ACKNOWLEDGMENTS

475

This work was funded by National Natural Science Foundation of China (31871786), Zhejiang

476

Public Welfare Technology Application Research Project (2018C37023), and Open Project of

477

Key Laboratory of Sustainable Utilization of Technology Research for Fishery Resource of

478

Zhejiang Province.

479 480

SUPPORTING INFORMATION DESCRIPTION

481

Stern–Volmer and binding parameters for the interaction of FX with WPI at three temperatures

482

(Table S1). Fluorescence emission spectra of whey protein isolate with different concentrations of

483

fucoxanthin at 300 K, 305 K, and 310 K (Figure S1). Amino acid residues with electrically charged

484

side chains involved in the interaction of whey proteins and fucoxanthin (Figure S2).

485 486

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632 633

Figure captions

634

Figure 1. Stern–Volmer (A) and binding model (B) calculated by intrinsic fluorescence of whey

635

protein isolate (WPI) quenched with different concentrations of fucoxanthin (FX, 5–15 μM) at three

636

temperatures (300, 305, 310 K). Plots of ANS fluorescence intensity versus concentration of WPI

637

with or without FX (C). Protein surface hydrophobicity (PSH) is determined the slope of the linear

638

regression model.

639 640

Figure 2. Particle size (A) and ζ-potential (C) measured immediately after vortex mixing different

641

concentrations of fucoxanthin (FX) in whey protein isolate (WPI) solution (0.1%, w/v). Effects of

642

standing time on particle size (B) and ζ-potential (D) of WPI solution (0.1%, w/v) upon adding FX.

643

The same letters in all figures represent no significant difference (p > 0.05).

644


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645

Figure 3. RMSD for the backbone of β-lactoglobulin (A), bovine serum albumin (B), and α-

646

lactalbumin (C) with fucoxanthin (FX) and their detailed interaction after molecular dynamic

647

simulation. The green dash lines represent hydrogen bonds, and the red eyelash models represent

648

hydrophobic interactions between FX and whey proteins.

649 650

Figure 4. Changes in radius of gyration (Rg), solvent accessible surface area (SASA), and secondary

651

structures of β-lactoglobulin (A, D, G), bovine serum albumin (B, E, H), and α-lactalbumin (C, F,

652

I) with fucoxanthin (FX) during process of molecular dynamics.

653 654

Figure 5. Positive (blue) and negative electrostatic potential (red) for whey proteins before and after

655

binding fucoxanthin (FX) calculated by APBS: (A) free β-lactoglobulin, (B) FX-binding β-

656

lactoglobulin, (D) free α-lactalbumin, (E) FX-binding α-lactalbumin, (G) free bovine serum albumin,

657

(H) FX-binding bovine serum albumin. Titration curves for β-lactoglobulin (C), α-lactalbumin (F),

658

and bovine serum albumin (I) with or without FX were also calculated using the H++ web server.

659 660

Figure 6. Size and ζ-potential of heteroprotein complex coacervates formed by whey protein isolate

661

(WPI) and lysozyme (Lyz) in the presence and absence of fucoxanthin (FX): effect of WPI/Lyz

662

ratio at a fixed pH of 6.5 (A), and effect of pH at a fixed WPI/Lyz ratio of 1:2 (B).

663 664

Figure 7. FT-IR of free fucoxanthin (FX), whey protein isolate (WPI), lysozyme (Lyz), FX-binding

665

WPI (WPI–FX), complex coacervates of WPI and Lyz (WPI–Lyz), and complex coacervates of

666

WPI–FX and Lyz (WPI–FX–Lyz).



667 668

Figure 8. Loading efficiency (A), thermal stability (B), storage stability (C), and digestion property

669

(D) of fucoxanthin-binding whey protein isolate (WPI–FX) and complex coacervates formed by

670

WPI–FX and lysozyme (Lyz). ** represents the significant at p 

Construction of Fucoxanthin Vector Based on Binding of Whey Protein

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