Aggregation of Fucoxanthin and Its Effects on Binding and Delivery

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Food and Beverage Chemistry/Biochemistry

Aggregation of Fucoxanthin and Its Effects on Binding and Delivery Properties of Whey Proteins Junxiang Zhu, Cong Wang, Jun Gao, Hao Wu, and Qingjie Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03046 • Publication Date (Web): 29 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Journal of Agricultural and Food Chemistry

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Aggregation of Fucoxanthin and Its Effects on Binding and

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Delivery Properties of Whey Proteins

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Junxiang Zhu,†,‡ Cong Wang,§ Jun Gao,‖ Hao Wu,*,‡ Qingjie Sun‡

5 6

†

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266001, People's Republic of China

8

‡ College

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266109, People's Republic of China

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

of Food Science and Engineering, Qingdao Agricultural University, Qingdao

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§

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Laboratory of Guangxi Colleges and Universities for Food Safety and Pharmaceutical

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Analytical Chemistry, School of Chemistry and Chemical Engineering, Guangxi

13

University for Nationalities, Nanning 530006, People's Republic of China

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‖

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Huazhong Agricultural University, Wuhan 430070, People's Republic of China

Guangxi Key Laboratory of Chemistry and Engineering of Forest Products, Key

Hubei Key Laboratory of Agricultural Bioinformatics, College of Informatics,

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* Corresponding author. Dr. Hao Wu

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E-mail address: [email protected]

19 20 21 22 3

ACS Paragon Plus Environment


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Abstract: In this study, aggregation of fucoxanthin and its effects on binding and

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delivery properties of whey proteins were explored. Initially, the H- and J-aggregates

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of fucoxanthin were successfully prepared by adjusting the water/ethanol ratio and

26

water dripping rate. The transition from J- to H-aggregates was observed over the

27

standing time. Then, the molecular arrangement of fucoxanthin H-aggregates was

28

analyzed using the point-dipole approximation model and molecular dynamics,

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showing their intermolecular distance and angle were about 5.0–6.7 Å and −35°–35°,

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respectively. The transformation of J- to H-aggregates was also observed during

31

molecular dynamics, with a shortened intermolecular distance, a reduced solvent

32

accessible surface area, an enhanced interaction force, and a narrowed dihedral angle.

33

Further, the interactions of whey proteins with different forms of fucoxanthin were

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investigated, indicating that both β-lactoglobulin and whey protein isolates could form

35

complexes with the monomers, H-aggregates, and J-aggregates of fucoxanthin. In terms

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of affinity, whey proteins bound fucoxanthin monomers more strongly than aggregates.

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Furthermore, the complexes comprising whey proteins and monomeric fucoxanthin had

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better delivery capabilities than aggregated fucoxanthin, manifested in encapsulation

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efficiency, physical stability, and bioaccessibility.

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Keywords: fucoxanthin, aggregates, whey proteins, interaction, molecular dynamics

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INTRODUCTION

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As a characteristic xanthophyll in edible brown seaweeds, FX is one of the most

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abundant carotenoids in nature.1 It has many therapeutic properties for human health

44

and disease management, such as antioxidant, antidiabetic, anti-obesity, and anticancer

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activities.2 However, due to its conjugated polyene chain, FX is very sensitive to heat,

46

light, and air in the food industry.3 Besides, FX has a poor human absorption

47

efficiency,4 and its bioavailability is lower than β-carotene,5 lutein,5 and astaxanthin.6

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Thus, FX is commonly encapsulated in some colloidal systems, which protect it from

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harmful conditions and improves its bioavailability in vivo. Compared to lipid-based

50

carriers, lipid-free vehicles have received considerable attention in the delivery of FX

51

owing to the avoidance of costly dispersing equipment and potentially toxic synthetic

52

surfactants. Many researchers have used biopolymers to design nanocarriers to improve

53

the stability and in vivo bioavailability of FX, such as chitosan−glycolipid complex and

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zein−caseinate nanoparticles.7,8

55

Similar to other hydrophobic carotenoids, the FX can exist as a self-aggregated form

56

when it encounters a hydrophilic environment.9 This process is largely driven by the

57

weak and reversible bonding by H-bridges, dipole forces, van der Waals interactions,

58

and hydrophobic effects, resulting in the formation of H-type (Figure 1A) and J-type

59

aggregates (Figure 1B).9 In fact, these aggregated carotenoids are readily generated

60

when constructing their delivery systems in an aqueous medium. Auweter et al. found

61

that the β-carotene formed H- and J-aggregates during the preparation of gelatin

62

nanoparticles to load them, resulting in a color-variable colloidal system.10 Karabuda 5



63

et al. further demonstrated that the relative concentration of these two aggregates in the

64

mixed system was a key factor in determining the color change from yellow to red.11

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Cao-Hoang et al. also found that the β-carotene encapsulated in polylactic acid

66

nanoparticles could generate a well-stabilized H-aggregates, while the β-carotene

67

embedded by Tween micelles formed J-aggregates with a poor stability.12 However, the

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aggregation of carotenoids in colloidal systems has not received considerable attention

69

and the interaction of carotenoid aggregates with biopolymers encapsulating them have

70

rarely been reported.

71

Whey proteins are the natural carriers in vivo, transporting essential micronutrients,

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amino acids, as well as immune system components. They have many functional

73

properties that enable them to be “building blocks” for the design of micro- and

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nanocarriers, such as binding property, gelation, emulsification, covalent modification,

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and complex coacervation.13 At present, many studies have used whey proteins to bind

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carotenoids to prepare nanocomplexes for delivery.14-19 However, some of these studies

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dissolved carotenoids in an organic solvent before being added to the protein

78

solutions,14-16 while others dissolved carotenoids in an aqueous buffer.17-19 This will

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allow carotenoids to interact with whey proteins in a dispersed or aggregated form.

80

However, the impact of this phenomenon on the ligand binding of whey proteins has

81

been ignored by researchers and needs to be studied.

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Recently, some reports indicated the native whey proteins could spontaneously interact

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with FX to produce nanocomplexes.20,21 In fact, this was a process in which FX was

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bound in the hydrophobic cavity of whey proteins driven by non-covalent forces. 6


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However, how the dissolved forms of FX affect its binding to whey proteins has not

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been reported. So, the aim of this work was to explore the role of FX aggregation in its

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interaction with whey proteins. First, the H- and J-type FX aggregates were prepared in

88

an aqueous ethanol solution, and their molecular arrangements were revealed by the

89

point-dipole approximation model and MD simulation. Then, the main component β-

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Lg in whey proteins and a commercial product WPI were selected to study their

91

interactions with different forms of FX, including monomers, H-aggregates and J-

92

aggregates. Furthermore, the encapsulation efficiency, physical stability, and in vitro

93

release property were assessed to investigate the capabilities of whey proteins to deliver

94

the monomeric and aggregated FX.

95

MATERIALS AND METHODS

96

Materials and Chemicals

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Purified β-Lg and FX standard were procured from Beijing Solarbio Science &

98

Technology Co., Ltd. (Beijing, China). WPI (Hilmar-9410, protein 92.9% dry basis)

99

was obtained from Hilmar Cheese Company, Inc. (Hilmar, CA, USA). FX was prepared

100

from Undaria pinnatifida based on the previous study and its concentration was

101

determined by an external standard method using HPLC.22 Pepsin from porcine gastric

102

mucosa (EC 3.4.23.1), pancreatin from porcine pancreas (EC 232-468-9), and porcine

103

bile salt were purchased from Sigma Chemical Company (St. Louis, MO, USA).

104

Solvents for HPLC including acetonitrile and methyl tert-butyl ether (MTBE) were

105

provided by Merck (Darmstadt, Germany). All other chemicals were of analytical 7



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reagent grade and procured from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,

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China).

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Formation of H- and J-aggregated FX

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The H- and J-aggregates of FX were prepared based on the previous study.23 The FX

110

was dissolved in ethanol with different initial concentrations (50, 100, and 150 μM).

111

Then, 1 mL of the prepared FX solution was mixed with 1 mL of deionized water.

112

Aliquots of water (100 μL) were successively added thereto over a period of time (200,

113

100, 20, 4 min) until the total volume was 4 mL, corresponding to the adding rates of

114

10, 20, 100, and 500 μL min−1. Owing to the light sensitivity of FX, samples were

115

prepared under dimmed light at room temperature and directly measured after

116

preparation. UV/vis spectra of FX in ethanol or ethanol/water binary solvents were

117

recorded in the range of 350–550 nm at 25 °C using a 2102PC spectrophotometer

118

(Shanghai Unico Instruments Co., Ltd., China).

119

Molecular arrangement calculation of FX aggregates

120

First, the oscillator strength (f) of the S0 → S2 transitions of FX was evaluated by using

121

the equation:24,

122

𝑓 = 4.319 × 10 ―9∫𝜎1𝜀(𝜎)d𝜎

123

Where ε was the molar extinction coefﬁcient (M−1 cm−1). σ was the respective

124

wavenumber (cm−1) determined by UV-vis spectroscopy.

125

The magnitude of the transition dipole moment of FX in ethanol (M, D), which was

126

necessary to calculate the molecular arrangement of FX aggregates, was then

𝜎

(1)

2

8


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determined by the following relation:25

128

𝑓=

129

Where, me was the mass of the electron, 9.1094 × 10–31 kg. c was the lightspeed,

130

2.9979×108 m s–1. h was Planck constant, 6.6261×10–34 J s. e was the quantity of electric

131

charge, 1.6022×10–19 C.

132

The distance between two FX molecules in their aggregated form (R, nm) was estimated

133

by a point-dipole approximation according to the following equation:26

134

2𝑉12 = 44𝜋𝜀0

135

Where, V12 was interaction energy (J), which was obtained as the difference between

136

the (0–0) band energies of the FX in a monomer state and in an aggregated state. ε0 was

137

the vacuum permittivity, 8.8542×10−12 C2 J−1 m−1. n was the refractive index of the

138

medium (ethanol = 1.36). N was the aggregation number. θ was the angle between the

139

FX molecules (Figure 1C). α and β were the angles between the FX molecules and the

140

line between the FX centers (Figure 1C).

141

MD simulation of FX aggregates

142

In addition to the point-dipole approximation calculation, the MD simulation was used

143

to study the molecular arrangement of FX dimer. First, the FX molecule (ID: 85552299)

144

was downloaded from the ZINC database, available at http://zinc.docking.org/. Its

145

structure was optimized by the B3LYP functional and the 6-31G* basis set, using the

146

Gaussian 16 program.27 The MD was performed using the Gromacs program (version

147

2018.3).28 An OPLS-AA force field was used to simulate monomeric and aggregated

8𝜋2𝑚𝑒𝑐𝜎12

|𝑀|2

3ℎ𝑒2

1

(

(2)

) ( )( )(cos 𝜃 ― 3cos 𝛼cos 𝛽)

𝑛2 + 2 1 𝑁 ― 1 3 𝑛2 𝑁

|𝑀|2 𝑅3

9


(3)


148

FX. Then, two FX molecules were placed in a cubic box (50 × 50 × 50 Å3), and the

149

remaining space was filled with ethanol and SPC/E water molecules, resulting in a final

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volume ratio of 50:50 and 25:75. The energy of the box was optimized using the

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steepest descent method, with a maximum step of 5 000, up to a maximum force Fmax

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of no more than 500.0 kJ mol−1 nm−1. The box was then equilibrated under the canonical

153

and isothermal−isobaric ensembles. After each equilibration for 0.5 ns, production runs

154

of 10 ns were performed. Geometry frames were saved every 10 ps. Finally, the PyMOL

155

package (version 1.8) was used for the visualization of MD results. The intermolecular

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distance, SASA, interaction energy, and dihedral angle were analyzed using the

157

Gromacs program (version 2018.3). The SESA was computed with a probe radius of

158

~1.4 Å using the Chimera package (version 1.12).29

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Fluorescence measurements

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All fluorescence measurements were performed using an F-4600 fluorescence

161

spectrophotometer (Hitachi Ltd., Tokyo, Japan). For three-dimensional fluorescence

162

measurement, FX was dissolved in ethanol or ethanol/water binary solvents to form its

163

monomer, H- and J-aggregates based on the above section. Then, the emission spectra

164

were acquired in the range of 400–750 nm with a scanning rate of 3000 nm min−1, and

165

the initial excitation wavelength was set to 400 nm in increments of 5 nm.

166

The β-Lg solution with a concentration of 5.0 μM was prepared using 20 mM PB (pH

167

7.4). A weighed amount of WPI powder was also dispersed in PB (20 mM, pH 7.4)

168

with gentle stirring at 25 °C for 2 h to ensure complete hydration of the protein. Then 10


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the solution was filtered by a 0.22 μm pore-size filter membrane and fixed to a protein

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concentration of 0.1% (w/v). For fluorescence quenching measurement, the intrinsic

171

fluorescence was measured by mixing 5 mL of the above protein solutions with

172

monomeric, H-, and J-aggregated FX. The excitation wavelength was set at 280 nm,

173

and the emission spectra were recorded in the range of 300–450 nm at 25 °C. The

174

excitation and emission slit widths were fixed at 10 nm. The spectral data were

175

processed using the well-known Stern–Volmer equation as follows:21

176

𝐹0 𝐹

(4)

= 1 + 𝐾SV[𝑄]

177

Where F0 and F represented the steady-state fluorescence intensities in the absence and

178

presence of FX, respectively. KSV was the Stern–Volmer constant (M−1). [Q] was the

179

concentration of the FX (M). Then, the equilibrium between the free and bound FX in

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sites of whey proteins was given by the following equation:21

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𝐹0 ― 𝐹

log

𝐹

= log𝐾𝑎 +𝑛log[𝑄]

(5)

182

Where Ka and n were the binding constant and the number of binding sites, respectively.

183

Encapsulation efficiency

184

The encapsulation efficiency of FX in β-Lg and WPI was determined separately as

185

described earlier with some modifications.8 The solutions of β-Lg and WPI with

186

monomeric or aggregated FX were centrifuged in Millipore (10 kDa, MWCO, 50 mL)

187

filters at 4000 g for 20 min and washed with PB (20 mM, pH 7.4) three times to remove

188

free FX. After the ultrafiltration centrifugation, the acetone/n-hexane (1:1, v/v) was

189

added to each sample at a volume ratio of 1:1, and vortexed for 10 s. Then the upper 11



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organic phase was collected, concentrated under a steam of nitrogen, and dissolved in

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acetonitrile for HPLC analysis (1260, Agilent Technologies Inc., Santa Clara, CA,

192

USA).22 The encapsulation efficiency of FX was calculated as follows:

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Encapsulation efficiency (%) =

Loaded FX (μmol) Total FX (μmol)

× 100

(6)

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Particle size determination

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The solutions of β-Lg and WPI with monomeric or aggregated FX were stored at 25 °C

196

for 3 d. Then, these samples were taken periodically, and their particle size was

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measured by a Zetasizer Nano ZS90 instrument (Malvern Instruments Ltd., Malvern,

198

Worcestershire, U.K.). The optic was used with a scattering detector angle of 90° and

199

the light source was a He/Ne laser with a wavelength of 632.8 nm. Each sample solution

200

was vortexed for 10 s, then taking 1 mL of sample in a plastic cuvette (1.0 cm path

201

length) within the sample holder of the analyzer. The average hydrodynamic diameter

202

(Dh) was calculated using the Stokes-Einstein equation. All measurements were

203

conducted at 25 °C.

204

In vitro digestion model

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In vitro digestion of the complexes of β-Lg/WPI with monomeric and aggregated FX

206

was performed following the Minekus et al. with the slight modification.30 To simulate

207

the gastric phase, 10 mL of sample was mixed with 7.5 mL of SGF stock solution (6.9

208

mM KCl, 0.9 mM KH2PO4, 25 mM NaHCO3, 47.2 mM NaCl, 0.1 mM MgCl2·6H2O).

209

Then, the pepsin (2 000 U mL−1 in final digestion mixture) and CaCl2 (0.075 mM in

210

final digestion mixture) was added and the pH was adjusted to 3.0 using 1.0 M HCl. 12


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The mixtures were incubated for 2 h in a shaking incubator (95 rpm, 37 °C). In the

212

following intestinal phase, 20 mL of gastric chyme was mixed with 11 mL of SIF stock

213

solution (6.8 mM KCl, 0.8 mM KH2PO4, 85 mM NaHCO3, 38.4 mM NaCl, 0.33 mM

214

MgCl2·6H2O), 5.0 mL of porcine pancreatin solution (800 U mL−1 in SIF), 2.5 mL of

215

porcine bile salt (12 mg mL−1 in SIF), 40 μL of CaCl2 (0.3 M). The pH was adjusted to

216

7.0 using 1.0 M NaOH, and the mixture was incubated for 2 h while shaken as

217

mentioned above. After completion of the intestinal phase, samples were made up to

218

50 mL with deionized water and centrifuged at 60 000 g for 40 min at 10 °C (Avanti J-

219

30, Beckman Coulter, Inc., CA, USA) to separate solids from the aqueous phase. Then,

220

an aliquot of the supernatant was retained, and the remainder was membrane-filtered

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(0.22 μm) to obtain the micellar phase. Both the unfiltered and filtered fractions were

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extracted using an equal volume of acetone/n-hexane (1:1, v/v) and vortexed for 10 s.

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Then, the upper organic phase was evaporated to dryness under nitrogen and dissolved

224

in acetonitrile to determine FX content using HPLC. All measurements were completed

225

on the same day. In vitro FX liberation and bioaccessibility referred to the percentage

226

of FX transferred from the test sample to the supernatant obtained after centrifugation

227

and to the micellar phase obtained by membrane filtration of the above-mentioned

228

supernatant, respectively.31

229

Statistical analysis

230

Data were expressed as the mean ± standard deviation of three separate experiments.

231

All statistical analysis was conducted using SPSS software (version 19.0, SPSS Inc., 13



232

Ammonst, New York, United States). The Shapiro–Wilk test was used to assess the

233

normality of data and the Levene test was used to check the homoscedasticity. The

234

difference between the heating treatments was evaluated using the one-way analysis of

235

variance (ANOVA) and Duncan's multiple comparisons. All statements of significance

236

were based on the 0.05 probability level.

237

Results and discussion

238

UV-vis and fluorescence spectra of FX aggregates

239

Similar to other carotenoids, FX had a conjugated polyene skeleton belonging to the

240

C2h point group. As shown in Figure 2A, its absorption bands in the visible spectral

241

range of 350−550 nm corresponded to an S0 → S2 (1Ag− → 1Bu+) electronic transition,

242

attributed to the 0–0, 0–1, and 0–2 vibrational transitions.32 The maximum absorption

243

peak (λmax) was around 445 nm and gradually decreased with an increasing ethanol

244

concentration without shifting, indicating that the spectral feature of FX was retained

245

and the FX molecules existed in a monodisperse state. The λmax of FX in Figure 2B and

246

2C was shifted with two opposite orientations by adding deionized water with different

247

rates. The absorption to shorter or longer wavelengths were caused by different

248

arrangements between the polyene chains in FX molecule, representing the spectral

249

characteristics of H- and J-aggregates.33 When dropping water rapidly (500 μL/min),

250

the spectrum of FX showed a hypsochromic shift from 445 nm to 435 nm, indicating

251

the predominant formation of the H-aggregates, in which transition dipoles were

252

oriented in a horizontal card-pack alignment (Figure 1A).33 Conversely, when water 14


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was slowly added (20 μL/min), the absorption spectrum of FX showed a bathochromic

254

shift from 445 nm to 466 nm with a new hump at 525 nm, suggesting that J-aggregates

255

were mainly formed and their dipole transitions were oriented in a head-to-tail

256

alignment (Figure 1B).33

257

The three-dimensional fluorescence subtracted from the solvent background in Figure

258

3 also provided some information on aggregated FX. As shown in Figure 3A,

259

monomeric FX in ethanol did not exhibit the significant Rayleigh scattering peaks.

260

Besides, an emission with a large Stokes shift relative to its own absorption was also

261

not observed, corresponding to the S1 → S0 (2Ag− → 1Ag−) transition around 700−750

262

nm.34 As previously reported, FX in carbon disulfide and tetrahydrofuran had a

263

maximum emission peak at 750 nm when excited at 475 nm and 430 nm,

264

respectively.35,36 In present study, the absence of the above fluorescent feature might

265

be due to the strong solvent dependence of the emission intensity of FX. Bautista et al.

266

showed that the peridinin, a structural analogue of FX, had a strong emission in solvents

267

having a low dielectric constant (e.g. carbon disulfide and n-hexane), while in solvents

268

with a high dielectric constant (e.g. methanol, ethanol, and acetonitrile), its emission

269

was weak, and the quantum yield was relatively low.37 After dilution with deionized

270

water, the three-dimensional fluorescence of FX were dominated by the apparent first-

271

order (λex = λem) Rayleigh scattering peaks in Figure 3B and 3C. This effect could be

272

attributed to the increase in molecular size caused by FX aggregation, which in turn led

273

to increased scattering.38

15



274

Factors influencing the formation of FX aggregates

275

As discussed above, when deionized water was dripped into the ethanol/water (50/50,

276

v/v) at different rates, the spectral characteristics of the H- and J-aggregates of FX could

277

be observed. So, in order to further study the factors affecting aggregation, the

278

absorbance values at 419 nm and 525 nm were selected to quantify the CAR of water

279

to ethanol. As shown in Figure 4A, the A419 of FX diluted with water at 500 μL/min

280

began to decrease densely when the water/ethanol ratio rapidly increased to 66.7/33.3

281

(v/v). This value was consistent with the inflection point of λmax in Figure 2B,

282

corresponding to the CAR of H-aggregation. As shown in Figure 4B, the A525 of FX

283

diluted with water at 20 μL/min started to rise as the water/ethanol ratio increased to

284

60/40 (v/v), which was considered to be the CAR of J-aggregation, well agreeing with

285

the redshift of spectra in Figure 2C. It is worth noting that the CAR of J-aggregates was

286

lower than that of H-aggregates. Similarly, Billsten et al. indicated a low water content

287

was necessary to form and maintain the J-aggregated zeaxanthin in water/ethanol

288

mixtures, and an increasing number of water molecules compelled the head-to-tail

289

arrangement to transform into the card-pack assembly that pushed the water molecules

290

away from the conjugated chains.39

291

The above results also suggested the J-aggregates of FX were difficult to form than H-

292

aggregates because it requires a slow growth in the water/ethanol ratio for elaborate

293

preparation. Thus, some factors affecting the formation of J-aggregates were further

294

studied. As shown in Figure 4C, the CAR of water/ethanol was reduced as the rate of

16


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295

water addition slowed, indicating the J-aggregates became earlier to produce. A

296

previous study reported the H-aggregates of astaxanthin oximium hydrochloride were

297

formed when water was continuously added to its methanol solution in small

298

increments, whereas the J-aggregates were produced when it was immediately

299

dispersed in water.40 It seemed that was contrary to the result of this study, which might

300

be due to the positively charged terminal rings of the astaxanthin oximium

301

hydrochloride. In their case, when the aggregates were self-assembled in a card-pack

302

form, the electrostatic repulsion between the molecules was greater than the head-to-

303

tail arrangement. Besides, data in Figure 4D showed the CAR of J-aggregates prepared

304

at initial FX concentrations of 150 and 100 μM were lower than that at 50 μM. The

305

result revealed the higher the initial concentration, the closer the distance between the

306

FX molecules in the binary solution, the more favorable for they were to form J-

307

aggregates. This was also similar to the case of zeaxanthin in ethanol/water solvent,

308

showing the J-aggregates were preferentially formed at an initial concentration as high

309

as 100 μM.39

310

Figure 5 depicted the normalized absorption spectra of FX aggregates dependent on

311

time variation. As shown in Figure 5A, after the formation of H-aggregates, the λmax of

312

FX shifted from 448 nm to 419 nm as the storage time extended, indicating the degree

313

of aggregation increased with time. After forming J-aggregates, the λmax in Figure 5B

314

shifted from 448 nm to 453 nm with a new peak appearing at 493 nm. After standing

315

for 96 h, its absorption spectrum had a hypochromatic shift as a whole (Figure 5B).

316

This result suggested the FX aggregates changed from a J-type form to a H-type form 17



317

in the hydrated ethanol solvent, which was consistent with the report of Avital et al.,

318

demonstrating the structural transition of zeaxanthin aggregates in Triton X-100

319

micelles.41

320

Calculation of FX aggregate alignment

321

After the successful preparation of FX aggregates, their molecular alignment was

322

studied by the point-dipole approximation model. Noteworthy, this model was only

323

suitable for calculating H-aggregates, because the spectral shift of J-aggregates was

324

mainly caused by the dispersive interaction forces, thus hindering approximation

325

calculation.23 In order to use the point-dipole model, the magnitude of the S0 → S2

326

transition dipole moment of FX was first numerically calculated. It was obtained from

327

the absorption spectrum in Figure 6, which was multi-peak deconvoluted using five

328

Gaussian functions. The fitting results showed that average wavenumber difference

329

between the fitted vibration peaks was about 1293 cm−1, which was between the C–C

330

stretching vibration (1150 cm−1) and the C=C double bond stretching vibration (1600

331

cm−1). This was consistent with the stretching vibration frequency of the carotenoid

332

conjugated polyene skeleton. As a result, using Eq. (1) and (2), the magnitude of

333

transition dipole moment of FX was calculated to be 12.5 D, which was smaller than

334

the reported values of 18.4 D in benzene at room temperature.42 The result appeared to

335

be a decrease in dipole moment of FX as the solvent polarity increased, which can be

336

attributed to the dependence of the electron structure of the donor/acceptor-substituted

337

polyenes on the solvent effect. Previously, Bublitz et al. performed Stark spectroscopic 18


Page 16 of 47

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338

experiments on some above polyene compounds, indicating that their ground-state

339

structure could become more or less dipolar depending on the solvent.43 With an

340

increasing solvent polarity, the polyene chain changed from an unperturbed state

341

toward a fully delocalized structure, and for even higher solvent polarity, toward the

342

limit of an again localized structure but with charge separation. In this process, the

343

dipole moment tended to decline.43 Frank et al. reported that the allene and carbonyl

344

groups in FX can act as electron donors and electron acceptors, respectively. Therefore,

345

the behavior of FX bore some resemblance to that of donor/acceptor polyenes.34

346

Further, since the 0–0 absorption band positions of FX monomer and H-aggregates

347

were 21 026 cm−1 and 23 256 cm−1, respectively, the interaction energy (V12) between

348

FX molecules forming the H-aggregate was calculated as 2230 cm−1. Then, the

349

approach of Hempel et al. was used to perform the initial approximation by setting N =

350

2, θ = 0°, and α = β = 90°.23 The intermolecular distance (R, Figure 1C) between the

351

planes of the polyene chains of two adjacent FX was calculated to be 6.8 Å. However,

352

owing to the steric hindrance of end groups, aligned FX molecules should be slightly

353

twisted (θ ≠ 0°) to avoid overlapping of their end groups. Based on some previously

354

proposed torsion angles of xanthophylls ranged from 20° to 35°,23 FX was also assumed

355

to have the same value due to the structural similarity of these compounds. As a result,

356

R = 6.4−6.7 Å was estimated for H-aggregates of FX.

357

MD simulation of FX aggregates

358

Since the point-dipole approximation model only calculated the molecular arrangement 19



359

of H-aggregates, the MD was applied to simulate the structure of H- and J-aggregated

360

FX in aqueous ethanol. Based on the experimental conditions, two FX molecules were

361

randomly located in a box filled with water/ethanol solvent (50/50 and 75/25, v/v) and

362

subjected to a 10-ns dynamic simulation. By extracting the MD trajectory of FX

363

molecules in water/ethanol (75/25, v/v), the result in Figure 7 indicated they could form

364

a head-to-tail J-dimer and then assembled into the card-pack H-dimer, well agreeing

365

with the result of Figure 5B. Inversely, two FX molecules in water/ethanol (50/50, v/v)

366

were well dispersed without any signs of significant aggregation (Figure S1).

367

In order to further elucidate the conformational transformation of FX J-aggregates to

368

H-aggregates, some time-dependent parameters in the simulation were studied. The

369

intermolecular distance of two FX molecules during MD was first discussed. The result

370

in Figure 8A showed the distances between five pairs of carbon atoms in the polyene

371

chain of FX in water/ethanol (75/25, v/v) were decreased as the MD time prolonged,

372

showing the FX molecules were close to each other. In the period of 6.0−8.0 ns, the

373

polyene chains are rapidly adjacent to each other, corresponding to the transition of J-

374

type to H-type aggregates. After 8.0 ns, the distance between the five pairs of carbon

375

atoms of FX H-aggregates basically maintained invariably. At 10.0 ns, the

376

intermolecular distance of FX H-dimer was about 5.0 Å (Figure S2), which was slightly

377

less than the R (6.4−6.7 Å) calculated by the point-dipole approximation model. This

378

might be because the FX molecules interleaved card-pack alignment due to steric

379

hindrance of the terminal groups. However, two FX molecules in water/ethanol (50/50,

380

v/v) had a relatively large distance between the conjugated polyene chains (Figure 8A), 20


Page 18 of 47

Page 19 of 47


381

which made it difficult to form non-bond interactions between molecules, meaning the

382

FX existed in a monomeric form. Under this dissolving condition, the intermolecular

383

distance between two FX molecules was about 30.0 Å after MD (Figure S2).

384

Commonly, SASA was used to describe the surface area of a simulated molecule with

385

its surrounding solvent environment. When aggregation occurred, the FX molecules

386

approached each other, causing their solvent-accessible surface to overlap and total

387

SASA to decrease (Figure 7). As shown in Figure 8B, the SASA value of coupled FX

388

in water/ethanol (75/25, v/v) was significantly reduced after 4.5 ns, owing to the

389

proximity between FX molecules (i.e., the formation of aggregates). As the MD

390

proceeded, SASA continued to decrease and remained essentially constant at a later

391

stage (8.0−10.0 ns), indicating the overlapping area was increasing and eventually

392

reaching equilibrium. This was due to the transition of a head-to-tail pattern into a card-

393

pack alignment, which was also consistent with Figure 8A. At 10.0 ns, H-aggregates of

394

FX was formed with the SASA value of 1866 Å2, which was smaller than J-aggregates

395

(SASA = 2054 Å2) at 6.0 ns (Figure S3). Moreover, the SASA of FX molecules in

396

water/ethanol (50/50, v/v) was higher than in water/ethanol (75/25, v/v) after 4.5 ns,

397

illustrating that their solvent-accessible surfaces were not intertwined and showed a

398

feature of monomeric distribution (Figure S1).

399

Further, considering non-bonded interactions such as van der Waals and electrostatic

400

forces, the LJ-SR and Coul-SR potential energy were calculated between FX molecules.

401

As shown in Figure 8C, the different ratios of water to ethanol resulted in the disparities

402

in the interaction of one FX with another, especially in LJ-SR energy. A high-water 21



403

ratio dissolution will cause FX to display relatively lower interaction energy, which was

404

advantageous for stabilizing the structure upon forming FX aggregates. It was observed

405

that the LJ-SR energy between the two FX molecules was rapidly decreased from 4.5

406

ns, at which time the FX aggregates belonging to the J-type arrangement began to be

407

produced (Figure 7). As the simulation progressed, the LJ-SR energy was further

408

reduced within 4.5−6.0 ns and tended to stabilize at the 8.0−10.0 ns, during which time

409

the H-aggregates eventually formed.

410

Besides the above parameters, the dihedral angles between FX molecules during MD

411

were investigated. In the case of water/ethanol (75/25, v/v) in Figure 9A, the FX−FX

412

dihedral angle of J-type orientation at 4.5−6.0 ns was mainly between 35° and 140°,

413

while H-aggregation at 8.0−10.0 ns showed the dihedral angle between −35° and 35°.

414

In terms of percentage relative frequency obtained from the histogram (Figure 9B), the

415

results showed 99.0% of the dihedral angle in H-aggregated FX pairs was observed

416

between −35° and 35°, which was in accordance with torsion angles (20−35°) of the

417

above-mentioned xanthophylls. In the histogram of J-dimer at 4.5−6.0 ns (Figure 9C),

418

the angular distribution was wider than H-dimer. The highest probability was observed

419

between 35−140°, accounting for 90.1%. Some of the FX J-dimer had the dihedral

420

angles greater than 90° or less than −90°, accounting for 23.2%. In addition, the case

421

of water/ethanol (50/50, v/v) indicated the dihedral angle between two FX molecules

422

in 8.0−10.0 ns (Figure 9B) and 4.5−6.0 ns (Figure 9C) varied significantly, fluctuating

423

within a range of less than −70° and great than 70°. This result demonstrated that the

424

dynamic motion of the FX molecules was random and consistent with the monomer 22


Page 20 of 47

Page 21 of 47


425

characteristics.

426

Binding of whey proteins with FX monomer and aggregates

427

As mentioned above, the H- and J-aggregates of FX were successfully prepared, and

428

their structures were also ascertained. Next, their interactions with β-Lg and WPI were

429

studied by the classical fluorescence quenching. The fluorescence data fitted by Eq. (4)

430

were shown in Figure S4, and the detailed parameters were summarized in Table 1. The

431

results showed the Stern–Volmer model could provide a sufficient description for the

432

fluorescence quenching (R2 > 0.99) and was suitable for studying the interaction

433

between FX aggregates and whey proteins. As the protein fluorescence originated from

434

tryptophan residue with an emission lifetime (τ0) around 10 ns, the bimolecular

435

quenching constant for quenching, kq, could be calculated from the ratio of KSV to τ0.14

436

The result showed and the kq values (~1012 M−1 s−1) well exceeded the diffusion-

437

controlled rate constant of various quenchers with a biopolymer (2.0 × 1010 M−1 s−1),

438

supporting a static quenching mechanism of β-Lg and WPI by FX monomer and

439

aggregates, also meaning that they formed complexes.19

440

After clarifying the static quenching mechanism, the log[(F0−F)/F] versus log[Q] was

441

plotted in Figure S5 to evaluate the binding capability of β-Lg and WPI with FX

442

monomer and aggregates. The related parameters were also listed in Table 1. Obviously,

443

all curves of binding affinity also showed good linear relationships (R2 > 0.99). The

444

binding constant Ka of β-Lg and WPI to FX monomer were 2.270 × 104 M−1 and 3.214

445

× 104 M−1, respectively. These values were higher than the Ka of two FX aggregates, 23



446

indicating a stronger affinity of whey proteins for the monomeric FX. An explanation

447

for the above results might be that the self-aggregation of ligand molecule obstructed

448

its placement into the binding site in proteins, resulting in a decrease in the binding

449

constant and the number of binding sites. The similar results have been reported by

450

Zsila et al. who studied the interaction of astaxanthin disodium disuccinate and

451

astaxanthin dilysinate tetrahydrochloride with HSA. They found that these derivatives

452

interacted with HSA in the monomeric form at low concentrations. Once the

453

concentration was too high, the effect of aggregation became apparent, thus inhibiting

454

the binding to HSA.44,45 The results in Table 1 also showed that Ka of FX H-aggregates

455

bound to β-Lg and WPI were 1.986 × 104 M−1 and 2.690 × 104 M−1, respectively, which

456

were higher than that of J-aggregates. This result indicated that H-aggregated FX was

457

more preferably bound by whey proteins than J-aggregated FX, which might be due to

458

differences in the molecular surface of ligands affecting their accessibility relative to

459

acceptor molecules. As discussed above, the SASA of J-dimer was larger than that of

460

H-dimer. Besides, we computed the total SESA of FX aggregates, which gave the

461

boundary of the molecular volume with respect to a specific solvent. The results in

462

Figure S3 showed the SESA of FX J-dimer was 1219 Å2, which was also higher than

463

the H-dimer (1173 Å2). The above results indicated that the FX molecules after card-

464

pack arrangement would have a smaller molecular surface area than the head-to-tail

465

structure. Consequently, H-aggregates of FX were more easily bound to the

466

hydrophobic cavities in whey proteins than J-aggregates.

24


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467

Delivery of FX monomer and aggregates by whey proteins

468

Based on the above results, β-Lg and WPI could bind FX aggregates to form a complex

469

similar to that formed by binding FX monomer. Then, the delivering capabilities,

470

including encapsulation efficiency, physical stability, and release in vitro, were studied

471

in the following experiments. As shown in Figure 10A, when FX with different

472

configurations was captured by whey proteins, a higher encapsulation efficiency (over

473

60%) was observed for WPI compared to β-Lg. This was similar to the recently reported

474

WPI nanoparticles prepared using an ethanol desolvation method, and the entrapment

475

efficiency of lycopene was 64.7 ± 3.6%.46 Furthermore, the encapsulation efficiency of

476

FX monomer and aggregates in whey proteins was ranked as follows, monomer > H-

477

aggregates > J-aggregates. For β-Lg, this difference was not significant (p > 0.05). For

478

WPI, the loading effect of J-aggregates was evidently lower than that of monomers (p

479

< 0.05). The above results might be related to the affinity of these FX aggregates with

480

whey proteins, which previously indicated that the ability of whey proteins to bind

481

aggregates was weaker than that to bind monomers (Table 1).

482

In order to further evaluate the effects of different FX aggregation patterns on the

483

physical stability of whey protein−FX complexes, the scale changes within three days

484

were evaluated. As shown in Figure 10B, the particle size of all samples showed a

485

tendency to increase with the prolonging of time. At the end of storage, this trend

486

became more pronounced (p < 0.05), suggesting the whey protein−FX complexes

487

experienced a sustained aggregation process. Abbasi et al. also prepared the WPI

25



488

nanoparticles to entrap vitamin D3, indicating that a long storage time will lead to an

489

increase in particle size.47 These β-Lg/WPI−FX nanocomplexes were not very

490

physically stable compared to the nanoparticles obtained by the antisolvent method,

491

whose particle size could be stabilized for three months.46 However, they also showed

492

great potential as building blocks to interact with other biopolymers to further enhance

493

the stability, like a recent study on microencapsulation of ergosterol by complex

494

coacervation using whey protein and chitosan.48 In addition, the complexes formed by

495

different forms of FX in Figure 10B indicated that the particle sizes of β-Lg and WPI

496

with the H- and J-aggregates were significantly larger than those with FX monomer

497

when newly prepared (p < 0.05). After storage, the proportion of particle size increase

498

of the sample containing FX aggregates was also significantly higher than that of the

499

monomer FX group (p < 0.05). These results showed the complex prepared by FX

500

aggregates with whey proteins had poor physical stability compared to the case of FX

501

monomer.

502

Finally, the release properties of whey proteins encapsulating FX monomer and

503

aggregates in the gastrointestinal tract were investigated by the in vitro digestion model.

504

The results in Figure 10C showed that the liberation of FX monomer in β-Lg (57.6 ±

505

4.8%) and WPI (56.6 ± 5.1%) was higher than that of FX H- and J-aggregates, although

506

not reaching statistical significance (p > 0.05). This result indicated the whey proteins

507

had similar gastrointestinal protective effects on different forms of FX. This result

508

indicated that whey proteins had similar gastrointestinal protective effects on different

509

forms of FX, mainly because natural β-Lg resisted the hydrolysis of pepsin but could 26


Page 24 of 47

Page 25 of 47


510

be digested by trypsin in the intestinal tract.49 Moreover, in addition to the case of H-

511

FX in β-Lg, the bioaccessibility of FX monomer in β-Lg (12.6 ± 3.8%) and WPI (14.9

512

± 4.7%) was significantly higher than that of FX H- and J-aggregates (p < 0.05),

513

respectively, which was unexpected considering their efficient liberation. This result

514

might be due to the fact that FX molecules were embedded as an aggregated form into

515

the whey proteins. A previous study on carrot roots and tomato fruits showed that the

516

bioaccessibility of carotenoids in these two food matrices was poor, which was related

517

to their large, solid-crystalline aggregates in the chromoplasts. This large aggregated

518

carotenoid had a small surface-to-volume ratio, so it was not easily dissolved in dietary

519

lipids during digestion, resulting in poor bioaccessibility.50

520

ABBREVIATIONS USED

521

β-Lg, β-lactoglobulin; CAR, critical aggregation ratio; Coul-SR, short-range

522

Coulombic potential energy; FX, fucoxanthin; HSA, human serum protein; LJ-SR,

523

short-range Lennard-Jones potential energy; MD, molecular dynamics; OPLS-AA,

524

optimized potential for liquid simulations-all atom; PB, phosphate buffer; SESA,

525

solvent excluded surface area; SASA, solvent accessible surface area; SGF, simulated

526

gastric fluid; SIF, simulated intestinal fluid; WPI, whey protein isolate

527

ACKNOWLEDGMENTS

528

This work was funded by National Key R&D Program of China (2018YFD0700303,

529

2017YFB0203405), National Natural Science Foundation of China (31401549,

530

41606184, 21873034), Special Funds for Taishan Scholars Project of Shandong 27



531

Province (ts201712058), Agriculture Scientific and Technological Innovation Project

532

of Shandong Academy of Agriculture Sciences (CXGC2016B10), China Scholarship

533

Council (201808370051).

534

SUPPORTING INFORMATION DESCRIPTION

535

Two fucoxanthin molecules with their solvent-accessible surface in water/ethanol

536

(50/50, v/v) simulated by molecular dynamics (Figure S1); Molecular arrangement of

537

two fucoxanthin molecules at 10.0 ns in water/ethanol solvents with volume ratios of

538

75/25 and 50/50 (Figure S2); Solvent excluded surface area (SESA) and solvent

539

accessible surface area (SASA) of fucoxanthin H-dimer and J-dimer in water/ethanol

540

(75/25, v/v) during molecular dynamics (Figure S3); Stern−Volmer model calculated

541

by intrinsic fluorescence of β-lactoglobulin (β-Lg) and whey protein isolates (WPI)

542

quenched with fucoxanthin (FX) monomer and aggregates (Figure S4); Binding model

543

calculated by intrinsic fluorescence of β-lactoglobulin (β-Lg) and whey protein isolates

544

(WPI) quenched with fucoxanthin (FX) monomer and aggregates (Figure S5).

545

NOTES

546

The authors declare no competing financial interest.

547

28


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548

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heat-processed orange-fleshed sweet potato. J. Agric. Food Chem. 2009, 57, 9693–9698.

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Photosynthetic Functions. In Frontiers of Optical Spectroscopy; Di Bartolo B.; Forte, O.;

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dynamics of the lowest excited states of carotenoids. J. Phys. Chem. B 2000, 104, 4569−4577.

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fucoxanthin: Implication for energy transfer in photosynthetic pigment systems. Photosynth.

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Wasielewski, M. R.; Frank, H. A. Excited state properties of peridinin: observation of a solvent

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dependence of the lowest excited singlet state lifetime and spectral behavior unique among

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protein binding and aqueous aggregation behavior of astaxanthin dilysinate tetrahydrochloride.

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700 35



701

FIGURE CAPTIONS

702

Figure 1 Card-pack H-aggregates (A) and head-to-tail J-aggregates (B) of FX.

703

Molecular arrangement of FX H-aggregates as illustrated by the point-dipole

704

approximation model (C).

705

Figure 2 Absorption spectra of FX in different solvents: Adding ethanol to reduce

706

water/ethanol (v/v) from 50/50 to 25/75 (A); Adding deionized water to increase

707

water/ethanol (v/v) from 50/50 to 75/25 with dripping rates of 500 μL/min (B) and 20

708

μL/min (C).

709

Figure 3 Three-dimensional fluorescence of FX in different solvents: Monomer in

710

ethanol (A); H-aggregates in water/ethanol (75/25, v/v) with diluted rates of 500

711

μL/min (B); J-aggregates in water/ethanol (75/25, v/v) with diluted rates of 20 μL/min

712

(C). The fluorescent signal of the corresponding solvent is subtracted as background.

713

Figure 4 Absorbance of FX in water/ethanol (50/50, v/v) diluted with ethanol or

714

deionized water with 500 μL/min (A) and 20 μL/min (B). Effects of dripping rate (C)

715

and initial concentration of FX (D) on absorbance at 525 nm of FX in water/ethanol

716

(50/50, v/v) diluted with deionized water. The dashed line is the critical water/ethanol

717

ratio at which aggregation occurs.

718

Figure 5 Effects of storage time (0−96 h) on absorption spectra of FX in water/ethanol

719

(75/25, v/v) with diluted rates of 500 μL/min (A) and 20 μL/min (B). The control

720

represents the absorption spectrum of FX in ethanol.

36


Page 34 of 47

Page 35 of 47


721

Figure 6 Spectral deconvolution of the absorption spectrum of FX in ethanol using five

722

Gaussian functions. Solid line shows experimentally observed absorption spectrum,

723

dash line is simulated spectrum, dot lines are results of spectral deconvolution, σ1 is

724

0−0 absorption band.

725

Figure 7 Two FX molecules with their solvent-accessible surface in water/ethanol

726

(75/25, v/v) simulated by MD. J-aggregates are formed at 4.5−6.0 ns, and then

727

gradually converted to H-aggregates from 6.0 ns to 8.0 ns. Finally, H-type aggregation

728

remains stable until the end of the MD.

729

Figure 8 Changes in distances between five pairs of carbon atoms in the polyene chain

730

of FX (A), total SASA of two FX molecules (B), and LJ-SR and Coul-SR energy

731

between FX molecules (C) during MD. The time when J-aggregates are converted to

732

H-aggregates is marked.

733

Figure 9 Changes in dihedral angle between FX molecules during MD (A). The

734

conformational trajectories for histogram statistics are extracted from 8.0–10.0 ns (B)

735

and 4.5–6.0 ns (C).

736

Figure 10 Encapsulation efficiency (A), physical stability (B), and in vitro release

737

property (C) of monomeric, H-type, J-type aggregated FX in β-Lg and WPI

738

nanocomplexes. Mono-FX, H-FX, and J-FX represent FX monomer, H-aggregates, and

739

J-aggregates, respectively.

37



Page 36 of 47

TABLES Table 1 Stern–Volmer constant (KSV), biomolecular quenching constant (kq), binding constant (Ka), and number of binding sites (n) for the interaction of β-Lg and WPI Systemsa β-Lg + mono-FX β-Lg + H-FX β-Lg + J-FX WPI + mono-FX WPI + H-FX WPI + J-FX a

with FX monomer and aggregates KSV (M−1) kq (M−1 R2 Ka (M−1) s−1) 4.696×104 4.696×1012 0.9990 2.270×104 4.389×104 4.389×1012 0.9930 1.986×104 3.866×104 3.866×1012 0.9924 1.306×104 4.483×104 4.483×1012 0.9966 3.214×104 3.978×104 3.978×1012 0.9965 2.690×104 3.649×104 3.649×1012 0.9990 2.218×104

n

R2

0.936 0.930 0.905 0.970 0.965 0.953

0.9942 0.9994 0.9969 0.9986 0.9994 0.9979

mono-FX, H-FX, and J-FX represent FX monomer, H-aggregates, and J-aggregates,

respectively.

38


Page 37 of 47


TABLE OF CONTENTS GRAPHIC

39



Figure 1 Card-pack H-aggregates (A) and head-to-tail J-aggregates (B) of FX. Molecular arrangement of FX H-aggregates as illustrated by the point-dipole approximation model (C).


Page 38 of 47

Page 39 of 47


Figure 2 Absorption spectra of FX in different solvents: Adding ethanol to reduce water/ethanol (v/v) from 50/50 to 25/75 (A); Adding deionized water to increase water/ethanol (v/v) from 50/50 to 75/25 with dripping rates of 500 μL/min (B) and 20 μL/min (C). 292x785mm (109 x 109 DPI)



Figure 3 Three-dimensional fluorescence of FX in different solvents: Monomer in ethanol (A); H-aggregates in water/ethanol (75/25, v/v) with diluted rates of 500 μL/min (B); J-aggregates in water/ethanol (75/25, v/v) with diluted rates of 20 μL/min (C). The fluorescent signal of the corresponding solvent is subtracted as background. 121x285mm (300 x 300 DPI)


Page 40 of 47

Page 41 of 47


Figure 4 Absorbance of FX in water/ethanol (50/50, v/v) diluted with ethanol or deionized water with 500 μL/min (A) and 20 μL/min (B). Effects of dripping rate (C) and initial concentration of FX (D) on absorbance at 525 nm of FX in water/ethanol (50/50, v/v) diluted with deionized water. The dashed line is the critical water/ethanol ratio at which aggregation occurs. 696x535mm (109 x 109 DPI)



Figure 5 Effects of storage time (0−96 h) on absorption spectra of FX in water/ethanol (75/25, v/v) with diluted rates of 500 μL/min (A) and 20 μL/min (B). The control represents the absorption spectrum of FX in ethanol. 280x391mm (109 x 109 DPI)


Page 42 of 47

Page 43 of 47


Figure 6 Spectral deconvolution of the absorption spectrum of FX in ethanol using five Gaussian functions. Solid line shows experimentally observed absorption spectrum, dash line is simulated spectrum, dot lines are results of spectral deconvolution, σ1 is 0−0 absorption band. 973x564mm (109 x 109 DPI)



Figure 7 Two FX molecules with their solvent-accessible surface in water/ethanol (75/25, v/v) simulated by MD. J-aggregates are formed at 4.5−6.0 ns, and then gradually converted to H-aggregates from 6.0 ns to 8.0 ns. Finally, H-type aggregation remains stable until the end of the MD. 1222x677mm (109 x 109 DPI)


Page 44 of 47

Page 45 of 47


Figure 8 Changes in distances between five pairs of carbon atoms in the polyene chain of FX (A), total SASA of two FX molecules (B), and LJ-SR and Coul-SR energy between FX molecules (C) during MD. The time when J-aggregates are converted to H-aggregates is marked. 179x240mm (300 x 300 DPI)



Figure 9 Changes in dihedral angle between FX molecules during MD (A). The conformational trajectories for histogram statistics are extracted from 8.0–10.0 ns (B) and 4.5–6.0 ns (C). 141x242mm (300 x 300 DPI)


Page 46 of 47

Page 47 of 47


Figure 10 Encapsulation efficiency (A), physical stability (B), and in vitro release property (C) of monomeric, H-type, J-type aggregated FX in β-Lg and WPI nanocomplexes. Mono-FX, H-FX, and J-FX represent FX monomer, H-aggregates, and J-aggregates, respectively. 99x243mm (300 x 300 DPI)


Aggregation of Fucoxanthin and Its Effects on Binding and Delivery

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