Alcalase Enzymolysis of Red Bean (adzuki) Ferritin Achieves

Jan 29, 2018 - Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Beijing 1000...
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Alcalase enzymolysis of red bean (adzuki) ferritin achieves nano-encapsulation of food nutrients in a mild condition Rui Yang, Yuqian Liu, Demei Meng, Christopher L. Blanchard, and Zhongkai Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05656 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on January 30, 2018

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Alcalase enzymolysis of red bean (adzuki) ferritin achieves

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nano-encapsulation of food nutrients in a mild condition

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Rui Yang a,b*, Yuqian Liu a, Demei Meng a, Christopher L. Blanchard c, Zhongkai Zhou a,*

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a

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Tianjin University of Science and Technology, Tianjin, 300457, People’s Republic of China.

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b

State Key Laboratory of Food Nutrition and Safety, College of Food Engineering and Biotechnology,

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology

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and Business University (BTBU), Beijing 100048, People’s Republic of China.

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c

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Australia.

ARC Industrial Transformation Training Centre for Functional Grains, Wagga Wagga NSW 2678,

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*Corresponding authors

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Dr. Rui Yang and Prof. Zhongkai Zhou

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College of Food Engineering and Biotechnology, Tianjin University of Science and Technology

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E-mail: [email protected]; [email protected]

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ABSTRACT: Classical methods to fabricate ferritin-nutrients shell-core nanoparticles usually apply

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extremely acid/alkaline pH transition, which may cause the activity loss of nutrients or the formation

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of insoluble aggregates. In this work, we prepared an extension peptide (EP) deleted red bean (adzuki)

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ferritin (apoRBF∆EP) by Alcalase 3.0T enzymolysis. Such enzymolysis could delete the EP domain

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and remain the typical shell-like structure of the ferritin. Meanwhile, the α-helix content of

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apoRBF∆EP was decreased by 5.5% and the transition temperature (Tm) was decreased by 4.1 °C.

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Interestingly, the apoRBF∆EP can be disassembled into subunits under a benign condition at pH 4.0

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and is assembled to form an intact cage protein when the pH was increased to 6.7. By using this

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novel route, the epigallocatechin gallate (EGCG) molecules were successfully encapsulated into the

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apoRBF∆EP cage with an encapsulation ratio of 11.6% (w/w) which was comparable with that by

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the traditional pH 2.0 transition. The newly prepared EGCG-loaded apoRBF∆EP exhibited a

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similarly protective effect on the EGCG upon simulated gastrointestinal tract and thermal treatment,

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as compared with the control. In addition, the EGCG-loaded apoRBF∆EP could significantly relieve

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the ferritin association induced by pH transition, which was superior to traditional method. The

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thinking of this work will be especially suitable for encapsulating pH-sensitive molecules based on

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ferritin in a benign condition.

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KEYWORDS: Alcalase; enzymolysis; ferritin; food nutrients; shell-core nanoparticle

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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Ferritins, a kind of shell-like protein that can store thousands of iron in the inner cavity, are

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widely distributed in animals, plants, and microorganisms.[1] Each ferritin consists of 24 similar

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homopolymer H or L type subunits.

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four-α-helix bundle with two antiparallel helix pairs (A and B, C and D) which are connected by a

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BC-loop between helices B and C. There is an E helix locating at one end of the bundle with 60° to

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the nearby 4-fold axis, which spatially forms the 4-fold channels. [4,5] These α-helix bundles fold to

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form a cage-like structure with inner diameter of 8 nm and external size of 12 nm, respectively.

[2,3]

Structurally, each ferritin subunit is comprised of a

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In recent years, the 8 nm nano-cavity of the ferritin has been sparkly applied for the

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encapsulation of food bioactive molecules by taking advantage of the reversible assembly character.

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Briefly, the reversible assembly can be described as follows: the ferritin cage must firstly be

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disassembled into ferritin subunits in an extreme acid pH condition at ≤ pH 2.0, and the reassembly

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should be achieved when the solution pH is adjusted to neutral. [1] By using this method, β-carotene,

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lutein, EGCG, proanthocyanidins, and rutin have been successfully encapsulated into the

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phytoferritin or recombinant ferritin. [6-10] However, it should be noted that the pH changes may bring

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about a large fraction of the ferritin to insoluble aggregates. Additionally, after the extreme pH

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transform, the integrity of the reassembled ferritin remains a question.[11] The extreme acid/alkaline

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conditions may abrogate the sensory properties, stability, and bioactivity of the certain pH-sensitive

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molecules, such as EGCG and rutin. Thus, to realize the successful encapsulation of the food

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nutrients in the ferritin cage and meanwhile to stabilize the targeted nutrients in a relative mild

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condition are challenges.

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One of the mostly used ferritin vehicle for the encapsulation of food nutrients is phytoferritin.

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Structurally, an obvious difference between the phytoferritin and vertebrate ferritin is that there is a

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specific domain, namely the extension peptide (EP), at N-terminal sequence of the phytoferritin

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(Figure 1a). The EP is located on the exterior surface of the ferritin shell and has been reported to be

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involved in the mineral core formation and iron release in pea seed ferritin.[12-13] In addition, the EP

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can stabilize the entire oligomeric conformation of phytoferritin by its interaction with a neighboring

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subunit on the shell surface. [14] Previous literature has also revealed that Alcalase was specifically

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for deletion of EP, and this deletion could relieve the ferritin aggregation and played an important

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role in the ferritin stability.[12] Inspired by these findings, how the absence of the EP influences the

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ferritin folding upon pH transition, and whether this deletion can affect the nano-encapsulation

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behavior of the EP deleted ferritin are worth investigating questions.

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The aim of this work is to investigate the disassembly and reassembly property of the apo-red

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bean (adzuki) ferritin (deprived of irons) by EP deletion with Alcalase enzymolysis (apoRBF∆EP),

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to prepare the epigallocatechin gallate (EGCG)-loaded apoRBF∆EP, and to evaluate their

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encapsulation behavior and condition as compared with the traditional method. The effects of EP

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deletion on the structure changes of the apoRBF, the morphology of EGCG-loaded apoRBF∆EP, the

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encapsulation ratio of the EGCG, and the stability of EGCG are emphasized. This work will be

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useful for fabrication of ferritin-nutrients nanoparticle based on a modified ferritin under a mild

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condition, which is especially suitable for the encapsulation of pH-sensitive molecules without

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undergoing extreme pH changes.

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MATERIALS AND METHODS 4

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Ferritin preparation and Native-PAGE and SDS-PAGE analysis

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Red bean (adzuki) ferritin (RBF) and apoRBF (deprived of irons) was prepared as previously

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described.[15] The molecular weight of apoRBF was estimated by Native-PAGE using a 12%

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polyacrylamide gradient gels running at 5 mA for 8 h at 4 °C. SDS-PAGE was performed under a

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reducing condition in 15% SDS-polyacrylamide gel. Gels were stained with bromophenol blue. The

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concentration of ferritin was determined according to the Lowry method by using Bull Serum

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Albumin as a standard sample.

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Preparation of EP deleted apoRBF (apoRBF∆EP)

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The Alcalase is a serine protease that can specifically hydrolyze the peptide bond near serine.

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The apoRBF∆EP was prepared by Alcalase 3.0T according to the reported method.[12] Typically,

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purified apoRBF (1.0 mg) was added to 0.18 mL of Alcalase 3.0T (Novozymes, Denmark) and

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incubated at 65 °C, pH 7.5, for 5 min followed by phenylmethanesulfonyl fluoride at a final

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concentration of 2 mM to stop the proteolysis reaction. To separate the apoRBF subunit with the EP

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deleted, the above solution was diluted with distilled water (200 mL) and then ultrafiltrated by using

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an YM-100 membrane device (Millipore Corp), resulting in the apoRBF∆EP. The apoRBF∆EP was

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applied for Native-PAGE and SDS-PAGE analysis to confirm the EP was successfully removed.

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Fabrication of holo RBF∆EP and iron release analysis

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HoloRBF∆EP was prepared by adding 5µL of FeSO4 (pH 2.0) to each apoRBF∆EP (1.0 µM, 50

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mM MOPS, pH 7.0) to make a molar ratio of 500:1 (FeSO4/apoRBF∆EP) by 5 increments with

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every 25 min intervals. Iron release from holoRBF∆EP was investigated as follows. Briefly, the 5

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reaction system (1 mL) contained 1.0 µM holoRBF∆EP, 500 µM ferrozine and 50 mM NaCl in 50

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mM Mops buffer (pH 7.0). Reactions were initiated by the addition of ascorbic acid (1 mM). The

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formation of [Fe(ferrozine)3]2+ was measured by recording the increase in absorbance at 562 nm, and

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iron release was measured using ε562 = 27.9 mM−1 cm−1.[16] The initial rate of iron release (vo) was

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calculated as previously described. [16] HoloRBF with the same iron loading amount was used as a

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control sample to detect the vo.

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Preparation of EGCG-loaded apoRBF and apoRBF∆EP

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EGCG (15.0 mg) was dissolved in deionized water (pH 6.5, adjusted by HCl) to make a 1.0 mM

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stock solution and stored in an amber bottle at 4°C. EGCG-loaded apoRBF∆EP nanoparticle was

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prepared by the reversible disassembly/reassembly method with some modifications. [1] Firstly, the

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pH value of the apoRBF∆EP solution (1.5 µM, 4.0 mL) was adjusted to pH 4.0 with HCl (1 M) for

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50 min to disassemble apoRBF∆EP into subunits; subsequently, EGCG stock solution was added to

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above solution with an apoRBF∆EP/EGCG ratio of 1 to 120, followed by stirring for 1 h (4 °C) in

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the dark to produce a homogeneous solution. The pH of the resulting mixture was then adjusted to

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6.7 with NaOH (1.0 M), followed by incubation at 4°C for 60 min to induce the reassembly

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encapsulation to generate the crude EGCG-loaded apoRBF∆EP nanoparticle. Then the products were

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dialyzed (MW 10 kDa cutoff) against MOPS buffer (20 mM, pH 6.7) with three buffer changes

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(every 1.5 h intervals). Finally, the suspension was further filtered through a 0.45-µm cellulose

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membrane filter to clarify the sample, resulting in the EGCG-loaded apoRBF∆EP nanoparticle.

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EGCG-loaded apoRBF nanoparticle as a control sample was also prepared by the reversible

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disassembly/reassembly method; differently, the denatured pH was adjusted to pH 2.0, followed by 6

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the same procedure as described for the preparation of EGCG-loaded apoRBF∆EP nanoparticle.

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Effect of different denatured pH values (2.0, 3.0, 40, 5.0, and 6.0) on the fabrication of the

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nanoparticles was also investigated. Under this conditions, the EGCG-apoRBF mixtures and EGCG-

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apoRBF∆EP mixtures were also prepared by mixing EGCG and apoRBF or apoRBF∆EP (1.0 µ M,

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5.0 mL) in a same EGCG/ferritin ratios (120:1) without disassembly of the protein cage.

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Fluorescence spectrofluorometry analysis

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The Fluorescence spectrofluorometry analysis of apoRBF and apoRBF∆EP was performed by

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using the RF-5301PC spectrofluorophotometer (Shimadzu, Japan). Path lengths for excitation and

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emission were 1.0 and 0.5 cm, and the excitation and emission wavelengths were 290 and 330 nm,

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respectively. A slit width of 10 nm and 5 nm was set for excitation and emission.

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HPLC analysis

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HPLC was performed by a SSI/LabAlliance HPLC system (Scientific Systems, Inc., PA, USA)

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with an UV detector and a Waters Xterra RP18 column (4.6×250 mm, 5µm) (Waters Corporation,

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MA, USA). To assay the EGCG concentration encapsulated in an apoRBF or apoRBF∆EP cage,

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EGCG loaded ferritin (2.5 mL) was adjusted to pH 2.0 or pH 4.0 by addition of HCl (1M) to

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disassemble the ferritin cage into subunits, resulting in the release of the EGCG, followed by

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transferring to an Amicon Ultra-3K centrifugal filter device (Pall Corp.). After centrifugation at 7000

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rpm for 20 min, free EGCG which penetrated the Ultracel membrane was determined by HPLC.

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Samples were eluted by the use of a mobile phase of methanol/water (99.9:0.1, v/v), the injection

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volume was 15 µL with a flow rate for the mobile phase of 0.7 mL/min, and wavelength was set to 7

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280 nm. The encapsulation ratio of EGCG was calculated according to Equation (1) as follows. Encapsulation ratio (%) = Encapsulated EGCG / EGCG totally used × 100%

(1)

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Circular dichroism (CD) analysis

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CD spectra of apoRBF and apoRBF∆EP were recorded with a MOS-450 spectrometer using

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quartz cuvettes with 1 mm optical path length. CD spectra were scanned at the far UV range (190–

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260 nm) with 3 replicates. The band width was set as 1 nm. The CD data were expressed in terms of

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mean residual ellipticity, (h), in deg cm2 dmol-1. Content of secondary structure was calculated

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according to a previously reported method. [17]

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TEM Analyses

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Transmission electron microscope (TEM) experiments were carried out as following: different

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samples including apoRBF, apoRBF∆EP, EGCG-loaded apoRBF, and EGCG-loaded apoRBF∆EP

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(0.5 µM) were respectively diluted with 50 mM Mops buffer (pH 6.7) prior to placing on the

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carbon-coated copper grids. The excess solution was removed with the filter paper. Then, the

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samples were stained using 2% uranyl acetate for 5 min. Transmission electron micrographs were

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imaged at 80 kV through a Hitachi H-7650 electron microscope.

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Dynamic Light Scattering (DLS) Analyses

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DLS experiments were performed by using a dynamic light scattering instrument (Malvern

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Instruments Ltd., Malvern, UK) at 25 °C. The OmniSIZE 2.0 software was used to calculate the size

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distribution of samples. The samples with a final concentration of 1.0 µM were allowed to stand for 1 8

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h prior to DLS measurement.

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Analysis of the stability of EGCG in ferritin

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The EGCG stabilities in apoRBF and apoRBF∆EP in simulated gastric/intestinal tract were

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investigated according to the reported method.[18] The gastric and intestinal incubation was

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continuously stirred for 2.0 h, and the lipid (0.2 mL) was collected for EGCG quantfication by HPLC.

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The thermal stability of EGCG in EGCG-loaded apoRBF∆EP was performed by placing 8.0 mL of

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EGCG-loaded apoRBF∆EP (1 µM ferritin, an equivalent of 11.6 µM EGCG) in a water bath (model

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DK-8D, Tianjin Honor Instrument Co., China) which was incubated in the dark mantled by

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aluminum foil at 30-80 °C for 6 h. The residual EGCG was calculted by HPLC. The thermal stability

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of EGCG in EGCG-loaded apoRBF after simulated gastric/intestinal tract and thermal treatment was

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also detected as a control sample. The retention ratios of EGCG after thermal treatmet or after

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gastric/intestinal tract treatment were calculated according to Equation (2) as follows:

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Retention ratio of EGCG (%)= residual EGCG (g)/encapsulated EGCG (g)×100%

(2)

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Statistical analysis

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All data were collected in triplicate and were presented as mean ± standard deviation (SD).

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Statistical significance of different treatments was determined using SPSS 13.0 software. A value of

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P