Alcalase Enzymolysis of Red Bean (adzuki) Ferritin Achieves

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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

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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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ACS Paragon Plus Environment


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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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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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Alcalase Enzymolysis of Red Bean (adzuki) Ferritin Achieves

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