Letter pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX-XXX
Thermally Induced Encapsulation of Food Nutrients into Phytoferritin through the Flexible Channels without Additives ABSTRACT: The cavity of phytoferritin provides a nanospace to encapsulate and deliver food nutrient molecules. However, tranditional methods to prepare the ferritin−nutrient complexes must undergo acid/alkaline conditions or apply additives. In this work, we provide a novel guideline that thermal treatment at 60 °C can expand ferritin channels by uncoiling the surrounding αhelix. Upon reduction of the temperature to 20 °C, food nutrient rutin can be encapsulated in apo-soybean seed ferritin (apoSSF) at pH 7.0 through channels without disassembly of the protein cage and with no addition of additives. Results indicated that one apoSSF could encapsulate about 10.5 molecules of rutin, with an encapsulation ratio of 8.08% (w/w). In addition, the resulting rutin-loaded SSF complexes were monodispersed in a size of 12 nm in aqueous solution. This work provides a novel pathway for the encapsulation of food nutrient molecules into the nanocavity of ferritin under a neutral pH condition induced by thermal treatment. KEYWORDS: thermal treatment, phytoferritin, encapsulation
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this reason, to find a novel route to encapsulate these kinds of compounds in the ferritin cavity is an urgent matter. In this work, we reported a successful case of nanoencapsulation of rutin molecules in apo-soybean seed ferritin (apoSSF) by thermal treatment. We proved that thermal treatment at 60 °C can expand the ferritin channels, and thus, the bioactive molecule rutin can be captured through channels at pH 7.0 without disassembly of the protein cage. Upon reduction of the temperature to 20 °C, rutin can be retained within the apoSSF cage, resulting in the rutin-loaded SSF nanocomplexes. This work provides a green and efficient method for preparation of ferritin−nutrient nanocomplexes without applying any acid/alkali agents or other additives, which will be beneficial for the pH- and additive-sensitive molecule encapsulation.
INTRODUCTION Shell-like proteins, such as viral capsid, DNA binding proteins from starved cell (Dps), and ferritin, have received considerable attention in food, nutition, and medicine domains. The unique structure and interface-modifiable properties facilitate extensive applications of these proteins in the encapsulation, delivery, and sustained release of the nutrients or drugs.1−8 To achieve these aims, the small core molecules should first be encapsulated in the inner cavity of protein cages to generate shell−core nanoparticles by destroying and reassembling of the proteins, such as by pH transformation.9,10 To develop a novel route to realize the encapsulation of bioactive molecules into the protein cage and, meanwhile, maintain the activity of nutrients is interesting and challenging. Ferritin protein is a ubiquitous iron storage and detoxification protein. Recently, as an intriguing protein vehicle, the ferritin cage is further extended to be used as a reactor, carrier, and template for the encapsulation and stabilization of food nutrients (such as β-carotene, lutein, and anthocyanin).2−4,9 Generally, each ferritin is composed of 24 subunits that selfassemble into a shell-like molecule with a 4−3−2 symmetry and is characterized by a spherical structure with inner and outer diameters of 8 and 12 nm.11−13 An important feature is the multipores of ferritin. One typical ferritin owns eight 3-fold and six 4-fold channels with pore size between 0.3 and 0.5 nm,14 and such pore-related channels build up the relationship between the inner cavity of ferritin and external environment. In addition, these channels only allow for metal ions with positive charges or small molecules to diffuse into the cavity as a result of electrostatic attraction, whereas relatively larger molecules cannot as a result of steric hindrance.15 At this stage, to realize the aim of encapsulation and delivery, nutritional compounds are usually encapsulated into the ferritin cage by the dissociation/reassembly characteristics of ferritin. The disassociation of the ferritin cage can be achieved under extremely acid/alkaline conditions (pH 2.0 or 11.0) or adding additives, and the reassembly is carried out when pH is adjusted to a neutral range.10,11 However, it must be noted that the extreme acid/alkaline conditions and the applied additives may be bad for the stability, bioactivity, and pharmacological activity of the pH- and additive-sensitive nutritional compounds. For © XXXX American Chemical Society
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MATERIALS AND METHODS
Materials. Soybean seed ferritin (SSF) and apoSSF (deprived of irons) were prepared as previously described.2 Holo-soybean seed ferritin (holoSSF) with 600 Fe3+/ferritin was prepared in the Supporting Information. Rutin and epigallocatechin gallate (EGCG) were purchased from Sigma-Aldrich, St. Louis, MO, U.S.A. All other reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and of analytical grade. Fabrication of Rutin-Loaded SSF Nanocomplexes. Rutin was dissolved in ethyl alcohol to make a stock solution (4.6 mM) and stored in the dark at 4 °C. There were two steps for the preparation of the rutin-loaded SSF nanoparticles. Briefly, apoSSF (2.0 μM, pH 7.0, 5.0 mL) was preincubated with thermal treatment at 60 °C for 30 min; subsequently, rutin stock solution was added to the above solution with apoSSF/rutin ratios of 1:130, followed by stirring to produce a homogeneous solution. Then, the incubation was performed at 60 °C for 60 min, followed by reducing the temperature to 20 °C in the dark to induce the formation of rutin-loaded SSF nanocomplexes. The resulting solution was dialyzed [molecular weight cut-off (MWCO) of 10 kDa] against 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (50 mM, pH 7.0) with three buffer changes (every 2.0 h Received: Revised: Accepted: Published: A
August 24, 2017 October 7, 2017 October 16, 2017 October 16, 2017 DOI: 10.1021/acs.jafc.7b03949 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Letter
Journal of Agricultural and Food Chemistry interval) to remove unbound rutin and ethyl alcohol. Finally, the suspension was further filtered through a 0.45 μm hydrophilic cellulose membrane filter to clarify the complexes. The encapsulation ratio of rutin was quantified by high-performance liquid chromatography (HPLC) (see the Supporting Information). Analysis. The iron release experiment was performed to detect the initial rates of iron release (υo). Circular dichroism (CD) was carried out to figure out the secondary structure changes of the rutin-loaded SSF complexes. Transmission electron microscopy (TEM) experiments were performed to analyze the morphology of complexes. Dynamic light scattering (DLS) was conducted to detect the hydrodynamic radius (RH). Ultraviolet/visible (UV/vis) spectroscopy was carried out to observe the absorbance of the samples. A thermal test of the retention ratio of rutin was performed by HPLC. These detailed operations were provided in the Supporting Information.
Figure 1. (a) Kinetics of iron release from holoSSF (600 irons/ protein) with different thermal treatments (60 and 20 °C) induced by ascorbic acid. Upon reduction of the temperature from 60 to 20 °C, the kinetic of iron release was also recorded. (b) Comparison of υo among six groups with different temperature treatments.
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RESUTLS AND DISCUSSION Ferritin Preparation. The obtained ferritin SSF is a heteropolymer of H-1 and H-2 subunits from soybean seed, and its yield was 3.1 ± 0.2 mg/kg. ApoSSF was prepared and used to encapsulate rutin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE) showed that obtained apoSSF consisted of two subunits, H-1 and H-2, with a molecular weight of about 28.0 and 26.5 kDa (Figure S1a of the Supporting Information), respectively. Native PAGE exhibited a single lane with a molecular weight of about 560 kDa (Figure S1b of the Supporting Information), which was the same as the typical value of ferritin, indicating that apoSSF can be used for further usage. Ferritin Channel Changes Induced by Thermal Treatment. The reason for applying thermal treatment in encapsulation is based on the evidence that the whole ferritin structure is stable against heat treatment (80 °C) for 10 min,16 while the ferritin channels are sensitive to experimental conditions, such as temperature changes.17,18 It is well-known that the temperature has a critical impact on protein structures and may result in aggregation as a result of misfolding or unfolding of the protein. Thus, how the thermal treatment influences the ferritin structure is an interesting question, and whether the ferritin channels are expanded so that nutrient molecules can permeate into the ferritin is worth investigating. To address this question, the iron release properties of ferritin are important indicators that could reflect the microenvironment change of the ferritin channels.9,19 The effects of thermal treatment on the ferritin structure and ferritin−small molecule interaction were investigated in this work. The initial rates of iron release (υo) could reflect the iron effusion efficiency from the ferritin cavity and was used as an index to represent Fe mineral reduction and transportation through channels induced by certain reductants or chelators. Herein, the effect of thermal treatment on υo of holoSSF (600 Fe3+/ferritin) was evaluated. It was found that 60 °C treatment for 30 min could significantly increase υo of ferritin induced by Vc compared to that without thermal treatment (20 °C) (Figure 1a); that is, υo of 0.28 ± 0.01 μM min−1 after 60 °C treatment was significantly higher than that upon 20 °C treatment (0.16 ± 0.02 μM min−1) (p < 0.05). A different form reports that the pathway of iron release in vertebrate ferritins was the 3-fold channels, and the pathway for iron release in plant ferritin is the 4-fold channels as a result of their hydrophilic residues in the E-helix around the channels.20 Thus, we confirmed that part of the increase of υo resulted from the expansion of the 4-fold channels of ferritin as a result of thermal treatment. We calculated that about 57.1% (0.04/0.07) of the
increasing rate of the release ratio directly stemmed from the partial unfolding of protein channels upon thermal treatment (detailed discussion in the Supporting Information). In addition, an interesting finding is the recovery of υo to 0.15 ± 0.01 μM min−1 when the treated temperature decreased to 20 °C (from 60 °C) (Figure 1a). We deduced that the local region, such as the ferritin channel, was flexible and is sensitive to environmental changes, and the expansion and recovery of the channel may be susceptible to the temperature changes. The effect of different temperature treatments (20−70 °C) on υo changes induced by Vc was further detected (Figure 1b). Results indicated that υo was continuously increased as the temperature increased from 20 to 60 °C, beyond which (70 °C) was a significant decrease of υo (p < 0.05), indicating that the ferritin channel might be expanded to the maximum upon 60 °C treatment. The 70 °C treatment showed a different manner on iron release, possibly a consequence of the collapse of the channel structure around the ferritin pores or even the association of the ferritin cage. Thus, the ferritin structure is differentially sensitive to different thermal treatments. Analysis of Ferritin by UV Absence and CD Spectrum with Thermal Treatment. The transition temperature (Tm) of protein thermal unfolding is an important factor that could reflect the thermal stability of the whole protein, and the thermal denaturation curves of apoSSF in the temperature range from 25 to 90 °C were measured by UV absorbance at 280 nm. It was found that Tm for apoSSF was 75.1 °C (Figure 2a). However, below 65 °C, the absorbance at 280 nm hardly changed, indicating that the spherical ferritin structure was stable under this condition. In terms of the local region or subdomain of ferritin, the result was different. The changes of the secondary structure of apoSSF with different thermal treatments were studied by CD spectroscopy. We found that 20−40 °C treatment did not change the CD spectrum of ferritin (Figure 2b and right column in Figure 2c), which is inconsistent with the UV absorbance results. However, continuing to increase the temperature to 60 °C resulted in a 6.2% decrease of the αhelix content and a 5.1% increase of the random coil content (Table S1 of the Supporting Information). When the treated temperature decreased to 20 °C (from 60 °C), the CD spectroscopy restored to the initial curve with the α-helix content recovered, which is consistent with the iron release result. An impressive finding is that 75 °C treatment even decreased about 22.5% of the α-helix content (right column in Figure 2c), indicating that the ferritin structure may be partially unfolded upon high-temperature treatment. B
DOI: 10.1021/acs.jafc.7b03949 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
the temperature was decreased to 20 °C, and the mixtures were dialyzed for 6 h, resulting in the rutin-loaded SSF complexes. Different from the insoluble state of rutin in water (pH 7.0) (middle in Figure 3a), the rutin-loaded SSF complexes showed
Figure 2. (a) Thermal denaturation curve of apoSSF in the temperature range from 20 to 90 °C measured by UV absorbance at 280 nm. (b) CD spectra of apoSSF upon different temperature treatments. (c) CD analysis (220 nm) of subdomain temperature transitions below global melting of ferritin between 5 and 70 °C (left column) and effect of the temperature on the α-helix content (%) in ferritin (right column).
Figure 3. (a) Picture of apoSSF (left), rutin molecules (middle), and rutin-loaded SSF nanoparticles (right) in aqueous solution (pH 7.0). (b) UV/vis spectra of apoSSF, rutin, and rutin-loaded SSF complexes. (c) TEM of rutin-loaded SSF complexes. (d) DLS of rutin-loaded SSF complexes.
Another important finding is that the melting of subdomains (α-helix content) can be observed by CD spectroscopy (left column in Figure 2c). The temperature of subdomain unfolding (with a transition midpoint of 53 °C) was far below the Tm (75.1 °C) of the whole protein disassembly. This local nature of the helix coil transition induced by heat treatment can also be confirmed by the significant increase of υo (Figure 1b). Thus, the transition temperature differences between the helical subdomain and global structure indicated that the local domains (such as the channel and “pore” domains of ferritin) may be partially uncoiled upon thermal treatment, which is accordance with the notion that the ferritin cage may be localized unfolding and was sensitive to environmental changes, such as the temperature.17,18 We inferred that the ferritin structure underwent a multistate denaturation process. The channel/pore domains of ferritin showed a head start of expansion (uncoiling), which was prior to the whole structure changes of protein (Figure 2). This phenomenon leads to further investigation of the interaction between ferritin and small nutrient molecules upon thermal treatment in the aqueous solution. Characterization of Rutin-Loaded SSF Complexes by UV/vis Spectroscopy, TEM, and DLS. In view of the expansion of ferritin channels induced by thermal treatment, the rutin molecules were added to the apoSSF solution upon 60 °C incubation. Rutin is a bioactive molecule present in plant and has been widely used in food and medicine as a result of its anti-inflammatory, antibacterial, antitumor, and antioxidant activities,21 which can be hardly resolved in aqueous solution. Briefly, apoSSF (pH 7.0) was preincubated at 60 °C for 30 min to expand the ferritin channels, followed by mixing with rutin with a molar ratio of 130:1 (rutin/apoSSF) for 60 min. Then,
good transparency with a typical yellow color of rutin (right in Figure 3a), suggesting that the rutin solubility was significantly improved as a result of the ferritin encapsulation. To confirm this observation, the rutin-loaded SSF complexes were analyzed by UV/vis spectroscopy (Figure 3b). Results indicated that free rutin showed two major absorption peaks at 265 and 360 nm, while the rutin-loaded SSF complexes had a maximal absorption at 260 nm and a maximal absorption at about 360 nm. In comparison to free rutin, the rutin-loaded SSF nanocomplexes had a blue shift by about 5 nm in the UV range. This shift of the absorption peak proved that there was a strong interaction between embedded rutin and residues in the inner surface of apoSSF. TEM is an important method that can observe the microcosmic morphology of the particles, and it was used to confirm that rutin molecules were successfully encapsulated in apoSSF. Similar to apoSSF (Figure S2a of the Supporting Information), the rutin-loaded apoSSF nanocomplexes also showed a spherical morphology with an exterior diameter of ∼12 nm (Figure 3c). It should be noted that a distinct difference was evident in the ferritin cores of these two different samples. Most apoSSF was filled with black uranium cores (Figure S2a of the Supporting Information), which was mainly because of the permeation of uranium into the ferritin cavity via the protein channels when the samples were prepared with uranyl acetate staining. Contrastively, the rutin-loaded SSF nanocomplexes lacked these black uranium cores (Figure 3c). This result was consistent with the reports that, if certain molecules were encapsulated in the ferritin cage, there would be no or a small proportion of uranium cores forming within the cavity as a result of the occupancy of the encapsulated materials and prevention of the entrance of uranyl acetate.3,9,11 In C
DOI: 10.1021/acs.jafc.7b03949 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
consistent with the CD results (panels b and c of Figure 2) that 60 °C could uncoil and expand the ferritin channel to such an extent that rutin molecules can be more allowable to pass through the ferritin pores. It should be noted that, to obtain the highest value, the needed time reduced to 60 min for the 60 °C treatment group, indicating a more efficient condition. However, too long of a duration of thermal treatment (100 min) was bad for the stability of rutin, which can be reflected by the decreases of the encapsulation ratio to 5.09% (60 °C) and 6.01% (50 °C). Additionally, we found that the encapsulation ratio by this method was a little lower but was comparable to that by the typical reversible assembly of the ferritin cage (10.9%) (Figure S4 of the Supporting Information), indicating that this method was effective in preparing ferritin−nutrient nanoparticles. Different from the disassembly and refolding processes of ferritin upon acid/alkaline transform treatment, the “pore gate” effect and the steric hindrance of the ferritin channels are important limiting factors that could significantly influence the rutin encapsulation by this new method. It should be noted that the biggest advantage of this method over the frequently used technology is that the complexes can be formed under a neutral pH condition; thus, this new method showed more applicability in encapsulation of the pH-sensitive molecules. The stability of rutin in the free rutin sample and rutinloaded SSF complexes upon different thermal treatments (4, 25, 50, and 80 °C) was evaluated (Figure S5 of the Supporting Information). It was found that retention ratios of 95.1 and 95.8% (mean values) remained in both samples under 4 °C treatment for 8 h, with no significant differences (p > 0.05). In contrast, 50 °C treatment retained 38.6% of rutin in rutinloaded SSF complexes, which is significantly higher than that (21.8%) in the free rutin sample (p < 0.05). Similar results were also obtained upon 25 °C treatment. Differently, a hightemperature treatment (80 °C) significantly damaged rutin in both samples with only trace amounts of rutin (3.1 versus 2.9%) (p > 0.05). We can infer that the apoSSF cage played an insulated effect on the encapsulated molecules and can effectively protect rutin against thermal treatment in a temperature range below 50 °C. High-temperature treatment at 80 °C could uncoil or disintegrate the ferritin cage (Figure 2), and rutin may be rapidly released, leading to rapid damage when exposed to heat. Applicability of the Thermally Induced Bioactive Molecule Encapsulation in Ferritin. To explore the application of this scheme to encapsulate other nutrient molecules, EGCG-loaded SSF nanoparticles were fabricated by adding EGCG molecules to apoSSF (EGCG/apoSSF = 150:1, molar ratio) solution, which was pretreated with 60 °C for 80 min. After dialysis against MOPS buffer (pH 6.8) at 25 °C for 8 h and centrifugation, the resulting solution was analyzed by TEM. The EGCG-loaded SSF nanoparticles were clear, and an EGCG encapsulation ratio of 12.8% was achieved. TEM showed that EGCG-loaded SSF nanoparticles were in a homogeneous state without blank uranium cores (Figure S6 of the Supporting Information), indicating that this scheme also showed good applicability for nanoencapsulation of EGCG molecules. Further work will be focused on the expansion of the applicability of this scheme in other molecules and improvement of the encapsulation ratio of the nutrients. On the basis of the above findings, we propose a possible mechanism of ferritin nanoencapsulation based on apoferritin by the feat of thermal treatment (Figure 4). The first step is the
addition, almost all of the rutin-loaded SSF nanocomplexes exhibit a full “white” morphology, which was different from that of the apoSSF−rutin mixtures with black uranium cores (Figure S2b of the Supporting Information). These results ruled out the possibility that rutin molecules were only bound to the surface of the ferritin cage when rutin-loaded ferritin nanoparticles were prepared. All of these results confirmed that some of the rutin molecules were successfully encapsulated within apoSSF. The size distribution of the particles in the aqueous phase is an important factor that could influence the further application of rutin-loaded SSF nanocomplexes, because large aggregates would be bad for their stability and delivery. DLS of the samples was analyzed to investigate the hydrodynamic radius (RH) of the particles. Similar to apoSSF, the main distribution of rutin-loaded SSF nanocomplexes was centered at 7.5 nm, which represented the monomers of the nanoparticles (Figure 3d), comprising of 90.9% of the total mass (Table S2 of the Supporting Information). Another distribution is that centered at 46.0 nm, which is polymers of complexes accounting for 9.1% of the total mass. Thus, the obtained rutin-loaded SSF nanoparticles were mostly homogeneously distributed, being inconsistent with the TEM observation (Figure 3c), indicating that the nanoparticles were suited for further usage. Effect of the Temperature and Incubation Time on the Encapsulation Ratio of Rutin. The rutin encapsulation ratio in apoSSF was quantified, and it was calculated that one apoSSF molecule could encapsulate about 10.5 molecules of rutin with an encapsulation ratio of 8.08% (w/w) upon 60 °C treatment when the applied rutin/ferritin molar ratios were fixed to be 130:1. To acquire more information on the encapsulation behavior, the effect of the treated temperature on rutin encapsulation was evaluated in Figure S3a of the Supporting Information. Seven groups were established, and the rutin/ferritin molar ratios were all fixed to be 130:1. Treatment with 20−40 °C resulted in a trace amount of encapsulation ratios, indicating that the rutin molecules could not pass through the channels to be encapsulated in ferritin in this condition. Incubation of rutin and apoSSF at 50 and 60 °C both showed significant increases in the encapsulation ratios, with values of 7.31 and 8.08%, respectively. However, treatment at 70 °C brought about a significant decrease of the encapsulation ratio to 2.09%. Although it has been revealed that ferritin from horse spleen was stable against thermal treatment and can be kept intact upon heating at 80 °C for 10 min,16 there was a failure of rutin encapsulation in apoSSF in the present condition. We inferred that the thermally induced collapse of the channel structure and even the association of ferritin may be important reasons. In addition, rutin may be partly degraded upon high-temperature treatment, resulting in the decrease of the encapsulation ratio. Thus, the relatively higher temperature (60 °C) is an important prerequisite that could determine whether the encapsulation of rutin into ferritin is feasible. The effect of the incubation time between apoSSF and rutin upon thermal treatments was also evaluated (Figure S3b of the Supporting Information), because the duration of thermal treatment may also influence the stability of rutin. It was found that, with the increases of the incubation time (20−100 min), 50 °C treatment could result in a continuous increase of the encapsulation ratios, which reached maximum values in 80 min. In contrast, a maximum value was obtained for the 60 °C treatment group, which was significantly higher than that for the 50 °C treatment group (p < 0.05). This finding was D
DOI: 10.1021/acs.jafc.7b03949 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
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Figure 4. Graphical representation of the fabrication of ferritin− nutrient nanocomplexes by temperature conversion without additives.
Methods of native PAGE and SDS−PAGE analyses, preparation of holoSSF and iron release experiment, praperation of rutin or EGCG-loaded ferritin complexes, HPLC analysis, UV/vis absorbance, CD analysis, TEM analysis, DLS analysis, and thermal stability analysis, supplementary discussion, Figures S1−S6, and Tables S1 and S2 (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected].
ferritin cage structure change induced by a high temperature (60 °C), which is evidenced by an increase of the iron release ratio and a decrease of the α-helical content (uncoiling). If ferritins are incubated with enough time (30 min), the ferritin pores expanded to such an extent that the small molecules can penetrate into the ferritin cage. Finally, the ferritin channel would restore to its normal size, and nutrient molecules can be captured within the cage when the temperature is reduced to 20 °C. As a benefit from these results, we deduce that the ferritin shell, especially the channels, is flexible and the pore size may be closely associated with the environmental changes. When advantages of this interesting guideline are taken, small nutrients can thus be encapsulated within the protein cages. The tested nutrients rutin and EGCG are pH-sensitive molecules in this work. EGCG molecules may degrade very quickly under an alkaline environment, and rutin has acid instability. The meaning of this work is to provide a novel pathway for the encapsulation of food nutrient molecules into the cavity of ferritin under a neutral pH condition, which can avoid damage of the pH-sensitive nutrient during pH transform. The ferritin-gated pores at the junctions of subunits provide a passageway for the nutrient entrance into the ferritin inner cavity. Thermal treatment at 60 °C can expand the ferritin channel and enhance the nanocapsulation of the rutin molecules under a relatively benign condition, which is especially suitable for the stability and bioactivity of the pHand additive-sensitive molecule encapsulation. How the core molecules permeated into ferritin is very interesting research and will be further investigated in our further work. The strategy presented here has the potential to be used in a series of cage-like proteins with channels to achieve the encapsulation in benign conditions without pH changes.7,22
ORCID
Rui Yang: 0000-0002-7281-7672 Funding
This work was supported by the National Natural Science Foundation of China (31501489), the Natural Science Foundation of Tianjin City (16JCQNJC14500), the basic research funding of the 13th Five-Year Plan, Education Commission Foundation of Tianjin (2017KDYB02) and Tianjin Research Program of Application Foundation and Advanced Technology (15JCZDJC34300). Notes
The authors declare no competing financial interest.
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Rui Yang*,†,‡ Jing Tian† Yuqian Liu† Zhiying Yang† Dandan Wu† Zhongkai Zhou*,†
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REFERENCES
† Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China ‡ Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Beijing 100048, People’s Republic of China
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03949. E
DOI: 10.1021/acs.jafc.7b03949 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry (12) Li, M.; Viravaidya, C.; Mann, S. Polymer-mediated synthesis of ferritin-encapsulated inorganic nanoparticles. Small 2007, 3, 1477− 1481. (13) Zhao, G. Phytoferritin and its implications for human health and nutrition. Biochim. Biophys. Acta, Gen. Subj. 2010, 1800, 815−823. (14) Lv, C.; Bai, Y.; Yang, S.; Zhao, G.; Chen, B. NADH induces iron release from pea seed ferritin: A model for interaction between coenzyme and protein components in foodstuffs. Food Chem. 2013, 141, 3851−3858. (15) Yang, R.; Chen, L.; Yang, S.; Lv, C.; Leng, X.; Zhao, G. 2D square arrays of protein nanocages through channel-directed electrostatic interactions with poly(α,L-lysine). Chem. Commun. 2014, 50, 2879−2882. (16) Stefanini, S.; Cavallo, S.; Wang, C.-Q.; Tataseo, P.; Vecchini, P.; Giartosio, A.; Chiancone, E. Thermal Stability of Horse Spleen Apoferritin and Human Recombinant H Apoferritin. Arch. Biochem. Biophys. 1996, 325, 58−64. (17) Liu, X.; Jin, W.; Theil, E. Opening Protein Pores with Chaotropes Enhances Fe Reduction and Chelation of Fe from the Ferritin Biomineral. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 3653− 3658. (18) Theil, E.; Liu, X.; Tosha, T. Gated Pores In The Ferritin Protein Nanocage. Inorg. Chim. Acta 2008, 361, 868−874. (19) Yang, R.; Chen, L.; Zhang, T.; Yang, S.; Leng, X.; Zhao, G. Selfassembly of ferritin nanocages into linear chains induced by poly(α,Llysine). Chem. Commun. 2014, 50, 481−483. (20) Masuda, T.; Goto, F.; Yoshihara, T. A novel plant ferritin subunit from soybean that is related to a mechanism in iron release. J. Biol. Chem. 2001, 276, 19575−19579. (21) Koda, T.; Kuroda, Y.; Imai, H. Protective effect of rutin against spatial memory impairment induced by trimethyltin in rats. Nutr. Res. (N. Y., NY, U. S.) 2008, 28, 629−634. (22) Meldrum, F. C.; Wade, V. J.; Nimmo, D. L.; Heywood, B. R.; Mann, S. Synthesis of inorganic nanophase materials in supramolecular protein cages. Nature 1991, 349, 684−687.
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