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Soybean ferritin forms iron containing oligomer in tofu even after heat treatment Taro Masuda J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03080 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 24, 2015
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Journal of Agricultural and Food Chemistry
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Soybean ferritin forms iron containing oligomer in tofu even
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after heat treatment
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Taro Masuda
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Laboratory of Food Quality Design and Development, Division of Agronomy and
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Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji,
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Kyoto 611-0011, Japan
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Corresponding Author,
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Taro Masuda, Ph. D.
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Laboratory of Food Quality Design and Development,
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Division of Agronomy and Horticultural Science,
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Graduate School of Agriculture, Kyoto University
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Fax: +81 774 38 3765
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Tel: +81 774 38 3765
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E-mail:
[email protected] 21
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Abstract
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Ferritin, a multimeric iron storage protein distributed in almost all living kingdoms, has
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been highlighted recently as a nutritional iron source in plant-derived foodstuffs,
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because ferritin iron is suggested to have high bioavailability. In soybean seeds, ferritin
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contributes largely to the net iron contents. Here, the oligomeric states and iron contents
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of soybean ferritin during food processing (especially tofu gel formation) were analyzed.
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Ferritin was purified from tofu gel as an iron-containing oligomer (approx. 1,000 Fe
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atoms per oligomer) which was composed of two types of subunits similar to the native
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soybean seed ferritin. Circular dichroism spectra also showed no differences in α-helical
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structure between native soybean ferritin and tofu ferritin. The present data demonstrate
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that ferritin was stable during the heat treatment (boiling procedure) in food processing,
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although partial denaturation was observed at temperatures higher than 80°C.
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Key words
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Ferritin, iron, tofu, soybean, subunit, oligomer, the secondary structure, heat
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denaturation
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Introduction Iron deficiency is one of the most serious global nutritional problems, affecting
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approximately 2 billion people worldwide. (1) Although the prevalence of
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iron-deficiency anemia is higher in developing countries, iron deficiency is also
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common among women and young children in industrial countries. There are two major
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strategies for preventing iron deficiency: (1) improving individuals’ iron intake, and (2)
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improving iron bioavailability, i.e., the iron supplementation and iron fortification of
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foods. Iron supplementation is generally targeted to high-risk groups such as pregnant
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women, with the use of ferrous iron salts (ferrous sulfate and ferrous gluconate). The
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other strategy, iron fortification, is a practical way to overcome iron deficiency in both
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developing and industrial countries. A new iron fortification strategy called
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“bio-fortification,” based on the selection and/or genetic modification of crop plants,
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was recently developed.
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From a nutritional point of view, iron can be divided into two types: heme and
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nonheme. Heme iron, which is generally considered an excellent nutritional iron source,
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is present in animal foodstuffs such as meat. Most of the iron in foods of plant origin is
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non-heme iron, which includes various forms of iron such as salts of phytate, citrate,
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and sulfate. In general, non-heme iron enters duodenal enterocytes from the intestinal
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lumen by divalent metal transporter 1 (DMT1)(2) after reduction to ferrous iron by
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duodenal cytochrome b (Dcytb).(3)
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Among the many types of non-heme iron species, iron deposited in ferritin has
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recently been paid considerable attention. Ferritin is a ubiquitous iron storage protein.
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The overall structure of ferritin is well-conserved among all species from bacteria to
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mammals, although its primary structures show great variation. In mammals, ferritin is 3
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composed of two types of subunits, H and L, and ferritin functions as an iron reservoir
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mainly in the liver and serum.(4) In plants, ferritin also functions as iron storage proteins,
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as well as a members of antioxidant proteins.(5)
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The contributions of ferritin to the iron contents of crops are highly varied. Generally,
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most of the identified plant ferritins are localized in plastids including chloroplasts and
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proplastids. It was suggested that large amounts of iron (40%-90%, depending on the
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reports)(6-8) are stored in ferritin in legume plants, whereas ferritin’s contribution to iron
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contents tends to be low in Gramineae plants such as rice and wheat, (9) i.e., most of the
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iron is present as salt with phytic acid.
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It was suggested that ferritin is a good nutritional iron source with high bioavailability,
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(10-13)
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as phytate and polyphenols,(14) and ferritin is absorbed via an independent transporter,
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(15-17)
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a third type of dietary iron with excellent bioavailability.(19) Based on the significance of
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ferritin iron as a dietary iron source, many attempts using bio-fortification to increase
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the iron contents of food crops have been conducted by ectopically introducing ferritin
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genes. (20-25)
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because iron deposited in ferritin can be isolated from inhibitory compounds such
not via the DMT1 or heme transporter.(18) It was thus proposed that ferritin iron is
Although the significance and advantages of ferritin as a nutritional iron source have
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been actively discussed, (26) the transition of ferritin protein oligomer and its iron
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content during the processing of food has yet to be investigated. Soymilk and tofu are
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important food products made from soybeans. Although the contributions of soybean
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seed storage proteins such as glycinin and β-conglycinin to the emulsification and
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gelation during food processing are well established, (27, 28) there is no information about
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soybean ferritin in processed soy-derived foods. 4
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As mentioned above, soybean is one of the better sources for dietary iron, and ferritin
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iron is a major form of soybean seed iron storage. Here, I present the structural features
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of ferritin from tofu and describe the transition of iron contents during the processing of
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soybeans to tofu. The results presented here demonstrate the structural stability of
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soybean ferritin and its contribution to the iron content of soy-derived foods.
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Materials and Methods
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Soybean seeds, soybean derived-foods and chemicals
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Soybean dry seeds (Glycine max Merr. cv. Toyomasari) harvested in 2013 were
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purchased from a local market. All of the reagents used were of analytical grade or, if
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not available, special grade. Iron standards (Wako Pure Chemical, Tokyo) for atomic
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absorption spectrometry were diluted using distilled water for high-performance liquid
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chromatography (HPLC) grade (Nacalai, Kyoto, Japan). Commercially available
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soymilk, tofu, fried tofu, natto and miso were purchased at local markets.
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Preparation of tofu
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First, 250 g of dried soybean seeds were immersed in 1 L of distilled water at room
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temperature for 20 h. Subsequently, the water-absorbed seeds were homogenized using
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an electric cooking mixer. The homogenate was poured to a pan in which 1 L of distilled
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water was boiling. Subsequently, this soy homogenate was boiled in the pan with gently
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mixing for 10 min. The soymilk was squeezed from the boiled soybean seeds using
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cooking cloth and stored at 4°C until the next procedure. A half-volume (1 L) of the
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prepared soymilk was heated to 80°C, and then 5.0 g of calcium sulfate dihydrate
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(Nacalai) was added to the soymilk to initiate the gelation. The resultant gel was 5
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gathered in a box and pushed gently to form tofu. The wept solution from the tofu,
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designated here as tofu supernatant, was also kept for further analyses. The moisture of
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tofu was determined by a moisture analyzer (MOC63u, Shimadzu, Kyoto, Japan).
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Purification and detection of soybean ferritin
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Ferritin was purified from dry soybean seeds, tofu and its supernatant by the following
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processes. The purification procedure of native soybean ferritin from dry soybean seeds
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was as described previously. (29) Ferritin was purified from tofu and its supernatant in a
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similar way. In brief, extraction buffer (20 mM TrisHCl, pH 7.7) was added to an equal
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volume of tofu (the average water content was 86.5%), followed by mixing with an
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electric cooking mixer and centrifugation (6,000 g, 15 min). The supernatant obtained
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here was designated as tofu extract. Subsequently, tofu ferritin was salted out by 30%
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saturated ammonium sulfate. The precipitant was dissolved in the extraction buffer
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followed by dialysis against the same extraction buffer. Ferritin was purified from the
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solution by anion exchange chromatography (Q-toyopearl, Tosoh, Tokyo) and size
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exclusion chromatography (SEC) (Superdex 200 pg 16/60, GE Healthcare, Piscataway,
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NJ). Recombinant human H chain ferritin (rHuHF) was used as a size standard in SEC
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analysis. The preparation of rHuHF was described elsewhere.(30) To determine the
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oligomeric state of tofu ferritin, the SEC column (flow rate was 0.8 ml/min) were
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calibrated using the elution volumes of protein standards (rHuHF (506,000), gamma
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globulin (158,000), bovine serum albumin (BSA) (66,300), ovalbumin (42,700), and
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chymotripsinogen A (25,700) (Fig. S1). The oligomeric states of tofu ferritins were
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estimated using this calibration.
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Ferritin was also purified from the tofu supernatant by the same procedure. Soybean 6
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ferritin was detected by sodium dodecyl sulfate-polyacrylamide gel (acrylamide
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concentration was 12.5%) electrophoresis (SDS-PAGE) followed by Coomassie
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Brilliant Blue R-250 staining and immunoblotting using anti-soybean ferritin
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anti-serum.(29) Horse-radish peroxidase (HRP)-labeled anti-rabbit IgG (Promega,
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Madison, WI) was used as the secondary antibody. The signal was visualized by using
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Chemilumi-one (Nacalai) and a laser imager (LAS-4000, GE Healthcare).
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For the estimation of the recovery of ferritin in the purification steps, a dot-blot
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analysis was performed using the diluted recombinant soybean ferritin H-1 (rSFER1)
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subunit as a standard. The N-terminal amino acid sequences of purified ferritins from
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soybean seeds, tofu and its supernatant were determined using the protein sequencer
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Procise HT (Life Technologies, Carlsbad, CA).
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Iron content analyses Lyophilized soy-derived food and milled soybean seeds were further dried at 120°C
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for 4 hr. Then, 0.5 g (dry weight) of each sample was wet-ashed with a solution of 14
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mL of HNO3 (60%, Nacalai) and 0.5 ml of H2O2 (Wako) for 5 hr at 250°C using a
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graphite block acid digestion system, Ecopre (Actac, Tokyo). The resulting digested
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solution of each sample was diluted with distilled water (HPLC grade, Nacalai),
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followed by the measurement of the iron concentration using an atomic absorption
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spectrophotometer (AAS) (AA-6800, Shimadzu) equipped with a graphite furnace
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atomizer. The iron concentrations of lab-prepared soy-milk, tofu supernatant and soluble
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fractions after the purification steps were analyzed by the AAS after dilution. The
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concentration of each sample was determined from an average of 3 independent samples.
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An analysis of variance (ANOVA) with Tukey-Kramer’s multiple comparison test was 7
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used to compare the iron contents among the various food products. Mean differences
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were considered significant at p
