Expression and Iron Storage Properties of Recombinant Human Ferritins in Escherichia Coli: Relevance for Functional Ferritins in Food Systems

Ferritin is an iron storage protein found in most living organisms. Human heavy chain (H-) and light chain (L-) ferritin were amplified from human heart cDNA library. Each ferritin gene was inserted down stream of the T7 promoter of bacterial expression, and finally four types of H-, L-, and co-expression vectors were constructed. Recombinant human ferritins were overexpressed and identified with SDS-PAGE and western blotting. The expression levels of recombinant ferritin proteins ranged about 29–36% of whole cell total protein. From atomic absorption spectrometry (AAS) analysis, the rate of iron uptake for H-ferritin was significantly faster than that for the HL-, LH-, and L‐ferritin, respectively. From AAS analysis, the levels of iron content in cells progressively increased in transformants with 0–30 mM ferric citrate in the media. Among these ferritin transformants, the highest amount of cellular iron was observed with H‐ferritin transformant. The total amounts of iron content in E. coli could be sequentially ranked as H-ferritin > HL-ferritin > LH-ferritin > L-ferritin > negative transformants. Expressed recombinant human ferritins indicated that their assembled subunits were able to store iron in the cells. The results of this study further enhances our understanding of iron uptake properties in vivo and suggest similar strategies for using food-grade ferritins for functional foods or iron-fortified food ingredients.     

铁蛋白铁释放机理的研究进展

铁蛋白是一种广泛存在于生命体中的铁贮藏蛋白,具有调节机体铁代谢平衡和去除二价铁毒性的双重功能。缺铁严重影响着全球近一半人的健康,研究表明,铁蛋白具有良好的补铁活性而且安全、高效,能够取代具有毒副作用的传统补铁试剂。因此,寻求并开发以铁蛋白为原料的新型补铁功能食品已成为一种趋势。为了更科学地应用于实践和开发,对铁蛋白理化性质及其生物学功能的阐明显得颇为重要。目前,关于铁蛋白铁释放机理的研究分为体外和体内两个方面,体外机理涉及还原剂和螯合剂的共同作用,而体内机理主要涉及降解途径和酶介导的还原释放途径。综述了国内外有关铁蛋白铁释放机理的研究进展,以期为新型补铁功能食品的开发提供理论依据。     

A novel homopolymeric phytoferritin from chickpea seeds with high stability

Iron deficiency is a major public health problem in the world. Phytoferritin as an alternative iron supplement has received increasing attentions recently. Plant ferritins are usually heteropolymers comprising two different H-type subunits, H-1 and H-2, while homopolymeric plant ferritin is rare. In the present study, a newly homopolymeric ferritin chickpea seed ferritin (CSF) was isolated and purified to homogeneity from chickpea seed (Cicer arietinum L.) by two consecutive anion exchange and size exclusion chromatography. SDS-PAGE result indicates that CSF only consists of 28.0 kDa (H-2) subunits, which was identified by Western blot analysis as well. N-terminal sequence and MALDITOF-MS analyses indicate that the subunit of CSF and H-2 subunit of heteropolymeric pea seed ferritin (PSF) share high identity in amino acid sequence. Subsequently, we demonstrated that homopolymeric CSF exhibits a higher catalyzing activity than heteropolymeric PSF at both low and high iron loadings. More importantly, the stability of homopolymeric CSF is much higher than all known plant ferritin due to its highest H-2 subunit content, which is favorable for its application as iron supplement.     

铁蛋白铁氧化沉淀机理研究进展

铁素的缺乏严重影响着全球近一半人的健康,传统的补铁制剂由于会时人体产生副作用逐渐被禁止使用,因此,寻找安全、高效的补铁制剂势在必行.铁蛋白是广泛存在于生物体中的一种铁贮藏蛋白,它具有去除铁的毒性以及调节铁代谢平衡的双重功能.研究表明,铁蛋白具有良好的补铁活性,所以它可以作为加工原料被开发成补铁型功能食品,为此,对铁蛋白理化性质及其生物学功能的阐明就显得颇为重要.目前,有关铁蛋白的基础研究主要集中于铁蛋白的铁氧化沉淀和还原释放机理的研究,相对于后者而言,铁蛋白铁氧化沉淀机理的研究比较清楚.本文综述了国内外有关铁蛋白铁氧化沉淀机理的研究进展,以期为以后新型补铁功能食品的开发提供理论依据.     

Ferritin cage for encapsulation and delivery of bioactive nutrients: From structure,property to applications

Ferritin is a class of naturally occurring iron storage proteins, which is distributed widely in animal, plant, and bacteria. It usually consists of 24 subunits that form a hollow protein shell with high symmetry. One holoferritin molecule can store up to 4500 iron atom within its inner cavity, and it becomes apoferritin upon removal of iron from the cavity. Recently, scientists have subverted these nature functions and used reversibly self-assembled property of apoferritin cage controlled by pH for the encapsulation and delivery of bioactive nutrients or anticancer drug. In all these cases, the ferritin cages shield their cargo from the influence of external conditions and provide a controlled microenvironment. More importantly, upon encapsulation, ferritin shell greatly improved the water solubility, thermal stability, photostability, and cellular uptake activity of these small bioactive compounds. This review aims to highlight recent advances in applications of ferritin cage as a novel vehicle in the field of food science and nutrition. Future outlooks are highlighted with the aim to suggest a research line to follow for further studies.     

Binding of proanthocyanidins to soybean (Glycine max) seed ferritin inhibiting protein degradation by protease in vitro

Biofortification with phytoferritin is considered as a promising approach to the eradication of iron deficiency anemia. However, phytoferritin is not stable enough to be against degradation by protease(s) in the gastrointestinal tract, thereby leading to its low bioaccessibility. Fortunately, binding of proanthocyanidins (PAs) to protein offers the opportunity to prevent phytoferritin from degradation by the protease(s). To test this idea, the interaction of PAs from grape seeds with soybean seed ferritin (SSF) was studied using a combination of fluorescence, CD spectra, stopped-flow light scattering (SLS), and dynamic light scattering (DLS). Results showed that PAs can indeed bind to SSF in a dose-dependent manner. Consequently, such binding can significantly inhibit the degradation of SSF by the protease(s) in simulated gastric fluid (SGF) at pH 4.0 when the mass ratio of PAs to SSF is more than 1:1. Similarly, the stability of SSF in simulated intestinal fluid (SIF) was also increased upon treatment with PAs. These findings raise the possibility that the bioaccessibility of phytoferritin to the gastrointestinal tract was improved in the presence of proanthocyanidins.     

Study on iron availability from prepared soybean sprouts using an iron-deficient rat model

During soya seeds germination in FeSO₄ solutions their phytoferritin content is multiplied. Prepared soybean sprouts have been proposed as a safe and easily available source of iron supplementation. The preparation was compared with FeSO₄ and ferritin isolates, using rats with induced iron deficiency anaemia. After the end of the 2-week supplementation experiment, it was observed that no statistically significant differences in haemoglobin concentration, mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration existed between those animals supplemented with sprouts enriched in ferritin, ferritin isolate and FeSO₄ and healthy animals forming the control group. Moreover, the examined preparation had a beneficial influence on the recreation of ferritin reserves in both the liver and the blood serum, and also did not induce negative alterations in general growth parameters of animals. Use of an easily obtainable ferritin iron source may be a profitable alternative in supplementation due to its wide availability and food preservative properties.     

Calcium, zinc, and iron bioavailabilities from a commercial human milk fortifier: a comparison study

Adding human milk fortifiers (HMF) to human milk (HM) is one way of overcoming the nutrient deficits found in the latter. In this study, the bioavailabilities of calcium, zinc, and iron in S-26/SMA HMF added to HM were compared with those in HM fortified with various bovine milk proteins: alpha-lactalbumin, colostrum, caseinate, casein phosphopeptides, and whey protein concentrate. The bioavailability of each mineral was assessed using an in vitro digestion/Caco-2 cell culture model. Calcium and zinc uptake by the cells was traced with radioisotopes; iron uptake was assessed via cell ferritin levels. Samples were prepared on an equal protein content basis and with added calcium, but no zinc or iron was added. Results revealed that calcium uptake from HM + S-26/SMA was not different from any of the HM fortified with the bovine milk proteins, except for unfortified HM and HM + colostrum in which calcium uptake was significantly lower (-89 and -38%, respectively). Uptake of zinc and iron were significantly higher for HM + S-26/SMA than for the other HM + fortifiers.     

铁蛋白纳米笼分子装载途径及食品活性物质递送的研究进展

铁蛋白(ferritin)是一种广泛存在于动物、植物和微生物中的多亚基笼形结构蛋白,具有调节体内铁代谢平衡的功能,同时可以保护细胞免受因各种环境胁迫而导致的细胞氧化损伤。近年来,随着研究的深入,铁蛋白独特的纳米笼形结构以及特殊的理化性质使其成为一种具有广泛应用前景的新型蛋白质纳米载体材料。文章对铁蛋白的分子结构和功能进行了简要阐述,介绍了铁蛋白纳米颗粒的制备方法,总结了铁蛋白装载外源性小分子的基本途径:基于可逆组装特性的分子装载途径和基于环境响应的通道"门控"特性的分子装载途径;并综述了铁蛋白作为纳米载体在食品生物活性物质应用中的最新研究进展,以期为铁蛋白纳米载体的开发及在食品领域的应用提供研究思路。     

Iron Speciation in Beans (Phaseolus vulgaris) Biofortified by Common Breeding

The iron storage protein ferritin is a potential vehicle to enhance the iron content of biofortified crops. With the aim of evaluating the potential of ferritin iron in plant breeding, we used species‐specific isotope dilution mass spectrometry to quantify ferritin iron in bean varieties with a wide range of total iron content. Zinc, phytic acid, and polyphenols were also measured. Total iron concentration in 21 bean varieties ranged from 32 to 115 ppm and was positively correlated with concentrations of zinc (P = 0.001) and nonferritin bound iron (P < 0.001). Ferritin iron ranged from 13% to 35% of total iron and increased only slightly in high iron beans (P = 0.007). Concentrations of nonferritin bound iron and phytic acid were correlated (P = 0.001), although phytic acid:iron molar ratio decreased with increasing iron concentration (P = 0.003). Most iron in high iron beans was present as nonferritin bound iron, which confirms our earlier finding showing that ferritin iron in beans was lower than previously published. As the range of ferritin iron content in beans is relatively narrow, there is less opportunity for breeders to breed for high ferritin. The relevance of these findings to the extent of iron absorption depends on resolving the question of whether ferritin iron is absorbed or not to a greater extent than nonferritin bound iron.     

Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk,rectal feces,and teat apices

Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates (“IM” and “TA”) were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen–derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.     

Carotenoids,but Not Vitamin A,Improve Iron Uptake and Ferritin Synthesis by Caco‐2 Cells from Ferrous Fumarate and NaFe‐EDTA

Due to the high prevalence of iron and vitamin A deficiencies and to the controversy about the role of vitamin A and carotenoids in iron absorption, the objectives of this study were to evaluate the following: (1) the effect of a molar excess of vitamin A as well as the role of tannic acid on iron uptake by Caco‐2 cells; (2) iron uptake and ferritin synthesis in presence of carotenoids without pro‐vitamin A activity: lycopene, lutein, and zeaxantin; and (3) iron uptake and ferritin synthesis from ferrous fumarate and NaFe‐EDTA. Cells were incubated 1 h at 37 °C in PBS pH 5.5, containing ⁵⁹Fe and different iron compounds. Vitamin A, ferrous fumarate, β‐carotene, lycopene, lutein, zeaxantin, and tannic acid were added to evaluate uptake. Ferritin synthesis was measured 24 h after uptake experiments. Vitamin A had no effect on iron uptake by Caco‐2 cells, and was significantly lower from NaFe‐EDTA than from ferrous fumarate (15.2 ± 2.5 compared with 52.5 ± 8.3 pmol Fe/mg cell protein, respectively). Carotenoids increase uptake up to 50% from fumarate and up to 300% from NaFe‐EDTA, since absorption from this compound is low when administered alone. We conclude the following: (1) There was no effect of vitamin A on iron uptake and ferritin synthesis by Caco‐2cells. (2) Carotenoids significantly increased iron uptake from ferrous fumarate and NaFe‐EDTA, and were capable of partially overcoming the inhibition produced by tannic acid. (3) Iron uptake by Caco‐2 cell from NaFe‐EDTA was significantly lower compared to other iron compounds, although carotenoids increased and tannic acid inhibited iron uptake comparably to ferrous fumarate.     

Response of Staphylococcus aureus isolates from bovine mastitis to exogenous iron sources

Staphylococcus aureus can survive in conditions of extremely low iron concentration. The ability of S. aureus to use two exogenous hydroxamate types of siderophores (desferrioxamine and ferrichrome) and four iron-containing proteins found in cattle (hemin, hemoglobin, ferritin, and lactoferrin) were tested on 16 reference and clinical isolates. For all strains tested, ferrichrome and desferrioxamine showed strong growth-promoting activities in a disk diffusion assay and in liquid medium. The heme proteins hemin and hemoglobin were also found to support growth in culture media lacking other iron sources, while lactoferrin failed to do so. On media containing the iron chelator dipyridyl, ferritin induced a growth inhibition effect that was further enhanced in the presence of lactoferrin in seven of the 13 tested strains. Staphylococcus aureus was able to bind hemin and the level of binding activity was not increased after growth in iron-rich or -poor media. Dot-blot competition tests showed that biotin-labeled lactoferrin binds to S. aureus, and this binding can be inhibited by unlabeled lactoferrin. Expression of lactoferrin-binding activity was independent of the level of iron in the medium and the iron saturation status of lactoferrin. For each strain tested, ligand blots showed lactoferrin-binding proteins of molecular weights ranging from 32 to 92 kDa. Possible functions of these lactoferrin-binding proteins could not be related to iron acquisition mechanism in S. aureus.     

血红素铁研究进展

缺铁性贫血是我国人群普遍存在的常见营养性缺乏病,传统的补铁剂存在着吸收率低、副作用大等问题。血红素铁是一种生物态铁,是动物血液中的天然色素和生物铁化合物,能够直接被人体肠粘膜吸收,生物利用率高,而且无副作用,可作为营养强化剂广泛使用,应用于化工、医药和食品行业。本文就血红素铁的功能、应用及其制备、纯化方法等作一综述。     

Co-products of beef processing enhance non-haem iron absorption in an in vitro digestion/caco-2 cell model

Beef processing produces high volumes of protein rich, low value, ‘waste’ co-products such as offal. Beef improves uptake of low bioavailable non-haem iron (found in vegetables, fortificants, supplements) and this effect is dubbed the ‘meat-factor’, although the underlying mechanism is not fully understood. Here, we investigate whether bovine co-products (kidney, lung, heart) not previously studied share this enhancing potential. This was determined by coupled in vitro digestion of co-products and subsequent caco-2 cell ferritin formation (an intracellular iron storage protein). In this study we show that bovine co-products significantly increase caco-2 cells’ response to non-haem iron from infant rice cereal. The presence of these co-products, (kidney, lung and heart), increased relative uptake (by 207.13%, 171.21%, 265.28%, respectively), to a greater extent than beef (30.23%). Our findings present a novel function for co-products of beef processing that may have potential as food ingredients to improve non-haem iron bioavailability, thus adding value.     

Supplemental inulin does not enhance iron bioavailability to Caco-2 cells from milk- or soy-based,probiotic-containing,yogurts but incubation at 37 °C does

The in vitro effects of supplemental inulin (4%) on iron (Fe) availability in two different probiotic-containing yogurts were examined. Milk or soy-based yogurts, with and without inulin, were incubated (37 °C) for 48 h or without any incubation before comparison by an in vitro gastrointestinal digestion/Caco-2 cell culture model was used to assess iron bioavailability. The dialysable Fe fraction, cell ferritin formation, and cell associated Fe were monitored. Supplemental inulin decreased dialysable Fe only in non-incubated milk-based yogurt. In both yogurts incubation by itself increased dialysable Fe, and inulin increased the latter only in soy-based yogurt. Cellular ferritin concentration were higher after exposure to non-incubated milk-based than soy-based yogurt, although, after incubation the latter induced the highest ferritin formation. These data suggest that inulin does not have a direct effect on Fe bioavailability in the small intestine, and that probiotic bacteria play an enhancing role on Fe bioavailability.     

鸡蛋卵转铁蛋白螯合铁及其对大鼠贫血模型的影响

将从鸡蛋中分离纯化的卵转铁蛋白与FeCl3结合,研究其在不同铁离子浓度、pH值、NaHCO3和温度条件下与铁离子的结合能力。通过色度仪、红外光谱仪、粒径分析仪、扫描电镜等仪器研究冷冻和喷雾干燥对螯合铁卵转铁蛋白理化性质的影响。在0.4 mmol/L FeCl3,120 mmol/L NaHCO3,pH 8.0,温度低于60℃时,卵转铁蛋白铁结合能力为91.5%,结合的铁含量为1.28μg/g。冷冻和喷雾干燥对螯合铁卵转铁蛋白的色度、粒径分布、完全溶解时间存在极显著的差异,而在水分活度和化学键结构方面无明显差异。建立缺铁大鼠模型,考察30 d内卵转铁蛋白对大鼠贫血的改善作用,结果表明:冷冻和喷雾干燥处理的螯合铁卵转铁蛋白对贫血大鼠体重和脏器指数升高有显著作用;对血常规中的红细胞计数、血红蛋白浓度、血细胞压积、红细胞平均体积均有正向促进作用(P<0.01),能够显著增加大鼠血液中血清铁含量,提高血清铁蛋白含量,降低血清总铁结合力(P<0.05),部分指标优于对照组硫酸亚铁。卵转铁蛋白可作为一种生物补铁剂显著改善大鼠的缺铁性贫血状况。     

Effect of high hydrostatic pressure (HHP) on structure and activity of phytoferritin

High hydrostatic pressure (HHP) has drawn considerable attention because of its potential application in food industry. Ferritin, an iron storage protein, is widely distributed in food made from legume seeds, which is highly stable due to its shell-like structure. Therefore, it is of special interest to know whether or not high HHP treatment has effect on this protein. In this study, the structure and activity of soybean seed ferritin (SSF) were examined by circular dichroism spectrum (CD), UV–VIS and fluorescence spectrophotometry in conjunction with stopped-flow light scattering upon treatment with HHP at 400 MPa for 10 min. Results revealed that such treatment has little effect on the primary and secondary structure of SSF, but pronouncedly altered its tertiary and quaternary structure. As a result, the protein aggregation property and iron release activity were dramatically changed, while its activity of iron oxidative deposition was kept unchanged.     

Effect of tannic acid on properties of soybean (Glycine max) seed ferritin: A model for interaction between naturally-occurring components in foodstuffs

There are many components with different properties co-existing in food, so interactions among these components are likely to occur, thereby affecting food quality. However, relatively little information is available on such interactions. In this study, we focus on the interaction between tannic acid (TA) and soybean seed ferritin (SSF), since they co-exist in many foodstuffs, and the consequence of this interaction. As expected, TA interacts with SSF, resulting in changes in the tertiary/quaternary structure of the protein, while having no effect on its primary and secondary structure. On one hand, such interaction leads to protein association, which markedly inhibited ferritin degradation by pepsin at pH 4.0 and trypsin at pH 7.5. On the other hand, iron release was faster with TA than with ascorbic acid, and such release has a negative effect on iron supplementation. These results help to understand the interactions of food components.     

NADH induces iron release from pea seed ferritin: A model for interaction between coenzyme and protein components in foodstuffs

Plant ferritin from legume seeds co-exists with coenzyme NADH (a reduced form of nicotinamide-adenine dinucleotide) in many foodstuffs. In the present study, the interaction of NADH with apo pea seed ferritin (PSF) was investigated by fluorescence resonance energy transfer (FRET), fluorescence titration, transmission electron microscope (TEM), and isothermal titration calorimetry (ITC). We found that NADH molecules bound on the outer surface of PSF close to the 4-fold channels, which was 1.58 nm from tryptophan residue (Trp). Consequently, such binding facilitates iron release from holo PSF, which might have a negative effect on PSF as an iron supplement, while NADH was oxidised into NAD⁺. However, the binding of NADH to the protein does not affect the entry of toxic ferrous ions into the apo protein shell, where these ferrous ions were oxidised into less toxic ferric ions. Moreover, NADH binding markedly affects the tertiary structure around Trp residues of PSF. These findings advanced our understanding of the interactions between different naturally occurring components in a complex food system.