铁蛋白:一种新型矿质元素营养强化剂载体

传统的铁、钙、锌等矿质元素营养强化剂存在诸多缺点,铁蛋白因其特殊结构和功能可作为这些矿质元素营养强化剂的载体。本文通过对铁蛋白的结构和功能的阐述,以及综述其作为载体在储存铁、钙、锌等金属离子方面的研究,指出其在食品领域将具有广阔的应用前景。     

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.     

Structure,Function, and Nutrition of Phytoferritin: A Newly Functional Factor for Iron Supplement

Ferritins are members of the superfamily of iron storage and detoxification proteins present in all living organisms and play important roles in controlling cellular iron homeostasis. In contrast to animal ferritin, relatively little information is available on the structure and function of phytoferritin. Phytoferritin is observed in plastids whereas animal ferritins are largely found in the cytoplasm of cell. Compared to animal ferritin, phytoferritin exhibits two major distinctive features in structure. First, phytoferritin contains a specific extension peptide (EP) at the N-terminal while animal ferritin lacks it. The EP is located on the exterior surface of protein, which recently has been found to act as a second ferroxidase center for iron-binding and oxidation, and regulate iron release during the germination and early growth of seedlings. Second, only H-type subunit has been identified in phytoferritin, which is usually a heteropolymer consisting of two different subunits, H-1 and H-2, sharing ～80% amino acid sequence identity. These two subunits in phytoferritin play a positively cooperative role in iron oxidative deposition in protein. Iron deficiency anemia (IDA) is the most common and widespread nutritional disorder in the world, so it is crucial to explore a safe and efficient functional factor for iron supplement. Fortunately, phytoferritin seems to be a suitable candidate. In legume seeds, more than 90% of iron is stored in the form of ferritin in amyloplasts. Recently, some studies at different levels have demonstrated that plant ferritin could be used as novel, utilizable, plant-based forms of iron for populations with a low iron status. This review focuses on recent progress in structure, function, and nutrition of phytoferritin.     

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.     

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.     

Ferritin synthesis by Caco-2 cells as an indicator of iron bioavailability: Application to milk-based infant formulas

The bioavailability of iron from milk-based infant formulas was estimated by an in vitro system including enzymatic digestion, iron uptake by Caco-2 cells and ferritin determination via an enzymoimmunoassay (ELISA). Positive correlations (p < 0.01) were found between the Fe(II) added to Caco-2 cells and ferritin synthesis and between the amount of dialyzed iron added to the cell culture and ferritin synthesis. The comparison of the bioavailability of iron from different milk-based formulas showed that adapted formulas having the same composition but differing in the iron salts added yielded similar ferritin levels. The same happened with follow-up formulas differing only in the presence or absence of bifidobacterium added. However, significant differences in the amount of ferritin synthesized were recorded between the two analyzed toddler formulas. Such differences could be attributed firstly to the ascorbic acid content and perhaps also to the manufacturing process involved, because one formula was in liquid form while the other was powdered.     

Effect of Various Food Additives on the Levels of 4(5)‐Methylimidazole in a Soy Sauce Model System

In this study, the effect of food additives such as iron sulfate, magnesium sulfate, zinc sulfate, citric acid, gallic acid, and ascorbic acid on the reduction of 4(5)‐methylimidazole (4(5)‐MI) was investigated using a soy sauce model system. The concentration of 4(5)‐MI in the soy sauce model system with 5% (v/v) caramel colorant III was 1404.13 μg/L. The reduction rate of 4(5)‐MI level with the addition of 0.1M additives followed in order: iron sulfate (81%) > zinc sulfate (61%) > citric acid (40%) > gallic acid (38%) > ascorbic acid (24%) > magnesium sulfate (13%). Correlations between 4(5)‐MI levels and the physicochemical properties of soy sauce, including the amount of caramel colorant, pH value, and color differences, were determined. The highest correlations were found between 4(5)‐MI levels and the amount of caramel colorant and pH values (r² = 0.9712, r² = 0.9378). The concentration of caramel colorants in 8 commercial soy sauces were estimated, and ranged from 0.01 to 1.34% (v/v).     

Preparation of iron‐fortified rice using edible coating materials

Iron‐fortified rice premix (IFRP) was prepared using soaking and spraying method followed by coating with hydroxypropyl methyl cellulose (HPMC), methyl cellulose (MC), combination of HPMC and MC, zein, palmitic acid (PA) and stearic acid (SA). Steaming caused a reduction in iron content in iron‐fortified rice premix than without steaming treatment. Iron content ranged from 1.33 to 7.11 and 1.61 to 4.49 mg g^?1, respectively, in IFRP prepared using soaking and spraying method. Retention of iron in IFRP samples coated with 9% PA, 7% and 9% SA, and combination of HPMC and MC at 2% level, respectively, after washing twice with distilled water was similar (P > 0.05). Retention of iron in these coated IFRP ranged from 87.34% to 89.39% (P > 0.05) as compared to 39.12% in uncoated IFRP. Sensory acceptability indicated the scope for the production of IFRP by spraying of iron solution with iron content 20.1 mg mL^?1, 180‐min tempering time and coating with 2% HPMC and MC followed by drying.     

Studies to analyse the relationship between IFNα2b gene dosage and its expression,using a Pichia pastoris‐based expression system

Human interferon α2b (hIFNα2b) is the most important member of the interferon family. Escherichia coli, yeasts, mammalian cell cultures and baculovirus‐infected insect cells have been used for expressing recombinant human interferon. Recently a Pichia pastoris‐based expression system has emerged as an attractive system for producing functional human recombinant IFNα2b. In this regard, gene dosage is considered an important factor in obtaining the optimum expression of recombinant protein, which may vary from one protein to another. In the present study we have shown the effect of IFNα2b gene dosage on extracellular expression of IFNα2b recombinant protein from P. pastoris. Constructs containing from one to five repeats of IFNα2b‐expressing cassettes were created via an in vitro multimerization approach. P. pastoris host strain X‐33 was transformed using these expression cassettes. Groups of P. pastoris clones transformed with different copies of the IFNα2b expression cassette were screened for intrachromosomal integration. The IFNα2b expression level of stable transformants was checked. The copy number of integrated IFNα2b was determined by performing qPCR of genomic DNA of recombinant P. patoris clones. It was observed that an increase in copy number generally had a positive effect on the expression level of IFNα2b protein. Regarding the performance of multicopy strains, those obtained from transformation of multicopy vectors showed relatively high expression, compared to those generated using transformation vector having only one copy of IFNα2b. It was also observed that an increase in drug resistance of a clone did not guarantee its high expression, as integration of a marker gene did not always correlate with integration of the gene of interest. Copyright © 2013 John Wiley & Sons, Ltd.     

Iron absorption and haemoglobin status of rats fed a ferrous bisglycinate‐fortified growing‐up milk

BACKGROUND: Infants and toddlers aged 6–24 months constitute one of the groups at highest risk of iron deficiency. A promising alternative for ferrous sulfate (FS) might be ferrous bisglycinate (FeAAC), which is less likely to cause sensory changes in the food vehicle. This work aims to compare the effect of FeAAC with that of FS, when added to a growing‐up milk, on the iron and haemoglobin status in weanling rats using a depletion–repletion model. RESULTS: After the repletion period no significant differences were found in iron absorption, Hb concentration, Hb iron, Hb regeneration efficiency (HRE), mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), serum iron, total iron binding capacity (TIBC), transferrin saturation (TS), and ferritin between the group fed a growing‐up milk fortified with FS and the group fed one fortified with FeAAC. Furthermore, iron absorption, HRE, MCV, MCH, serum iron, and TS were significantly (P < 0.05) higher in groups fed either type of iron‐fortified growing‐up milk compared to the control group (AIN‐93G diet). CONCLUSION: A growing‐up milk supplemented with FeAAC showed an improvement in the iron absorption and haemoglobin status at the same level as those of FS. Copyright © 2009 Society of Chemical Industry     

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.     

Development of a three‐tier in vitro system,using Caco‐2 cells,to assess the effects of lactate on iron uptake and transport from rye bread following in vitro digestion

A three‐tier Caco‐2 cell system was developed to assess simultaneously iron dialysability, uptake and transport across the Caco‐2 monolayer from an in vitro digested food matrix. The effect of lactate (0–200 mmol L⁻¹) on iron absorption from rye bread subjected to simulated peptic (pH 5.5) and pancreatic digestion (pH 6.5) was investigated to model absorption pre and post the sphincter of Oddi. Lactate increased dialysability (11.8%, P < 0.05) in peptic digests whereas it reduced it in pancreatic digests (4.9%, P < 0.001). Iron uptake from the peptic digests was in the region of 39–76 pmol mg⁻¹ protein whereas it decreased from 281 to 51 pmol mg⁻¹ protein in pancreatic digests. Iron transport was calculated for the peptic digests from [¹⁴C]polyethylene glycol movement and only at 200 mmol L⁻¹ lactate was there any detectable transcellular transport (180 pmol mg⁻¹ protein, P < 0.05). Iron absorption was positively correlated to dialysable iron for both digests (R² = 0.48 and 0.41, respectively, P < 0.01) and the effect of lactate was therefore associated mainly with iron bioaccessibility. The three‐tier system showed the potential to obtain detailed insight into each step involved in iron transport across the monolayer from a food mixture. Copyright © 2006 Society of Chemical Industry     

Differences in hydrolytic abilities of two crude lipases from Geotrichum candidum 4013

The fungus Geotrichum candidum 4013 produces two types of lipases (extracellular and cell‐bound). Both enzymes were tested for their hydrolytic ability to p‐nitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols). Higher lipolytic activity of extracellular lipase was observed when triacylglycerols of medium‐ (C12) and long‐ (C18) chain fatty acids were used as substrates. Cell‐bound lipase preferentially hydrolysed trimyristate (C14). The differences in the abilities of these two enzymes to hydrolyse p‐nitrophenyl esters were observed as well. The order of extracellular lipase hydrolysis relation velocity was as follows: p‐nitrophenyl decanoate > p‐nitrophenyl caprylate > p‐nitrophenyl laurate > p‐nitrophenyl palmitate > p‐nitrophenyl stearate. The cell‐bound lipase indicates preference for p‐nitrophenyl palmitate. The most striking differences in the ratios between the activity of both lipases (extracellular : cell‐bound) towards different fatty acid methyl esters were 2.2 towards methyl hexanoate and 0.46 towards methyl stearate (C18). The Michaelis constant (K_m) and maximum reaction rate (V_max) for p‐nitrophenyl palmitate hydrolysis of cell‐bound lipase were significantly higher (K_m 2.462 mM and V_max 0.210 U/g/min) than those of extracellular lipase (K_m 0.406 mM and V_max 0.006 U/g/min). Copyright © 2010 John Wiley & Sons, Ltd.     

Bioavailability of iron and zinc from multiple micronutrient fortified beverage premixes in Caco-2 cell model

Abstract: Iron and zinc deficiencies are the most prevalent nutrient deficiencies worldwide. They often coexist as the dietary factors, especially phytate, which impairs iron absorption also affects zinc absorption. Therefore, suitable strategies are required to control multiple micronutrient deficiencies in populations that subsist on high‐phytate foods such as the whole wheat flour based Indian bread (chapatti). The objective of the study, therefore, was to test the bioavailability of iron and zinc in 2 multiple micronutrient beverage premixes in the absence and presence of chapatti. The premix‐1 contained iron, zinc, and vitamin A while premix‐2 contained all micronutrients in premix‐1, plus folic acid and ascorbic acid. Ferritin induction and ⁶⁵Zn uptake were assessed using coupled in vitro digestion/Caco‐2 cell line model as the surrogate markers of iron and zinc bioavailability, respectively. The results show that iron bioavailability from premixes‐1 and 2 was similar in the absence of chapatti. However, premix‐2 showed significantly higher iron bioavailability compared to premix‐1 in the presence of chapatti. In contrast, the zinc uptake was similar from both premixes‐1 and 2 in the absence or presence of chapatti. These results suggest that both the premixes provide bioavailable minerals, but premix‐2 appears to be promising in the presence of foods that have high phytate.     

Dephytinisation of Sangak and Barbari bread made from different extraction rate flours increases iron and zinc bioaccessibility in Caco‐2 cells

Bread is a staple food in many countries and an important source of iron and zinc. The bioavailability of these minerals is generally low because of the content of phytic acid. Traditional Iranian breads were prepared with flours of different extraction rates, Sangak at 93% and Barbari at 82%. Breads were dephytinised by addition of Aspergillus niger phytase during in vitro digestion. The effect upon iron and zinc bioaccessibility in the Caco‐2 cell model was investigated. The cellular uptake of iron and zinc was lower from Sangak, compared to Barbari, despite higher mineral content in Sangak. Dephytinisation of both breads increased iron uptake in the Caco‐2 cells (0.65 vs. 1.64 in Sangak and 0.77 vs. 1.97 ng mg^?1 protein in Barbari). Zinc uptake increased from 0.98 to 2.8 in Sangak and from 1.4 to 2.9 ng mg^?1 protein in Barbari. Thus, dephytinisation substantially improves iron and zinc bioaccessibility.     

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。     

Bioavailability of a Heme-Iron Concentrate Product Added to Chocolate Biscuit Filling in Adolescent Girls Living in a Rural Area of Mexico

ABSTRACT: A heme-iron concentrate product derived from swine hemoglobin was used to enrich the chocolate-flavored filling of biscuits and the bioavailability of this source of heme-iron was assessed in adolescent girls. The placebo control (PC) group consisted of 35 teenagers with the highest baseline hemoglobin concentrations. The supplemented groups were randomized to receive biscuits fortified with iron sulfate (IS, n = 37) or heme-iron concentrate (HIC, n = 40). Both groups were supplemented with 10.3 mg Fe/d for 7 wk. Blood chemistry and hematology analyses were performed at baseline and at the end of the study. The baseline prevalence of anemia (hemoglobin <12g/dl) in the entire group was 3.9% and by the end of the study it had fallen to 2.3%. The hemoglobin levels in both supplemented groups increased (P < 0.05) during the study period from 13.6 and 13.5 g/dl for HIC and IS, respectively, at baseline to 14 g/dl at the end of the study. Serum ferritin concentrations decreased by the end of the study in both the PC and IS groups (P < 0.05), but not in the heme group. In conclusion, iron bioavailability from HIC-fortified biscuits was calculated to be 23.7% higher than that observed for IS, as shown by the differences observed in serum ferritin levels during the study. The iron contained in the heme-iron concentrate was well absorbed and tolerated by the adolescents included in the study.     

A comparison of various extractants for the estimation of extractable zinc in cacao-growing soils of south-western Nigeria

Forty-six soil samples from cacao plantations located in South-western Nigeria were analysed for extractable zinc by four methods, namely, ammonium acetate, dilute hydrochloric acid, magnesium chloride and diethylenetriamine pentaacetic acid (DPTA). Guinea corn (sorghum) was grown as a test crop. Relations among soil test methods, plant zinc uptake, and soil properties were examined by simple and multiple correlation analyses. The Zn extracted was in the following order: 1.0N NH₄OAc>0.1N HCl>2N MgCl₂>0.005M DTPA. The only correlation between extracts that was significant at the 5% level was between NH₄OAc and DTPA extractable Zn. No soil properties except iron and aluminium oxide were significantly correlated with soil test values. Silt, clay and silt plus clay were correlated at 5, 5 and 1% respectively with Zn uptake, but pH was not. Zinc uptake by the plant was more closely correlated with 2N MgCl₂ extractable Zn (r=+0.389) than with 1N NH₄OAc extractable Zn (r=+0.291); 0.1N HCl extractable Zn (r=+0.079); or with DTPA extractable Zn (r=+0.220). The best prediction of uptake by plants was observed in multiple regression analyses involving combination of MgCl₂ extractable Zn, pH, organic matter, and silt plus clay fraction (r=+0.601).     

The fate of forage plant DNA in farm animals: a collaborative case-study investigating cattle and chicken fed recombinant plant material

 The fate of ingested recombinant plant DNA in farm animals (cattle and chicken) being fed a diet containing conventional maize or recombinant Bacillus thuringiensis toxin-maize (Bt-maize) is described. The probability of the detection by polymerase chain reaction of chloroplast-specific gene fragments of different lengths (199 bp and 532 bp) and a Bt-maize-specific fragment [truncated version of CryIA(b)] is shown. First data indicated that only short DNA fragments (<200 bp) derived from plant chloroplasts could be detected in the blood lymphocytes of cows. In all other cattle organs investigated (muscle, liver, spleen, kidney) plant DNAs were not found, except for faint signals in milk. Furthermore, Bt-gene fragments possibly recording the uptake of recombinant maize, were not detected in any sample from cattle. However, in all chicken tissues (muscle, liver, spleen, kidney) the short maize chloroplast gene fragment was amplified. In contrast to this, no foreign plant DNA fragments were found in eggs. Bt-gene specific constructs originating from recombinant Bt-maize were not detectable in any of these poultry samples either. Received: 23 February 2000 / Revised version: 20 March 2000