An iron binding assay to measure activity of known food sequestering agents: studies with buttermilk solids

A method enabling the measurement and differentiation of both ferrous (Fe²⁺) and ferric (Fe³⁺) binding was developed to assess the relative sequestering power of known iron chelators and buttermilk solids (BMS). Fe²⁺ and Fe³⁺ chelation by BMS (0.1–10.0 mg), phytic acid, ethylenediaminetetraacetic acid (EDTA) and citric acid (0.1 mM) was measured using a modification of a ferric-thiocyanate method. Fe²⁺ and Fe³⁺ was determined from the amount of unbound ferric-thiocyanate following incubation with chelator. The assay was optimized for polarity of solvent and pH. Fe²⁺ and Fe³⁺ binding by sequestering agents was, in descending order of affinity to be: phytic acid>EDTA>BMS>citric acid. Known food iron chelators had a higher affinity towards Fe³⁺, while BMS had a higher affinity towards Fe²⁺. BMS also demonstrated an excellent iron solubilization property through iron binding activity.     

EFFECT OF ACID PRETREATMENT ON THE STABILITY OF CITRIC AND MALIC ACID COMPLEXES WITH VARIOUS IRON SOURCES IN A WHEAT FLAKE CEREAL1

An in vitro incubation at pH 2 of citric and malic acids with each of five iron sources [(hydrogen (HRI) and electrolytically reduced elemental iron (ERI), ferric chloride (FeCl₃), ferrous sulfate (FeSO₄) and ferric orthophosphate (FOP)] at a 10:1 molar ratio (acid:iron) was evaluated for its effect on iron solubiliza-tion in a wheat flake cereal subjected to a sequential gastrointestinal pH treatment from endogenous pH (E) to 2 to 6. Citric acid maintained significantly more complexed and ionic iron (Fe⁺³) in solution than malic acid, with and without incubation, through the final stage of the sequential pH treatment with each of the five iron sources. However, the malate treated samples were affected less than the citrate by acidification from pH E to 2, with less of the soluble complexed iron being insolubilized and significantly more ionic iron (Fe⁺²) being produced. Incubation significantly enhanced the iron solubilizing capacity of citrate at all stages of the sequential pH treatment with HRI and at pH E with ERI, FeCl₃ and FeSO₄, As well, incubation increased the iron solubilization properties of malate at all stages with FeSO₄ and at E and 2 with HRI and ERI. These results indicate that acid incubation to form an organic acid-iron chelate, particularly with elemental iron, has the potential to improve cereal iron fortificant bioavail-ability.     

Development of caramel colour with improved colour stability and reduced 4-methylimidazole

ABSTRACT

This study investigated the effect of chemical substances, such as Mg²⁺ (as magnesium sulphate), Zn²⁺ (as zinc sulphate), Fe²⁺ (as iron sulphate), ascorbic acid, and citric acid, on the formation of 4-methylimidazole (4-MI) and colour stability of caramel colour. The 4-MI concentration in the caramel colour without chemical substances was 963.10 μg/g, and this decreased the most (67.7%) with the addition of 0.1 molar ratio of citric acid. Colour stability was evaluated by measuring the total colour change (ΔE) after storage, and heating in pH 2, 4, and 7 solutions and 50% solution in ethyl alcohol. Among the chemical substances added to reduce 4-MI in the caramel colour, Mg²⁺ (0.1 molar ratio) and ascorbic acid improved the colour stability more than the others.     

Influence of dietary phenolic acids on redox status of iron: Ferrous iron autoxidation and ferric iron reduction

We investigated the effect of dietary phenolic acids on the oxidation of Fe²⁺ caused by molecular oxygen. All phenolic acids bearing 3,4-dihydroxy (catechol) or 3,4,5-trihydroxy (galloyl) moiety formed chelates with ferric iron and significantly increased the rate of Fe²⁺ autoxidation. The carboxylate group and catechol substitution instead of galloyl moiety facilitated the ferrous ion oxidation more effectively. Caffeic acid and protocatechuic acid, the strongest accelerators of Fe²⁺ autoxidation, were able to facilitate autoxidation at concentrations lower than 1% of the initial amount of Fe²⁺. Therefore chelates of these catecholic acids with iron displayed ferroxidase-like activity. Conversely, when we started from ferric ions, catechols partially formed ferrous ions in the presence of ferrozine. Thus, catecholic acids formed stable chelates with iron, in which ferric ion is the dominant species, but the redox cycling of iron between Fe²⁺ and Fe³⁺ in chelates probably plays a crucial role in the catalysis of ferrous iron autoxidation. Interestingly, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid and vanillic (4-hydroxy-3-methoxybenzoic) acid protected ferrous ions from autoxidation as effectively as ascorbic acid and cysteine. These monophenolic acids, differently from ascorbic acid and cysteine, were not able to reduce ferric ions. Syringic (3,5-dimethoxy-4-hydroxybenzoic) acid did not alter the redox state of iron, only in a large excess over metal, syringic acid slightly inhibited ferrous ions autoxidation and partially reduced ferric ions. Therefore, the effects of syringic acid at high concentration were similar but much lower to those of ascorbic acid and cysteine. The biological importance of ferroxidase-like activity of polyphenols, especially the influence on iron absorption, is also discussed.     

Effect of Acid Pretreatment on the Stability of Ascorbic Acid Complexes with Various Iron Sources in a Wheat Flake Cereal

The effect of acid incubation of ascorbic acid with each of five iron sources (ferrous sulfate, ferric chloride, ferric orthophosphate, hydrogen and electrolytically reduced iron) on iron solubilization in a wheat flake cereal was evaluated. Incubation produced more soluble iron at pH 2 but not necessarily at the endogenous pH of the cereal nor at pH 6. At pH 2, Fe⁺² rather than Fe ⁺³ was produced, apparently by a reduction of bound Fe ⁺³ and subsequent release of Fe⁺². At pH 6, the soluble iron was mainly in a complexed form. This indicates that acid incubation with ascorbate might facilitate bioavailability of iron if it were incorporated by fortification techniques.     

Influence of irradiation time and solution concentration on the photochemical degradation of EDDHA/Fe3+: effect of its photodecomposition products on soybean growth

BACKGROUND: Ethylenediamine‐N, N′‐bis(2‐hydroxyphenylacetic acid (EDDHA) is one of the most efficient iron‐chelating agents employed to relieve iron chlorosis in plants. It has been well known for decades that this compound is photosensitive, but in spite of this fact its degradation pathways are virtually unknown. The aim of this work was to evaluate how the length of sunlight exposure and the concentration of irradiated EDDHA/Fe³⁺ solutions influence the photostability of the chelate at constant pH. Moreover, the possible toxic effect of the chelate photodegradation products, elsewhere proposed, on soybean growth has been tested. RESULTS: The photodecomposition of the chelate increased as the time of sunlight exposure increased, and resulted in a partial decomposition of the organic ligand. Moreover, EDDHA/Fe³⁺ photodecomposition was highly correlated with the concentration of solution exposed. Plants did not present differences in recovery from chlorosis among treatments with and without decomposition products. CONCLUSIONS: EDDHA/Fe³⁺ undergoes photodegradation, like other aminopolycarboxylic acids, being more degraded as solution concentration decreases and exposure time increases. The photodecomposition products salicylic acid, salicylaldehide and Salicylaldehyde ethylenediamine diimine tested did not have negative effects on soybean growth, at least in the short‐term hydroponic experimental design tested. Copyright © 2011 Society of Chemical Industry     

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).     

EFFECT OF PACKAGING MATERIALS ON THE QUALITY OF IRON-FORTIFIED WHOLEMEAL FLOUR DURING STORAGE

ABSTRACT

The effect of packaging materials on the physicochemical and rheological characteristics of iron‐fortified wholemeal flour (WMF) during storage was determined. WMF was fortified with three fortificants, namely ferrous sulfate (30 ppm), ferrous sulfate + ethylenediamine tetraacetic acid (EDTA) (20 + 20 ppm) and elemental iron (60 ppm). Each flour was also fortified with 1.5 ppm folic acid. Moisture, flour acidity and peroxide value increased during storage, while protein and fat contents decreased. Highest conversion of Fe²⁺ into Fe³⁺was observed in flour fortified with ferrous sulfate (2.72%), followed by that fortified with ferrous sulfate + EDTA (1.49%) and elemental iron (1.06%). Water absorption and dough viscosity of iron‐fortified flours increased during storage. The flour containing ferrous sulfate was most acceptable regarding sensory characteristics, followed by samples containing ferrous sulfate + EDTA. Fortified flours were more stable during storage than unfortified. Addition of EDTA increased the stability of flours and fortificants. The fortified flours stored in polypropylene bags proved more stable than those stored in the tin boxes.

PRACTICAL APPLICATIONS

The main role of packaging is to protect the product during handling, distribution and storage against environmental and mechanical hazards. The success of a fortification program depends on the stability of micronutrients and food to which these are added. Chemical changes during storage badly affect chapatti making and sensory properties. Exposure of the fortificant to any factor including heat, moisture, air or light, and acid or alkaline environments during processing, packaging, distribution, or storage affects its stability. Flour containing elemental iron and ferrous sulfate with EDTA remained stable up to 42 days. The unfortified flour and flour containing ferrous sulfate remained stable for 21 days in tin boxes and 28 days in the polypropylene bags. Wheat flour milling industry would be benefited from this research if government is keen to launch iron fortification program in the country to curb iron deficiency anemia among population.

     

Accumulation of Iron in Lactic Acid Bacteria and Bifidobacteria

Lactobacillus acidophilus, L. delbrueckii var. bulgaricus, L. plantarum and Streptococcus thermophilum, all used extensively in the food industry, were tested for their ability to internalize and/or oxidize ferrous iron (Fe²+). For comparison some experiments were performed with bifidobacteria, B. thermophilum and B. breve. All organisms except L. bulgaricus could transport Fe²⁺ into the cell, where it was partially oxidized to the ferric form (Fe(III)). In addition, L. acidophilus and L. bulgaricus could oxidize Fe²⁺ to Fe(III) extracellularly through the elaboration of H₂O₂ into the medium when the experiments were carried out in air. L. bulgaricus elaborated H₂O₂ only in the presence of glucose, whereas L. acidophilus released H₂O₂ in absence of glucose. We concluded that lactic acid bacteria, like bifidobacteria, can exert some beneficial effects in animal organisms, or in food processing and storage, by making Fe²⁺ unavailable to harmful microorganisms.     

Measurement of antioxidative activity in metal ion‐induced lipid peroxidation systems

This study aims to investigate the acceleration effects of various prooxidants on linoleic acid peroxidation and to assess the feasibility of applying these model systems by measuring the antioxidant activities of butylated hydroxyanisole (BHA), α‐tocopherol and rosemary extracts. According to our results, Fe²⁺ and Fe³⁺ exhibited more stimulatory effects than other prooxidants on the peroxidation of linoleic acid. An additional amount of ascorbic acid can reduce Fe³⁺ to Fe²⁺, resulting in more severe peroxidation. However, ascorbic acid displays antioxidant activity with increasing concentration. The peroxidation of linoleic acid induced by Fenton reaction is less than that induced by iron ions and was increased by addition of ascorbic acid to the system to maintain the regeneration of Fe²⁺ and the production of hydroxyl radicals. BHA shows the strongest inhibitory effects on the peroxidation of linoleic acid in all systems. The antioxidant activity of α‐tocopherol is less than that of rosemary extracts in the iron ion‐induced peroxidation systems. However, α‐tocopherol shows stronger inhibitory effects than rosemary extracts in the Fenton reaction‐induced peroxidation system at a concentration less 100 mg kg⁻¹. These systems can be useful in screening antioxidants from natural sources because of the short reaction time and clear difference between each antioxidant. © 1999 Society of Chemical Industry     

Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription

The metal ions, Cu^2+/+ and Fe^3+/2+, are essential co-factors for a wide variety of enzymatic reactions. However, both metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins. Although copper transport into yeast cells is apparently independent of iron, the known dependence on Cu²⁺ for high affinity transport of Fe²⁺ into yeast cells has established a physiological link between these two trace metal ions. In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly or acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis. Yeast cells lacking a functional PEP3 or PEP5 gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe²⁺ transport, also repressible by exogenous copper ions. The inability of these same vacuolar mutant strains to repress FET3 mRNA levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution or redox states of Fe^3+/2+ in the presence of elevated extracellular concentrations of copper ions. Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions. © 1997 John Wiley & Sons, Ltd.     

Growth Inhibition of Listeria monocytogenes by Sodium Polyphosphate as Affected by Polyvalent Metal Ions

Sodium polyphosphate (SPP, average chain length = 13) increased the lag time of L. monocytogenes Scott A (Lm) in brain-heart infusion broth (BHI). Polyvalent metal ions (1–10 mM) reversed inhibition of Lm growth by 0.5% SPP (nominal 3.6 mM). 10 mM Ca²⁺ or Mg²⁺, 5 mM Fe³⁺, 2 mM Mn²⁺ or 1 mM Zn²⁺ added to SPP-containing BHI, pH 6.0, at 19°C resulted in growth comparable to control cultures. Fe²⁺ partially restored growth; Ni²⁺, Co²⁺, Cu²⁺ or Al³⁺ were ineffective. SPP inhibited growth at 28°C in BHI, pH 5.0, and Lm grew upon addition of Ca²⁺, Mg²⁺ or Mn²⁺, but not Zn²⁺ or Fe³⁺. Addition of 0.5% SPP to mineral-rich foods, such as pureed beef, green beans or sweet potatoes, did not delay growth.     

Characterization of purified xylanase from finger millet (<Emphasis Type="Italic">Eleusine coracana-</Emphasis>Indaf 15) malt

Xylanase (E.C. 3.2.1.8) was purified to apparent homogeneity from 96 h finger millet (Eleusine coracana, Indaf-15) malt by a three step purification procedure via ammonium sulphate fractionation, DEAE-cellulose ion exchange and Sephadex G-75 gel permeation chromatographies with a recovery of 4.0% and fold purification of 60. Xylanase, having a molecular weight of 29 ± 2 kDa was found to be monomeric on SDS-PAGE. pH optimum of the enzyme was found to be in the range of 5.0–5.5. The activation energy was 25 kJmol⁻¹. Xylanase showed maximum stability at 35 °C in a pH range of 5.0–6.0. K _m and V _max of purified xylanase were found to be 0.2% and 4.5 μmol min⁻¹, respectively. Metal ions such as Ca²⁺, Mg²⁺, Mn²⁺, Cu²⁺, Fe²⁺, Ag²⁺ and Ni²⁺ enhanced xylanase activity at 5 mM concentration. p-chloromercuribenzoate, citric, oxalic and boric acids inhibited the enzyme in concentration dependent manner. The mode of action of xylanase was found to be “endo” as determined by the analysis of products liberated from larchwood xylan by ESI-MS and H¹NMR. In vitro studies using Bifidobacterium and Lactobacillus sp. confirmed the prebiotic activity of the xylo-oligosaccharides.     

LIPID OXIDATION IN TURKEY MEAT AS INFLUENCED BY SALT,METAL CATIONS AND ANTIOXIDANTS1

Turkey breast or thigh muscle was mixed with 2% pure salt, rock salt, or pure salt plus 50 ppm of one or a combination of copper, iron or magnesium. Efficacy of 2 antioxidants was tested. Lipid oxidation was monitored during refrigerated and frozen storage of raw and cooked turkey by the thiobarbituric acid (TBA) test. TBA results indicated that the most significant prooxidant effect was caused by salt plus Cu²⁺ and Fe²⁺ followed by salt plus Fe³⁺ or Cu²⁺ alone. Tenox 6 was an effective antioxidant in the presence of copper and iron ions. Thigh meat was more susceptible to oxidation than breast meat. Cooking had a significant prooxidant effect as measured by TBA.     

Modified barley straw as a potential biosorbent for removal of copper ions from aqueous solution

Barley straw (BS), a very low-cost material, has been utilized as a biosorbent material for the removal of copper (Cu²⁺) ions from aqueous solutions after treatment with citric acid. Barley straw was thermochemically modified with citric acid (CA–BS) for the purpose of improving the Cu²⁺ ion sorption capacity of the straw. Biosorption studies have been carried out to determine the effect of pH, adsorbent concentration, contact time, extent of modification, and adsorbate concentration on the biosorption capacity of Cu²⁺ ions by the esterified straw. The equilibrium sorption capacities of Cu²⁺ were 4.64 mg/g and 31.71 mg/g for BS and CA–BS, respectively. The optimum pH for the removal of Cu²⁺ ions by CA–BS was around pH 7.0 and the removal of Cu²⁺ ions was 88.1%. Langmuir, Freundlich, Scatchard and D–R (Dubinin–Radushkevich) isotherms have been used to characterize the observed biosorption phenomena of Cu²⁺ ions on CA–BS. The carboxyl groups on the surface of the modified barley straw were primarily responsible for the sorption of Cu²⁺ ions.     

Purification and enzymatic characteristics of a novel polyphenol oxidase from lotus seed (Nelumbo nucifera Gaertn.)

A polyphenol oxidase (PPO) from lotus seed was purified by the procedures including ammonium sulphate precipitation and affinity chromatography. The apparent molecular mass was 38.6 kDa by SDS‐PAGE. Kinetic studies showed that the K_m and V_max values for catechol were 6.04 mm and 416.67 U, respectively. The PPO performed optimal activity in 20 °C and pH 7.0. The enzymatic activity could be mainly maintained up to 50 °C and pH 4.0–7.0. The activity could be inhibited by various inhibitors including thiourea, urea, sodium hydrogen sulphite, EDTA·2Na, SDS, citric acid, guanidine hydrochloride, ascorbic acid, sodium sulphite and sodium thiosulphate. The metal ions Ba²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺ and Zn²⁺ could inhibit the activity of PPO, while Cu²⁺ performed obvious enhancement. The enzymatic properties of PPO could probably provide practical application in inhibiting the PPO activity and preventing enzymatic browning in the process of picking, transportation, processing and storage of fresh lotus seeds.     

Optimization of Trace Metals Concentration on Citric Acid Production by Aspergillus niger NRRL 2001

Citric acid is nowadays produced by submerged fermentation of Aspergillus niger. The process yield depends on the composition of the medium, as well as on the microorganism strain. In this work, the effect of Fe⁺³, Zn⁺², and Mn⁺² on citric acid production by A. niger NRRL 2001 is presented. The culture medium composition was glucose (120 g/L) KH₂PO₄ (1.0 g/L); K₂HPO₄ (1.0 g/L), MgSO₄.7H₂O (0.5 g/L), (NH₄)₂SO₄ (3.0 g/L). The ions Fe⁺³, Zn⁺², and Mn⁺² had their concentrations changed according to an experimental design. The experiments were carried out in an orbital shaker at 200 rpm and 30°C. The strain produced an extracellular polysaccharide that was also quantified. The optimum experimental condition was found using 7.0 mg/L of Fe⁺³ and 6.5 mg/L of Zn⁺² in absence of Mn⁺². No oxalic acid formation was observed using this experimental condition. Metal contents were not significant for the production of the polysaccharide. The highest production rate (2.95 g L⁻¹ day⁻¹) was reached after 10 days of fermentation. After this period, the productivity decreased slightly. In 20 days, the citric acid production rate (2.44 g L⁻¹ day⁻¹) was 82% of the highest productivity. The conversion into citric acid increased continuously, yielding 45.8% in 20 days of fermentation.     

Effect of natural antioxidants on stored freeze-dried food product formulated using horse mackerel (Trachurus trachurus)

The physical and chemical properties of Trachurus trachurus (horse mackerel) as well as a food product in the presence and absence of natural antioxidants, were investigated. The rate of lipid oxidation in the freeze-dried horse mackerel product stored at room temperature (22 °C) was investigated, using peroxide value (PV), thiobarbituric acid reactive substances (TBARS) and hexanal concentration to determine primary, secondary and tertiary oxidation products. Natural antioxidants were used to slow down oxidation processes and to improve nutritional and eating quality. These included vitamin E + vitamin C + citric acid (250, 250, 100 mg kg⁻¹, respectively), vitamin E + vitamin C + citric acid + rosemary (250, 250, 100, 250 mg kg⁻¹, respectively) and rosemary (250 mg kg⁻¹). Biochemical analysis such as PV and TBARS measurements showed that the combination of vitamin E + vitamin C + citric acid (250, 250, 100 mg kg⁻¹) were found to be the most significant (P ≤ 0.01) in controlling the rate of lipid oxidation in freeze-dried food followed by rosemary (250 mg kg⁻¹). There was an initial rise in the PV of all samples, which dropped after 8 weeks, because of formation of secondary oxidation products. The rise in PV was much higher in the control samples with no antioxidants added, compared with the antioxidant-treated samples. Combinations of E + C + citric acid treatment group followed by rosemary alone were the most significant in lowering PV, hence, reducing formation of primary oxidation products (P ≤ 0.01). Once the primary oxidation products were lowered, TBARS levels (secondary oxidation products) increased in control samples after 12 weeks; however, in E + C + citric acid treatment group and rosemary-treated samples, this increase was significantly lower (P ≤ 0.01). There was a rise in hexanal concentration for up to 8 weeks, which dropped only in the antioxidant-treated groups and continued to rise in the control samples.     

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     

Purification of diamine oxidase and its properties in germinated fava bean (Vicia faba L.)

Background: γ‐Aminobutyric acid (GABA) is a non‐protein amino acid with bioactive functions for human health. Diamine oxidase (DAO, EC 1.4.3.6) is one of the key enzymes for GABA formation. In the present study, this enzyme was purified from 5 day germinated fava bean and its properties were investigated in vitro. Results: The molecular mass of the enzyme estimated by Sephadex G‐100 gel filtration was 121 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) displayed a single band at a molecular mass of 52 kDa. The enzyme had optimal activity at 40 °C and retained its activity after being incubated at 30 °C for 30 min. It showed higher activity at pH 6.5 than at other pH values. The enzyme was significantly inhibited by Mg²⁺, Cu²⁺, Fe³⁺, aminoguanidine, ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid disodium salt (EDTA‐Na₂), L ‐cysteine and β‐mercaptoethanol. The K_m value of DAO was 0.23 mmol L^?1 for putrescine and 0.96 mmol L^?1 for spermidine. However, the enzyme did not degrade spermine. Conclusion: DAO from germinated fava bean was purified. The optimal reaction temperature and pH of the enzyme were mild. The enzyme had higher affinity to putrescine than to spermidine and spermine. Copyright © 2011 Society of Chemical Industry