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.     

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.     

Effects of soluble ferri-hydroxide complexes on microbial neutralization of acid mine drainage

Heterotrophic respiration of ferric iron by Acidiphilium cryptum was investigated in anoxic microcosms with initial media pH values from 1.5 to 3.5. No organic carbon consumption or iron reduction was observed with an initial pH of 1.5, indicating that A. cryptum may not be capable of iron respiration at this pH. Significant iron reduction was observed at pH 2.5 and 3.5, with different effects. When the initial pH was 3.5, pH increased to 4.7-5.5 over 60 days of incubation with simultaneous production of 0.4 g L(-1) Fe2+. However, at an initial pH of 2.5, no significant change in pH was observed during iron respiration, although the accumulation of soluble ferrous iron was significantly higher, averaging 1.1 g L(-1) Fe2+. The speciation of the ferric iron electron acceptor may explain these results. At pH values of 3.5 and higher, precipitated ferric hydroxide Fe- (OH)3 would have been the primary source of ferric iron, with reduction resulting in net production of OH- ions and the significant increases in media pH observed. However at pH 2.5, soluble complexes, FeOH2+ and Fe(OH)2+, may have been the more prevalent electron acceptors, and the alkalinity generated by reduction of complexed iron was low. The existence of charged ferri-hydroxide complexes at pH 2.5 was verified by voltammetry. Results suggest that initiation of bacterial iron reduction may result in neutralization of acid mine drainage. However, this effect is extremely sensitive to iron speciation within a relatively small and critical pH range.     

Characterization of Solution Properties of Four Iron Sources in Model Systems by Solubility Studies and IR/VIS Reflectance Spectrophotometry

The hydrolysis of iron and the effect of aging on the resolubilization of iron hydroxides under simulated gastric pH conditions were measured and spectroscopy was evaluated as a predictor of these changes. Model systems of ferrous sulfate (FeZSO₄), ferric sulfate (Fe₂(SO₄)₃), ferric chloride (FeCl₃) and hydrogen-reduced elemental iron (HRFe), with and without ascorbic acid, were analyzed at intervals over 10 wk storage for both soluble iron and iron resolubilized from insoluble iron hydroxide polymers. Irreversible hydrolysis, resulting in polymers with ordered structures, occurred in the FeSO₄ and HRFe systems. Spectra of air-dried precipitates correlated with solution behavior of iron providing a possible predictive technique. Added ascorbic acid increased the levels of soluble iron in all systems.     

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.     

IRON SOLUBILITY FROM SEAFOODS WITH ADDED IRON AND ORGANIC ACIDS UNDER SIMULATED GASTROINTESTINAL CONDITIONS

Iron absorption from foods is dependent on the type of food consumed and other factors such as ligands which may have enhancing or inhibiting effects on iron absorption. In this study, iron (Fe²⁺ or Fe³⁺) and two organic acids (ascorbate and citrate) were added to three seafood surimis (scallops, prawns, and cod) and then subjected to simulated gastrointestinal conditions (pH 2, 4, and 6) to evaluate iron solubility. Soluble iron decreased with increasing pH in all samples and control. In cod, a combination of ascorbic and citric acid (1:2) was most effective to increase soluble iron; however, in scallops and prawns citric acid alone was best. After sequential pH and enzyme treatments, it was found that the effectiveness of iron solubilization depended primarily upon the type of seafood present and to a lesser extent the oxidation state of the iron salt.     

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.     

A Maltooligosaccharides Producing α-Amylase from Bacillus subtilis SDP1 Isolated from Rhizosphere of Acacia cyanophylla Lindley

Maltooligosaccharides producing amylases are required in the food industry, especially in breadmaking. The Bacillus subtilis strain SDP1 amylase hydrolyses starch to produce maltotriose and maltotetraose along with maltose after prolonged reactions of 5 h. Bacillus subtilis strain SDP1 was isolated from the rhizosphere of Acacia cyanophylla Lindley from the Çukurova region of Turkey. The highest enzyme production was achieved with soluble starch as the carbon and yeast extract as the nitrogen source and at pH 7.0 and 37°C. Under optimized culture conditions, 68.49 U/mL activity was obtained. SDP1 α-amylase had molecular weight of 61 kD. The optimum pH of the enzyme was 7.0 and was highly active at pH ranging from 5.0 to 9.0. The optimum temperature of the crude enzyme was 60°C, and it retained 83% and 74% of its initial activity after 1 h and 2 h incubation periods, respectively, at 50°C. While, Mn⁺² has a stimulatory effect on the activity, Ca⁺², Mg⁺², Na⁺ did not effect the enzyme activity. Fe⁺³, Ni⁺², Cu⁺² and Co⁺² had an inhibitory effect on SDP1 amylase activity.     

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.     

Substrate Specificity and Inhibition of Polyphenoloxidase (PPO) From a Dwarf Variety of Banana (Muss Cavendishii, L.)

Banana polyphenoloxidases oxidize specifically o-diphenols. The Km values were low for dopamine, epinephrine, and norepinephrine. It was lower for L-dopa as compared with D-dopa. The most effective inhibitor was ascorbic acid followed by cysteine and then sodium metabisulfite. Diethyldithiocarbamate, at pH 7.0, was not as effective as the above inhibitors. Ammonium ion and the oxidized β-nicotine adenine dinucleotide acted as activators whereas Fe⁺², Fe+³, Al⁺³, Ca⁺² and Zn⁺² showed various degrees of inhibition. On the other hand, C⁺¹, Cu⁺², Mg⁺² and Mn⁺² did not affect the enzyme activity. Mercaptoethanol (17 mM) completely inactivated the enzymes. On dialysis 30% of the activity was restored with regeneration of two isozymes.     

Iron fortification of skim milk: Minerals and 57Fe Mössbauer study

Reconstituted skim milk was fortified at 2, 5, 10 and 20 mmol Fe kg^?1 with ferric and ferrous chloride iron with or without reversible acidification by injection of CO₂ under pressure. Carbonation improved transfer of iron from the soluble to the colloidal phase and accelerated ferrous iron oxidation. ⁵⁷Fe Mössbauer spectra of the freeze-dried casein micelles collected by centrifugation demonstrated that iron is present in a distorted octahedral coordination and is chelated by phosphate rather than citrate. Milk iron fortification induced several changes in the mineral component of the casein micelles, which are explained by the formation of a ternary complex: inorganic phosphate–iron–organic phosphate.     

Characterization of an acidophilic and thermostable α‐amylase from Alicyclobacillus sendaiensis NUST

This paper describes the characterization of an acidophilic and thermostable α‐amylase from Alicyclobacillus sendaiensis NUST. The MW of this enzyme was estimated to be 56 kDa by SDS–PAGE. The enzyme was stable over a range of pH from 2.5 to 5.5 with an optimum around 3.5. Maximum activity of the α‐amylase was observed at pH 3.5 and 85°C in the presence of soluble starch as substrate. The enzyme activity was decreased by Mg²⁺, Cu²⁺, Zn²⁺, Al³⁺, K⁺, Li⁺, Ag⁺, urea, EDTA, trichloroacetic acid and Tween 60 and inhibited by Hg²⁺, Ce²⁺ and SDS, whereas the activity was increased by Mn²⁺, DTT, and β‐mercaptoethanol. Ca²⁺and Fe²⁺ did not affect the enzyme activity.     

Effect of Ethylenediaminetetraacetic Acid (EDTA) and Metal Ions on Growth of Staphylococcus aureus 196E in Culture Media

The effects of ethylenediaminetetraacetic acid (EDTA) and its salts, alone or in combination with Mg²⁺, Fe³⁺, Zn²⁺ or Ca²⁺, on growth of S. aureus strain 196E, were studied. EDTA or its Na⁺ or K⁺ salts (0.8–1.7 mM), but not its Ca²⁺ or Fe³⁺ salts, inhibited cell growth in Brain Heart Infusion broth. Bacteriostatic effects in Casamino Acids-Yeast Extract (CasY) broth were produced by 0.17 mM EDTA. Addition of Fe³⁺, Zn²⁺, or Ca²⁺ to EDTA-containing broth eliminated inhibition at an EDTA: cation molar ratio of 1:1, while Mg²⁺ required a sevenfold concentration. Data suggest that EDTA exerts its inhibitory effect by chelating calcium and/or other essential cations which form complexes with comparable stability constants with EDTA.     

Ascorbate enhances iron uptake into intestinal cells through formation of a FeCl3–ascorbate complex

It has been well documented that ascorbate enhances iron uptake, with a proposed mechanism based on reduction to the more absorbable ferrous form. We have performed a study on the effects of ascorbate on ferric iron uptake in the human epithelial Caco-2 cell-line. Ascorbate increased uptake in a concentration-dependent manner with a significant difference between iron uptake and reduction. Uptake kinetics are characteristic of a non-essential activator and the formation of an Fe³⁺–ascorbate complex. This investigation provides evidence that ascorbate enhances the apical uptake of ferric iron into Caco-2 cells through the formation of a Fe³⁺–ascorbate complex.     

Early Stage Formation of Iron Oxyhydroxides during Neutralization of Simulated Acid Mine Drainage Solutions

The phases and stability of ferric iron products formed early during neutralization of acid mine drainage waters remain largely unknown. In this work, we used in situ and time-resolved quick-scanning X-ray absorption spectroscopy and X-ray diffraction to study products formed between 4 min and 1 h after ferric iron sulfate solutions were partially neutralized by addition of NaHCO(3) ([HCO(3)(-)]/[Fe(3+)] < 3). When [HCO(3)(-)]/[Fe(3+)] = 0.5 and 0.6 (initial pH ～ 2.1 and 2.2, respectively), the only large species formed were sulfate-complexed ferrihydrite-like molecular clusters that were stable throughout the duration of the experiment. When [HCO(3)(-)]/[Fe(3+)] = 1 (initial pH ～ 2.5), ferrihydrite-like molecular clusters formed initially, but most later converted to schwertmannite. In contrast, when [HCO(3)(-)]/[Fe(3+)] = 2 (initial pH ～ 2.7), schwertmannite and larger ferrihydrite particles formed immediately upon neutralization. However, the ferrihydrite particles subsequently converted to schwertmannite. The schwertmannite particles formed under both conditions aggregated extensively with increasing time. This work provides new insight into the formation, stability and reactivity of some early products that may form during the neutralization of natural acid mine drainage.     

A novel potentiometric pH electrode based on sulfated natural Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> and analytical application in food samples

A novel solid state potentiometric pH electrode based on sulfated natural Fe₃O₄ silicone was fabricated. The optimum potentiometric performances such as Nernstian response, response time, selectivity, life-time and reproducibility of pH electrodes were investigated by using a computer-controlled potentiometric device. Moreover, the potentiometric performance of the solid state pH electrodes was studied with different mixtures sulfated natural Fe₃O₄, silicone and graphite powder. The best potentiometric behavior of proposed pH electrode was obtained with a composition of 20% (w/w) sulfated natural Fe₃O₄, 40% (w/w) graphite powder and 40% (w/w) silicone. The sub-Nernstian response for pH electrode was exhibited with a slope of 30.8?±?1.4 mV/pH (r?=?0.9963) from pH 2 to pH 12. In addition, the dynamic response time was found as 10 s in acidic medium and further the proposed pH electrode can be used for at least 1 year without any significant slope of the pH–potential curve. The selectivity coefficient of pH electrode was interpreted according to fixed interference method in the presences of Na⁺, Li⁺, K⁺, NH₄⁺, SO₄^2?, CH₃COO^? and NO₃^? ions. The reproducibility of pH electrode was calculated in pH 4 and pH 6 phosphate buffers and it was found as 0.24 RSD (%) and 0.27 RSD (%) respectively. The proposed pH electrode was used to determine of pH in acid?bases titration compared with glass pH electrode and is highly stable in corrosive systems including HF solution. Terminally, the pH value was successfully determined in some soft drinks and milk samples by proposed solid state pH electrode at 95% confidence level with satisfactory agreement compared with glass pH electrode.     

Use of the stable isotope (57) Fe to track the efficacy of the foliar application of lignosulfonate/Fe(3+) complexes to correct Fe deficiencies in cucumber plants

BACKGROUND: During the last decade, environmental concerns regarding the use of recalcitrant synthetic chelates to overcome iron chlorosis has increased and new ligands such as lignosulfonates (LS) have been evaluated. However, the efficacy of these products is variable. In this work a hardwood (eucalyptus) and softwood (spruce) LS were compared to try to relate their physico‐chemical characteristics and their efficacy. Also two more products derived from the eucalyptus lignosulfonate were tested. RESULTS: All the LS tested presented a good ability to complex Fe, but only the spruce LS was capable to maintain significant amounts of soluble Fe above pH 8. According to the FTIR data, structural changes related to the Fe source (Fe²⁺ or Fe³⁺) used to form the complex occurred in the LS molecule and might influence their efficacy. Cucumber (Cucumis sativus L. cv Ashley) chlorotic plants were used to test lignosulfonate efficacy when applied through foliar sprays in comparison with FeSO₄ and EDTA/⁵⁷Fe³⁺. The ⁵⁷Fe content of plants sprayed with LS was very low in respect to the EDTA treatment, but this was not reflected in the biomass and re‐greening rates. Eucalyptus LS modifications improve its efficacy for iron chlorosis recovery to levels similar to those found for the spruce LS. Two applications of the LS are recommended. CONCLUSIONS: Lignosulfonates did not require surfactants for their application; they did not burn the leaves, and had a stimulating effect on the vegetative growth of the plants. So these by‐products could be a good alternative when applied through foliar sprays for cucumber plants. Copyright © 2010 Society of Chemical Industry     

Heat shock-induced thermotolerance inListeria monocytogenes13-249 is dependent on growth phase,pH and lactic acid

The effect of heat shock on the thermotolerance ofListeria monocytogenes13-249 was compared as a function of growth phase (log and late stationary phase), pH (5.4, 5.8, 6.2) and heating medium (broth vs minced beef). Lactic acid was added to tryptic phosphate broth (TPB⁺) to simulate the level found in beef. Furthermore, the specific effect of lactic acid was investigated, comparing results obtained in broth with and without lactic acid with pH adjusted to 5.4 and 7.0. In late stationary cultures, heat shock produced only marginal differences in thermotolerance in TPB⁺and beef regardless of pH. For log phase cultures, the increase in heat shock induced thermotolerance (HSIT) depended on pH in both TPB⁺and beef with gradually larger increases at rising pH. At the highest pH tested (pH 7.050 in TPB⁺and pH 6.2 in beef) the increase in HSIT was 5.7-fold and 2.4-fold, respectively. These increases were highly significant (P< 0.001). If no lactic acid was present, the HSIT increased at both pH 5.4 for 7.0 for log phase cultures. This indicates that the presence of lactic acid in naturally occurring levels will have a specific effect on the response ofL. monocytogenesto heat shock. The D₆₀-values ofL. monocytogenes13-249 were generally higher (2—6-fold) in minced beef than in TPB⁺.     

Determination of phytic acid in feeds and faeces of pigs and poultry by capillary isotachophoresis

A method for phytic acid determination in the feeds and faeces of pigs and poultry has been developed on the basis of capillary isotachophoresis. Phytic acid was extracted by 0.95 M HCl and separated from interfering compounds by iron precipitation. Complete formation of ferric phytate required 7 mol FeCl₃ mol⁻¹ phytic acid. Residual Fe³⁺ was estimated colorimetrically by the tiron reagent, and ferric phytate was dissolved in 1.5 M NaOH at 9 mol NaOH mol⁻¹ Fe precipitated. Analyses were carried out using an electrolyte system with Cl⁻ as the leading anion, bis‐tris‐propane, and 2‐morpholinoethanesulphonic acid as the terminating anion. The recovery of phytic acid (added to hen faeces) using this procedure was 962 ± 24 g kg⁻¹. The limit of determination of phytic acid was 0.3 µmol ml⁻¹ extract. The amount of phytic acid in feeds ranged from 8.3 to 10.8 g kg⁻¹ on a dry matter basis. Phytic acid P represented 112 g kg⁻¹ total P in faeces of young pigs (40–60 kg) fed a feed with supplemental phytase (490 U kg⁻¹), 153 g kg⁻¹ total P in faeces of finishing pigs and 185 g kg⁻¹ total P in faeces of non‐lactating sows. Excreta of laying hens contained 23.7 g phytic acid kg⁻¹ dry matter (362 g kg⁻¹ total P). The isotachophoretic method is sufficiently simple and reproducible to be used for routine analyses of feeds and faeces. © 2000 Society of Chemical Industry     

The Behavior and Stability of Iron-Ascorbate Complexes in Solution

The thermodynamic stability constants for ferric and ferrous ascorbate complexes, change in net redox potential with pH, and efficacy of bathophenanthroline as an iron reagent in the presence of ascorbate were determined. The stability constants for the ferric and ferrous ascorbate complexes were found to be in the range 1.90 × 10³ to 2.61 × 10⁴ and 7.69 × 10^-3 to 6.95 × 10^-2, respectively. The stability of ferric ascorbate over a wide range of pH values suggests its potential use as an iron additive in foods. Redox potential decreased with an increase in pH. Bathophenanthroline analysis of these solutions for free iron proved to have limitations in the presence of ascorbate.