Nitric oxide exchange in nitrosylmyoglobin

Summary The exchange of nitric oxide in nitrosylmyoglobin, the heme pigment of nitrite-cured meat, has been studied using nitrogen-15 labelling in aqueous solution under conditions (pH, concentration of ascorbate and nitrite) similar to those prevailing in meat during the curing process, and has been found to have a half-life of approximately 2 h at 40° C. One nitric oxide molecule is coordinated to the iron(II) centre of a myoglobin molecule and, in weakly acidic aqueous solution under anaerobic conditions, the exchange rate of the bound nitric oxide is proportional to the concentration of nitrosylmyoglobin, nitrite and hydrogen ion. The rate of exchange has a moderate temperature dependence, corresponding to an activation barrier ofH =47±3 kJ·mol^–1 at 25° C and pH 5.9, a value dramatically lower than that found for the enthalpy of activation for the oxidation of nitrosylmyoglobin by molecular oxygen,H =110 kJ·mol^–1. The difference in temperature dependence between the exchange and the autoxidation is discussed in relation to the function of nitrosylmyoglobin as antioxidant in cured meat products.

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Stickoxidaustausch in Stickoxidmyoglobin

Zusammenfassung Der Stickoxidaustausch im Häm-Farbstoff Stickoxidmyoglobin in Nitrit-gesalzenem Schinken wurde unter Verwendung einer Stickstoff-15-Markierung untersucht, und zwar in wäßriger Lösung und unter Verhältnissen (pH, Konzentration von Ascorbat und Nitrit), welche den Verhältnissen während des Einsalzens im Schinken ähnlich sind. Die Halbwertszeit des Austausches wurde bei 40 °C bei ungefähr 2 h festgestellt. Ein Stickoxid ist dem Eisen (II) des Myoglobins koordiniert; in schwach saurer Lösung und bei Sauerstofffreiheit ist die Austauschgeschwindigkeit des gebundenen Stickoxids proportional zur Konzentration von Stickoxidmyoglobin, Nitrit und Wasserstoffion. Die Austauschgeschwindigkeit ist etwas temperaturabhängig und entspricht einer Aktivierungsbarriere vonH =47±3 kJ·mol^–1 bei 25 °C und pH 5,9. Dieser Wert ist signifikant niedriger als der für die Aktivierungsenthalpie für Stickoxidmyoglobin-Oxydation durch molekularen Sauerstoff,H =110 kJ·mol^–1, festgestellte Wert. Der temperaturabhängige Unterschied zwischen Austausch und Autoxidation wird im Verhältnis zur Funktion des Stickoxidmyoglobins als Antioxidant in Schinken und ähnlichen Produkten diskutiert.

     

Nitric Oxide Complex of Iron(II) Myoglobin Converted from Metmyoglobin by Staphylococcus xylosus

With Staphylococcus xylosus FAX-1, metmyoglobin in MRS broth (pH 5.8) was found to undergo conversion to hexacoordinate nitric oxide (NO) complex of Fe(II) myoglobin. When the pH of the MRS culture containing myoglobin changed from 5.8 to 4.0, it affected the conversion from hexacoordinate to pentacoordinate NO complex of Fe(II) myoglobin. This conversion process was reversible. Salami without nitrite or nitrate addition was prepared by inoculating S. xylosus FAX-1, and pentacoordinate NO complex of Fe(II) myoglobin (nitrosylmyoglobin formed in cured meat) was formed in the salami.     

添加葡萄球菌和微球菌对广式腊肠亚硝酸盐残留量和色泽研究

本文旨在研究葡萄球菌和微球菌对广式腊肠亚硝酸盐残留量和色泽的影响及其机制。首先将分离筛选自广式腊肠的两株优良特性菌株(葡萄球菌H33B和微球菌X142B)接种至腊肠测定相关指标,然后通过紫外扫描图谱来确定菌株是否具有转化高铁肌红蛋白能力。结果表明,接种单菌和混合菌株都能够降低腊肠中的高铁肌红蛋白含量和亚硝酸盐残留量,并且能够增加亚硝基肌红蛋白含量,其中以接种葡萄球菌和微球菌2:1时效果最好,与对照组差异明显(p0.05);紫外扫描图谱显示接种葡萄球菌的培养基中溶液出现了亚硝基肌红蛋白的特征吸收峰,并且溶液中的亚硝酸盐含量最低。这些结果表明葡萄球菌H33B具有转化高铁肌红蛋白的能力。因此添加葡萄球菌H33B的腊肠,由于其具有转化高铁肌红蛋白的能力,会形成更多的还原性肌红蛋白与亚硝酸盐反应,不仅进一步减少了亚硝酸盐含量还改善了色泽。     

KINETIC ANALYLSIS OF THE FORMATION OF NITROSYLMYOGLOBIN

The formation of nitric oxide myoglobin (nitrosylmyoglobin) was followed in buffered solutions in which the concentrations of ascorbate, nitrite, chloride, myoglobin and hydrogen ion were varied systematically to determine their effect on the rate constants. The rate of formation of nitrosylmyoglobin was zero order with respect to the pigment. The orders for the other reactants were determined by plotting the zero order rate constants as functions of varying orders of each reagent to determine which order gave a linear plot. The results were used to develop a mechanism and a mathematical expression for the reaction. Two reaction sequences involving different nitrosating species were involved; 1, direct action of nitrous acid and 2, the formation of nitrosyl chloride. Both species then nitrosated ascorbate and ascorbic acid, by different mechanisms. The nitric oxide for nitrosylmyoglobin formation came from the nitrosated ascorbate.     

Quantum chemical calculations of sulfate adsorption at the Al- and Fe-(hydr)oxide-H20 interface-estimation of gibbs free energies

Quantum chemical calculations were performed to estimate relative Gibbs free energies of sulfate adsorption on variably charged Al- and Fe-(hydr)oxide clusters. Innersphere bidentate bridging and monodentate adsorption were predicted to be exergonic on positively charged Al- and Fe-(hydr)oxides (ranging from -19 to -124 kJ mol-1). However, inner-sphere and H-bonded adsorption on neutral Al- and Fe-(hydr)oxides was predicted to be endergonic (ranging from +5 to +61( kJ mol-1)). Atthe highest positive surface charge, bidentate bridging adsorption was most thermodynamically favorable. At intermediate positive surface charge, bidentate bridging and monodentate adsorption energies were equivalent on Al-(hydr)oxides; monodentate adsorption was more thermodynamically favorable on Fe-(hydr)oxides as compared with bidentate bridging adsorption. The predicted thermodynamic favorability of sulfate adsorption on Al- and Fe-(hydr)oxides was directly related to positive surface charge and indirectly related to the HO-/SO42- exchange stoichiometry, chi. Predicted Gibbs free energies of bidentate bridging and monodentate sulfate adsorption on an Fe-(hydr)oxide cluster (charge = +1, chi = 1) agreed reasonably well with published experimental estimates of sulfate adsorption on geothite (predicted values -34 and -52 kJ mol-1, respectively, and experimental range -36 to -30 kJ mol-').     

Formation and identification of nitrosylmyoglobin by Staphylococcus xylosus in raw meat batters: A potential solution for nitrite substitution in meat products

Staphylococcus xylosus and Pediococcus pentosaceus isolated from Chinese dried sausage were assessed for their ability to convert metmyoglobin into nitrosylmyoglobin in Mann–Rogosa–Sharp broth model systems and raw pork meat batters without the addition of nitrite. The results showed that samples in model systems with S. xylosus cultures had an absorption spectra that is typical of nitrosylmyoglobin, an obvious pink colour (judged by visual inspection) and a significantly higher a*-value than the control samples or samples inoculated with P. pentosaceus. In raw meat batters, the a*-values of the S. xylosus samples were almost the same as those for the meat with nitrite added. The complementary analysis of meat batter samples by photochemical information from UV–vis, electron spin resonance and resonance Raman spectroscopy revealed that the existing status of the myoglobin in meat batters inoculated with S. xylosus was mainly pentacoordinate nitrosylmyoglobin. This study provides a potential solution for nitrite substitute in meat products.     

The effects of lactate on nitrosylmyoglobin formation from nitrite and metmyoglobin in a cured meat system

Two experiments were conducted to determine the effects of lactate on nitrite during meat curing. In the first experiment, using a model system, eight reaction components including nitrite and lactate, were used to assess the effect of each component on metmyoglobin reducing activity by excluding one component at a time. Excluding lactate, nicotinamide adenine dinucleotide (NAD), l-lactate dehydrogenase (LDH) or phenazine methosulfate (PMS) resulted in no reducing activity. A second experiment, utilising a meat mixture, investigated the effects of lactate (0%, 2%, 4% or 6%), nitrite (0 or 156 ppm), and packaging (oxygen-permeable or vacuum) on residual nitrite, meat colour and pH. Addition of lactate reduced residual nitrite in the meat mixtures. Both experiments support the hypothesis that lactate generates NADH which then reduces metmyoglobin to deoxymyoglobin. The resulting greater concentration of reduced myoglobin subsequently reacted with nitrite to produce more nitric oxide, reducing nitrite concentration and accelerating curing reactions.     

Relationship between nitrate/nitrite reductase activities in meat associated staphylococci and nitrosylmyoglobin formation in a cured meat model system

Quantitative determination of catalase, nitrate reductase, nitrite reductase and nitric oxide synthase activities (NOS) was performed on 11 different bacterial strains, mainly staphylococci, isolated from fermented sausages, bacon brine or cured meat products. All except one strain possessed catalase activity in the range from 1.0 to 6.1 μmol min^− 1 ml^− 1. Ten out of 11 bacteria strains showed nitrate reductase activity in the range between 50 and 796 nmol min^− 1 ml^− 1 and nine showed nitrite reductase activity in the range between 6 and 42 nmol min^− 1 ml^− 1. No evidence of NOS activity of the selected strains was detected. In a colour formation assay containing myoglobin all strains affected nitrosylmyoglobin (MbFe^IINO) formation in assays containing nitrite, whereas only strains having nitrate reductase activity generated MbFe^IINO in assays containing nitrate as the sole nitrosylating agent. The quantitative nitrate and nitrite reductase activity did not fully explain or correlate well with the observed rate of formation of MbFe^IINO, which seemed to be more affected by the growth rate of the different strains. The mechanism of the reduction of nitrite into NO of strains not having nitrite reductase activity remains to be fully elucidated, but could be due to a dual-mode action of nitrate reductase capable of acting on nitrate.     

Production of cured meat color in nitrite-free Harbin red sausage by Lactobacillus fermentum fermentation

Lactobacillus fermentum was substituted for nitrite to produce cured pink color in a Chinese-style sausage. Treatments included inoculations (10⁴, 10⁶, and 10⁸ CFU/g meat) followed by fermentation at 30 °C for 8 h and then at 4 °C for 16 h. Control sausage (with sodium nitrite, 60 mg/kg meat) was cured at 4 °C for 24 h without L. fermentum. The UV–Vis spectra of pigment extract from L. fermentum-treated sausage were identical to that of nitrosylmyoglobin (NO-Mb) formed in nitrite-treated control. The NO-Mb concentration and the colorimetric a^* value of sausage treated with 10⁸ CFU/g meat of L. fermentum essentially replicated those in nitrite-cured meat. Free amino acid content in sausage treated with L. fermentum was greater and the pH slightly lower compared with the nitrite-cured control sample. This study showed that L. fermentum has the potential to substitute for nitrite in the sausage production.     

Effect of pH and temperature on the thermostability of prickly pear (Opuntia ficus-indica) yellow-orange pigments

The colour stability of the yellow-orange pigment (lambda max = 476 nm) of prickly pear (Opuntia ficus-indica) fruit was determined as a function of temperature and pH. The experiments were carried out at three different temperatures (50, 70 and 90 degrees C) with pigment solutions at pH values ranging from 2-7. The degree of pigment retention decreased with increasing temperature as a function of increasing thermal exposure time with least pigment degradation at pH 5. The reaction rate constants were determined as 0.0062, 0.0383 and 0.1102 min-1 for a thermal degradation reaction rate of pseudo-first order. The activation energy was calculated as 65.1 kJ.mol-1.     

Effect of Iron Form, Temperature, and Inoculation with Clostridium botulinum Spores on Residual Nitrite in Meat and Model Systems

The effect of iron form (ferrous, ferric, heme), temperature and botulinal spores on nitrite level was determined in meat. In model systems, ferritin iron was also included, and ascorbate was used as a reducing agent. Reduced hemoglobin caused the most rapid nitrite depletion in both systems. Ferrous iron caused faster nitrite depletion in model systems than in meat. Ferrous iron reduced nitrite readily in model systems at 27°C, but not at 5°C. Ferritin iron did not affect nitrite level. In meat at 27°C, nitrite depletion was much faster in inoculated samples. Protein-bound nitrite levels were higher in meat with added ionic iron. In cured meat with added ionic iron, iron-NO-protein complexes may form, lowering the amount of nitric oxide (NO) available to inhibit botulinal spore outgrowth.     

Microbial formation of nitrite-cured pigment,nitrosylmyoglobin, from metmyoglobin in model systems and smoked fermented sausages by<Emphasis Type="Italic"> Lactobacillus fermentum</Emphasis> strains and a commercial starter culture

Two Lactobacillus fermentum strains (JCM1173 and IFO3956) were evaluated for their ability to generate nitrosylated derivatives of myoglobin either in broth media or fermented sausages. For comparison, a commercial starter culture was also included. All bacteria species investigated converted brown metmyoglobin into red myoglobin derivatives when incubated separately in broth, but only the two lactobacilli showed a signal for nitrosylmyoglobin as measured by electron spin resonance spectroscopy. In smoked sausages with added bacteria culture the highest amount of nitrosylmyoglobin was observed in the centre of sausage with added L. fermentum, but colour formation in sausages with 60 ppm of nitrite added was more pronounced. An outer peripheral zone of all fermented sausages contained levels of nitrosylmyoglobin comparable to nitrite-cured sausages. Nitrogenous gasses from smoke may, however, cause this zone to be formed. Depending on a further optimisation of the processing parameters, the bacteria's ability to generate NO could form the basis for production of cured meat products without the use of nitrite/nitrate.     

A rapid method for the determination of nitrate and nitrite by chemiluminescence

Nitrite and nitrate + nitrite can be determined by selective chemical reduction to nitric oxide which is measured using a chemiluminescence analyser. The reducing agents are sodium iodide in acetic acid for nitrite and ferrous ammonium sulphate-ammonium molybdate for nitrate + nitrite. The concentrations of the reducing agents have been optimized to obtain the maximum yield of nitric oxide and the minimum coefficient of variation. Under these conditions, it is possible to inject repeated samples into the refluxing reducing agents and to obtain rapid evolutions of nitric oxide from which the determinations can be made. Nitric oxide has also been produced using the nitrite reagents from organic nitrites, a S-nitrosothiol, a pseudonitrole and N-nitrosamines. Similarly, an organic nitrate and some C-nitroso compounds respond to the method for nitrate but only to the extent of a yield of nitric oxide of about 10% of the theoretical. Very low or zero responses were evident from aliphatic and aromatic C-nitro compounds but not omega-N-nitroarginine which gave a large yield of nitric oxide using the reagents for nitrate. In general, however, concentrations of nitrate will be in considerable excess of those of related compounds which would interfere with the determinations. Nitrate can be determined either by difference in its mixtures with nitrite or by prior removal of the nitrite using ascorbic acid provided oxygen and nitric oxide are removed by degassing with nitrogen.     

Mitochondrial enzyme pathways and their possible role during curing.

The velocity of oxidation of exogenous ferrocytochrome c by nitrite under anaerobic conditions in the presence of skeletal muscle mitochondria is dependent upon pH over at least the range 5.6-6.7, increasing markedly as the pH is lowered. A product of the reaction is the complex formed between nitric oxide and ferricytochrome c. At levels up to 20 mM, nitrite inhibits aerobic cytochrome oxidase action; at higher concentrations, however, a partial resuscitation of the oxidation of ferrocytochrome c occurs, the enhancement of reaction velocity being considerably greater at pH 6.0 than at 6.5. Mitochondrial respiration is also inhibited by nitrite but no similar resurgence was, however, observed and thus the oxidation of ferrocytochrome c by high levels of nitrite is considered to be a direct non-enzymic action. Under anaerobic conditions, the rate of increase of the velocity constant of the oxidation of ferrocytochrome c with nitrite concentration in the presence of muscle mitochondria similarly decreased with rise of pH over the same range. The permeability of the muscle mitochondrion to nitrire has been demonstrated by swelling studies and by the rapid conversion of endogenous ferrocytochrome a3 into its nitrosyl-derivative. Over longer periods of anaerobic incubations of mitochondria with nitrite, oxidation of endogenous cytochromes occurs with the formation of nitrosylferricytochrome c. Above a nitrite concentration of 0.3 mM, the mitochondrial enzyme system probably involved is increasingly inhibited but by a concentration of 30 mM a direct non-enzymic oxidation has intervened. Commercial vacuum packed bacons were examined by electron microscopy. Mitochondria were clearly recognisable although they contained fewer cristae than those observed in fresh meat.     

Oxidation of ferrocyanide by birnessite

The Fe-CN complexes ferrocyanide, [FeII(CN)6]4-, and ferricyanide, [FeIII(CN)6]3-, which are contaminants in soil and groundwater, form a redox couple, [FeII(CN)6]4- <==> [FeIII(CN)6]3- + e-, E(H) = 356 mV. We studied the oxidation of [FeII(CN)6]4- by birnessite, delta-MnIVO2, in batch experiments as influenced by [FeII(CN)6]4- concentration, pH, and reaction time. Additionally, stopped-flow experiments were carried out at five temperatures (10-30 degrees C) and four pH values (pH 4.1-5.3). In the batch experiments, [FeII(CN)6]4- was completely oxidized to [FeIII(CN)6]3-, and oxidation did neither depend on time for t > 2 min, nor on concentration (0.12-0.47 mM), nor on pH (pH 3.3-9.9). Lasting adsorption of Fe-CN complexes on the birnessite surface or precipitation of manganese ferricyanide were not detected. Manganous ions resulting from the reductive dissolution of birnessite did not precipitate as manganese oxide because an identical decrease of Mn solution concentrations was observed under air and under a N2 atmosphere. Two processes were detected by the stopped-flow experiments. The first rapid one with an activation energy of approximately 60 kJ mol(-1) was attributed to short-term adsorption and simultaneous oxidation of [FeII(CN)6]4- on the birnessite surface. The second slower process with an activation energy of approximately 20 kJ mol(-1) was attributed most probably to diffusion of the reaction product Mn2+ into the interior of the birnessite, which creates fresh reaction sites at the outer surface.     

Contribution of nitrite and nitrate to the colour and flavour of cured meats

The formation of nitric oxide myoglobin from nitrite and myoglobin involves a complex series of reactions not all of which are completely understood even now, and the stability of the cured colour, so important from the marketing point of view, continues to be investigated. The amount of nitrite necessary for complete formation of nitric oxide myoglobin is very small and the presence of no more than 25 mg/kg of nitrite in the cured meat is enough to ensure an adequately stable colour. At least four times this level is essential to produce a full development of the typical cured flavour. Very little is known of the mechanism of the reactions leading to the formation of cured flavours in cooked products or of the identity of the volatile substances responsible for it.     

Effect of stress induced by suboptimal growth factors on survival of Escherichia coli O157:H7.

This study investigated the growth and survival of E. coli O157:H7 exposed to a combination of suboptimal factors (22 degrees C, 7 degrees C, -18 degrees C/0.5% NaCl, 5.0% NaCl/pH 7.0, pH 5.4, pH 4.5/addition of lactic acid) in a simulation medium for red meat (beef gravy). Prolonged survival was noted as the imposed stress was more severe, and as multiple growth factors became suboptimal. At a defined temperature (7 degrees C or -18 degrees C), survival was prolonged at the more acid, more suboptimal pH (pH 4.5 > pH 5.4 > pH 7.0) while at a defined pH (pH 4.5), better survival was observed at 7 degrees C than at 22 degrees C. This suggests that application of the hurdle concept for preservation of food may inhibit outgrowth but induce prolonged survival of E. coli O157:H7 in minimal processed foods. At both 22 degrees C and 7 degrees C, the addition of lactic acid instead of HCl to reduce pH (to pH 4.5) resulted in a more rapid decrease of E. coli O157:H7. High survival was observed in beef gravy, pH 5.4 at -18 degrees C (simulation of frozen meat)-reduction of log 3.0 to log 1.9 after 43 days--and in beef gravy, pH 4.5 and 5% NaCl at 7 degrees C (simulation of a fermented dried meat product kept in refrigeration)--less than 1 log reduction in 43 days. In these circumstances, however, a high degree of sublethal damage of the bacterial cells was noted. The degree of sublethal damage can be estimated from the difference in recovery of the pathogen on the non-selective TSA medium and the selective SMAC medium.     

Fluorocytosine causes uncoupled dissipation of the proton gradient and behaves as an imperfect substrate of the yeast cytosine permease.

At pH 5-6 ATP-depleted washed cell preparations of strain NC233-10b[pII4-9], in which the cytosine permease was overexpressed, absorbed cytosine, hypoxanthine or fluorocytosine stoichiometrically with, respectively, about 1, 1.4 and 5 proton equivalents. The cellular pH fell proportionately. The membrane depolarization caused by each compound was assayed in the presence of glucose with a voltage-sensitive dye and increased in the same order. Fluorocytosine significantly lowered the growth yield that a 'petite' strain of the yeast formed at limiting glucose concentrations. At pH 5.6 with extracellular [K+] below 1 mM, each of the three substrates was accumulated about 200-fold from a dilute solution at the expense of the proton gradient. This concentration ratio corresponds to a solute gradient (delta mu(s)) of 13 kJ mol-1. Raising [K+]o systematically lowered the substrate accumulation ratio and delta muH. The mean ratio delta mu(s)/delta muH was 0.82 for all three substrates. It was concluded that whereas the behaviour of cytosine approximated to that expected for a symport of unit proton stoichiometry, the absorption of protons with fluorocytosine and, to a lesser extent, hypoxanthine, was only partly conserved as useful work. A possible mechanism of this novel phenomenon is outlined.     

Isolation and characterization of heat stable proteinases from Pseudomonas isolate AFT 21

Pseudomonas strain AFT 21 produced three heat stable extracellular proteinases in milk and nutrient broth at 7 or 21 degrees C, but the proportions depended on medium and cultivation temperature. The three proteinases were EDTA- and o-phenanthroline-sensitive metalloenzymes and were not inhibited by N-ethylmaleimide or phosphoramidon. Proteinases I and II showed maximum activity at pH 7-7.5 and proteinase III at pH 8.5. All three enzymes showed maximum activity at 45-47.5 degrees C, but had relatively high (19-27% of maximum) activity at 4 degrees C. They were unstable at 55 degrees C in phosphate buffer, pH 6.6, or synthetic milk ultrafiltrate (SMUF) containing 12 mmol Ca2+, but were stabilized by short preheating at 100 degrees C. They were extremely heat stable in both phosphate buffer and SMUF, pH 6.6, at 70-150 degrees C. Their D-values at 140 degrees C were 69, 54 and 80 s respectively. The Z-values for Pseudomonas AFT 21 proteinase III in phosphate buffer and SMUF were 29.7 and 30.3 degrees C respectively; the corresponding activation energies for inactivation were 8.7 x 10(4) J mol-1 and 9.2 X 10(4) J mol-1.     

High-pressure effects on oxidation of nitrosylmyoglobin

The rate of oxidation of nitrosylmyoglobin by oxygen decreases with increasing hydrostatic pressure. At 15 °C in air-saturated solution with ionic strength 0.16 and pH 6.8 (tris-buffer), the first-order rate constant is smaller by a factor of 5 at 300 MPa compared to ambient conditions. The pressure-effect on rate is not primarily caused by protein denaturation, as the presence of urea (up to 4 M) at ambient pressure increases the rate of oxidation. From rate/pressure data a volume of activation of +8 ml·mol(-1) and a compressibility coefficient of activation of -3 × 10(-8) ml·mol(-1) Pa(-1) is estimated.