RATE OF NITRIC OXIDE FORMATION FROM NITRITE AS AFFECTED BY CHLORIDE ION CONCENTRATION

The role of chloride ions in nitrite reactions was assessed by measuring the rates of nitric oxide formation in a simplified system consisting of sodium nitrite, chloride, ascorbate, horse myoglobin and bovine serum albumin in acetate buffer. The addition of chloride ions accelerated the formation of nitric oxide, the rate increasing linearly with chloride ion concentration. The chloride ion effect was observed to be greater with increasingly acidic conditions. With exposure to air nitric oxide myoglobin conveted to metmyoglobin, but remained stable when held under nitrogen.     

Nitric oxide exchange in nitrosylmyoglobin

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 degrees 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 of delta H+- = 47 +/- 3 kJ.mol-1 at 25 degrees 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, delta 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.     

FORMATION OF NITRIC OXIDE MYOGLOBIN BY NICOTINAMIDE ADENINE DINUCLEOTIDES AND FLAVINS

SUMMARY –Formation of nitric oxide myoglobin was studied under anaerobic conditions. NADH (reduced nicotinamide adenine dinucleotide) and NADPH (reduced nicotinamide adenine dinucleotide phosphate) as reducing agents alone did not produce nitric oxide myoglobin from metmyoglobin and nitrite. However, in the presence of FMN (flavin mononucleotide), either NADH or NADPH readily produced nitric oxide myoglobin. FAD (flavin adenine dinucleotide) and riboflavin also were effective for the formation of nitric oxide myoglobin by NADH. In the absence of myoglobin, the NADH-FMN system did not reduce nitrite to nitric oxide. Similarly, a diaphorasemethylene blue system readily produced nitric oxide myoglobin, but in the absence of myoglobin it did not reduce nitrite to nitric oxide. On the other hand, nitric oxide myoglobin was produced from deoxymyoglobin by the action of nitrite in the absence of reducing agents under anaerobic conditions. Deoxymyoglobin can directly reduce nitrite to nitric oxide; the NADH-FMN and diaphorase-methylene blue systems may participate in the formation of nitric oxide myoglobin by means of reduction of metmyoglobin to the deoxy form.     

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.     

Formation of Nitric Oxide Myoglobin: Mechanisms of the Reaction with Various Reductants

SUMMARY– The concentration, temperature and pH dependences of the formation of nitric oxide myoglobin (NOMb) from metmyoglobin nitrite (MetMb·NO₂) were determined for nitrite and the reductants, ascorbic acid, cysteine, hydro-quinone, nicotinamide adenine dinucleotide (NADH) and glyceraldehyde. The reaction for all reductants except glyceraldehyde involves the production of a nitroso-reductant intermediate which breaks down to release nitric oxide. The latter forms a nitric oxide metheme complex (Fe⁺⁺⁺) which is then reduced to the ferrous state (Fe⁺⁺). With cysteine and NADH there is a second pathway which probably involves the direct reduction of MetMb NO₂. Ascorbate and hydro-quinone form nitroso intermediates that are stabilized in alkali. The effects of oxygen, ethylenediaminetetraacetic acid and cytochrome c on the reaction were determined. Oxygen slows or inhibits the reaction, while the latter two have no effect on the reaction as studied.     

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.     

An Improved Method for Preparation of Nitric Oxide Myoglobin

We developed an improved method for preparation of bovine nitric oxide myoglobin using the following starting materials: 0.1 mM purified metmyoglobin; 0.1 mM (7 ppm) sodium nitrite; and 1.76 mM (350 ppm) sodium ascorbate. The method requires complete deoxygenation of the reacting system. NOMb prepared in this manner is a source of cured meat pigment which contains a minimum of impurities, since the nitrite reacts quantitatively with the myoglobin in this method.     

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。     

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.     

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.     

Nonenzymic browning reactions in boiled grape juice and its models during storage

Reaction orders, rate constants and activation energies were evaluated for 5-hydroxymethyl furfural (HMF) accumulation and brown pigment formation (BPF) in pekmez and its model systems stored at 55, 65 and 75°C over 10 days at pH 4.0. The model systems and their compositions were decided by considering the major pekmez components. Accumulation of HMF and BPF were the highest in pekmez and they were the smallest in model 1. ANOVA showed that the effects of temperature on both accumulation of HMF and BPF were significant (p<0.05). Results obtained from non-linear regression analysis indicated that reaction order was 0.5 for 5-HMF accumulation and was zero for brown pigment formation in the model systems. Reaction orders were zero for both HMF accumulation and BPF in pekmez. Calculated activation energies for HMF and brown pigment formation were in the range of 49.7–103 kJ mol⁻¹ and 116–132 kJ mol⁻¹ respectively.     

Capacity of skeletal muscle for the formation of nitrosylhaemoproteins during the curing of meat

The effects of total haem pigment content of pork muscle on the potential for the formation of nitric oxide pigment during curing are reported. A linear relationship between the amount of endogenous pigment present and yield of nitric oxide pigment was found. Although the conversion to nitric oxide pigment was far from complete, the reducing capacity of muscle was more than adequate to convert all the haemoprotein present to nitric oxide pigment. The role of myoglobin and haemoglobin in these conversions is discussed.     

Thermodynamic and Kinetic Stability Constants of Selected Carboxylic Acids and Iron

Thermodynamic stability constants for complexes of lactic, malic, succinic and citric acids with both ferrous and ferric iron were determined. The rate of transfer of iron to apotransferrin, which has been defined as the kinetic stability constant, was also determined for both these and ascorbic acid complexes. The thermodynamic stability constants for malate and succinate were similar to ascorbate while that for lactate was somewhat lower. The kinetic stability constants in ascending order were as follows: lactate, ascorbate and malate, succinate and citrate. However, data from this study indicated that the slope of the Initial portion of the iron exchange curves might be a better measure of potential bioavailability. In descending order the slopes were: ascorbate, lactate, malate, citrate, and succinate.     

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.

     

EFFECT OF MYOGLOBIN ON THE MUSCLE LIPASE SYSTEM

The effect of different concentrations of myoglobin on the enzyme systems involved in lipid degradation were determined. Lipases, including both acid and neutral lipases and acid phospholipase, were inhibited by myoglobin. The neutral lipase was more susceptible to the inhibitory effect of myoglobin (35% of its initial activity at 1 mg/mL of myoglobin), followed closely by acid phospholipase; acid lipase activity was affected to a lesser extent (less than 20% inhibition). This inhibition was also observed on the esterase activities, being greater on the neutral esterase activity (38% of its initial activity at 1 mg/mL of myoglobin) than on the acid one. In view of these results, myoglobin could be considered as an endogenous inhibitor which would determine the lipolytic activity in different type of muscles and therefore, the rate of generation of the free fatty acids.     

Preparation of the Cooked Cured-Meat Pigment, Dinitrosyl Ferrohemochrome, from Hemin and Nitric Oxide

The cooked cured-meat pigment dinitrosyl ferrohemochrome was synthesized from hemin, an iron (III) porphyrin, and gaseous nitric oxide in buffered solutions. Reductants used were ascorbic acid, isoascorbic acid, sodium ascorbate, and sodium dithionite. The purity of the pigment so obtained was better than 97%. Sodium ascorbate was the most effective reducing agent. Application of this pigment to meats reproduced the characteristic color of nitritecured products on thermal processing.     

Lability and Reactivity of Nonheme Protein-Bound Nitrite

Nitrite treated nonheme protein was separated from the free nitrite of the reaction mixture by Sephadex G-15 column chromatography. Spectral analysis (absorbance at 330 nm) indicated that tryptophyl residues of the protein had been modified with the NO group. The protein-nitrite complex was not stable, decomposing faster at lower pH and higher temperature. As the extent of the decomposition increased with time, an increasing amount of nitrite was detected by the AOAC method, indicating that the Griess reagent reacted with nitrite after it was regenerated from the NO group. When myoglobin was incubated with the nitrosated protein, nitrosyl hemochrome could be extracted from the reaction mixture in the presence of ascorbic acid. The results indicate the potential reversibility of the protein-nitrite reaction.     

Kinetics of the Thermal Degradation of Patulin in the Presence of Ascorbic Acid

Degradation of the mycotoxin patulin between 25 and 85 °C without and with added ascorbic acid was studied, and the effectiveness of linear and nonlinear models for predicting reaction rates was compared. In agreement with previous reports, ascorbic acid significantly increased (P ≤ 0.05) the rate of patulin degradation at all temperatures studied. The data for patulin degradation in the absence of ascorbic acid were adequately modeled using a zero‐order linear kinetic model. However, the predictive abilities of zero and higher‐order linear models were not adequate to describe the more complex reactions that likely occurred when ascorbic acid was added. In contrast, the nonlinear Weibull model adequately described the patulin‐ascorbic acid reaction throughout the temperature range studied. Zero‐order rate constants and Weibull scale values for each of the respective reactions followed the Arrhenius law. Activation energies of 58.7 ± 3.9 and 29.6 ± 1.9 kJ mol^?1 for the reaction without and with ascorbic acid, respectively, confirmed decreased patulin stability in the presence of ascorbic acid and suggested that the mechanisms for the 2 degradation reactions were different.     

Conversion of Metmyoglobin to Bright Red Myoglobin Derivatives by Chromobacterium violaceum, Kurthia sp., and Lactobacillus fermenturn JCM1173

Lactic: acid bacteria (LAB) and various bacteria isolated from the environment were screened on microbiological media for the ability to convert brown metmyoglobin to more desirable bright red derivatives. Of 1,550 environmental isolates tested, two isolates (Kurthia sp. K-22 and Chromobacterium violaceum K-28) were recovered which consistently converted metmyoglobin to more desirable color derivatives as characterized spectrophotometrically. Strains K-22 and K-28 produced oxymyoglobin and nitric oxide myoglobin, respectively. Of 347 LAB tested, one strain (Lactobacillus fermentum JCM1173) was identified that generated nitric oxide myoglobin. These data indicate certain bacteria can effectively convert metmyoglobin to more red forms, and establish the potential of bacterial systems for preserving or improving rhe color of meat products.     

Kinetics of anthocyanin degradation and polymeric colour formation in black carrot juice concentrates during storage

The changes in anthocyanins (ACNs) and polymeric colour of black carrot juice concentrate (BCJC) samples were monitored during storage at ?23, 5 and 20 °C for 319 days and at 30 °C for 53 days. While ACN degradation was fitted to a first‐order reaction model, polymeric colour formation was fitted to a zero‐order reaction model during the storage. Half‐life periods for ACN degradation in BCJCs were 603, 137 and 29 days at 5, 20 and 30 °C, respectively. The reaction rate constants for polymeric colour formation were 0.0207, 0.1435 and 0.5581%/days at 5, 20 and 30 °C, respectively. HPLC‐MS analyses of BCJC showed that cyanidin‐3‐galactoside‐xyloside‐glucoside‐ferulic acid (56%) was the major ACN, followed by cyanidin‐3‐galactoside‐xyloside (19%) and cyanidin‐3‐galactoside‐xyloside‐glucoside‐sinapic acid (10%). Cyanidin‐3‐galactoside‐xyloside‐glucoside‐ferulic acid was the most stable ACN in BCJC at storage temperatures. BCJCs should be kept at sub‐freezing temperatures to minimise ACN degradation.