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.     

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.     

Cured colour development during sausage processing

A study of colour evolution in dry cured sausage manufactured using industrial technology was made. Parameters which define changes related to nitrosation during curing were determined. The main changes in the colour characteristics of Spanish sausage took place during the fermentation stage. pH, nitrate and nitrite concentration, pigment nitrosation index, pigment discoloration index, a(?), b(?), C(?) and H(?) values decreased during this stage. However, the nitrosation of the myoglobin pigment continued during the whole curing process. The percent conversion of total pigments to the cured nitric oxide heme pigment form was about 70% in the minced mix, and it increased gradually to about 90% in the final product. During fermentation nitrites reacted with myoglobin (Mb) to form nitrosomyoglobin (NOMb) and metmyoglobin (MetMb), which reduced to NOMb during the drying process.     

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.     

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.     

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.     

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.     

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.     

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: 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.     

Nitric oxide inhibits the formation of zinc protoporphyrin IX and protoporphyrin IX

The aim of this study was to elucidate the mechanism by which curing agents, especially nitrite, inhibit the formation of zinc protoporphyrin IX (ZPP) in dry-cured hams such as Parma ham. The oxidation–reduction potential of model solutions was increased by the addition of nitrite, but it was not clear whether the formation of ZPP is inhibited by the oxidizing property of nitrite. The effect of nitric oxide (NO) produced from nitrite on the formation of ZPP was examined. The amount of ZPP formed was decreased by the addition of NO donors. The amount of protoporphyrin IX (PPIX), which is the precursor of ZPP, was also decreased by the addition of NO donors. It is concluded that NO produced from nitrite inhibited the formation of PPIX and ZPP was therefore not formed in cured meat products with the addition of nitrite or nitrate.     

Citrus Co-Products as Technological Strategy to Reduce Residual Nitrite Content in Meat Products

ABSTRACT:  Sodium or potassium nitrite is widely used as a curing agent in cured meat products because it inhibits outgrowth and neurotoxin formation by  Clostridium botulinum , delays the development of oxidative rancidity, develops the characteristic flavor of cured meats, and reacts with myoglobin and stabilizes the red meat color. As soon as nitrite is added in the meat formulation, it starts to disappear and the nitrite that has not reacted with myoglobin and it is available corresponds to residual nitrite level. Health concerns relating to the use of nitrates and nitrites in cured meats (cooked and dry cured) trend toward decreased usage to alleviate the potential risk to the consumers from formation of carcinogenic compounds. Recently, some new ingredients principally agro-industrial co-products in general and those from the citrus industry in particular (albedo [with different treatments], dietetic fiber obtained from the whole co-product, and washing water used in the process to obtain the dietetic fiber) are seen as good sources of bio-compounds that may help to reduce the residual nitrite level in meat products. From these co-products, citrus fiber shows the highest potential to reduce the residual nitrite level, followed by the albedo and finally the washing water. The aim of this article is to describe the latest advances concerning the use of citrus co-products in meat products as a potential ingredient to reduce the nitrite level.     

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.     

Prevention of Clostridium outgrowth in heated and hermetically sealed meat products by nitrite – a review

This paper discusses several hypotheses regarding the inhibition of Clostridium in cured meat. It focuses on the binding of iron, as there is some evidence that inhibition of Clostridium botulinum outgrowth in nitrite-cured meat products is mainly due to iron binding in such a way that it is no longer available for outgrowth of Clostridium spores. This strong binding also explains the antioxidative properties of nitrite in these products. The binding of iron, in all probability, requires no more than a moderate amount of nitrite which, after binding as nitric oxide to the haem group, is unavailable for reactions leading to N-nitroso compounds either. Other reactions of nitrite, which also could lead to Clostridium inhibition, are not discussed in detail here.     

A review of nitrite and chloride chemistry: Interactions and implications for cured meats

Concerns for the health-related implications of sodium nitrite and sodium chloride in cured meats have lead to reductions in use of both ingredients and there is evidence of chemical interaction between nitrite and chloride in food systems which have implications for cured meats. This review considers the role of chloride in nitrite chemistry and extends this consideration to evaluation of potential changes in cured meat products. Microbiological control depends on nitrite reactivity and a chloride effect has been demonstrated in Clostridium botulinum spore outgrowth and toxin formation. Changes in flavour of cured meats or in development of cured colour are of lesser concern but may take place. More information on specific inhibitory mechanisms by nitrite is needed.     

Kinetics of nitrite evaluated in a meat product

The evaluation of the efficiency with which the reactions involving nitrite proceed in mortadella and of the effect exercised on their kinetics by some variables (ingoing amount of sodium nitrite and temperature) is the purpose of this work. Kinetics parameters were calculated at each level of nitrite added (40, 70, 100 and 150 mg/kg) and at five temperature (55°, 60°, 65°, 70° and 72 °C). While the colour formation reaction is favoured by low activation energy, it becomes crucial to enable nitrite to proceed according to direct reduction thus preventing an increase in nitrate concentration as well as an excess of nitric oxide in the product. Kinetics data suggest that this scope is performed when the product achieves the temperature of 65 °C as fast as possible with an ingoing amount of sodium nitrite of 70 mg/kg.     

Zn-porphyrin formation in cured meat products: Effect of added salt and nitrite

Zn-porphyrin (Zn-pp) was quantified by fluorescence spectroscopy in the cured and dry cured meat products: Parma ham, Iberian ham, dry-cured ham with added nitrite, cooked ham with added nitrite, raw ham meat, raw bacon and Karree-Speck. The highest amount of Zn-pp was found in dry-cured Parma ham and Iberian ham, while the use of nitrite as curing agent was found to inhibit completely the formation of Zn-pp in meat products. A positive correlation between both Zn content and Fe content and the logarithmic transformed Zn-pp content (measured as fluorescence intensity I_fl) was found for the different cured and dry cured meat products, with correlation coefficients of 0.79 (p < 0.001) and 0.71 (p < 0.01), respectively. Log I_fl correlates best with the Zn content, indicating that the formation of Zn-pp is proportional to the Zn content. A model system with vacuum packed pork in brine with different added levels of sodium chloride with or without nitrite and Zn acetate was investigated in order to further elucidate the mechanism of Zn-pp formation. Zn-pp increased with time (up to 42 days investigated) in non-cured meat and for meat cured solely with NaCl lower than 9%. Addition of nitrite or Zn(II) in the curing brine was found to inhibit formation of Zn-pp confirming the observations from the various cured meat products. It is suggested that a chloride anion assisted dissociation of iron from myoglobin could be rate-determining for Zn-pp formation in meat products.     

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.     

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.     

Effect of processing of corn for production of masa,tortillas and tortilla chips on the scavenging capacity of reactive nitrogen species

The effect of processing conventional and pigmented corn into masa, tortilla and tortilla chips on the ability to scavenge nitric oxide (NO) and peroxynitrite (ONOO^?) was investigated. The level of retention of nitric oxide and peroxynitrite (mediated for inhibition of nitrite formation scavenging capacity for masa, tortilla and tortilla chips) ranged 56.2–78.2%, 67.4–45%, 40.3–62.0% (for nitric oxide) and 38.6–81.7%, 23.3–47.7%, 19.3–67.2% (for inhibition of nitrite formation), respectively. The antinitrosative activities were affected significantly (P > 0.05) by nixtamalisation, but not by processing masa into tortilla and tortilla chips (P > 0.05). The yellow variety and its corresponding products showed the greatest capacity to scavenge nitric oxide and inhibition of nitrite formation among conventional varieties and the purple variety ranked highest among the pigmented varieties.