Nitrite acts as a colorant flavorant antioxidant and antibotulinal agent in cured meat

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In ancient times, meat was preserved with saline desert sands and sea salts, both of which contain nitrate [5]. The reddening effect of nitrate in preserved meat was mentioned as far back as the late Roman era, but it wasn't until the early twentieth century that the bacterial reduction product, nitrite, was identified as the agent responsible for coloring and curing meat [5]. In meat curing, nitrate functions as a reservoir for nitrite, which acts not only to color meat, but also acts as a flavorant, an antioxidant and an anti-microbial agent [14]. These days, it is generally accepted that most of these effects are due to the action of NO, which is generated by the reduction of nitrite [15]. However, in recent years the use of nitrite has faced considerable negative pressure, because its reaction with amines in meat has been shown to produce nitrosamines, which are known carcinogens and possibly mutagens [16-18].

The characteristic red pigment in cured meat has been attributed to nitrosylmyoglobin, which is formed by the reaction of muscle tissue myoglobin with nitrites in the curing agent [18,19]. However, although nitrite and/or NO are generally accepted as playing a crucial role in this process, the reaction mechanism of myoglobin with nitrite and/or the reduction mechanism of nitrite to NO in cured meat remain under some debate. In fresh minces, nitrite has been proposed to be enzymatically reduced to NO by the cell respiratory system [20]. In addition, various reductants that are present in or added to fresh meat (e.g. ascorbate and cysteine) can reduce nitrite to NO via non-enzymatic routes [21]. In cell-free systems, the addition of nitrite to oxyhemoglobin has been demonstrated to transform proteins to their met-forms [22], indicating that nitrites can be considered causative agents for methemoglobinemia, particularly in infants [23-25]. In contrast, metmyoglobin has been shown to react with excess nitrite at pH < 7 to yield a nitrimyoglobin with an unusual nitrovinyl group on its heme-side chain [26,27]. Nitrimyoglobin is a green pigment involved in the phenomenon called "nitrite burning" or "nitrite greening" in improperly cured meat. Thus, the color of nitrite-treated meat appears to be controlled by multiple factors, including the utilized agents, pH, temperature and the presence/absence of other additives.

In nitrite-cured meat, NO appears to play a crucial antioxidant role. For example, undesirable lipid oxidation in nitrite-cured meat is inhibited by S-nitrosothiol and nitrosylmyoglobin [28,29], both of which are formed via nitrite-derived NO [30]. In addition, the cysteine-containing nitrosyl iron complex, which is produced by the reaction of cysteine, iron(II) salt and NO, showed inhibitory activities against lipid peroxidation [31]. S-nitrosothiol, nitrosylmyoglobin and nitrosyl iron complex with cysteine all have reactive NO groups, are able to release an NO molecule and may undergo a transnitrosation reaction under appropriate physiological conditions [31-34]. Further, NO can undergo rapid radical-radical combination reactions with other radical species, leading to quenching of those species; thus it can act as a terminating species for radical chain reactions during lipid oxidation [35,36]. Thus, the chemical reactivity of nitrite-derived NO are largely responsible for the antioxidant activity of nitrite in cured meat.

Nitrite has also been shown to exert significant anti-botulinal effects in cured meat [37-42]. Initially, this anti-botulinal activity was thought to be due to the interaction of nitrite as nitrous acid with thiol-containing constituents of the bacterial cell [43]. However, in clostridia, nitrite was found to directly interact with pyruvate-ferredoxin oxidoreductase, an iron-sulfur cluster that contains enzymes necessary for botulinal energy production in some clostridial vegetative cells. The interaction of nitrite with pyruvate-ferredoxin oxidoreductase was found to inhibit the phosphoroclastic system in Clostridium sporogenes and Clostridium botulinum [44,45]. In terms of an action mechanism for this effect, Reddy et al. (1983) reported that iron-sulfur proteins in vegetative cells of C. botulinum reacted with nitrite to form iron-NO complexes, resulting in destruction of the iron-sulfur clusters. Inactivation of iron-sulfur enzymes (e.g. ferredoxin) by NO binding was shown to inhibit the growth of C. botulinum [7]. Additional studies identified the active iron-NO complex in this mechanism as a dinitrosyl dithiolato iron complex [46,47]. However, although iron-thiol-nitrosyl complexes are extremely inhibitory toward botulinal growth, Payne et al. (1990) reported that there was no correlation between botulinal growth inhibition and the content of iron-thiol-nitrosyl complexes, suggesting that direct inhibition and/or destruction of iron-sulfur enzymes may not be the principal basis of the anti-botulinal activity of nitrite [8,9].

Ingested nitrate may be reduced to nitrite, which plays numerous roles in the stomach

Nitrate, an essential plant nutrient, is ultimately metabolized to form plant proteins. High concentrations of nitrate are found in fresh, green, leafy vegetables such as spinach and lettuce [23], while most other non-vegetable food products contain relatively low nitrate contents. When nitrate-containing food is ingested, the nitrate is absorbed from the small intestine, and nearly a quarter of it is transported to the salivary glands and re-secreted into the mouth [48-52]. In the mouth, bacteria on the dorsum of the tongue reduce about 30% of the salivary nitrate to nitrite [48-53], and may further reduce this nitrite to NO under anaerobic conditions [51]. Under a fasting condition, the salivary nitrite concentration is approximately 50 /M [12], and this rises to as high as 2 mM after ingesting food with a high nitrate content such as green lettuce [13], as described above. When salivary nitrite enters the stomach, it is reduced to NO in the acidic media (pH 1-2) through following reactions [54]:

In the stomach, nitrite can be totally converted to nitrous acid because its pKa is lower than the pH in the gastric juice, subsequently leading to the generation of reactive nitrogen oxide species (RNOS) such as N2O3, NO and NO2 (N2O4). In addition, ascorbic acid is actively secreted within the gastric juice of the healthy stomach and is stabilized under acidic conditions [55]. Since ascorbic acid rapidly reduces nitrite to NO, salivary nitrite can be efficiently converted to NO in the stomach [56-59]. High concentrations of NO resulting from enterosalivary recirculation of dietary nitrate have been detected within the lumen of the stomach by employing a variety of methods [60-63]. NO and other RNOS derived from salivary nitrite have been demonstrated to play critical roles in the physiology of the normal stomach [64].

However, these molecules may also play pathophysiological roles. For example, salivary nitrite-derived N2O3 and NO2 may act as nitrosating agents in the stomach, reacting with a variety of dietary amines to yield N-nitrosamines, which are chemical carcinogens known to cause gastric cancer, likely via their ability to deaminate DNA bases and inactivate DNA repair enzymes [65,66]. Recently, particularly high levels of NO were reported in the gastroesophageal junction (GEJ) and cardia, where salivary nitrite first encounters gastric acid [63]. These NO molecules diffuse into the adjacent gastric tissues, increasing local glutathione consumption in the tissue [67] and enhancing the formation of DNIC [68]. These findings suggest that high levels of NO may be involved in the high prevalence of mutagenesis and neoplasia at the GEJ.

In contrast to their pathophysiological roles, salivary-derived nitrite and the resulting NO are likely to play a protective role in the stomach, guarding against ingested pathogens and maintaining gastric mucosal integrity by improving mucosal blood flow and mucus secretion [69]. Although nitrite has limited anti-microbial activity at neutral pH, this activity is profoundly enhanced in acidic media such as gastric juice. Acidified nitrite exhibits strong bactericidal activity against Candida albicans, Salmonella enteritidis, Salmonella typhimurium, Yersinia enterocolitica, Shigella sonney, Escherichia coli O157:H7 [61,70] and E. coli CM120 [71]. In contrast, Helicobacter pylori and five lactobacilli species have been shown to be relatively resistant to acidified nitrite [71,72]. The anti-microbial activity of acidified nitrite appears to be influenced by many local environmental factors, including the presence of ascorbic acid, thiocyanate and chloride, the oxygen concentration and the culture medium [64,71]. In the stomach, other constituents of gastric juice such as amino acids, peptides and proteins are also likely to affect the level of anti-microbial activity. Although the antimicrobial mechanisms of salivary-derived nitrite are not well understood, several studies have suggested the involvement of RNOS arising from nitrite under acidic conditions [69,71,73].

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