The Fading Colour of Cured Meat By Eben van Tonder November 2022
Preventing the fading of the cured meat colour is of the greatest importance. Here are my notes on the subject. “Color chemistry has been one of the most studied and well-understood aspects of nitrite usage and a number of investigations exploring detailed cured meat colour chemistry reactions have been reviewed owing to the depth of our current understanding (Sebranek and Fox, 1985; Townsend and Olson, 1987; Pegg and Shahidi, 2000; Sebranek, 2009). The complexity of these reactions, however, underscores what is still not yet known about nitrite chemistry. Many factors contribute to the impressive complexity of this pale yellow crystalline substance. It is well accepted that the production of nitric oxide from nitrite is a required step for cured colour. The highly reactive ion, nitrite, itself does not fix the pigment causing cured meat colour. Rather, it forms nitrosylating agents by several different mechanisms which then have the ability to transfer nitric oxide that subsequently reacts with myoglobin to produce cured meat colour. Further, several significant factors affect the many nitrite curing reactions including meat system pH, the number of reductants present, temperature, and time (Sebranek, 2009).” (Sindelar, 2011)
A. Aspects Influencing Cured Meat Colour
I first turn my attention to work done by Cole (1961) to review some of the important aspects influencing colour stability in cured meat.
“Meat having a low pH had less residual nitrite after curing and retained its color better than did meat having a high pH. Brooks (1938) observed that the reaction between nitrite and hemoglobin in the presence of a reducing agent was slow over the pH range of 7.2 to 7.8 but rapid in the range of 5.2 to 6.6. Conditions of pH and salt concentration present in muscle during curing were found to be adequate for a rapid reaction. According to Duisberg and Miller (1943), the effect of pH is related to the inability of the pigment to take up nitric oxide below pH 4.0 rather than to the instability of nitric oxide hemoglobin at low pH values. These workers considered the optimum pH for curing to be about 5.2. Hornsey (1959) found that maximum conversion of pigment to the nitroso derivative occurred in meat of low pH.” (Cole, 1961)
“The rate of oxidation of nitric oxide myoglobin is a function of pH and nitrite concentration. Walsh and Rose (1956) found that in the absence of nitrite, nitric oxide myoglobin was stable in the dark in a pH range of 5.0 to 7.5. In the presence of 200 PPM of sodium nitrite, the rate of oxidation of nitric oxide myoglobin was markedly accelerated below pH 6.3. Urbain and Jensen (1940) had previously found that the oxidation of nitric oxide myoglobin was inhibited by an increase in pH. On the basis of pH effect, Walsh and Rose (1956) concluded that nitric oxide myoglobin was oxidized by nitrous acid below pH 6.3 in the presence of nitrite.” (Cole, 1961)
“Earlier work on the reaction of nitrite with blood (Haldane et al. 1897, Brooks, 1937; Keilin and Hartree, 1937) showed that reducing agents were required to maintain the pigment in the ferrous or reduced form and to reduce nitrite to nitric oxide. Reducing agents serve a similar purpose in the production of cured meat pigment.” (Cole, 1961)
“Reducing substances are normally present in muscle tissue. Brooks (1936, 1938) observed that muscle tissue maintained an oxidation potential of – 0.2 volts in the absence of oxygen. The oxygen uptake of pork muscle was about the same as beef muscle. Bacon, however, had a somewhat lower oxygen consumption (Brooks, 1936). Among the components of fresh tissue, Bender et al. (1958) found 2.1 percent reducing sugars (as glucose) on a dry weight basis. Sulfhydryl groups released from protein during heat processing are a source of reducing substances in meat. Watts et al. (1955) observed that the development of cured meat color paralleled the appearance of free sulfhydryl groups. Erdman and Watts (1957) found that cured meat maintained both color and sulfhydryl groups during low-temperature storage. Prolonged heat treatment can destroy sulfhydryl groups in meat. Fraczak and Padjdowski (1955) indicated that 80°C. was the critical temperature for the decomposition of sulfhydryl groups in meat.” (Cole, 1961)
“Another source of reducing agents in meat is additives introduced during processing. Greenwood et al. (1940) found that sugars improved the color of cured meat by establishing reducing conditions and preventing the oxidation of nitric oxide hemoglobin to methemoglobin in the presence of microorganisms. Ascorbic acid and related compounds have been widely used in recent years to improve the color of cured meats. Watts and Lehman (1952a) found that 0.1 percent ascorbic acid added to meats caused better color development when the meat was heated at 70°C. or frozen at -17°C. These workers (1952b) observed that hemoglobin did not react with ascorbic acid in the absence of oxygen. Ascorbic acid reduced methemoglobin and promoted the reduction of nitrite to nitric oxide. In the presence of oxygen, an undesirable side reaction occurred in which the green pigment choleglobin was formed. According to Hollenbeck and Monahan (1953) the beneficial effect of ascorbic acid in curing meat is due to the reduction of nitrogen dioxide to nitric oxide. Kelley and Watts (1957) observed that cysteine, ascorbic acid and glutathione were capable of promoting the formation of nitric oxide hemoglobin, regenerating this pigment on surfaces of faded meat and protecting surfaces of cured meat from fading when exposed to light.” (Cole, 1961)
“Since nitrite and nitrate are oxidizing agents in acid solution while ascorbic acid is a reducing agent, the compatibility of these compounds in a curing mixture is of some concern. Henrickson et al. (1956) reported ascorbic acid protected cured meat color from fading but was not completely stable with nitrite. Hollenbeck and Monahan (1955) concluded that moisture and temperature were important in controlling the reaction between ascorbate and nitrite in dry curing mixtures. In solutions, pH and temperature determined the rate of reaction. A very slow rate of reaction was observed at a pH of 6.5 to 7.0 in meat brines having high salt concentration.” (Cole, 1961)
“When Siedler and Schweigert (1959) studied the effect of reducing agents on the production of denatured globin nitric oxide nyohemochrome in model systems, they observed that ascorbic acid caused a significant loss of metmyoglobin at 60° and 70° C. Nitrite protected the heme from destruction by ascorbic acid and cysteine, but was less effective in the presence of the latter. Dithionite was the only reductant capable of forming the cured meat pigment at 60° C. while all reductants formed the pigment at 70° C. The yields of pigment at 70° C. were dependent on nitrite concentration when cysteine was the reductant, but not when ascorbic acid was the reductant.” (Cole, 1961)
Effect of metallic ions
“Acceleration of the reduction of methemoglobin by ascorbic acid in the presence of cupric or ferrous ions was observed by Gibson (1943). Addition of 8-hydroxyquinoline, which complexes copper, o\which complexes ferrous ion, reduced the rate of reduction of methemoglobin by ascorbic acid by as much as 50 percent. Weiss et al. (1953) found metallic ions increased the formation of nitric oxide hemoglobin in the order Cu> Fe>Zn. The metal chelating agent, ethylenediaminetetraacetic acid, inhibited nitric oxide hemoglobin formation. Cupric and ferrous ions accelerated fading in the absence of ascorbic acid, but protected the color of solutions of nitric oxide hemoglobin in the presence of ascorbic acid. Metallic ions had no effect in solutions of hemoglobin reduced with sodium dithionite. Siedler and Schweigert (1959) noted that ferrous ions had a nitrite sparing effect under certain conditions. The yields of cured meat pigment obtained with cysteine and a low nitrite level were increased by the addition of low levels of ferrous salts.” (Cole, 1961)
B. Factors Causing Loss of Color by Cured Meats
“A reversible relationship between moisture loss and color change in the absence of air was reported by Winkler (1939). Color change was explained on the basis of pigment concentration. According to Urbain and Ramsbottom (1948), discolorations due to dehydration can be controlled by proper packaging.” (Cole, 1961)
Color loss by autoxidation
“Urbain and Jensen (1940) observed that nitric oxide hemoglobin was susceptible to oxidation in the presence of oxygen. A combination of high pH (8.5) and low temperature (0o C.) prevented oxidation of nitric oxide hemoglobin for 13 days. Temperature had little effect on oxidation of nitric oxide hemoglobin at higher hydrogen ion concentrations.” (Cole, 1961)
Walsh and Rose (1956) described one of the mechanisms of oxidation of nitric oxide myoglobin as a slow autoxidation in air. They visualized the following reaction as occurring :
MbNO + l/2 O2 -> metMb + NO2–
While the autoxidation was independent of pH, oxidation of nitric oxide myoglobin by nitrous acid was markedly influenced by pH.” (Cole, 1961)
Color loss by photooxidation
” (Cole, 1961)The requirement for both light -and oxygen to produce cured meat fading has been shown by Urbain and Ramsbottom (1948), Allen (194-9) and Hornsey (1957). Cured meat remained stable toward light if packaged under vacuum in gas impermeable films.” (Cole, 1961)
“The reversibility of fading was first observed by Urbain and Ramsbottom (1948). These workers noted that cured meat vacuum packaged in impermeable films decreased in color the first day of storage; thereafter, the color increased to the initial level. Color was reformed by the reducing activity of the meat and the curing agents present. Watts et al. (1955) reported that faded cured meat which had residual free sulfhydryl groups and excess nitrite would recover some of the faded color upon being stored in the dark.” (Cole, 1961)
“The effect of light on fading depends on the time and intensity of illumination (Hockman, 1946; Allen, 1949; Archer and Bandefield, 1950; Taylor and Pracejus, 1950; Kraft and Ayres, 1954; Hornsey, 1957). There is some controversy in the literature regarding the effect of various spectral regions of light on fading. Allen (1949) reported that different portions of the visible spectrum adjusted to the same intensity were equally effective in fading cured meats. Although Hockman (1946) considered intensity to be a more important factor than wave length, he suggested a yellow or amber light for cured meat display. On the other hand, Taylor and Pracejus (1950) observed that various types of light sources differed in their fading power, and Archer and Bandefield (1950) reported that shorter wave lengths of visible light were more active in causing fading than were the longer wave lengths. The work of Taylor and Pracejus (1950), Kraft and Ayres (1954) and Hashimoto and Yasui (1956) indicated that ultraviolet light as well as visible light was effective in fading cured meat. Kampschmidt (1955) reported the wave lengths effective in fading of cured meat were those which were absorbed by nitrosomyoglobin, or approximately 350 to 600 rnu. This confirmed the finding of Urbain and Ramsbottom (1948) that wrapping cured meat in a red film to filter out light below 600 mu protected cured meat from fading.” (Cole, 1961)
“Three separate mechanisms for the oxidation of nitric oxide myoglobin were proposed by Walsh and Rose (1956). These are, l) the oxidation of pigment by nitrous acid at low pH, 2) autoxidation in air, and 3) photooxidation which increased in rate with increasing light intensity. These workers failed to observe a mixture of oxymyoglobin and metnçroglobin forming in the photooxidation of nitric oxide myoglobin and therefore rejected a dissociation mechanism such as was advocated by Urbain and Jensen (1940) and Kampschmidt (1955)» They proposed as a possible mechanism of photooxidation an activated nitric oxide myoglobin which could either deactivate to form nitric oxide myoglobin or lose an electron to oxygen and become oxidized to metrayoglobin and nitrite. Inasmuch as Walsh and Rose studied the native pigment rather than the denatured pigment found in cooked meat, their proposal may not relate to the mechanism of cured meat fading.” (Cole, 1961)
“Draudt and Deatherage (1956) observed in manometric studies that a portion of the nitric oxide of denatured globin nitric oxide irçyohemochrome was oxidized to nitrite and nitrate. Carbon dioxide was also found as one of the oxidation products. The increase in oxygen uptake was proportional to the decrease in color of the pigment. In a study of meat components which could cause loss of color, oleic acid and rancid fat were found to affect color of cured meat pigment while methyl laurate and methyl oleate had no effect on color.” (Cole, 1961)
C. Relation of Sulfhydryl Groups to Oxidative Processes
Sulfhydryl-containing compounds have been involved in a wide variety of free radical reactions. Waters (1948, p. 73) states:
The fact that thiols can act as chain carriers in autoxidation, and can add on to defines by the peroxidecatalyzed radical mechanism, are further indications of the transient existence of neutral thiol radicals.
In relation to enzyme reactions, Waters concluded (1948, p. 283):
In view of the considerable amount of evidence which proves that free thiol radical, R-S*, can be formed either by the dissociation of disulfides, or by the one-electron oxidation of thiols by ferric and cupric salts, it is not unlikely that these enzymes catalyze chain reactions by way of the oxidation and reduction of thiol groups to free thiol radicals in the same way as thiophenol can act as a chain carrier in autoxidation, and amyl disulfide as an autoxidation catalyst.
A sulfur-containing protein could become an active catalyst if a minute proportion only of its disulfide links could momentarily dissociate to the radical form and thereupon dehydrogenate a vicinal (i.e. and absorbed) metabolite and start a reaction chain in biological systems, just as the alkyl disulfides can initiate the dehydrogenation of tetralin.
Tobolsky and Mesrobian (1954, p. 22) proposed that organic hydroperoxides might be formed by the dehydrogenation of a sulfhydryl group, yielding a free radical thiol. Free radical thiols have been used to catalyze the decarboxylation of aldehydes (Barrett and Waters, 1953) and free radical exchanges (Cohen and Wang, 1955). Free radical chain transfer reactions catalyzed by thiols are important in the manufacture of synthetic high polymers such as synthetic rubber and plastics (Walling, 1957, p. 152). (Cole, 1961)
Sulfhydryl groups and fat oxidation
“The effect of sulfhydryl groups on the oxidation of fats was first observed by Meyerhof (1923) who found that frog muscle in the presence of thioglycolic acid absorbed large amounts of oxygen. When the muscle was extracted with hot alcohol or ether, oxygen absorption ceased. The same reaction with lecithin was traced to the linolenic acid component of lecithin. Meyerhof attributed this reaction to a transfer of oxygen by sulfhydryl groups to the unsaturated fatty acids. The inhibition of the lecithin-sulfhydryl system by cyanide was regarded as evidence of trace metal activation. Copper accelerated the oxidation of lecithin by thioglycolic acid; iron and manganese had no effect on lecithin in the presence of cysteine.” (Cole, 1961)
“Szent-Gyorgyi (1924a) was unable to find a fatty acid peroxide as a product in the oxidation of linolenic acid by thioglycollic acid. He proposed ( 1924b) that sulfhydryl groups bound molecular oxygen in the form of an active peroxide.” (Cole, 1961)
“In attempting to discover the metabolic significance of glutathione, Hopkins (1925) studied the reaction between this sulfhydryl-containing compound and fats. In systems of glutathione and linoleic or linolenic acid at pH 3-4» sulfhydryl concentration remained constant while oxygen in excess of that required to oxidize glutathione to its disulfide was taken up and a corresponding amount of fatty acid oxidized. Hopkins agreed with the Meyerhof postulate of a peroxide formed by two thiol groups. During decomposition by the thiol peroxide, oxygen was purportedly transferred to the fatty acid and reduced sulfhydryl groups were regenerated. At neutral pH the reaction was altered and sulfhydryl groups were oxidized. When glycerides were used, an induction period of from one to three hours was observed. This interval could be reduced by increasing the glutathione concentration. Hopkins suggested that an autocatalyst in very low concentrations developed slowly in the reaction mixture. He recognized the role of metallic ions in the system and considered that they might be part of the autocatalyst. According to Hopkins, thiols acted as “pseudo” catalysts by assisting in the formation of the autocatalyst.” (Cole, 1961)
“Allott (1926) considered that some of Hopkins’ results could be attributed to autoxidation of the fatty acids or glycerides. He found the activity of glycerides could be increased by aeration and some samples showed definite uptake of oxygen in the absence of glutathione. Tait and King (1936) reported the oxygen uptake at acid reactions by lecithin and reduced glutathione was greater than that of neutral fat or fatty acids * and also greater than that obtained from lecithin after hydrolysis.” (Cole, 1961)
“Scarborough and Watts (1949) reported the prooxidant effect of ascorbic acid and cysteine in aqueous fat systems in the absence of phenolic antioxidants. These workers inferred that the prooxidant activity of ascorbic acid and cysteine was related to their property of being reversibly oxidized. Watts and Wong (1951) found ascorbic acid could catalyze the oxidation of linoleic and linolenic acids, but not of oleic acid. The prooxidant activity of ascorbic acid was inhibited by ethylenediaminetetraacetic acid. Holtz (1936) had previously reported the oxidation of linoleic and linolenic acids catalyzed by ascorbic acid was inhibited by cyanide. Deutsch et al. (1941) reported the oxidation of phospholipids catalyzed by ascorbic acid at pH 4 was inhibited by hydroquinone. Phospholipid oxidation catalyzed by ascorbic acid was increased by iron but was not inhibited by cyanide (Elliot and Libet, 1944). Ottolenghi (1959) studied the activity of ascorbic acid and metals on mitochondrial lipids and concluded that a co-oxidation of ascorbic acid and unsaturated fat was mediated by a metallic ion. The rate of the reaction was governed ty the concentrations of ascorbic acid, metal and lipid. The peroxidation of lipids in isolated mitochondria was also reported by Tappel and Zalkin (1959a) who concluded that the oxidation was due to hematin catalysis. Sulfhydryl groups lost during oxidation accounted for only 20 percent of the observed oxygen consumption. Low concentrations of antioxidants inhibited fat peroxidation in mitochondria (Tappel and Zalkin, 1959b).” (Cole, 1961)
“The decomposition of lipid peroxides by protein sulfhydryl groups was studied by Dubouloz and Fondarai (1953). Free sulfhydryl groups in proteins were rapidly destroyed when peroxide was present in excess. Three to five atoms of peroxide oxygen disappeared for each sulfhydryl group originally present. A similar reaction was observed in liver tissue (Dubouloz et al., 1954).” (Cole, 1961)
“Work previously cited in relating sulfhydryl groups to fat oxidation has shown that trace metals are important in sulfhydryl and ascorbic acid catalyzed oxidations. These reports indicated that a reaction occurs between metals and reducing agents.” (Cole, 1961)
“Mathews and Walker (1909) reported that iron at a level of 6 X 10-6 molar could double the speed of oxidation of cysteine. They believed that the mechanism of this reaction involved the formation of an intermediate compound of a ferric salt with cysteine. Dixon and Tunnicliffe (1923) found the optimum pH for the autoxidation of glutathione, cystine and thioglycollic acid was 7.4 while the reaction was inhibited at pH 4.0.” (Cole, 1961)
“The presence of the disulfide form of the sulfhydryl compound accelerated the reaction. Warburg and Sakuma (1923) demonstrated that the so-called autoxidation of cysteine was caused by trace metal impurities. By careful purification of cysteine, autoxidation was reduced to a low level. Addition of one microgram of iron to 20 mg. of pure cysteine resulted in a 10-fold increase in the amount of oxidation. The oxidation was inhibited by cyanide or pyrophosphate. Ferric and ferrous iron were equally effective in promoting the oxidation of cysteine (Harrison, 1924.” (Cole, 1961)
“Harrison believed that sulfhydryl compounds were unable to combine directly with molecular oxygen unless they had been activated by iron. Meldrum and Dixon (1930)believed that the autoxidation of glutathione was not accelerated by the addition of iron alone but depended on the presence of some substance able to form catalytically active complexes with the metal. The mechanism of the metal catalyzed oxidation of cysteine was described by Michaelis (1929). Ferrous salts reacted with cysteine at pH 7 to 8 to give ferritricysteine. This underwent an internal oxidation-reduction to form ferrocysteine plus cystine which, in turn, reacted with two molecules of cysteine to yield free cystine and ferrotricysteine. A cycle was established which ended in the oxidation of all the cysteine to cystine. Because of the oxidation-reduction nature of the iron-cysteine complex, Michaelis and Barron (1929) suggested that an active chemical system could be produced with cysteine, a metallic ion and an oxidant.” (Cole, 1961)
“In the catalytic oxidation of glutathione by selenite, selenium diglutathione was an active intermediate (Tsen and Tappel, 1958). The work of Dixon and Tunnicliffe (1923) and Meldrum and Dixon (1930) also indicated that a metal complex with the disulfide form of a sulfhydryl compound was the active catalyst. Albert (1950, 1952) reported that cysteine-metallic ion complexes were more stable than other amino acid-metallic ion complexes. Amino acids combined most strongly with cupric ion followed in decreasing order by nicklous, zinc, cobaltous, ferrous and manganous ions. Hughes (1950) reported that the mercury-sulfur bond is stronger than the bond between mercury and any other group found in proteins.” (Cole, 1961)
Iyroan and Barron (1937) found that glutathione was readily oxidized in the presence of a catalyst such as copper, hemin or hemochromogen, but was not oxidized by atmospheric oxygen when dissolved in metal-free buffer. Qyanide inhibited the oxidation of glutathione by copper, but the hemin catalyzed oxidation of glutathione was inhibited only at high concentrations of cyanide.
Photochemical oxidations involving sulfhydryl groups
The basic requirement for photochemical reactions is the absorption of radiant energy. Since most compounds which contain a free sulfhydryl group do not absorb visible light (Anslow and Foster, 1932), photochemical reactions involving sulfhydryl groups must be activated either by ultraviolet light or visible light in the presence of a sensitizer which can transfer the photo energy to the sulfhydryl group. Weiss and Fishgold (1936) exposed various sulfhydryl compounds to ultraviolet radiation and detected the production of hydrogen corresponding to the amount of the disulfide formed. Sulfhydryl compounds were also found to react with fluorescent dyes in the dark; fluorescence was quenched by the sulfhydryl groups.
“Certain amino acids can be oxidized by visible light in the presence of a sensitizing agent. Weil et al. (1951) reported tyrosine, tryptophane, histidine, methionine and cystine were photooxidized in the presence of methylene blue. In the photooxidation of blood protein hydro lyzates that had been sensitized by methylene blue, riboflavin, eosin, or protoporphyrin, only the aromatic, heterocyclic and sulfurcontaining amino acids were photooxidized (Vodrazka and Sponar, 1957). Brin and Krasnovskii (1957) described the chlorophyll sensitized oxidation of ascorbic acid and cysteine. They postulated a mechanism of reaction involving the photoreduction of the sensitizer in the light, followed by reaction of the photoreduced pigment with oxygen.” (Cole, 1961)
“Repke (1956) observed that ultraviolet radiation and daylight (in the presence of photosensitizers) inhibited the activity of hydrogen transporting enzymes in the skin. Inactivation reportedly resulted from oxidation of enzyme sulfhydryl groups. The oxidation of sulfhydryl groups by ultraviolet radiation was reported by Kofman (1957). The amount of free sulfhydryl groups in solutions of native egg albumin exposed to ultraviolet light decreased 30 percent in two to three hours following irradiation.
Ginsburg et al. (1957) reported peroxides were formed in animal tissues subjected to X-irradiation. The reactivity of sulfhydryl groups in the enzyme systems of animals subjected to ionizing radiations was increased while the sulfhydryl concentration remained unchanged. Some modification of the sulfhydryl groups was believed to have occurred. Barron (1954) reviewed literature regarding the production of free radicals from sulfhydryl compounds subjected to ionizing radiations in the presence of oxygen. A high yield of oxidizing compounds can be formed in this manner.
Fox et al (1958) reported the production of a green pigment, identified as sulfmyoglobin, during the irradiation of meat extracts. The pigment was formed in maximum concentration at pH 5.3 with addition of cysteine to the meat extracts. A two-step reaction was believed to have occurred: (l) The production of hydro sulfide ion from the thiol-containing compound followed by (2) a reaction between hydrosulfide ion, myoglobin and an oxidant. Hydrogen peroxide was not considered to be the active oxidant since only cholemyoglobin was obtained by the use of hydrogen peroxide in the reaction mixture.” (Cole, 1961)
D. The Relationship between Residual Nitrite and Stable Cured Colour over time
Alahakoon (2015) reviewed the curing reaction as followed. “Nitrite . . . is considered a multifunctional food additive that forms nitric oxide during the curing process. Formation of nitric oxide from the intermediates is facilitated by reductants such as ascorbate. It has been recognized that nitrous acid (HNO2) is formed from nitrite under acidic conditions such as that in postmortem muscles (Pegg & Shahidi, 2000). According to Honikel (2004), dinitrogen trioxide (N2O3) is formed from nitrous acid and will subsequently form nitric oxide or will react with other substrates in meat. Nitric oxide will react with iron of both myoglobin (Fe+2) and metmyoglobin (Fe+3) to form cured color (Pegg & Shahidi, 2000). Comminuted meat quickly turns into brown color with the addition of nitrite due to metmyoglobin formation since nitrite acts as a strong heme pigment oxidant and is, in turn, reduced to nitric oxide. Nitric oxide reacts with metmyoglobin and subsequent reduction reactions convert the oxidized heme to reduced nitric oxide myoglobin for typical cured color subjected to cooking (Pegg & Shahidi, 2000).” (Alahakoon, 2015)
“Nitric oxide reaction with myoglobin forms the nitrosylmyoglobin complex, which outline the basis for unique cured meat color (Parthasarathy & Bryan, 2012). Nitrosylmyoglobin is bright red in color (Parthasarathy & Bryan, 2012) and is an extremely unstable compound. During thermal processing, it is converted to a stable, attractive reddish-pink compound – nitrosohemochrome – because of the denaturation of the protein moiety of the myoglobin pigment (Parthasarathy & Bryan, 2012).” (Alahakoon, 2015)
“Basically, a very small quantity of nitrite is required for the development of the cured color in meats, usually approximately 2–14 ppm. However, the level of residual nitrite in cured meats gradually decreases owing to oxidation during storage time. As a result, the meat starts to lose its cured color and become faded.” (Shakil, 2022)
Sindelar quotes Sebranec who refers to the 2-14ppm inclusion ratio of nitrite that is able to develop the cured colour. They caution that “significantly higher levels are required to prevent rapid fading and non-uniform curing while also maintaining cured color throughout an extended shelf life (Sebranek and Bacus, 2007).” (Sindelar, 2011) Alahakoon (2015) says that “as the residual nitrite levels in cured meat products gradually decline due to oxidation- and light-induced fading over the storage period (Cassens, 1997b), a residual nitrite level of 10-15 ppm is generally recommended as a reservoir primarily for the regeneration of cured meat color (Sindelar & Milkowski, 2011).
The level of residual nitrite is related to the original ingoing nitrite and Sindelar (2011) reported that “investigating the consumer acceptance of hams manufactured with varying levels of nitrite (0, 25, 75, and 125 ppm), DuBose et al. (1981) reported that no significant (P > 0.05) differences existed for color among the 25, 75, and 125 ppm nitrite containing samples while all were found different (P < 0.05) than the sample containing 0 ppm nitrite. A similar study conducted by Hustad et al. (1973) reported the only differences found between wieners having varying levels of nitrite (0, 50, 100, 150, 200, and 300 ppm) were when comparisons were made to the 0 ppm added nitrite treatment. Sebranek et al. (1977) investigating the consumer acceptance of frankfurters cured with varying levels (0, 26, 52, and 156 ppm) of nitrite found frankfurters containing 156 ppm nitrite to be more acceptable (P < 0.05) for color, flavor, and overall acceptability than all other nitrite concentrations. The researchers concluded that nitrite concentration was of critical importance for consumer acceptance of products possessing cured meat characteristics. The aforementioned research are examples of the extensive research studies that supported that minimum levels between 25 and 50 ppm of nitrite were likely sufficient for acceptable cured meat color in most meat and poultry products. However, higher levels would be necessary to achieve and maintain acceptable cured meat color, especially during a long product shelf-life period.” (Sindelar, 2011)
“When nitrite is added to meat systems, it reacts with or binds to a number of chemical components such as protein (Cassens, 1997b). Much of the nitrite added during the product manufacturing is either depleted through a series of reactions or physically lost during certain manufacturing steps. Typically, between 10 and 20 percent of the originally added nitrite normally remains after the manufacturing process and those levels continue to decline during storage (Pérez-Rodríguez et al., 1996; Cassens, 1997b). These levels of nitrite, referred to as residual nitrite, slowly decline over the storage life of cured meat products until they are often nondetectable (Skjelkvåle and Tjaberg, 1974; Eakes and Blumer, 1975; Honikel, 2004).” (Sindelar, 2011) As mentioned above, “to maintain a cured meat color throughout extended shelf-life, it is generally accepted that a small amount (10–15 ppm) of residual nitrite is needed to serve as a reservoir for the re-generation of cured meat pigment lost from oxidation and lightinduced fading (Houser et al., 2005).” (Sindelar, 2011)
Higher levels of sodium nitrite (>600 ppm/kg of meat) and low pH value may lead to nitrite burn (discolouration) where meat shows a green color due to the formation of nitrihemin, a green-brown pigment. (Shakil, 2022)
E. Relationship Between Residual Nitrite and Antimicrobial Efficacy
Function follows form in the case of meat colour. Residual nitrite is related to colour stability, but it is also related to antimicrobial efficacy. “The effectiveness of antimicrobial activity is dependent on various factors like pH, residual nitrite level, salt concentration, Fe content, reductants presence, storage temperature, etc.. At acidic pH, nitrite hinders the growth of unwanted microorganisms more effectively.” (Sindelar, 2011)
“Nitrite attacks bacteria at numerous sites by blocking metabolic enzymes, restricting oxygen absorption, and breaking the gradient of protons. Furthermore, nitric oxide binds to iron and reduces the availability of iron which is required for enzyme activity as well as bacterial metabolic activity and development. Because of the strong reactivity of Fe and nitrite, heme ion centers of enzymes and Fe-sulfur complexes are the major target of nitrite. The antibacterial activity of nitrite may be due to the peroxynitrite (ONOO) formation and nitric oxide formation from nitrite. Acid catalysis may cause oxymyoglobin to be autoxidized, generating superoxide radicals. The interaction of nitric oxide with superoxide radicals as well as the reaction of nitrite with hydrogen peroxide can produce peroxynitrite. Under physiological environments, peroxynitrite and peroxynitrous acid (ONOOH) stay in equilibrium. These two compounds are strong oxidants as well as nitrating agents. They penetrate the bacterial cells by passive anionic diffusion and disrupt the microorganisms by causing protein and lipid oxidation or by damaging DNA. Nitric oxide (NO) can also inhibit microbial growth by forming protein-bound dinitrosyl iron complexes when it reacts with iron-sulfur proteins, which are engaged in critical physiological activities including energy metabolism & DNA synthesis.” (Sindelar, 2011)
“Various kinds of microorganisms have various metabolic pathways and antioxidant defense strategies, and certain microorganisms are found to be resistant to the oxidative stress of peroxynitrite and peroxynitrous acid. Furthermore, the antibacterial action of nitrite in Gram-positive anaerobic bacteria has been shown to be more effective than in Gram-negative aerobic bacteria.” (Sindelar, 2011)
“Most of the nitrite applied to cured meat products is used to suppress C. botulinum, with only a little amount (about 25 ppm) required for color development. Suppression of C. botulinum development and toxin generation rises when nitrite levels rise. The level of additional nitrite is thought to have a greater influence on inhibiting C. botulinum than that of the residual nitrite during storage, implying that the production of antimicrobial compounds as a consequence of nitrite-related reactions might be noteworthy. The growth of starter cultures and bacteriocin production have been shown to be inhibited when the nitrite concentration was 100 ppm in sausage (fermented using Lactococcus lactis). An estimation predicts that when the nitrite content in sausage fermented with Lactococcus lactis reached 100 ppm, the development of starter cultures and bacteriocin synthesis
were suppressed. Several other estimates suggest that pathogens including Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus and E. coli grow slower in the presence of nitrite at levels found in cured meats and poultry products.” (Sindelar, 2011)
F. Strategies to Enhance Meat Colour
Stegeman (2008) summarised the strategies to enhance the meat colour besides the reliance on residual nitrite as follows. “Next to nitrites, other additives can be used to obtain a pink colour in cooked meat products. These products must be divided in two categories:
- Vegetables, spices and herbs that contain nitrate, like leek and celery;
- Products that have a red colour or that enhance the red colour of meat.
In the first category, the nitrate level of the vegetables is the important factor. Nitrate must be reduced towards nitrite. This reduction step is mostly carried out by increased temperature and adding nitrate reducing microorganisms. Moreover, nitrite still will be present in the product after stimulated reduction of nitrate by microorganisms.
The controllability of the process, however, becomes more precarious:
- Often, vegetables with high nitrate levels are added to the raw ingredients and in a successive step nitrate is reduced to nitrite. The extra reduction step requires a temperature increase. During this step, extra microorganism may grow. Although the meat product will be pasteurised after this, it is not mentioned in open literature whether or not possible toxin formation during the reduction step may taken place.
- An alternative procedure is performing the reduction step separately. The vegetables are reduced by microorganisms to form nitrite before they are added to the meat raw material. During this extra step, which focuses on the conversion of nitrates in nitrites, possible other undesired reactions may take place.
- The concentration of nitrate in these additives may change over time, in particularly in organically grown ingredients. Therefore, too, the final amount of nitrite in the product may vary, and eventually, it may exceed the amount of nitrite ordinarily used.
In the second category, fermented rice (Angak), beetroot juice, or other organically produced red colouring agents are suggested. Next to that, non-organic additives like commercial colouring additives and synthesized preformed cooked cured meat pigments have been reported in literature. The latter mentioned ingredients are not allowed in organic products. Furthermore, it is questionable whether the organic additives may be applied, since they merely have a cosmetic function and are no real functional additive or processing aid.
In addition, vitamin E (α-tocopherol) is mentioned either as a feed ingredient in animal production or as an additive in meat products to slow down oxidation and enhance redness, enabling the use of a reduced amount of nitrite in the final product. Whereas the addition of vitamin E to organic meat products is not allowed, it is also debatable whether it is allowed as a feed additive to organically grown animals if they are merely added to stabilize the colour of the final meat product.” (Stegeman, 2008)
Alahakoon (2015) also summarised the plant-based alternatives to nitrites. They write that “phenolic compounds, organic acids, and flavonoids are the key antimicrobial and antioxidant compounds in most plant extracts. These compounds can damage the cell membrane, which may lead to the leakage of cellular components, thereby inactivating or destroying microorganisms (Oussalah, Caillet, Saucier, & Lacroix, 2006). The antioxidant property of these compounds can be attributed to their characteristic to function as donors in the free radical chain reaction of lipid oxidation (O’Grady, Maher, Troy, Moloney, & Kerry, 2006). The antioxidant and antimicrobial properties of cranberry may be attributable to its organic acid content (citric acid, quinic acid, and malic acid), and to the presence of anthocyanins, flavonol glycosides, and proanthocyanidins (Chen, Zuo, & Deng, 2001; Lee, Reed, & Richards, 2006). In addition, the residues of tomato processing industries, including seeds and peels, contain highly biologically active compounds such as carotenoids (e.g., lycopene, b-carotene, phytoene, phytofluene, and lutein; Choksi & Joshi, 2007). Moreover, carotenoids in tomato are among the most important groups of natural pigments used as food colorants (Francis, Barringer, & Whitemoyer, 2000). Both cranberry and tomato extracts showed pH reductions that increase the amount of nitrite involved in the curing reactions when added together with nitrite and thus reduce the residual nitrite concentration. Hence, the decreased pH observed in these studies due to cranberry and tomato extracts may have accelerated the nitric oxide production that led to the depletion of residual nitrite (Pegg & Shahidi, 2000; Xi, Sullivan, Jackson, Zhou, & Sebranek, 2011).” (Alahakoon, 2015)
“Cranberry powder is a source of natural antimicrobial agents, particularly effective against L. monocytogenes growth in natural and organic processed meats (Qiu & Wu, 2007). Although lemon and lime powders, and grape seed extract are less effective against L. monocytogenes, they have the potential to control this organism in cured cooked meat when combined with cranberry powder (Xi et al., 2011). These authors further reported that nitrite (150 ppm initial nitrite) along with cranberry powder at 1, 2, and 3% concentration reduced the growth of L. monocytogenes by 2-4 log CFU/g as compared with only nitrite. However, the disadvantage regarding cranberry products is that it is slightly acidic in nature as they contain organic acids, which may eventually limit the amount of cranberry product to avoid quality defects (Xi et al., 2011). Therefore, selection of the optimum concentration of this product for supplementation to processed meat is a crucial factor.” (Alahakoon, 2015)
Deda, Bloukas, and Fista (2007) examined the quality parameters of frankfurters produced with different levels of sodium nitrite and tomato paste. Frankfurters with low levels of sodium nitrite (50 and 100 ppm) and 12% tomato paste showed the highest redness, whereas frankfurters with 12% tomato paste alone showed the lowest levels of residual nitrite. Therefore, the amount of nitrite added to frankfurters can be reduced from 150 to 100 ppm when combined with 12% tomato paste without any negative effect on the quality of the final product. In addition, Eyiler and Oztan (2011) stated that tomato powder retarded the oxidation reaction and improved the consumer acceptability in case of frankfurters. Furthermore, these authors observed that increase in the amount of tomato powder resulted in increased redness values in the final product, which is in agreement with the results of Deda et al. (2007). In addition, tomato powder has been shown to reduce the residual nitrite level in frankfurters as well as to act as a natural colorant (Eyiler & Oztan, 2011). Hayes, Canonico, and Allen (2013) stated that tomato pomace powder, when incorporated at a concentration of 1.5%, did not have any detrimental effects on the physicochemical properties of pork luncheon roll. The pork luncheon roll formulated with 50 ppm nitrite and 1.5% tomato pomace powder had similar or enhanced sensory attributes and no negative effects on the texture, sensory qualities or the microbial stability as compared to those formulated with 100 ppm nitrite alone. Several studies that tested tomato-based ingredients in meat products reported lower contents of nitrite and thiobarbituric acid retaining the properties of typical cured meat products (Sebranek & Bacus, 2007). Celery powder was originally available in its nitrate form; however, subsequently, preconverted celery powder was developed by some processors, in which nitrate was converted to nitrite since the incubation step increased the processing time (Sebranek & Bacus, 2007). It is considered that commonly used preconverted celery powder contains 10,000e15,000 ppm sodium nitrite (Sindelar, Terns, Meyn, & Boles, 2010). However, the addition of celery powder to processed meat is generally limited to 0.2-0.4% of the formulation weight because at levels higher than this, off flavors may develop (Sindelar et al., 2007). Since celery powder contains a significant amount of naturally occurring nitrate it will not be the best alternative source of nitrite for meat without using in combination with nitrate reducing bacterial cultures to produce a standard cured meat product. However, celery powder has only a very little amount of pigments and a mild taste that does not detract the flavor of meat.”
Recently, spray-dried Swiss chard powder was used as a natural source of nitrate. This product is similar to celery powder and contains 3.0-3.5% nitrate and as for celery powder, this product should also be used at a concentration of 0.15-0.3% (Sebranek, Jackson-Davis, Myers, & Lavieri, 2012). High concentrations may negatively affect the sensory attributes. The main advantage of Swiss chard powder is that it does not contain allergens (Sebranek et al., 2012). In addition, Gabaza, Claeys, Smet, and Raes (2013) showed that fresh and dried spinach can be used as a source of nitrate and that Staphylococcus carnosus can convert nitrate
to nitrite. Sebranek and Bacus (2007) reported that the nitrate level of spinach juice is approximately 3227 ppm. The residual nitrite content of fermented spinach-treated pork samples (50 g/L) was lower than that of nitritetreated samples (Gabaza et al., 2013).
Phenolic compounds, organic acids, and flavonoids are the key antimicrobial and antioxidant compounds in most plant extracts. These compounds can damage the cell membrane, which may lead to the leakage of cellular components, thereby inactivating or destroying microorganisms (Oussalah, Caillet, Saucier, & Lacroix, 2006). The antioxidant property of these compounds can be attributed to their characteristic to function as donors in the free radical chain reaction of lipid oxidation (O’Grady, Maher, Troy,
Moloney, & Kerry, 2006). The antioxidant and antimicrobial properties of cranberry may be attributable to its organic acid content (citric acid, quinic acid, and malic acid), and to the presence of anthocyanins, flavonol glycosides, and proanthocyanidins (Chen, Zuo, & Deng, 2001; Lee, Reed, & Richards, 2006). In addition, the residues of tomato processing industries, including seeds and peels, contain highly biologically active compounds such as carotenoids (e.g., lycopene, b-carotene, phytoene, phytofluene, and lutein; Choksi & Joshi, 2007). Moreover, carotenoids in tomato are among the most important groups of natural pigments used as food colorants (Francis, Barringer, & Whitemoyer, 2000). Both cranberry and tomato extracts showed pH reductions that increase the amount of nitrite involved in the curing reactions when added together with nitrite and thus reduce the residual nitrite concentration. Hence, the decreased pH observed in these studies due to cranberry and tomato extracts may have accelerated the nitric oxide production that led to the depletion of residual nitrite (Pegg & Shahidi, 2000; Xi, Sullivan, Jackson, Zhou, & Sebranek, 2011).” (Alahakoon, 2015)
The antioxidative components in garlic are S-alkenyl cysteine sulfoxide and other sulfur components such as diallyl disulphide and diallyl trisulphide (Sasse, Colindres, & Brewer, 2009). Antibotulinal properties of the majority of spice extracts can be attributed to their constituents such as eugenol, isoeugenol, D-borneol, citronellol, menthol, cinnamic acid aldehyde, and rosemarin acid (Ueda,
Yamashita, & Kuwabara, 1982). Moreover, the application of spices such as rosemary, thyme, sage, and garlic can reduce the content of heterocyclic aromatic amines, thereby reducing the formations of carcinogens in cooked cured meat (Murkovic, Steinberger, & Pfannhauser, 1998). The active constituents in sage and rosemary are rosmanol, rosemadial, carnosol, carnosic acid, and epirosmanol (Murkovic et al., 1998).
Cui, Gabriel, and Nakano (2010) used combinations of sodium nitrite and spice extracts from sage, clove, and nutmeg and found them to successfully inhibit the growth of C. botulinum (sage) or inactivate (clove and nutmeg) the organism. The combined antibotulinal efficacy of nutmeg, sage, and clove extracts observed in this meat model system can be useful in the development of minimally processed meat products, particularly those with low levels of sodium nitrite (approximately 10 ppm; Cui et al., 2010). Ismaiel and Pierson (1990) noted diverse antibotulinal activities of sodium nitrite (50-100 ppm) and oregano essential oil (400 ppm) in ground pork. Furthermore, Nevas, Koronen, Lindstrom, Turkki, and Korkeala (2004) examined the antibacterial properties of essential oils derived from several spices against 12 bacterial strains including Escherichia coli, L. monocytogenes, Salmonella
Typhimurium, C. botulinum, C. perfringens etc. The authors found that oregano, savory, and thyme essential oils showed the broadest range of antibacterial activity by inhibiting the growth of all tested organisms. However, C. botulinum and C. perfringens were the most sensitive among all organisms. Since spice extracts could not provide all the functions that nitrite alone could do, it can be suggested to use spice extracts in combination with an appropriate amount of nitrite.” (Alahakoon, 2015)
“Balentine, Crandall, O’Bryan, Duong, and Pohlman (2006) demonstrated that processed meat treated with rosemary at a concentration of 3000 ppm could maintain the red color for longer period and showed lower TBARS value. Doolaege et al. (2012) investigated the effects of different doses of rosemary extracts (0, 250, 500, and 750 ppm) combined with low sodium nitrite levels (40, 80, and 120 ppm). The addition of rosemary extract positively retarded lipid oxidation in liver pate. Furthermore, it was found that the concentration of sodium nitrite added to liver pate could be reduced from 120 to 80 ppm when rosemary extract was added at all three concentrations, without any negative effect on lipid oxidation, antioxidant level, and color stability.” (Alahakoon, 2015)
“Incorporation of citrus co-products in meat products is another recent trend adopted for reducing the residual nitrite concentration. The extracts of citrus co-products are rich in dietary fibers, antioxidant fibers, and bioactive compounds such as organic acid and polyphenols which can be used as functional ingredients in meat products (Perez-Alvarez, 2008).” (Alahakoon, 2015)
“Fernandez-Gines, Fernandez-Lopez, Sayas-Barbera, Sendra, and Perez-Alvarez (2003) investigated the effects of different concentrations of orange dietary fiber (0.5, 1, 1.5, and 2%) on the residual nitrite levels in a bolognatype cooked sausage. The maximum reduction (69.57%) in the residual nitrite level was obtained with 2% orange dietary fiber in combination with 0.02% oregano essential oil (Garcia-Martinez, 2009). The incorporation of orange dietary fiber into dry-cured meat products resulted in reduced residual nitrite levels as compared with that in the control and a higher redness value at levels >5 g/kg (Fernandez-
Lopez et al., 2007).” (Alahakoon, 2015)
“Several researchers studied the use of lemon albedo (raw or cooked, dehydrated raw or dehydrated cooked) in cooked and dry cured meat products for reducing the levels of residual nitrite. Reduction in residual nitrite levels due to the bioactive constituents of raw and cooked lemon albedo (Fernandez-Gines, Fernandez-Lopez, Sayas-Barbera, Sendra, & Perez-Alvarez, 2004). Aleson-Carbonell, Fernandez-Lopez, Sendra, Sayas-Barbera, and Perez- Alvarez (2004) investigated the influence of various concentrations (0, 25, 50, 75, and 100 g/kg) of raw and cooked lemon albedo on the residual nitrite levels in dry-cured sausages
and found that raw albedo was more effective in reducing the residual nitrite content and delaying lipid oxidation at all tested concentrations. Samples treated with 50 g/kg dehydrated raw albedo and 75 g/kg dehydrated cooked albedo showed sensory properties similar to those of the control. However, it is important to select the best concentration since higher concentrations may
exert a negative effect on sensory attributes.” (Alahakoon, 2015) I give their table presenting other potential plant-based alternatives for nitrite that can be used effectively in meat and meat products below.
G. What about using Meat with High Levels of Heam Iron?
Right at the start of my career in curing, I added blood to meat that I cured, arguing that it would enhance the colour. Stegeman (2008) had something to say about ,y notion. He noted, that “ingoing nitrite concentrations may have to be increased for products manufactured from raw material containing high levels of heam iron, like liver, blood and spleen. The amount of heam iron may play a role since iron may bind nitrite, disabling its functionality.”
H. How to Stabilise the Meat Colour?
“Stabilization of the red pigment is obtained through denaturation of the globin moiety of the molecule (Li.icke, 1985; Demeyer et
al, 1986). It is often assumed that the presence of nitrate is required for optimal colour stability (Puolanne, 1977, 1986). Also, “residual nitrite (as a source of NO to inhibit NOMb dissociation) and ascorbate (to inhibit MMb and N oxide formation) are considered essential for colour stability (Ranken, 1981).” (Alley, 1992)
I. The Accumulation of NO3–
Alley (1992) evaluated “the effects of nitrate and ascorbate on colour formation and stability in dry sausages prepared using ‘back slopping’. The latter procedure involves the use of finished fermented sausage as inoculum (starter) for a new production.”
In this work they experiment with “various levels of KNO3 with or without sodium ascorbate (NaAsc), added with various levels of NaNO2 to a sausage mix prepared in the sequence (% w/w): beef(30.6) and pork (30.6) ( – 5°C); glucose (0.7); pepper (0.09); nutmeg (0.01); ascorbate and starter (1 min 40 s; – 2°C), followed by lard (35) ( – 20°C) (30 min 30 s; – 5°C) and finally NaC1 containing nitrite and/or nitrate (2.85) (4 min 20 s; – 7°C). As starter, 1% of a mixture of 2 two-day-old sausages (from two companies), containing approximately 107/g and 105/g of lactobacilli and micrococci respectively, was used.” The sausages were “fermented for 2 days (20-24°C; 92-98% RH) and then transferred to drying chambers for further drying up to 21 days (gradual decrease to 13 or 16°C and 80 or 83% RH).” The use of starter culture resulted in a lowering of the pH.
They found that “Nitrite was rapidly depleted in all experiments, whereas nitrate accumulated. No clear effect of ascorbate was observed on these changes. From molar changes, it can be calculated that net nitrate production accounts for 62% + 15% (mean+ SE) of nitrite disappearance up to 24h after stuffing. At later stages, other reactions clearly become more important. Such reactions are numerous and complex: generalizing, Cassens et al. (1979) stated that myoglobin accounted for 5-15% of the nitrite originally added, nitrate 1-10%, residual nitrite 5-20%, gas 1-5%, sulfhydryl 5-15%, lipid 1-5% and protein 20-30%. Clearly, only nitrate production from nitrite is involved in net nitrate changes, as nitrate when added alone, did not disappear: up to 17 days after stuffing, nitrate recovery was 97 +/– 3% and 90 +/– 5% (mean value +/– SE of eight determinations) in series s. 5 and s. 6 of Experiment 2 respectively. These data suggest that micrococci present in the starter sausage are not sufficiently active, competitive or numerous to induce nitrate reduction, before pH becomes inhibitory.
Let’s look at a summary of the experiments.
I think it is fair for me to say that this inverse relationship between nitrate and nitrite under these conditions are the most nbeautiful consequince of curing I have ever seen!
Colour and colour stability
The substitution of nitrite for nitrate clearly brightens the surface colour (increased L), whereas it is clear that the surface is always considerably darker than the interior. However, brightening of the surface is not an effect of nitrate per se, but rather the reflection of lower nitrite addition, as evidenced in Experiment 3: increasing both nitrite and nitrate addition lowered L from an average value of 47.5 to 46.5. Sausages were brighter here than in other experiments. It should be mentioned that the different experiments are not comparable to each other, because, for example, of differences in drying rate. It should be remembered that such small differences in colour coordinates as discussed here, are much more apparent to the eye. Surface darkening, because of higher nitrite addition, is most probably, due to an extensive formation of MMb during the mixing of ingredients and immediately after stuffing. Further acidification and surface drying fixes the MMb colour on the surface. Other possible explanations are the formation of brown metmyoglobin nitrite (Fox, 1966) followed by its fixation through drying; and even globin denaturation by the excess of nitrite added. Dry matter content was 73.1% +/- 1.0 and 54.6 % +/- 0.3% for the 1 cm sausage edge and for the centre respectively (mean values +/- SE for s. 1, 2, 3 and 4 of Experiment 1). Results on colour formation in Experiment 3 support that hypothesis: the series with lower nitrite addition (s. 1, 2 and 3: 74 mg/kg) showed bright red surface colour 3 days after stuffing, whereas at higher nitrite addition (s. 4, 5 and 6:148 mg/kg) surface colour was still greyish.
These differences were also apparent for the interior (Table 4). The presence of a micrococci starter seemed to improve reddening at high concentrations of both nitrite and nitrate, but luminosity was lower (underlined values in Table 4). Addition of ascorbate does not seem to affect these changes to any clear extent. In this respect it should be noted that less than 100 mg/kg of nitrite may be sufficient for both colouring and antimicrobial purposes in dry sausage when combined with lowered water activity and pH (Hotchkiss and Cassens, 1988).
Colour stability as measured here is mainly affected by the use of ascorbate and is reflected by the decrease in a value following illumination. The average interior a value was 13.0+/- 1.0 and 13.5+/- 1-0 for sausages prepared without and with ascorbate, respectively. These values were respectively reduced however by 7.9+/- 1.0 and 5.5+/- 0.7 after 24h of illumination, this difference being clearly significant (mean values of six determinations +/- SE).
Redox potential and ascorbate depletion
Nitrite levels clearly affected early values of redox potential and ascorbate recovery as illustrated in Table 5. Increasing nitrite concentrations clearly decreases ascorbate recovery. Obviously, reduction of MMb, possibly also involved in ascorbate-consuming reactions, is not sufficient to improve surface colour (Table 3). The establishment of a more reducing environment however promotes colour stability (see also Ranken, 1981).
Making sense of it
Let’s consider the conclusion of Alley (1994) to make sense of it all. They write, “Nitrate is not reduced to nitrite in sausages prepared using a starter sausage (‘back slopping’) probably because of lack of (active?) micrococci.
Nitrite is rapidly depleted and nitrate accumulates in such sausage fermentation. In the initial stages of fermentation, nitrate accumulation accounts for over 50% of nitrite disappearance. In such sausages, oxidation of nitrite to nitrate is coupled to rapid oxidation of MbO2 and Mb to MMb, giving a greyish surface and interior colour. Later MMb is reduced and NOMB is formed, reponsible for the bright red colour. At high nitrite concentrations (say over 100mg/kg) surface MMb formation exceeds the potential for subsequent reduction, hampered by surface drying. Rate of reddening (NOMb formation) is not affected by the presence of nitrate or ascorbate. A slight improvement was observed in the presence of a micrococci starter, but this effect was negligible compared with that of nitrite concentration. Substitution of nitrate for nitrite does not affect colour, except for the lowering of nitrite concentration. Colour stability is mainly promoted by residual ascorbate and not affected by substitution of nitrate for nitrite. Ascorbate depletion and redox potential are increased by increasing nitrite levels.
Alahakoon, A. U., Jayasena, D. D., Ramachandra, S., Jo, C.. (2015) Alternatives to nitrite in processed meat: Up to date. Trends in Food Science & Technology, Volume 45, Issue 1, 2015, Pages 37-49, ISSN 0924-2244, https://doi.org/10.1016/j.tifs.2015.05.008. (https://www.sciencedirect.com/science/article/pii/S0924224415001429)
Alley G, Cours D, Demeyer D. Effect of nitrate, nitrite and ascorbate on colour and colour stability of dry, fermented sausage prepared using ‘back slopping’. Meat Sci. 1992;32(3):279-87. doi: 10.1016/0309-1740(92)90091-H. PMID: 22059814.
Cole, Morton Sylvan, “Relation of sulfhydryl groups to the fading of cured meat” (1961). Retrospective Theses and Dissertations. 2402. https://lib.dr.iastate.edu/rtd/2402
Sindelar, J. J., Milkowski, A. L.. (2011) Sodium Nitrite in Processed Meat and Poultry Meats: A Review of Curing and Examining the Risk/Benefit of Its Use. American Meat SWcience Association White Paper Series.
Shakil, Mynul Hasan, Anuva Talukder Trisha, Mizanur Rahman, Suvro Talukdar, Rovina Kobun, Nurul Huda, and Wahidu Zzaman. 2022. “Nitrites in Cured Meats, Health Risk Issues, Alternatives to Nitrites: A Review” Foods 11, no. 21: 3355. https://doi.org/10.3390/foods11213355
Stegeman, D., Verkleij, T.J.. (2008) Reducing the amount of nitrites in the production of pasteurized organic meat products. Agrotechnology and Food Sciences Group.