Reaction Sequence: From nitrite (NO2-) to nitric oxide (NO) and the cooked cured colour.
By: Eben van Tonder
3 July 2017

I dedicate this work to Oscar Klynveld.  The MD of Woody’s Consumer Brands (Pty) Ltd, my partner and friend who undertook this great work with me when we decided to make a living off these mechanisms.

Index

SUMMARY AND INTRODUCTION

THE FORMATION OF NITRIC OXIDE

ITS DISCOVERY

PRIORITY OF NITRIC OXIDE

REDUCTION OF NITRATE

INTERACTION BETWEEN NITRITE AND HEME PROTEIN

THE FORMATION OF NITROUS ACID

NITROSATING SPECIES TO NITRIC OXIDE

THE REACTION OF NITRIC OXIDE WITH MYOGLOBIN

THERMAL TREATMENT

SUMMARY OF CHEMICAL STATE, COLOUR and NAME – FERROUS AND FERRIC

OVERALL IMPACT OF TEMPERATURE

IMPACT OF REDUCING AGENTS

IMPACT OF TIME

THE IMPACT OF pH

MYOGLOBIN CONCENTRATION

BACTERIAL/ ENZYMATIC CREATION OF CURED COLOUR

CONCLUSION

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SUMMARY

In this article, we deal with the cured colour formation in meat curing.

INTRODUCTION

I am not a trained chemist or hold a degree in food science.  I am an entrepreneur who makes my living through meat processing and is curious about the science of my trade.  I write about it in order to learn.  Here I set out to review the different factors that impact on meat curing by following the chemical reaction sequences to identify practical measures to employ in the factory to ensure proper curing.

Curing involves the development of an appealing reddish/ pinkish cured colour, meat preservation, the prevention of rancid fat formation and a particular cured taste.  In this article, we deal with the chemistry of curing in its relationship to colour development.

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THE FORMATION OF NITRIC OXIDE

ITS DISCOVERY

For a full chronology of the people responsible for the discovery of the meat proteins and the chemical constituents associated with meat curing, including nitric oxide, refer to my article, Fathers of Meat Curing.

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PRIORITY OF NITRIC OXIDE (NO)

The colour of cured meat is brought about by the reaction of nitric oxide (NO).  Nitric oxide plays an equally central role in the nitrogen cycle as a key intermediate.  The nitrogen cycle is the collective name given to several interconnected processes by which nitrogen, which exists as a stable gas of two nitrogen atoms (dinitrogen) joined together are converted into ammonia (NH₃) which is then converted into amines.  An amine is a derivative of ammonia, NH₃, where one or more of the hydrogen atoms are replaced by either an alkyl or aryl group.

An important amine is amino acids, the building block of life.  Every cell in the body contains amino acids where they are used to build proteins.  From muscles to simple structures like cell membranes consists of proteins.

When an organism decays, ammonia (NH₃) is produced and is oxidised to nitrites and nitrates during nitrification. The one direction of the process of nitrification exists where ammonia and subsequently amines are formed.  A reverse process also exists of denitrification where “nitrate is used instead of oxygen as an electron acceptor for energy production, and reduced to gaseous nitrogen oxides (NO, N₂O, N₂).

Denitrification is important in the nitrogen cycle, but also in bacon curing.  The reduction of nitrate to nitrite and nitrite to nitric oxide are all performed by denitrifying bacteria.  These bacteria are classed as facultative anaerobes which mean that they are capable of existing in an environment with oxygen or without.  The “molecular unit of currency” of intracellular energy transfer is ATP (adenosine triphosphate) and it requires the oxygen to make ATP.  When oxygen is however not available, these special class of organisms is able to switch to fermentation or anaerobic respiration.  (Knowles, J. R. (1980).  The denitrification trait is usually active under low oxygen tension or when nitrogen oxides are available as electron acceptors.

Denitrification plays an important role in the reduction of nitrate (NO3-) to nitrite (NO2-) which was the first step in curing at a time when saltpetre was used in curing brines as the starting ingredient en route to nitric oxide formation. Nitrite is the second step in the chemical reaction sequence of curing that existed for millennia.  Since the early 1900’s, nitrite has been added directly in the form of sodium nitrite in order to reduce curing time, achieve greater consistency and to limit the amount of nitrite in cured meats due to health concerns.

Today, in our curing plants, we begin at step two of the ancient curing process, namely with nitrite which exists in the ionic form in the aqueous curing brinene’s widely used.  How does this now end up as nitric oxide, attached to the hem part of the meat protein to give the cured meat colour?  Here matters get very complex and fascinating all at once with enormous application in the meat curing industry.

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REDUCTION OF NITRATE

By 1750, in Europe, the use of saltpetre (potassium nitrate) with salt in curing brines were universally practised.  (Bacon Curing) It was thought that the potassium or sodium nitrate was responsible for the cured colour formation in meats.  Polenke (1891) found nitrite in a curing bring that he made with Saltpeter (nitrate) only.  (Saltpetre)

Denitrifying bacteria was identified by E. Meusel in 1875 and the term coined by Gayon and Dupetit in 1882.  The microbial reduction of nitrate (NO3-) to nitrite (NO2-) was well known by 1891 when Polenske wrote and he drew on this knowledge.   K. B. Lehmann (1899) and his understudy, KIßKALT, confirmed that nitrite is responsible for the curing of meat and not the nitrates.  In order for meat curing to take place, the reduction of NO3- to NO2- by denitrifying bacteria has to take place first.  (Fathers of Meat Curing)

The reaction is represented as follows:

The prolific British physiologist and philosopher, John Scott Haldane finally showed that it was not ultimately nitrite that was responsible for the cured colour formation, but nitric oxide (NO).  In 1901 he demonstrated that nitrite is further reduced to nitric oxide (NO) in the presence of muscle myoglobin and forms iron-nitrosyl-myoglobin. It is nitrosylated myoglobin that gives cured meat, including bacon and hot dogs, their distinctive red colour and protects the meat from oxidation and spoiling.  NO cures meat.

In the same way, as bacteria convert nitrate to nitrite, nitrite is also converted to nitric oxide (NO) through bacterial action where NiR enzymes reduce nitrite by one electron.  (Nan Xu, Jun Yi, and George B. Richter-Addo; 2010)  It is represented as follows: 

Microbial reduction is, however not the main way that nitrite in converted to nitric oxide in conventional meat curing.  In fermented meat curing systems where nitrate is used, the reduction of nitrate to nitrite through microbial enzyme activity is the bottleneck process.  In contrast to this, the curing mechanism in nitrite-cured meat is “less dependent on microbial action and seems to be purely chemical.”  (Editor-Toldr, F;2015: 203)

We will encounter the NiR enzymes again when we look at the mechanisms of nitrite reduction in a living organism (in vivo) and consider this, along with microbial reduction to NO when we look at the curing mechanics of Jinhau, Anfu, Westphalian and Parma Hams where only salt is used along with very long curing times.

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INTERACTION BETWEEN NITRITE AND HEME PROTEIN

A special relationship has been thought to exist between hem proteins and nitrite since Gamgee.  On 7 May 1868, Dr. Arthur Gamgee from the University of Edinburgh, brother of the famous veterinarian, Professor John Gamgee (who contributed to the attempt to find ways to preserve whole carcasses during a voyage between Australia and Britain), published a groundbreaking article entitled, “On the action of nitrites on the blood.”  He observed the colour change brought about by nitrite.  He wrote, “The addition of … nitrites to blood … causes the red colour to return…”

The researchers J. S. Haldane, R. H. Makgill and A. E. Mavrogordato studied the action of nitrites on blood further and found that nitrites convert the haemoglobin of the blood not simply into methemoglobin (iron in the heme group – Fe³⁺ (ferric), not Fe²⁺ (ferrous) as in normal hemoglobin); cannot bind oxygen, unlike oxyhemoglobin; colour – brown), but into a mixture of methaemoglobin and nitric oxide haemoglobin (J. S. Haldane, et al.; 1896: xviii) or nitrosohaemoglobin which has a red colour.  This was found to happen in the absence of oxygen and equimolar quantities if substances capable of reducing methaemoglobin and nitrites are not present.  In the case of myoglobin, and given these conditions, only metmyoglobin with its brown colour is produced.  (Lawrie, R. A., Ledward, D.; 2006: 257)

The discovery of Gamgee, supported by the work of Haldane and his co-workers was profound.  It turns out that nitrite is a highly reactive compound.  The example of the action of nitrite on haemoglobin and myoglobin in the absence of air and reductants, mentioned above, illustrates this fact.  Nitrite functions as an oxidising, reducing or nitrosylating agent which is the covalent incorporation of a nitric oxide moiety into another (usually organic) molecule.  (J.G. Sebranek, J.N. Bacus / Meat Science 77 (2007) 136–14)  In meat, nitrite can be converted into nitrous acid, nitric oxide, and nitrate.  When nitrite comes into contact with meat, the first reaction that takes place is that it acts as a strong oxidizing agent on myoglobin to forms metmyoglobin (iron in the heme group changes to – Fe³⁺ (ferric), from Fe²⁺ (ferrous) as in normal hemoglobin) with its brown colour.  The first visual effect of adding sodium nitrite to meat is therefore a colour change to brown.

In order for curing of meat to take place, it is, however, necessary for nitric oxide to be created.  This is a longer and much more complex process which is why time is required for curing to happen.

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THE FORMATION OF NITROUS ACID

We have already seen how nitrate in a curing brine is reduced through the action of denitrifying bacteria into nitrite.  In order to speed up curing and make the process more controlled, sodium nitrite has been used directly in curing brines since WWI.  Nitrite itself is not a nitrosylating agent (an agent that transfers nitric oxide) in meat.  Intermediaries are first formed such as .  (Sebranek and Bacus,  2007)

If an acidic oxide reacts with water, it forms an acid and if it reacts with a base, it forms a salt.  In nature, nitrite (an oxide of a non-metal, nitrogen) neutralises a base, such as potassium or sodium and in the process forms a salt (potassium or sodium nitrite) and in water, it forms nitrous acid ().  When the salt dissolves in water, the molecules of potassium or sodium nitrite separate into either sodium cations () or potassium cations () and nitrite anions ().

In water, potassium nitrite (KNO₂) dissolves and undergoes the following reaction.

Water is  and OH^–.  The  reacts with  to form a weak acid.  The  reacts with the  to form a strong base.  The  therefore does not affect the pH of the solution since it is a weak conjugate acid, but the  will.  It will act as a base.  It is a conjugate base since it came from the acid,  . We write the reaction of  with water as follows.  We first follow the reactions that start with the formation of nitrous acid.

We can also represent it as follows.

The acid dissociation constant or pKa of  is 3.6.  This means that it is a weak acid and most of the ions will exist either as hydrogen or nitrite ions.  The pH of meat (i.e., usually between 5.5 – 6.5) is well above the pKa of , and it is expected that about 99% of the nitrite exist as its anion  (Toldrá, F., 2015:  21).    This means that of all the nitrite added, between 0.1% and 1% is found in the form of the reactive acid.  (Pegg, R. B. and Shahidi, F.;2000: 39)  It is thought that the remainder of the nitrite anion plays no role in the curing process. (Dikeman, M. and Devine, C.; 2014:  201)

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NITROSATING SPECIES TO NITRIC OXIDE

The next step in such a reaction sequence  “is the generation of either a nitrosating species or the neutral radical, nitric oxide (NO).”  (Sebranek, J. and Fox, J. B. Jn.; 1985:  1170)  A nitrosating species is a molecular entity that is responsible for the process of converting organic compounds into a nitroso (NO) derivatives, i.e. compounds containing the R-NO functionality.

->  Nitronium and nitrous acidium ions
We first look at a nitrosating species that do not exist in any relevant quantities in meat since the pH of meat is generally too low for its formation.  It is important, however, since it is the strongest nitrosating species.  These species are forms of a positively charged (electrophilic) nitrogen oxide, either in its simplest form, the nitrosonium ion, , or as part of a larger molecule, the ion, nitrous acidium ().  (Sebranek, J. and Fox, J. B. Jn.; 1985:  1170)

It is not certain if nitrosonium ion exists free in solution.  It possibly only exists as nitrous acidium ions.  At the pH of meat, however, the principal reactive species is dinitrogen trioxide ().  (Sebranek, J. and Fox, J. B. Jn.; 1985:  1170)

->  HNO2 to N2O3 to NO 

Nitrous acid is again the starting point of this particular reaction sequence, which, in aqueous solutions, exists in equilibrium with its anhydrate, dinitrogen trioxide   

The equilibrium equation is:

(Williams, D. L. H..  2004: 1, 2)

In certain chemical structures, electrons are able to move around to help stabilise the molecule, called resonance structures.  Dinitrogen trioxide is such a structure where, in aqueous solutions, the molecule is stabilised through resonance. (Sebranek, J. and Fox, J. B. Jn.; 1985:  1170)

An electron poor site is created by the charge shift which is strongly electrophilic.  This means that it is strongly attracted to electrons.  This site will, in other words, nitrosate a nucleophilic site.  A nucleophilic site donates electrons to an electrophile to form a chemical bond where a  group will be attached.  (Sebranek, J. and Fox, J. B. Jn.; 1985:  1170)

In general, a lower pH accelerates the formation of . (Dikeman, M. and Devine, C..  2014: 417)  Nitric oxide may be formed through either a dismutation or a reduction reaction.  The following reaction takes place in a strong acid and is a good example of a rare reaction involving three molecules due to the low probability of three molecules colliding to create the reaction.  (Sebranek, J. and Fox, J. B. Jn.; 1985:  1171)

The following two sequences are the major sources of nitric oxide in meat, especially in light of the fact that meat is a rich source of reductants. (Sebranek, J. and Fox, J. B. Jn.; 1985:  1171) These reductants that react with dinitrogen trioxide are found naturally in muscle tissue as well as added reductants, such as ascorbate, to form nitric oxide.  (Krause, B. L.; 2009: 9)  They both pick up from the formation of dinitrogen trioxide.

where Rd is such reductants as ascorbate, sulfhydryl groups, hydroquinones, etc. (Sebranek, J. and Fox, J. B. Jn.; 1985:  1171)

“Nitric acid is an electron pair donor and forms a very stable complex with transition metals.”  (Dikeman, M. and Devine, C..  2014: 417) Such a complex of a central metallic atom or ion, especially with a transition metal, is called a coordinate covalent complex.  The metal centre is called the coordination centre and is surrounded by an array of bound molecules or ions, that are in turn known as ligands or complexing agents.”

The coordinate-covalent complexes of nitric oxide with the haem pigment of meat called either nitrosylmyoglobin, or dinitrosylhaemochrome (same thing, different names) form the pink and red colours of cured meats.  (Sebranek, J. and Fox, J. B. Jn.; 1985:  1171)

-> Backward reaction
Nitric oxide is, however, “readily oxidised, which accounts in part for the instability of cured meat colour in air.” (Sebranek, J. and Fox, J. B. Jn.; 1985:  1171)

or with water to form nitrous or nitric acid.

“These are both backward reactions, regenerating previous reactants in the sequence (, ).  It will be noted that nitrate is produced in this recycling (the last reaction) and, since it is relatively unreactive, it acts as a sink to remove nitrite from the system. This constant recycling results in a semi-stable equilibrium of reactants, intermediates, and products.  While these reactions are the major chemical reaction sequences, there are many other reactions that can and do take place. Even nitrous oxide (, laughing gas has been identified in the gasses above curing mixes.   (Sebranek, J. and Fox, J. B. Jn.; 1985:  1171)

The one pathway to NO formation is then through the anhydrate of  , dinitrogen trioxide, ”  (Pegg, R. B. and Shahidi, F.;2000: 39), which exists in equilibrium with the two oxides, of NO and NO2. (Toldrá, F., 2015:  21)

The hydration of nitrous acid is an important time-consuming reaction (Krause, B. L.; 2009: 9) and from the vantage point of the meat curing operation, resting the product or allowing for enough curing time after the curing brine has been added, is critical.  A good processing sequence is injecting the meat, tumbling it, resting it for at between 12 and 24 hours (depending on the temperature), tumbling it again to pick up brine that leached out during the maturing or colour development stage and then smoking it.  Where meat grids are being used, another option will be to inject, tumble, fill into grids and then resting it, but for this one would require additional trolleys.

-> HNO2 and NaCl to NITROSYL CHLORIDE -> NOCl

There are however other additives or conditions that influence the reduction of nitrite to nitric oxide.  The most important additive that influences nitric oxide formation is salt, due to the formation of nitrosyl chloride (NOCl), which is a more powerful nitrosating agent than dinitrogen trioxid. (Dikeman, M. and Devine, C..  2014: 417)  It was Ridd (1961) who first reported that nitrous acid and hydrochloric acid will generate nitrosyl chloride (NOCl).   (Ridd, J. H.; 1961: 418)

Both dinitrogen trioxide and nitrosyl chloride start from nitrous acid.  The reaction formation of nitrosyl chloride from nitrous acid can be represented as follows:

Note the reaction between the two anions   and  to form .  The reaction is due to the ability of electronegative anions to form resonance stabilised, charge separated molecules from nitrous acid.  They are more reactive than dinitrogen trioxide,  and less reactive than the nitrous acidium ion. (Sebranek, J. and Fox, J. B. Jn..  1985)

->  Five important nitrosating species to NO

There are five nitrosating species that has been identified from literature that is of interest to us related to meat curing.  Species 1 being the strongest and species 5 being the weakest.

Species 1:

Source:  “From smoke which has many other phenolic compounds”

Species 2:

Source:  From curing salt

Species 3:

Source:  Found in the air.

Species 4:

Source:  Nitrous acid anhydride

Species 5:

Nitrose derivatives of citrate, acetate, sulphate, phosphate.

Sources:  Cure ingredients, weakly reactive under certain conditions.

Despite the fact that nitrosyl chloride () is responsible for most of the nitric oxide in meat curing, considerable attention has been given to nitrous oxide formation.

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THE REACTION OF NITRIC OXIDE WITH MYOGLOBIN

Reversible cured colour formation before heat treatment.

Microorganisms present in the brine is capable of reducing nitrite to nitric oxide (NO), or by the surviving activity of enzyme systems of the muscle itself or by added reductants added through one of the intermediaries described above.  In order for cured colour development, the following reaction takes place.

The brine of a high pH is injected into the meat of a low pH.  Oxidising capacity of nitrites increases as the pH decreases.  Several views exist on how NO now reacts with the protein.

So, according to this view, the following takes place.  This new process has been suggested by the researchers Killday et al (1988).

1. oxidation of myoglobin to metmyoglobin by nitrite, which is itself reduced to nitric oxide (NO);
2. formation of the unobserved intermediate nitrosylmetmyoglobin;
3. rapid autoreduction to nitrolymyoglobin radical cation;
4. further reduction to nitrosylmyoglobin;
5. formation of nitrosylmychromogen and incorporation of a second mole of nitrite into the denatured protein on heating.  (Killday, et al, 1988)

NO + bright red Oxymyoglobin or myoglobin -> brown metMb 

a.   reacts with oxymyoglobin (, bright red, ) or myoglobin by oxidising it.

b.  and forms metmyoglobin (metMb, brown, )

In the process, the ion itself can be reduced to NO.  Myoglobin and oxymyoglobin () are oxidised to metMb (metMb, OH, brown, ) by nitrite.  The hem iron atom exists in either the ferrous (2+) state of the ferric (3+) state.  Lacking a covalent complex, either state can coordinate water.   In myoglobin,  is coordinated to the hem atom.  Adding nitrite () and a proton ()   —–>  OH coordinated to the heme atom, NO and  , forming metmyoglobin (metMb, brown, ).

These products can now combine with each other again to form an intermediary, called nitrosylmetmyoglobin ().

This reaction is represented as follows.

Nitrosylmetmyoglobin is unstable.  Over time, due to the influence of endogenous or exogenous reductants in the postmortem muscle tissue, it now autoreduces to the relatively stable the  form of nitrosylmyoglobin or nitrosomyoglobin (NOMb, cured colour, non-heated, ).

They suggest that nitrosylmetmyoglobin () is better described as an imidazole-centred protein radical which autoreduces to a nitrosylmyoglobin radical cation.

The reaction is represented as follows:

Møller and Skibsted (2001) also suggest that the sequence begins with the well known and observable oxidation by nitrite of oxymyoglobin (, bright red, ) or myoglobin to form metmyoglobin (metMb, brown, ). NO now reacts with the metmyoglobin to form nitrosylmyoglobin ().   It is then reduced by endogenous reductants such as NADH or by exogenous reductants such as ascorbate or erythorbate to yield .

They note the following problem with the alternative mechanism suggested by Killday et al (1988) which involves the autoreduction of the intermediate yielding an imidazole-centered protein radical that is, according to this view, reduced by electron donation from a reducing group in the protein.  They are not clear how this autoreduction will occur at low pH where the proximal histidine will be protonated and autoreduction will involve pentacoordinate NO-heme.  They note that at high pH, the hydroxide attack mechanism will dominate.  (Møller and Skibsted, 2001)

Irreversible cured colour formation without heat treatment

“Parma ham is traditionally produced using only sodium chloride without addition of nitrate or nitrite and develops a deep red colour, which is stable also on exposure to air. The identity of the pigment of Parma ham has not been established, but bacterial activity has been explored as responsible for transformation into nitrosylated heme pigments.  In one study, the stability of the pigment isolated from two different types of dry-cured ham (made with or without nitrite) was compared to that of the NO derivative of myoglobin formed by bacterial activity. Heme pigment from Parma ham made without nitrite was more stable against oxidation than the pigment from dry-cured ham with added nitrite.” (Møller and Skibsted, 2001)

“Heme pigments extracted from Parma ham and a bacterial (Staphylococcus xylosus) formed NO-heme derivative had similar spectral characteristics (UV/ vis spectra and ESR). ESR spectroscopy of heme pigment isolated from salami inoculated with bacteria had NO in a predominant pentacoordinate NOheme environment, whereas MbFeIINO, formed from nitrite and ascorbate, exclusively showed hexacoordinated iron, a difference which could be due to the decrease in pH during fermentation.”  (Møller and Skibsted, 2001)

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THERMAL TREATMENT

Before thermal treatment, the colour of cured meat is due to NOMb.  “Nitrosylmyoglobin is a ferrous mononitrosylheme complex in which the reduced iron atom is coordinated to four nitrogen atoms of the protoporphyrin-IX plane, one nitrogen atom of the proximal histidine residue of globin (fifth coordinate position) and a NO group (sixth coordinate position).  The NOMb pigment can be produced by the direct action of NO on a deoxygenated solution of Mb, but in conventional curing, it arises from the action of nitrite” as described in this article.  (Pegg, R. B. and Shahidi, F; 2000: 42)

“Upon thermal processing, globin denatures and detaches itself from the iron atom, and surrounds the hem moiety.  Nitrosylmyochromogen or nitrosylprotoheme is the pigment formed upon cooking and it confers the characteristic pink colour to cooked cured meats.”  (Pegg, R. B. and Shahidi, F; 2000: 42)

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SUMMARY OF CHEMICAL STATE, COLOUR and NAME – FERROUS AND FERRIC

FROM http://meat.tamu.edu/ansc-307-honors/meat-color/

Chemical state of myoglobin — Ferrous or Fe++ (covalent bonds)

Compound	Color	Name
:H2O	Purple	Reduced myoglobin or deoxymyoglobin
:O2	Red	Oxymyoglobin
:NO	Cured pink	Nitric oxide myoglobin
:CO	Red	Carboxymyoglobin

Chemical state of myoglobin — Ferric or Fe+++ (ionic bonds)

Compound	Color	Name
-CN	Red	Cyanmetmyoglobin
-OH	Brown	Metmyoglobin
-SH	Green	Sulfmyoglobin
-H2O2	Green	Choleglobin

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OVERALL IMPACT OF TEMPERATURE

Brine Temperature.

“One precaution in the handling of brines containing nitrite and erythorbate is to keep the temperature below 10°C. At higher temperatures, erythorbate will rapidly reduce nitrite to NO gas, which escapes from from brine injection, resulting in poor or no cured colour development in the cooked product.”  (AMSA, 2012: 8)

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IMPACT OF REDUCING AGENTS

The importance of reducing agents in meat curing systems have been known since the work of Haldane, et al (1897) when they studied the impact of nitrite on blood.  Others confirmed this such as Brooks (1937) and Keilin and Hartree (1937).  Reducing agents are needed to maintain the pigment in the ferrous or reduced form and to reduce nitrate to nitric oxide.

Oxidation Potential of Muscle Tissue

It was established very early that endogenous reductants are present in the muscle tissue. Brooks (1936, 1938), for example, 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).” (Cole, 1961)

Reducing Sugars

“Among the components of fresh tissue, Bender et al. (1958) found 2.1 percent reducing sugars (as glucose) on a dry weight basis.”  (Cole, 1961)  Examples of a reducing sugar are dextrose and lactose and a non-reducing sugar is sucrose.

Sulfhydryl Groups

“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.”  Free sulfhydryl groups are the strongest reducing groups released during the process of denaturing.  “These groups are normally tied up in many native proteins in intramolecular linkage.  As the protein molecule unfolds during denaturation, these linkages are broken and active sulfhydryl groups appear.  The denaturation is followed by coagulation, the sulfhydryl groups are progressively tied up again, this time in intermolecular linkage.  Thus, free sulfhydryl groups go through a maximum during the heat coagulation of many native proteins, including myosin and egg albumin.”  (Watts, et al,  1955)  

“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)

Exogenous Reductants

“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 Hollerbeck 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)

“The reduction of methemoglobin to hemoglobin by ascorbic acid was demonstrated by Gibson (1943) and was found to be catalyzed by iron and copper salts. Ivanova (1950) reported that both ascorbic acid and glutathione reduced methemoglobin to hemoglobin in vitro. Since nitrite and nitrate are oxidizing agents in acid solution while ascorbic acid is a reducing agent, the compatability 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 y0° C. were dependent on nitrite concentration when cysteine was the reductant, but not when ascorbic acid was the reductant.” (Cole, 1961)

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IMPACT OF TIME

We have seen that nitrite functions as an oxidising, reducing or nitrosylating agent which is the covalent incorporation of a nitric oxide moiety into another (usually organic) molecule.  (J.G. Sebranek, J.N. Bacus / Meat Science 77 (2007) 136–14)  In meat, nitrite can be converted into nitrous acid, nitric oxide, and nitrate.  When nitrite comes into contact with meat, the first reaction that takes place is that it acts as a strong oxidizing agent on myoglobin to forms metmyoglobin (iron in the heme group changes to – Fe³⁺ (ferric), from Fe²⁺ (ferrous) as in normal hemoglobin) with its brown colour.

The first visual effect of adding sodium nitrite to meat is, therefore, a colour change to brown.  In order for curing of meat to take place, it is, however, necessary for nitric oxide to be created.  This is a longer and much more complex process which is why time is required for curing to happen.  The starting point of many of these reactions to NO is the conversion of nitrite into nitrous acid and the hydration of nitrous acid is, itself, a time-consuming reaction.

It is normal to “rest” meat after injection and even tumbling for a minimum of 12 hours before it is hanged for smoking or filled into grids or moulds.  (see Restructuring)

In general, the warmer it is, the faster the curing reaction will take place.  It is also true that the smaller the particle size, the faster the curing will take place.  It should be kept in mind that temperature is probably the most important factor in managing bacterial growth.  It is therefore suggested that a cool temperature is maintained in the curing plant and that a special curing area should exist where the meat cures without any handling at a higher temperature before in is removed into a colder area again for hanging and or filling into grids.  (see Restructuring)

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THE IMPACT OF pH

Sebranek (1974) stated that greater loss of nitrite occurred in aqueous solutions at lower pH when held at room temperature. A small decrease in pH can be quite important. Fox et al. (1967) indicated that a pH decrease of 0.2 unit in meat will double the rate of colour formation due to nitrite-myoglobin interaction. Generally, as pH decreases from 6.5 to 5.5, cured colour development is more rapid and complete (Fox and Thomson, 1963). Residual nitrite that remains in the tissue after cure processing (including cooking) serves as a reservoir for nitric oxide for continued stabilisation of the colour pigment and other components of muscle.

Olsman and Krol (1972) and Olsman (1974) developed kinetic data describing nitrite depletion with respect to pH, however, at a low pH, the linearity of the depletion rate is lost because d log [HNO^] the change in the slope — with storage time progressively increases with decreasing pH. As a result, the kinetics of nitrite loss with respect to pH is between first and second order.

The idea of lowering pH to reduce residual nitrite has been developed and utilized in meat products in attempts to inhibit the formation of nitros amines (Goodfellow, 1979). A drastic reduction of residual nitrite was achieved by lowering the pH in a fermented sausage using a starter culture (Zaika et al., 1976). Another area where pH effects may become evident is in a variety of smoke applications. Sink and Hsu (1977) showed a lowering of residual nitrite in a liquid smoke dip process for frankfurters when the pH also was lowered. They suggested that phenolic compounds from smoke may contribute to lowering the pH of the products as well as the residual nitrite. The effects of smoke in nitrite reduction seem to be a combination of pH decrease and direct nitrosation of phenolic compounds (Knowles, 1974). Some chemical acidulants such as acetic acid, glucono-delta-lactone, citric acid and sodium acid pyrophosphate have been used to reduce pH (Goodfellow,1979). The van Slyke reaction (nitrous acid reacts with the alpha amino group) has been suggested as being responsible for nitrite
decomposition in the presence of acids (Bard and Townsend, 1971).

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MYOGLOBIN CONCENTRATION

One may think that the concentration of nitric oxide in meat is the only important requirement for good colour development, but this will be a mistake.  Equally important is the presence of myoglobin.

The main pigment in meat is myoglobin (Mb) and it is the concentration of myoglobin  (Mb) that determines the overall redness of meat and to a lesser extent by haemoglobin.    The greater the Mb level, the more intense the colour of the meat.  (Pegg, R. B. and Shahidi, F; 2000: 23, 24)

“Myoglobin is an iron- and oxygen-binding protein found in the muscle tissue of vertebrates in general and in almost all mammals. It is related to haemoglobin, which is the iron- and oxygen-binding protein in blood, specifically in the red blood cells.”  (Nelson DL, Cox MM; 2000: 206)  In humans, myoglobin is only found in the bloodstream after muscle injury.   It is the structure and chemistry of the iron atom that is the key to understanding the reactions and colour changes that Mb undergoes.  (Pegg, R. B. and Shahidi, F; 2000: 26)

“The concentration of myoglobin in meats is dependent upon the age and activity of the animal. (Millikan, 1939; Poel, 1949; Lawrie, 1950, 1953) Certain muscles appear to contain more myoglobin than do others. The myoglobin concentration was found to be higher in skeletal muscles than in cardiac muscles of dogs, horses (Drabkin, 1950), cattle and pigs (Watson, 1935; Lawrie, 1950). “The average myoglobin concentration of beef muscle is 3.7 mg per gram while pork muscle averages 0.79 mg per gram of meat for light muscle and 1.44 mg per gram for dark muscle.”  (Cole, Morton Sylvan, 1961: 2)

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BACTERIAL/ ENZYMATIC CREATION OF CURED COLOUR

Can meat be cured without nitrite?  There is a long and a short answer to this.  The short answer is that if you want to achieve curing in a short time period and not use sodium nitrate or nitrite, either directly or indirectly through the use of plant juices that is replete with nitrate or nitrite (after bacterial reduction, done under controlled conditions by the producers of these juices), curing will not take place.  If you use only sodium chloride, what you will have is salted meat and managing the risk involved in such a product is tricky.  (See Clostridium Botulinum)  For meat curing to take place, nitric oxide is required.

The long answer is that it is possible since the muscle itself contains various sources of nitric oxide but this requires long curing time.  Often as long as 18 months or even longer.  The main way of producing nitric oxide has been found in the case to be through bacterial action through enzymatic mechanisms.  In this section, we briefly look at these.

Introduction to enzymes and bacteria – history and important characteristics

– history 

One of the most fascinating fields of research is bacteria and the enzymes they produce.  It was Anselme Payen and Jean-François Persoz, chemists at a French sugar factory who discovered the first enzyme, diastase, in 1833. They extracted it from a malt solution.

The Swedish chemist Jon Jakob Berzelius in 1835 called the chemical action of enzymes catalytic.  “It was not until 1926, however, that the first enzyme was obtained in pure form, a feat accomplished by James B. Sumner of Cornell University. Sumner was able to isolate and crystallise the enzyme urease from the jack bean. His work was to earn him the 1947 Nobel Prize.”  (www.worthington-biochem.com)

“John H. Northrop and Wendell M. Stanley of the Rockefeller Institute for Medical Research shared the 1947 Nobel Prize with Sumner. They discovered a complex procedure for isolating pepsin. This precipitation technique devised by Northrop and Stanley has been used to crystallise several enzymes.”  (www.worthington-biochem.com)

– important characteristics

An enzyme accelerates the rate of reaction in which different substrates are converted to products through the formation of what is called and “enzyme-substrate complex.”  An enzyme is very specific in terms of its activity. Generally speaking, each enzyme will speed up (catalyses) only one type of reaction and it will only do this for one type of substrate. This highly specific mechanism is often referred to as a “lock and key” mechanism. Enzymes are in other words highly specific and discriminate between slightly different substrate molecules. Another important feature of enzymes is that their function as a catalyst is at an optimal level over a narrow range of temperature, ionic strength and pH. (www.natureclean.com)

Bacteria are single-celled living organisms.  They are typically enclosed in a rigid cell wall with a plasma membrane.  Internally, they do not have well-defined organelles such as a nucleus.  Bacteria have the ability to produce many different types of enzymes.

They respond to their environment. In general, they can produce enzymes that degrade a wide variety of organic materials such as fats, oils, cellulose, xylan, proteins, and starches. The materials listed are all polymers that must be reacted with more than one type of enzyme to be efficiently degraded to their basic building blocks.  To accomplish this, a specific “team” of enzymes are provided to attack each type of polymer. “For example, there are three different classes of enzymes (endocellulases, exocellulases, cellobiohydrolases) that are required to degrade a cellulose polymer into basic glucose units. All three types of enzymes are referred to as cellulases, but each class attacks a specific structure or substructure of the polymer. Acting individually, none of the cellulases is capable of efficiently degrading the polymer. Bacteria can produce the complete “team” of enzymes that are necessary to degrade and consume the organic materials present in their environment at any given time. Moreover, bacteria can produce multiple “teams” at the same time.”  (www.natureclean.com)

A further important feature of bacteria’s enzyme production is that it begins as soon as the bacteria begin to grow. “The cells must obtain nutrients from their surroundings, so they secrete enzymes to degrade the available food. The quantities of enzymes produced vary depending on the bacterial species and the culture conditions (e.g., nutrients, temperature, and pH) and growth rate. Hydrolytic enzymes such as proteases, amylases, and cellulases, etc. are produced in the range of milligrammes per litre to grammes per litre.”  (www.natureclean.com)

These particular conditions required for bacteria to multiply is equally important.  Bacteria require a particular environment to thrive closely associated with temperature and pH.

Contrary to bacteria, enzymes are not living organisms.  They have a limited half-life (minutes to days, depending on conditions). Like bacteria, they have optimal and less favourable conditions which determine the efficacy of their function.  “They are proteins that are biodegradable and are subject to damage by other enzymes (proteases), chemicals, and extremes of pH and temperature. An important difference between enzyme-based products and bacterial products is that the enzymes can’t repair themselves or reproduce. Living bacteria, however, produce fresh enzymes on a continuous basis and can bounce back following mild environmental insults.”  (www.natureclean.com)

Bacterial/ enzymatic creation of NO–Mb (Fe²⁺)

NO is responsible for the colour formation in both nitrite cured meat and meat that has been cured with new systems without nitrite.  Morita et al. found that NO formation in such nitrite-free system is achieved from L-arginine due to nitric oxide synthase (NOS) in either Staphylococci or Lactobacilli.  (Gasasira, et al, 2013)  The nitric oxide producing enzyme in cells is called, nitric oxide synthase (NOS)”. It converts l-arginine into l-citrulline and nitric oxide (NO). (www.sciencedirect.com)

This introduces us to the amazing world of l-arginine.  Arginine is an amino acid.  “Amino acids are a basic group of structural and biologically active organic compounds.  Amino acids are identified by the presence of amine ( ) and carboxylic acid (-COOH) functional groups.”  They are the basic building block of protein.  The only thing more abundant in human muscle, cells, tissue and organs is water.  The French chemists Louis-Nicolas Vauquelin and Pierre Jean Robiquet isolated a sample from asparagus that they named ‘asparagine,’ thus, identifying the first amino acid. (Wallach, J.;2014)

Nine of the amino acids are considered as essential.  These can not be synthesised or made by the human body and must be consumed daily or else it will lead to disease.  These nine are histidine, lysine, isoleucine, leucine, methionine, henylalanine, valine, threonine, tryptophan. A second class of amino acids is conditional amino acids meaning that they are usually not essential except in a time of stress or illness.  These are cysteine, glutamine, tyrosine, glycine, ornithine, proline, and serine and the amino acid of interest, arginine.

Arginine was isolated in 1886 by the Swiss chemist Ernst Schultze from a lupin seedling.  The name arginine comes from the fact that early researchers got the arginine from an amino-acid mix using silver, from an arginine-silver compound.  Silver in Latin is Argentum and from there the word arginine.  It is more specifically called L-Arginine and the L refers to the fact that it is a Laevorotatory, i.e. formed anti-clockwise ( having the property of rotating the plane of a polarised light ray to the left, i.e. anticlockwise facing the oncoming radiation).  (Jester, F.  2015)

The functionality of arginine includes a key role in complete and effective cell division, wound healing, facilitating the biological use and the excretion of ammonia, immune system support, and the availability of storage hormones.  It is an important dietary requirement since arginine is required for the synthesis or the production of nitric oxide (NO), the reduction of healing time following trauma, and is particularly useful for bone trauma; it reduces blood pressure and increases blood flow through obstructed blood vessels. Two well-known uses of arginine are in toothpaste that relieves dental pain and in treating erectile dysfunction. (Wallach; 2014.)  From the list above, I want to draw attention to the role it plays in managing blood flow and blood pressure, all related to the arteries.

By looking at the functional benefits of arginine in the body, one cal already deduces that nitric oxide is the component in arginine that facilitates many of these benefits.  It achieves this dramatic result by widening the arteries which increase the efficiency of the heart and lowers the blood pressure.  L-Arginine turned out to be the precursor for the formation of nitric oxide (NO).  (Jester, F.  2015)

In fact, the nutrient amino acid L-arginine is the direct precursor for the only essential way that NO is produced in the body.   Arginine circulates in the blood through the body and the enzyme, nitric oxide synthase, “controls a reaction in which a terminal nitrogen atom of arginine is combined with an oxygen molecule to form NO and the amino acid L-citrulline.”   (Block, W)

We can now restate the discovery of Morita et al. with the benefit of important background.  They found that NO formation in nitrite-free curing systems is achieved from L-arginine due to nitric oxide synthase (NOS) in either Staphylococci or Lactobacilli.  (Gasasira, et al, 2013) We have seen that the nitric oxide producing enzyme in cells is called, nitric oxide synthase (NOS) and converts l-arginine into l-citrulline and nitric oxide (NO). (www.sciencedirect.com)

“Arihara et al. found that Lactobacillus fermentumJCM1173 can transform Mb (Fe³⁺) (Mb, Myoglobin) into cured meat pigment NO-Mb (Fe²⁺) . Morita studied 10 L. fermentum strains and found that all of them can transform Mb (Fe³⁺) into bright red NO–Mb (Fe²⁺) in the MRS (Man–Rogosa–Sharpe) culture medium and L. fermentum IFO3956 had an outstanding transforming ability, which can utilize NOS to form NO from L-arginine . Another study on production of cured meat color in nitrite-free sausages by L. fermentum showed that nitrosylmyoglobin could be generated when L. fermentum AS1.1880 was inoculated into the meat batter, and the formation of a characteristic pink color with an intensity comparable to that in nitrite-cured sausage can be achieved using 10⁸ CFU/g of the culture.  (www.sciencedirect.com)

This is without any question one of the most exciting fronteirs of meat curing.

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CONCLUSION

The formation of the cured colour in meat is the result of highly complex chemical processes.  We do not fully understand it.

My son is 19 and we have to decide where he is going to study next year.  The plan is that while he studies meat science, he learns the practicalities of the meat business by working part time in our factory and at an abattoir.  Franz Loibl, a master butcher, suggested to us that Tristan first learns deboning before he goes into sausage production and curing.  He told me, “not to become an expert deboner, but to know and understand how a carcass is put together and the different muscles.”

This article fulfils the same function to me.  It is not to know everything there is to know about every single mechanism and reaction involved in curing and become a masters student in meat science and biochemistry but to know that these processes exist.  To be aware of its complexity and to have an appreciation for the impact of time, temperature, pH, micro, particle size, myoglobin concentration and a basic understanding of reduction and oxidation chemistry.  Writing about it is the best way for me to learn and become more familiar with the field.  It keeps me focused, working through the different aspects of the subject at hand and exposing us to the cutting edge research of enzymatic and bacterial controlled curing systems.  In the final analysis, I am loving it!  Discovering these things is part of what has become to me, the greatest journey on Earth!

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(c)  eben van tonder

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Random research notes to be incorporated at a later stage:

1. Notes on the chemistry of meat

The bright red colour of fresh meat is due to oxymyoglobin (MbO2).  Mb has a great affinity for O2.  It is the reaction of myoglobin with O2 that results in oxymyoglobin (MbO2) and produce the red colour which the consumer associate with freshness.  (Pegg, R. B. and Shahidi, F; 2000: 31)

Myoglobin is able to store O2 in the blood to transport it through the body and to release it where it is needed.  This means that it is able to react rapidly with O2 and reversibly.  The surface of the meat “blooms to a bright red colour within minutes of exposure to air.”  (Pegg, R. B. and Shahidi, F; 2000: 31)

We, however, know that this a short-lived phenomenon. “MbO2’s stability depends on a continuing supply of O2 because the enzymes involved in oxidative metabolism rapidly use the available O2.”  With time, the small layer of MbO2 present on the surface of the meat propagates downward, but the depth to which O2 diffuses depends on several factors, such as the activity of oxygen-utilizing enzymes (i.e. O2 consumption rate of the meat), temperature, pH, and external O2 pressure.  Consequently, maintaining the temperature of meat near freezing point minimises the rate of  enzyme activity and O2 utilisation and helps maintaining a bright red colour for the maximum possible time.”  (Pegg, R. B. and Shahidi, F; 2000: 31)

“In contrast, the interior tissue of meat is purple-red in colour.  This the colour of Mb, sometimes called deoxy-Mb, and the colour persists as long as reductants generated within the cells by enzyme activity are available.  When these substances are depleted, the hem iron is oxidized to the ferric state (Fe 3+ from ferrous, Fe 2+).”  (Pegg, R. B. and Shahidi, F; 2000: 31)

“The brown pigment formed, which is characteristic of the colour of meat left standing for a period of time, is called metmyoglobin (metMb).  It is generated by removal of a superoxide anion from the hematin and its replacement by a water molecule gives a high-spin ferric hematin.    The ferric ion, unlike its ferrous counterpart, has a high nuclear charge and does not engage in strong pi bonding.    Therefore, metMb is unable to form an oxygen adduct.”  (Pegg, R. B. and Shahidi, F; 2000: 31)

“In fresh meat there is a dynamic cycle such that in the presence of O2, the three pigments Mb, MbO2, and metMb are constantly interconverted; all forms are in equilibrium with one another.  Care is exercised by the retailer to reduce the likelihood of metMb formation, as its presence downgrades the quality of fresh meat.  When metMb is denatured by thermal processing, meat remains brown in colour, but this denatured pigment can be oxidized further to form yellow, green or colourless porphyrin-derived substances by bacterial action or photochemical oxidation.”  (Pegg, R. B. and Shahidi, F; 2000: 32)

The best way to view this is to see it represented in a diagram.

In other words, it turns into oxymyoglobin and a reductant in the meat or in the brine reduces it to either myoglobin or oxymyoglobin.  When the reducing agents have been depleted, the meat colour remains brown.

2. The larger the Ka the greater the ionization of the acid, the stronger it is, the lower the pH. In the other words, pH inversely proportional to Ka. (utdallas.edu)

3. Kb – the base ionization constant is the equilibrium constant for ionization of a base in an aqueous solution. Chemical reaction:

(utdallas.edu)

4. pOH: The pOH of an aqueous solution, which is related to the pH, can be determined by the following equation: pOH=−log[OH−] (chemwiki.ucdavis.edu)