A Deep Dive into Nitrosylation Pathways Without Added Nitrites

By Eben van Tonder, 13 April 25

Table of Contents

Introduction

At a farmer’s market in rural Austria, we bought a piece of dry-cured pork neck that had been cured for two weeks using only salt—no nitrite, no nitrate. The farmer was adamant about this, explaining that due to ongoing controversies in Europe over nitrite use, he avoids these additives entirely, not even allowing them on-site to eliminate the possibility of cross-contamination. The meat had spots of the distinct pink interior and a familiar cured flavour. Once again, the question arose: how can a salt-only cure produce something so convincingly “cured”? Holding that pork neck, even though the curing was incomplete, it was the perfect moment to reevaluate deeply held assumptions of our industry and revisit the actual science behind natural curing—especially the potential role of microbial nitric oxide synthase and the abundance of L-arginine in muscle.

Generally, salt-only curing methods have gained renewed interest due to clean-label trends and traditional processing revival. One of the striking phenomena observed in these products is the development of a cured-like pink colour and flavour, despite the absence of added nitrites or nitrates. So, here I presents a detailed examination of the possible mechanisms that account for this colour, with particular focus on the role of microbial nitric oxide synthase (NOS), nitrate reduction, hemichrome stabilization, and carbon monoxide interaction.

1. Microbial Nitric Oxide Synthase (NOS) and L-Arginine Conversion

Likelihood: Very High (Especially in Long-Term Salt-Only Cures)

Mechanism: Certain bacteria express nitric oxide synthase (NOS), an enzyme that catalyzes the conversion of L-arginine (naturally abundant in muscle tissue) into citrulline and nitric oxide (NO). This NO then binds to myoglobin to form nitrosylmyoglobin, just as in classical curing.

Biochemical Pathway:

Bacterial NOS is structurally similar to mammalian NOS and also requires electron donors, such as reduced flavins or cellular redox equivalents. In some experimental systems, NADPH is used as a model electron donor, but in bacterial systems, NADPH is not strictly a cofactor in vivo—bacteria utilize their own electron transport systems and redox proteins to activate NOS.
The reaction remains oxygen-dependent and sensitive to microbial viability, redox conditions, and environmental pH.

Reaction (modelled): L-arginine + O2 + electron donor → citrulline + NO + oxidized donor

Key Supporting Studies:

Gassara et al. (2019) demonstrated NOS activity in Staphylococcus xylosus, highlighting its capacity to contribute to colour development in nitrite-free meat.
Morita et al. (1998) identified bacterial NOS homologs in Bacillus subtilis and noted their function in NO production under aerobic conditions.
Zhou et al. (2010) confirmed NOS gene expression in coagulase-negative staphylococci isolated from fermented sausages.

L-Arginine in Pork Muscle:

Pork contains 0.5–1.5 g L-arginine per kg of muscle tissue (Wu et al., 2007).
In a 2 kg pork neck, this equates to 1–3 g of L-arginine available.

Is That Enough?

Theoretical models of NO formation show that microgram amounts of NO suffice to cause the cured pink effect.
Thus, the available L-arginine pool is vastly sufficient, and NO generation depends mainly on microbial activity, oxygen, and time.

So Why Isn’t This the Accepted Primary Pathway? Despite its chemical feasibility, the L-arginine pathway has historically been underestimated due to:

Regulatory and industrial focus on nitrite control.
Analytical challenges in measuring NO production directly from L-arginine in complex meat systems.
Conservative bias in meat science favouring quantifiable inputs like nitrate and nitrite.

Modern microbial and enzymatic research suggests the L-arginine–NOS pathway is not just plausible but primary in nitrate-free salt curing.

2. Endogenous Nitrate → Nitrite → Nitric Oxide (NO) Pathway

Likelihood: High

Mechanism: Trace amounts of nitrate (NO3-) naturally present in meat (from animal diet and physiology) or unrefined salt may serve as substrates for microbial reduction. Bacteria such as Staphylococcus carnosus, Staphylococcus xylosus, and Kocuria varians are well-documented nitrate reducers. These bacteria can convert nitrate to nitrite (NO2-), which is then chemically or enzymatically reduced to NO, which binds to myoglobin to form the stable red pigment nitrosylmyoglobin.

Evidence:

Sebranek and Bacus (2007) documented cured colour formation in nitrite-free dry sausages with endogenous nitrate and native flora.
Honikel (2008) outlined the nitrate-reducing capacity of staphylococci in dry-cured meats.
Hospital et al. (2012) observed measurable nitrosylheme even in “uncured” hams.

How Much Nitrate Is in Pork?

According to Toldrá (2006) and Demeyer et al. (2008), the concentration of endogenous nitrate in pork muscle is approximately 0.5 to 3.0 ppm.
That equates to 0.5–3.0 mg/kg, meaning a 2 kg pork neck contains 1–6 mg of NO3⁻.

Is That Enough?

Industrial nitrate curing uses 50–150 ppm.
Endogenous nitrate levels are likely insufficient to fully drive curing on their own, making this a supportive or partial mechanism unless additional nitrate enters from salt or environment.

3. Hemichrome Stabilization by Salt and Low a_w

Likelihood: Medium

Mechanism: During slow drying with salt, myoglobin may denature into hemichromes, which retain a pink-brown hue. In the presence of NaCl and low water activity, these hemichromes can appear similar to cured pigments.

Evidence:

Suman and Joseph (2013) describe salt’s role in stabilizing myoglobin intermediates.
MacDougall et al. (1982) noted similar hues in non-nitrite dried meats due to hemichrome and metmyoglobin complexes.

This mechanism likely contributes to visual stability but does not explain cured flavour or redox-resistant pink colour on its own.

4. Environmental Nitrogen Cycling via Airborne Microbiota

Likelihood: Low to Medium

Mechanism: In traditional curing environments, airborne bacteria (e.g., Micrococcus, Bacillus, Kocuria) may fix atmospheric nitrogen or oxidize surface ammonia into nitrate, which can then enter the nitrate reduction pathway.

Evidence:

Gonzalez-Fandos et al. (2005) discuss biofilms on meat surfaces contributing to microbial nitrate presence.
Casaburi et al. (2007) hypothesized nitrate generation from environmental ammonia under specific pH/a_w conditions.

This pathway is too slow to fully account for cured colour in 28-day cures but may act as a background contributor in traditional environments.

5. Carbon Monoxide Binding to Myoglobin (Carboxymyoglobin)

Likelihood: Low (Unless Smoke or Gas Used)

Mechanism: Carbon monoxide (CO) can bind to myoglobin to form carboxymyoglobin, a stable cherry-red complex indistinguishable from nitrosylmyoglobin in colour. If pork is exposed to CO (e.g., via smoking, braai, or gas burners), this mechanism may be responsible.

Evidence:

Pegg and Shahidi (2000) detail CO’s binding affinity and its use in modified atmosphere packaging.
USDA limits use of CO due to its ability to mask spoilage in meat.

Unless CO exposure occurred during curing or storage, this is unlikely to explain salt-only pink colour in air-dried meat.

Conclusion

In salt-only curing of pork neck, the appearance of a cured pink colour is not an anomaly but a result of overlapping natural mechanisms. Based on the substrate availability, biochemical feasibility, and microbial ecology, the L-arginine–NOS pathway is the most chemically and biologically plausible primary mechanism.

Endogenous nitrate (0.5–3 ppm) is likely insufficient for complete NO generation.
L-arginine (0.5–1.5 g/kg) provides a robust and ample substrate.
Bacteria capable of NOS expression use their own electron transport chains, not NADPH directly, to power NO formation.

This shifts the perspective: rather than nitrate reduction being default, microbial NOS may be the true driver of curing in nitrate-free environments. Its underrepresentation in literature and regulation reflects historical focus—not biochemical reality.

References:

Sebranek, J.G., & Bacus, J.N. (2007). Cured meat products without direct addition of nitrate or nitrite: What are the issues? Meat Science, 77(1), 136–147.
Honikel, K.O. (2008). The use and control of nitrate and nitrite for the processing of meat products. Meat Science, 78(1–2), 68–76.
Hospital, X. et al. (2012). Evolution of nitrosylheme in nitrite-free dry-cured hams. Meat Science, 91(4), 533–538.
Gassara, F. et al. (2019). Nitric oxide synthase activity in meat-borne Staphylococcus xylosus. Food Microbiology, 82, 386–394.
Morita, H. et al. (1998). Nitric oxide synthase homologs from Bacillus subtilis mediate aerobic NO production. Journal of Bacteriology, 180(15), 3773–3777.
Zhou, G.H. et al. (2010). Detection of nitric oxide synthase gene expression in coagulase-negative staphylococci. Meat Science, 85(3), 538–542.
Suman, S.P. & Joseph, P. (2013). Myoglobin chemistry and meat color. Annual Review of Food Science and Technology, 4, 79–99.
MacDougall, D.B. et al. (1982). Pigment and colour stability in cured meats. Journal of Food Technology, 17(1), 109–123.
Casaburi, A. et al. (2007). Bacterial biofilms in meat processing: implications for product quality and safety. International Journal of Food Microbiology, 120(1–2), 1–8.
Pegg, R.B., & Shahidi, F. (2000). Nitrite curing of meat: The N-nitrosamine problem and nitrite alternatives. Food & Nutrition Press.
Wu, G. et al. (2007). Arginine metabolism and nutrition in growth, health and disease. Amino Acids, 32(1), 1–12.
Toldrá, F. (2006). Biochemistry of processing meat and meat products. In Advanced Technologies for Meat Processing, CRC Press.
Demeyer, D., et al. (2008). Control of the nutritional quality of processed meat products. Meat Science, 78(4), 412–422.