Natural Nitric Oxide Curing in Meat: Enzymatic Pathways Using L-Arginine and NOS-Expressing Bacteria

By Eben van Tonder, 14 April 2025

Natural Nitric Oxide Curing in Meat: Enzymatic Pathways Using L-Arginine and NOS-Expressing Bacteria

Harnessing Microbial Nitric Oxide Synthase (NOS) Activity

Introduction

This paper builds on field observations made after purchasing a pork neck (Nacken) at a farmer’s market in Austria. The product had been cured using salt only and dried for two weeks, yet exhibited clear signs of successful curing, including a uniform pink interior and characteristic aroma. However, it became apparent that while curing had occurred, the process was not fully completed and was quickly reversed when the cut was exposed to oxygen. This prompted further investigation into the underlying mechanisms, with a specific focus on the L-arginine pathway as the most probable driver of nitric oxide (NO) formation in salt-only cured meat. (A Deep Dive into Nitrosylation Pathways Without Added Nitrites)

The work presented here complements and extends findings previously published on Earthworm Express in the article A Deep Dive into Nitrosylation Pathways Without Added Nitrites (EarthwormExpress.com), providing a mechanistic explanation for the variability observed in salt-only curing outcomes. In particular, this paper elucidates why salt-only curing often results in incomplete or reversible curing and highlights the central role of microbial nitric oxide synthase (NOS) activity in producing and stabilising NO-myoglobin complexes.

A key feature of the enzymatic curing process is its dependency on oxygen. Most known microbial NOS systems, particularly those expressed by Staphylococcus carnosus and Staphylococcus xylosus, require at least microaerobic conditions to catalyse the conversion of L-arginine to NO. Consequently, anaerobic microenvironments—either caused by excessive moisture, poor airflow, or early colonisation by facultative anaerobes—may inhibit curing or cause its reversal. This interplay between salt, microbial ecology, oxygen availability, and enzymatic potential is central to understanding when and why natural curing succeeds.

This paper therefore presents both a protocol and an explanation: it outlines the necessary conditions to optimise the NOS-L-arginine pathway and, in doing so, explains why salt-only dry curing may work inconsistently unless these specific microbial and environmental parameters are met.

Raw Material Selection

Pork neck (Nacken) is a suitable choice due to its high myoglobin content, marbling, and moisture retention—factors essential for supporting bacterial growth and NO-myoglobin formation. Freshness is critical; initial microbial populations influence subsequent flora dynamics. According to Bredie et al. (2020), spoilage bacteria present at the start can suppress desirable flora via nutrient competition and inhibitory metabolite production. An initial pH above 5.6 is preferable to ensure a hospitable environment for NOS-expressing bacteria (Wickramasinghe et al., 2019).

Salt and Sugar Levels

A salt concentration of 2.5–3.0% NaCl is recommended. This range maintains microbial stability while permitting the growth of beneficial flora (Honikel, 2008). Inclusion of dextrose at 0.3–0.8% supports lactic acid bacteria and Staphylococcus spp., enhancing microbial NOS activity (Talon et al., 2004).

pH Control and Drying Environment

Avoid rapid acidification in the initial curing phase, as fast pH drops can curtail NOS expression. Ideal environmental parameters begin at 10–14°C with 85–90% relative humidity (RH). After 3–5 days, gradually reduce RH to 75–80% and temperature to around 12°C. This supports prolonged metabolic activity, essential for stable NO production (Leroy et al., 2006).

Starter Cultures and Microbial Strategy

Where philosophy permits, inoculating with Staphylococcus carnosus or Staphylococcus xylosus at 10⁶ CFU/g ensures reliable NOS expression (Gaspar et al., 2018). These may be incorporated via brine or dry rub. If relying on wild flora, meat should be sourced from outdoor-raised animals, and antimicrobial interventions (e.g., acid rinses) should be avoided. Unrefined solar-dried sea salt may introduce trace nitrate and support flora diversity (Parolari et al., 2017).

Drying Time and Airflow

Drying should last 14 to 28 days depending on product size and humidity control. Gentle airflow is necessary to avoid case hardening and to promote uniform moisture loss, allowing sustained microbial activity throughout the interior (Toldrá & Reig, 2011).

Natural Colour Stabilisers

To enhance NO-myoglobin stability, natural antioxidants such as rosemary extract or ascorbic acid may be used at up to 200 ppm. These stabilisers help maintain the cured colour without disrupting NOS pathways (Riel et al., 2020).

Key Indicators of Success

  • Uniform pink-red internal colour (NO-myoglobin presence).
  • Mild, tangy aroma indicative of lactic fermentation.
  • Firm, pliable texture without hardened surfaces.
  • Absence of grey or greenish discolouration.

Spoilage Organisms and Inhibition of Natural NO Pathways

Spoilage bacteria pose a critical threat to the success of natural L-arginine curing. Key mechanisms include:

  1. Microbial Competition: Pseudomonas, Enterobacteriaceae, and Clostridium species can outcompete beneficial flora, altering pH and secreting antagonistic compounds (Lorenzo et al., 2014).
  2. L-arginine Depletion: Arginase or deaminase enzymes from spoilage organisms divert L-arginine from the NO pathway (Domínguez et al., 2016).
  3. Putrefactive Compound Accumulation: Compounds like cadaverine and H₂S destabilise NO-myoglobin and lead to undesirable colours and odours (Talon et al., 2004).
  4. Anaerobic Microenvironments: Rapid oxygen depletion by spoilage flora can inhibit oxygen-dependent NOS enzymes in Staphylococcus spp. (Gaspar et al., 2018).
  5. Enzyme Inhibitors and pH Drift: Proteases, lipases, and rapid pH changes can impair NOS activity and block NO formation (Wickramasinghe et al., 2019).

Conclusion

Microbial NOS-mediated L-arginine curing is a promising clean-label strategy for producing cured meat without direct nitrite addition. However, its success depends on a synergistic control of microbial ecology, biochemical substrates, and physical conditions. Pork neck proves an excellent test material, but results are contingent upon the exclusion of spoilage organisms, balanced salt and sugar addition, gradual drying, and careful monitoring of environmental variables.

References

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