Lysine, Proline, and Hydroxyproline Deamination: Pathways for Nitrosamine Formation in Food and Digestion (Considering Vitamin C/Erythorbate & 120 ppm Nitrite Limit)

Notes By Eben van Tonder, 30.1.25

Table of Contents

Introduction: Lysine, Proline, Hydroxyproline, and the Hallstatt Curing Method

In modern meat curing, regulations mandate the use of antioxidants such as Vitamin C (ascorbic acid) or sodium erythorbate to inhibit nitrosamine formation. These antioxidants reduce residual nitrite (NO₂⁻) and prevent secondary amines from reacting with nitrosating agents. Despite this, nitrosamine risks still exist under certain conditions, especially when:

Temperatures exceed 200°C (leading to thermal deamination of amino acids).
Nitrite levels approach 120 ppm, the maximum permitted concentration in many regulations.
Amino acids like lysine, proline, and hydroxyproline undergo enzymatic or bacterial deamination, forming ammonia and secondary amines.

The Hallstatt Curing Method and Nitrosamine Formation

The Hallstatt curing method, as explored in EarthwormExpress, suggests that enzymatic and bacterial deamination can generate ammonia, which, in the presence of bacteria, can be converted to nitrite. This raises the question of whether this process contributes to natural curing, but it also introduces the potential for nitrosamine formation if secondary amines are present.

Relevance to Skin-Containing Sausages
Products like Krainerwurst, South African “Russian” sausages, and Australian Kransky incorporate high collagen content from skin, increasing proline and hydroxyproline levels. Since proline is a direct precursor to nitrosamines, evaluating the real nitrosamine risk in these products—with and without Vitamin C/erythorbate—is critical.

1. Pathways of Lysine, Proline, and Hydroxyproline Deamination Leading to Secondary AminesThermal Deamination (Above 200°C)

– When heated above 200°C, lysine, proline, and hydroxyproline undergo oxidative deamination, forming amine derivatives that can become nitrosamine precursors.

Lysine:
- Produces pyrrolidine, a precursor for N-nitrosopyrrolidine (NPYR).
Proline:
- Has an intrinsic secondary amine (-NH-) and directly forms nitrosamines when exposed to nitrite.
Hydroxyproline:
- Can inhibit NPYR formation, reducing nitrosamine risk.

💡 Effect of Vitamin C/Erythorbate:
– Vitamin C/erythorbate reduces residual NO₂⁻, lowering nitrosamine risk.
– Converts NO₂⁻ to NO, limiting nitrosating agents.

– Risk Assessment (Thermal Processing & Nitrosamines)

Without Vitamin C/Erythorbate: High nitrosamine risk, especially with proline.
With Vitamin C/Erythorbate: Reduced risk due to controlled NO₂⁻ conversion.

Enzymatic or Acidic Deamination (Stomach pH ~2-3, Body Temp ~37°C)

– Lysine can decarboxylate to form cadaverine, which reacts with nitrites to form nitrosamines.
– Proline and hydroxyproline are less prone to this reaction in the stomach.

💡 Effect of Vitamin C/Erythorbate:
– Limits stomach nitrosation by reducing NO₂⁻ availability.
– Prevents secondary amines from reacting with nitrites.

– Risk Assessment (Digestion & Nitrosamines)

Without Vitamin C/Erythorbate: Moderate risk in the stomach if nitrites are present.
With Vitamin C/Erythorbate: Low risk due to nitrite inhibition.

2. Nitrosamine Formation in Skin-Containing Sausages (Krainerwurst, Russian, Kransky)

– Krainerwurst, South African Russian, and Kransky contain high collagen content, which means:
– Proline levels are elevated, increasing NPYR formation risk when heated.
– Nitrite concentrations are capped at 120 ppm, meaning limited NO₂⁻ is available for nitrosation.

💡 Effect of Vitamin C/Erythorbate:
– Prevents nitrosation of proline, significantly reducing risk.
– Faster nitrite conversion ensures safer cured products.

– Risk Assessment (Collagen-Rich Sausages & Nitrosamines)

Without Vitamin C/Erythorbate: Moderate risk if cooked at high temperatures (>180°C).
With Vitamin C/Erythorbate: Low risk, especially if not fried excessively.

3. Bacon Frying and Nitrosamine Formation

– Bacon frying (180–220°C) leads to lysine and proline deamination, increasing nitrosamine potential.
– Vitamin C/Erythorbate lowers residual nitrite, reducing nitrosamine formation.

💡 Effect of Vitamin C/Erythorbate:
– Reduces NPYR, NDMA, and NTHZ formation in fried bacon.

– Risk Assessment (Bacon Frying & Nitrosamines)

Without Vitamin C/Erythorbate: High nitrosamine risk due to heat and nitrite.
With Vitamin C/Erythorbate: Moderate risk, but frying at lower temperatures (150-170°C) is still recommended.

4. Nitrosamine Risks in Non-Fried Ham (Sandwiches, Roasted Ham)

– If heated (120-160°C, e.g., a roasted ham sandwich), no significant thermal deamination occurs.
– Residual nitrite can still react in the stomach, forming some nitrosamines.

💡 Effect of Vitamin C/Erythorbate:
– Significantly reduces in vivo nitrosamine formation by neutralizing NO₂⁻.

– Risk Assessment (Non-Fried Ham & Nitrosamines)

Without Vitamin C/Erythorbate: Moderate risk, especially in stomach conditions.
With Vitamin C/Erythorbate: Low risk, particularly when consumed with fruits or vegetables.

5. Nitrosamine Formation During Curing (With 120 ppm Nitrite Limit)

– Nitrite limits (≤120 ppm) already reduce nitrosamine potential.
– Vitamin C/Erythorbate further reduces NO₂⁻, ensuring nitrosamines remain minimal.

– Risk Assessment (Curing Process & Nitrosamines)

Without Vitamin C/Erythorbate: Low risk, unless cooked at very high temperatures.
With Vitamin C/Erythorbate: Minimal risk, even in cooked products.

Conclusion: Evaluating the Real Risk of Nitrosamine Formation

– Skin-containing sausages (Krainerwurst, Russian, Kransky) have higher proline content, increasing nitrosamine potential.
– However, Vitamin C or erythorbate significantly reduces nitrosamine risk by limiting NO₂⁻ reactivity.
– Frying at high temperatures (>200°C) still poses a risk, but proper cooking techniques further reduce exposure.

Key Takeaways:
– With Vitamin C/Erythorbate, nitrosamine formation is significantly reduced.
– Nitrite limits (120 ppm) are effective in controlling risk.
– For bacon, avoid excessive frying (>200°C).
– For hams and sausages, combining with fresh vegetables (Vitamin C) provides further protection.

Final Verdict: The real risk of nitrosamine formation is minimal with modern regulations, as long as cooking temperatures are controlled. But the fact that temperature should be controlled is also true of all cooking!