By Eben van Tonder, 2 Feb 2025

Introduction

Cured meat colour is normally attributed to nitrosylmyoglobin, the pigment formed when nitrite derived nitric oxide binds the ferrous iron of myoglobin. Parma ham receives no added nitrite or nitrate. It is cured with sea salt alone, over a minimum of twelve months and often longer. Its stable red colour therefore cannot come from nitrosylmyoglobin. Wakamatsu, Nishimura and Hattori identified the actual pigment in 2004. It is zinc protoporphyrin IX, abbreviated ZnPP, a metalloporphyrin in which zinc occupies the position normally held by iron in the heme ring (Wakamatsu, Nishimura, & Hattori, 2004, Meat Science, 67(1), 95 to 100).

This article sets out what the peer reviewed literature actually establishes about ZnPP. It covers the identity of the heme donor, the competing enzymatic and non enzymatic pathways proposed for its formation, the inhibitory action of nitrite, and the current state of research into ZnPP as a colouring tool for nitrite free products. Because the mechanism remains an active research question rather than a settled fact, the article distinguishes consistently between what has been demonstrated and what remains proposed or contested.

1. Identification of ZnPP as the Parma Ham Pigment

Hornsey described the colour of cured meat as early as 1956, well before nitrosylmyoglobin was confirmed as its chemical basis (Hornsey, 1956, Journal of the Science of Food and Agriculture, 7(8), 534 to 540). Parma ham’s colour puzzle persisted because no nitrite is present to generate the expected pigment. Wakamatsu, Nishimura and Hattori resolved this in 2004 by isolating and characterising the red pigment directly from Parma ham and confirming it as ZnPP through spectroscopic analysis (Wakamatsu, Nishimura, & Hattori, 2004, Meat Science, 67(1), 95 to 100). Møller, Adamsen, Catharino, Skibsted and Eberlin confirmed the identification independently by mass spectrometry in 2007, removing any doubt about the pigment’s chemical identity (Møller, Adamsen, Catharino, Skibsted, & Eberlin, 2007, cited in Wakamatsu, 2022, Meat Science, 192, 108905). ZnPP has since been reported to occur in larger quantities than residual heme in several dry cured hams, not only Parma ham (Bou, Llauger, Arnau, & Fulladosa, 2018, Meat Science, 139, 192 to 200).

2. The Heme Donor Question: Myoglobin or Hemoglobin

Early work assumed myoglobin was the source of the heme that becomes ZnPP, because myoglobin is the dominant heme protein in mature muscle (Wakamatsu, Okui, Ikeda, Nishimura, & Hattori, 2004, Meat Science, 68(2), 313 to 317). This assumption has since been revised. Wakamatsu and colleagues showed that adding myoglobin to a pork homogenate model did not increase ZnPP formation in a dose dependent way, whereas adding haemoglobin did (Wakamatsu, Okui, et al., cited in Zhai, Wang, Hayakawa, Kumura, & Wakamatsu, 2022, Food Chemistry, 395, 133604). Haemoglobin releases its heme group more readily than myoglobin, and haemoglobin addition raised non heme iron levels in the homogenate, while myoglobin addition did not (Zhai et al., 2022, Food Chemistry, 395, 133604).

On the basis of this evidence, Wakamatsu concluded in a 2022 review that the water soluble fraction of ZnPP in Parma ham is derived mainly from haemoglobin rather than myoglobin (Wakamatsu, 2022, Meat Science, 192, 108905). Approximately seventy five percent of water soluble ZnPP was found bound to haemoglobin (Wang, Hayakawa, Kumura, & Wakamatsu, 2021, cited in Abe et al., 2024, Food Chemistry). This point matters because much of the earlier literature, including popular summaries of the subject, still describes myoglobin as the sole precursor. That description is now outdated for the water soluble portion of the pigment, even though myoglobin remains a source of the water insoluble fraction, discussed in section 6.

3. Proposed Mechanisms of ZnPP Formation

The literature has not settled on a single mechanism. Three pathways have been proposed, and current reviews treat them as operating in parallel rather than as competing alternatives (Becker, Westermann, Hansson, & Skibsted, 2012, cited in Khozroughi, Jander, Schirrmann, Rawel, & Schlüter, 2017, Food Chemistry).

3.1 The Enzymatic Pathway: Ferrochelatase

Ferrochelatase, abbreviated FECH, is the terminal enzyme of heme biosynthesis, classified as EC 4.99.1.1. Its normal physiological function is the insertion of ferrous iron into protoporphyrin IX to form heme (Dailey et al., cited in Chau, Ishigaki, Kataoka, & Taketani, 2011, Journal of Agricultural and Food Chemistry, 59(22), 12238 to 12245). Under the low oxygen, low pH, high salt conditions of dry curing, this same enzyme has been shown to run in reverse. Chau, Ishigaki, Kataoka and Taketani purified the iron removal enzyme responsible for converting myoglobin heme into ZnPP in raw ham and found it to be identical to ferrochelatase (Chau, Ishigaki, Kataoka, & Taketani, 2007, Biochemistry). Recombinant ferrochelatase, combined with NADH cytochrome b5 reductase, catalysed an NADH dependent removal of iron from hemin and from intact hemoproteins in the same study. This was the first demonstration of ferrochelatase catalysing its own reverse reaction, opening a route by which the enzyme both removes iron from heme and, separately, inserts zinc into the resulting empty porphyrin ring (Chau et al., 2011, Journal of Agricultural and Food Chemistry, 59(22), 12238 to 12245).

Ferrochelatase activity is not constant during processing. It decreases as Parma ham matures, and it varies between muscle types within the same ham (Wakamatsu, 2022, Meat Science, 192, 108905). Paganelli and colleagues showed that limited proteolysis of myoglobin, carried out by pepsin in their experimental model, opens a channel in the myoglobin globin structure that allows ferrochelatase to access the heme and carry out the iron to zinc exchange while the enzyme remains bound to the globin (Paganelli et al., 2016, Food Chemistry, 210, 491 to 499). This partly explains why the extent of myoglobin degradation, and not the mere presence of the protein, correlates with the amount of ZnPP that forms (Khozroughi, Jander, Schirrmann, Rawel, & Schlüter, 2017, Food Chemistry).

3.2 The Non Enzymatic Pathway

A separate, purely chemical pathway has been proposed and demonstrated in model systems. Under anaerobic conditions, zinc ions can substitute directly for ferrous iron in the porphyrin ring without enzymatic assistance, provided the heme moiety has first dissociated from its protein carrier (Becker, Westermann, Hansson, & Skibsted, 2012, cited in Wu et al., cited in Kim et al., 2025, Food Science of Animal Resources). This reaction is inhibited by oxygen, by nitrite or nitric oxide, and by heating. Reviews published since 2014 treat the enzymatic and non enzymatic routes as running side by side rather than as rival explanations, since both have been demonstrated experimentally and both appear to contribute to the ZnPP pool found in a mature ham (Kim et al., 2025, Food Science of Animal Resources; De Maere et al., cited in Kim et al., 2025).

3.3 The Bacterial Pathway

A third pathway involves the resident microorganisms of the meat matrix. Wakamatsu, Nishimura and Hattori observed in their original 2004 work that certain bacteria isolated from incubated pork homogenate degrade meat proteins, including heme pigments, and that this degradation can be accompanied by metal exchange (Wakamatsu, Nishimura, & Hattori, 2004, Meat Science, 67(1), 95 to 100). This observation has since been developed into a deliberate strategy for nitrite free colour development, discussed in section 7.

4. The Role of Proteolysis and Lipolysis

Parma ham maturation is accompanied by continuous proteolysis, carried out mainly by endogenous cathepsins that remain active even in the final stages of processing, and by lipolysis (Sforza et al., cited in Silvestri, Pinu, Ferranti, Musio, & Chambery, 2014, Journal of Mass Spectrometry). Silvestri and colleagues tracked ZnPP concentration across fifteen months of maturation using fluorescence spectroscopy and found it increased steadily and in step with the accumulation of low molecular weight peptides, a marker of ongoing proteolysis (Silvestri et al., 2014, Journal of Mass Spectrometry). Their conclusion was direct: degradation of myoglobin is essential for heme transmetallation to occur. Fatty acid release through lipolysis has a related effect. Free fatty acids released during maturation appear to promote ferrochelatase activity, and this relationship has been reinforced by both field studies of Serrano ham and by in vitro experiments (cited in Bou et al., PubMed record 34920405). Bou, Llauger, Arnau and Fulladosa confirmed in commercial Parma hams that ZnPP content correlates with both the proteolysis index and the degree of marbling of the muscle, tying the biochemistry directly to variables that a processor can observe on the cutting floor (Bou, Llauger, Arnau, & Fulladosa, 2018, Meat Science, 139, 192 to 200).

5. Effect of pH, Temperature and Muscle Type

ZnPP formation is strongly pH dependent, and the literature identifies two distinct optimal pH ranges rather than one. Wakamatsu identified pH values of approximately 4.75 and 5.5 as the two conditions under which ZnPP formation is favoured in pork model systems (Wakamatsu, 2022, Meat Science, 192, 108905). Khozroughi and colleagues found substantially more ZnPP formed at pH 4.75 than at pH 5.5 in the longissimus lumborum of pork, and linked this to the greater extent of myoglobin degradation occurring at the lower pH (Khozroughi et al., 2017, Food Chemistry). Temperature has an inverse relationship with ferrochelatase activity and with ZnPP stability. Wakamatsu observed a near linear decline in ZnPP concentration as storage temperature rose from ten to seventy degrees Celsius, with a cumulative reduction close to seventy percent, and a further drop when temperature reached sixty degrees (Wakamatsu, cited in Wu et al., 2024, Foods). Muscle fibre type and myoglobin content also affect the outcome, since ferrochelatase activity and heme availability both vary between muscles (Wakamatsu, cited in Khozroughi et al., 2017, Food Chemistry).

6. Water Soluble and Water Insoluble ZnPP

ZnPP in Parma ham does not exist as a single free molecule. It occurs largely as complexes with heme proteins. Zhai, Wang, Hayakawa, Kumura and Wakamatsu established that most of the water soluble ZnPP in Parma ham exists bound to haemoglobin, forming what they term ZnPP-Hb, and that this complex forms non enzymatically once ZnPP has been generated (Zhai, Wang, Hayakawa, Kumura, & Wakamatsu, 2022, Food Chemistry, 395, 133604). A smaller water soluble fraction exists as ZnPP bound to myoglobin, termed ZnPP-Mb. Between thirty and fifty percent of total ZnPP in Parma ham is water insoluble and remains bound to myoglobin and haemoglobin in a form that resists extraction (Wang et al., 2021, cited in Abe et al., 2024, Food Chemistry). Abe and colleagues showed that the water extractability of the ZnPP-myoglobin complex rises with muscle pH, meaning that the proportion of pigment that behaves as soluble versus bound depends on the acidity profile of the specific ham (Abe, Zhai, Toba, Masumo, Hayakawa, Kumura, & Wakamatsu, 2024, Food Chemistry).

7. Nitrite Inhibition of ZnPP Formation

Nitrite suppresses ZnPP formation, and recent work has clarified that this is a direct effect on the enzyme rather than only a competitive occupation of the iron binding site by nitric oxide. Schivazappa and colleagues measured ferrochelatase activity directly in sausages treated with a ZnPP forming starter culture, in untreated controls, and in nitrite treated batches. Ferrochelatase activity reached 134.5 units per gram in the inoculated batch and 102.0 units per gram in the control, but fell to 9.75 units per gram in the nitrite treated batch, a reduction the authors describe as statistically significant (Schivazappa, Simoncini, Pinna, Faccioli, & Zambonelli, 2024, Meat Science, 213, 109477). Kauser-Ul-Alam and colleagues confirmed separately that none of the twenty five lactic acid bacteria strains they screened formed any ZnPP in the presence of nitrite, regardless of their ZnPP forming capacity in its absence (Kauser-Ul-Alam, Hayakawa, Kumura, & Wakamatsu, 2021, Meat Science, 176, 108467). Together these findings support a mechanism in which nitrite acts on the enzymatic system itself, in addition to any effect on free iron availability through nitrosylmyoglobin formation.

8. Applications: Working Toward Nitrite Free Colour Systems

Research aimed at using ZnPP formation deliberately, rather than waiting for it to occur naturally over many months, has followed two main routes.

8.1 Ferrochelatase Sourced from Animal By-Products

Because ferrochelatase is present in liver and other internal organs, several groups have investigated organ tissue as a direct source of catalytic activity. Wakamatsu and colleagues compared the ZnPP forming properties of various pig and chicken by-products, with a view to using them as functional ingredients (Wakamatsu, Murakami, & Nishimura, 2015, Animal Science Journal). Abril, Sanchez-Torres, Bou, Benedito and Garcia-Perez examined how drying pork liver affects the ferrochelatase activity retained in the tissue, relevant to using dried liver as a colour forming ingredient (Abril, Sanchez-Torres, Bou, Benedito, & Garcia-Perez, 2022, LWT, 171, 114128). A related study used ZnPP rich pork liver homogenate directly as a colouring ingredient in nitrite free liver pâté, incubating the homogenate anaerobically before incorporation (cited in PMC article, PMC10887533).

8.2 Lactic Acid Bacteria as ZnPP Forming Starter Cultures

A separate and more developed line of research has screened food grade lactic acid bacteria for their intrinsic capacity to generate ZnPP, with the intention of using them as starter cultures. Asaduzzaman and colleagues screened bacterial isolates for ZnPP forming ability and identified two non edible strains with strong activity, then confirmed that inoculating one of them into minced meat produced colour approaching that of nitrite cured meat (Asaduzzaman, Ohya, Kumura, Hayakawa, & Wakamatsu, 2020, Meat Science, 165, 108109). Kauser-Ul-Alam and colleagues extended this to food grade, edible strains, screening twenty five lactic acid bacteria isolates and identifying strains that grew well anaerobically in three percent salt at pH 5.5 to 6.5, conditions consistent with fermented meat processing (Kauser-Ul-Alam, Hayakawa, Kumura, & Wakamatsu, 2021, Meat Science, 176, 108467). Salt slightly reduced ZnPP formation in these strains, while nitrite abolished it entirely, as noted in section 7. Among specific species, Lactococcus lactis subsp. cremoris was shown to produce ZnPP under both aerobic and anaerobic conditions, and to improve the visual redness of fermented meat products in application trials (Kauser-Ul-Alam, Toba, Hioki, Hayakawa, Kumura, & Wakamatsu, 2020, Foods, 9(11), 1583). A more recent study identified Weissella viridescens strains with notably higher ferrochelatase activity than other lactic acid bacteria screened, alongside Lactobacillus pentosus (cited in PMC11640533). Lactiplantibacillus plantarum strain YR07 has similarly been shown to raise ferrochelatase activity and ZnPP concentration in fermented sausage trials (cited in ScienceDirect record S2212429225017778).

9. Regulatory and Commercial Status

ZnPP based colour systems have not reached commercial scale in the way that nitrite curing has. This reflects the practical difference between a compound that forms reliably within hours, as nitrosylmyoglobin does, and one whose formation depends on pH, temperature, muscle composition, proteolytic activity, and in some approaches the successful establishment of a specific bacterial culture, over a timescale of days to months under traditional dry curing. Nitrite itself remains permitted and regulated as a curing agent by food safety authorities including the EFSA, the USDA, the FDA and Codex Alimentarius, and part of that standing rests on its established antimicrobial role against Clostridium botulinum (Ferysiuk & Wójciak, cited in Kim et al., 2025, Food Science of Animal Resources). ZnPP does not perform this antimicrobial function. This point is worth stating clearly, because Clostridium control in a nitrite free product is not an unresolved problem waiting on ZnPP. Whole muscle dry cured products such as Parma ham already control Clostridium botulinum without nitrite, through salt diffusion and equalisation, reduction of water activity, controlled temperature, sufficient processing time, raw material handling and process hygiene. Fermented products achieve the same result through acidification and starter culture competition. A ZnPP based colour system is therefore best understood as addressing colour alone, sitting alongside whichever of these established hurdles already governs the product, rather than as a component that must itself solve Clostridium control. The lactic acid bacteria and enzyme sourced approaches described in section 8 represent research towards that goal. None has yet been reported in the literature as a marketed commercial curing system.

10. Conclusion

ZnPP is established, through direct isolation and independent mass spectrometric confirmation, as the pigment responsible for the red colour of Parma ham. Beyond that point, several aspects of the mechanism remain the subject of active investigation rather than settled fact. Haemoglobin, not myoglobin, now appears to be the principal donor of the heme that becomes water soluble ZnPP, though myoglobin remains a source of the water insoluble fraction. Ferrochelatase has been shown, in a genuine reversal of its normal biosynthetic function, to remove iron from heme, and a separate non enzymatic pathway operates in parallel under anaerobic, low pH conditions. A bacterial contribution has also been demonstrated and is now being developed deliberately through starter culture screening. Nitrite suppresses the process directly at the level of ferrochelatase activity, in addition to any competing effect through nitrosylmyoglobin formation. Proteolysis, lipolysis, pH, temperature and muscle type all measurably affect the extent of pigment formation. Research toward practical nitrite free colouring systems built on this chemistry, whether through lactic acid bacteria or through ferrochelatase sourced from animal tissue, is ongoing, but no such system has yet reached commercial application.

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