Zinc Protoporphyrin IX in Meat and Meat Products: Formation, Application, and Future Perspectives

ZnPP is a metalloporphyrin with a unique chemical structure that distinguishes it from other pigments such as nitrosylhemochrome and nitrosomyoglobin (Wakamatsu et al., 2009). In contrast to these pigments, which involve iron coordination with nitric oxide (NO), ZnPP is characterized by zinc coordination with PPIX. This distinctive feature enables ZnPP to impart a vibrant red coloration, particularly in nitrate- and nitrite-free cured products such as Parma ham and Iberian ham (Bou et al., 2018; De Maere et al., 2014; Wakamatsu et al., 2009).

As shown in Fig. 1, ZnPP formation involves substitution of the central iron atom in PPIX with zinc ions. PPIX is a key precursor in heme biosynthesis and is essential for the function of hemoglobin and myoglobin, which are responsible for oxygen transport and storage in blood and muscle tissues, respectively (Wakamatsu et al., 2010). The transformation of PPIX into ZnPP can occur naturally during meat aging, prolonged storage, or under controlled processing conditions. For instance, enzymatic pathways involving ferrochelatase (FECH) play a critical role in facilitating this substitution, particularly under low-oxygen conditions, which are typical in dry-curing methods (Adamsen et al., 2003; Wakamatsu, 2022).

Fig. 1. Structural comparison of (A) protoheme, (B) protoporphyrin IX, and (C) Zinc protoporphyrin IX.

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The substitution of iron with zinc not only alters the structural properties of PPIX but also enhances its functional characteristics. Compared to heme, ZnPP exhibits greater stability against oxidative degradation and thermal damage, rendering it particularly suitable for applications in nitrite-free meat processing. For example, studies have demonstrated that ZnPP retains its coloration even under extended curing and storage conditions, thereby maintaining the quality and shelf life of the products (Adamsen et al., 2004; Llauger et al., 2024). These distinctive structural and functional properties of ZnPP also influence its spectroscopic characteristics, thereby enabling precise identification in meat products.

In addition to its structural and functional advantages, ZnPP exhibits distinct spectroscopic and fluorescent characteristics. ZnPP exhibits a distinctive absorption spectrum that plays a crucial role in its identification and quantification in meat products. Its Soret band is observed at approximately 409–418 nm, with Q-band peaks varying according to the meat matrix and curing conditions. For instance, in Parma ham, the Soret band was observed at 409 nm, whereas Q-band peaks occurred at 509, 553, 600, and 637 nm, confirming ZnPP as the primary pigment in these products (Wakamatsu et al., 2004a). Furthermore, studies on acetone/water extracts of matured Parma and Iberian ham identified a Soret band at 418 nm and Q-band peaks at 546 and 584 nm, confirming the role of ZnPP as the dominant pigment in nitrite-free curing processes (Adamsen et al., 2004). These absorption characteristics clearly distinguish ZnPP from myoglobin derivatives such as oxymyoglobin, deoxymyoglobin, and nitrosomyoglobin (Møller et al., 2003).

In addition to its distinct absorption spectrum, ZnPP exhibits unique fluorescence properties. Upon excitation at 420 nm, ZnPP emits a characteristic fluorescence peak near 590 nm, which distinguishes it from other heme pigments (Khozroughi et al., 2017; Wakamatsu et al., 2004b). This fluorescence is directly attributed to the zinc-porphyrin complex structure, offering insights into the stability and role of ZnPP in nitrite-free products. Wakamatsu et al. (2004a) provided initial empirical evidence that the stable red color characteristic of traditional Parma ham is attributable to ZnPP, thereby challenging previous assumptions that assigned the color to an unidentified myoglobin derivative (Morita et al., 1996). Moreover, fluorescence analysis revealed that ZnPP is more stable in low-oxygen environments, which are typical of dry-cured meats, facilitating its widespread application in products such as Parma ham (Wakamatsu et al., 2004b; Wakamatsu et al., 2006).

The fluorescent properties of ZnPP have facilitated advancements in meat processing and quality control. Techniques, such as purple LED imaging, have enabled researchers to visualize the distribution of ZnPP in products such as Parma ham, revealing its higher concentration in intermuscular and subcutaneous fat tissues than in lean tissues. These findings have provided critical insights into ZnPP formation and migration during curing, contributing to the consistent coloration of nitrite-free meat products (Wakamatsu et al., 2006). By utilizing these properties, meat processors can monitor and optimize curing processes, ensuring a stable product quality and extended shelf life (Wakamatsu, 2022).

The formation of ZnPP in meat products is predominantly driven by the enzymatic activity of FECH (EC 4.99.1.1), a mitochondrial enzyme that catalyzes the substitution of zinc for iron in PPIX (Becker et al., 2012; Wakamatsu et al., 2015). Under typical physiological conditions, FECH exhibits strong specificity for ferrous iron, thereby facilitating heme biosynthesis. However, during the curing of meat products, particularly in low-oxygen environments, FECH preferentially incorporates Zn into PPIX, leading to the formation of ZnPP (Bou et al., 2020; Parolari et al., 2016). This process is influenced by a combination of intrinsic factors, including pH, temperature, and the availability of metal ions. Optimal activity for ZnPP synthesis has been observed within a pH range of 5.5 to 8.0, with slight variations depending on the source of the enzyme and processing conditions (Abril et al., 2021; De Maere et al., 2016). For instance, studies have reported maximum FECH activity in porcine liver extracts at neutral to slightly basic pH levels, while acidic conditions around pH 5.5, have been demonstrated to support efficient ZnPP formation under specific circumstances (Llauger et al., 2023; Parolari et al., 2016).

The role of substrate availability is of equal significance, as the relative concentrations of zinc and iron in the meat matrix directly influence the enzyme substrate specificity. Ferrous iron functions as a competitive inhibitor of zinc insertion, with higher zinc availability enhancing ZnPP accumulation (Becker et al., 2012; Bou et al., 2020). These conditions are frequently observed in dry-cured meat products, in which the curing process promotes a low-oxygen environment, thereby reducing the bioavailability of iron and facilitating zinc incorporation (De Maere et al., 2018; Wakamatsu et al., 2010). The interaction between these factors emphasizes the importance of controlled processing conditions for optimizing ZnPP formation.

Recent advancements in enzymatic extraction techniques are highly promising for enhancing ZnPP formation. Ultrasound-assisted extraction methods can significantly increase FECH activity, with studies indicating a 33% improvement in enzymatic efficiency compared with conventional methods (Abril et al., 2021; Abril et al., 2022). These findings suggest that ultrasound treatment not only accelerates the extraction process but also preserves the functionality of the enzyme, rendering it a promising approach for industrial applications. Furthermore, pork liver, a rich source of FECH, has garnered attention for its role in promoting ZnPP synthesis, offering a sustainable and efficient alternative for producing natural pigments in meat products (Abril et al., 2021; Bou et al., 2020).

The presence of myoglobin, a heme protein that is abundant in meat, contributes to an additional level of complexity in ZnPP formation. Although myoglobin degradation is not strictly necessary for ZnPP synthesis, its oxidation state and interaction with FECH significantly influence the enzymatic pathway (Becker et al., 2012; Khozroughi et al., 2017). Myoglobin functions as a precursor for PPIX, and its structural state can modulate the efficiency of zinc incorporation, further emphasizing the importance of elucidating the protein dynamics in the ZnPP formation process (Llauger et al., 2023; Parolari et al., 2016).

Based on these insights, ZnPP synthesis can be optimized to enhance the natural coloration of meat products in the meat industry, particularly in the context of nitrite-free processing. This approach aligns with increasing consumer demand for clean-label products, underscoring the importance of endogenous enzymatic pathways in achieving both functional and aesthetic qualities in meat products (Bou et al., 2020; Parolari et al., 2016).

In addition to the enzymatic formation of ZnPP, non-enzymatic pathways contribute significantly to its production, particularly in traditional dry-cured meat products, where enzymatic activity is limited. The primary mechanism involves the substitution of iron with zinc in the porphyrin ring of heme proteins, notably hemoglobin and myoglobin (Zhai et al., 2022; Zhai et al., 2023). According to previous reports, the predominant substrate for ZnPP formation is ferriheme, which dissociates from oxidized heme proteins. This process requires the reduction of ferriheme to ferroheme, thereby enabling zinc to replace iron within the porphyrin structure (Zhai et al., 2023).

Complexes of ZnPP with hemoglobin (ZnPP-Hb) and myoglobin (ZnPP-Mb) have been observed, with ZnPP-Hb identified as the predominant water-soluble ZnPP complex in Parma ham (Zhai et al., 2022). These findings underscore the importance of heme protein dynamics in non-enzymatic formation of ZnPP. Moreover, the formation of ZnPP through non-enzymatic pathways is influenced by environmental factors including anaerobic conditions, pH, and temperature. For instance, Becker et al. (2012) demonstrated that a non-enzymatic transmetallization reaction occurs in low-oxygen environments wherein the iron in myoglobin is replaced by zinc. Similarly, Wakamatsu et al. (2019) identified mildly acidic pH conditions (4.9–5.5) as optimal for metal exchange in myoglobin to facilitate efficient ZnPP formation.

De Maere et al. (2016) further emphasized the critical role of pH and production time in promoting ZnPP synthesis in nitrite-free meat products. Their findings revealed that ZnPP formation increased significantly during the later stages of curing under appropriate pH conditions. Notably, non-enzymatic formation of ZnPP frequently occurs concurrently with enzymatic pathways, suggesting a synergistic interaction between the two mechanisms (Becker et al., 2012; De Maere et al., 2016; Zhai et al., 2023).

These insights into the non-enzymatic pathways of ZnPP formation not only deepen our understanding of meat coloration processes, but also underscore the potential for optimizing natural pigment production in nitrite-free meat products.

Among lactic acid bacteria (LAB) strains, Lactococcus lactis subsp. cremoris and Leuconostoc mesenteroides produce more intense red colora in salt-added minced meat, with ZnPP autofluorescence serving as a reliable indicator of the presence of ZnPP (Kauser-Ul-Alam et al., 2021). Moreover, LAB strains exhibit optimal ZnPP-forming activity under mildly acidic conditions (pH 5.5–6.5) and anaerobic environments, which closely resemble the conditions present in fermented meat products (Wakamatsu et al., 2020; Wu et al., 2023). Sodium chloride marginally inhibits ZnPP formation, whereas sodium nitrite completely suppresses the process, underscoring the suitability of LAB for nitrite-free curing systems (Kauser-Ul-Alam et al., 2021; Wakamatsu et al., 2020).

Leuconostoc strains have been isolated from fermented meat products and exhibited significantly enhanced ZnPP formation under vacuum-packed and salted conditions, providing a viable alternative to traditional curing agents (Wu et al., 2023). These findings are consistent with studies indicating that LAB are the primary contributors to ZnPP synthesis in various meat matrices, not only improving color but also ensuring safety and stability during fermentation (Asaduzzaman et al., 2020; Wu et al., 2023). Furthermore, LAB-derived ZnPP formation occurs both aerobically and anaerobically, further emphasizing the versatility of these bacteria in diverse processing environments (Wakamatsu et al., 2020).

The application of LAB in nitrite-free meat processing presents a promising approach to address consumer demand for natural clean-label products, while maintaining desirable sensory attributes. These findings underscore the potential of LAB as an effective substitute for nitrites, offering both functional and safety advantages in meat production.

The pH of meat during curing significantly influences the formation of ZnPP, with distinct mechanisms exhibiting different optimal pH levels. For instance, pH 4.75, promotes ZnPP formation through myoglobin degradation, while pH 5.5, facilitates direct zinc incorporation without significant myoglobin breakdown (Wakamatsu et al., 2019). Furthermore, the processing temperature and time significantly affect ZnPP formation depending on the pH level. De Maere et al. (2016) observed that ZnPP formation increased significantly at pH>4.9 in nitrite-free dry fermented sausages, achieving optimal ZnPP levels during prolonged fermentation under these conditions. Similarly, Ishikawa et al. (2006) reported maximum zinc chelatase activity in porcine heart extracts at pH 5.5, exhibiting comparable effectiveness under both anaerobic and aerobic conditions. This versatility demonstrates the potential of ZnPP formation across diverse processing environments.

Benedini et al. (2008) demonstrated that ZnPP formation in pork muscle was significantly enhanced by the presence of NaCl and ATP, with the zinc chelatase activity being most efficient at pH 5.5–6.0. This suggests that the inclusion of NaCl and ATP in curing formulations can synergistically optimize ZnPP synthesis. Consequently, understanding these pH-dependent mechanisms is essential for optimizing ZnPP formation in nitrite/nitrate-free dry-cured meat products. Such optimization ensures desirable color characteristics and improved product stability and consumer acceptance.

Muscle type, meat source, and meat composition are among the most critical factors that influence ZnPP formation. These factors determine the biochemical and enzymatic environments that regulate ZnPP synthesis. Slow-twitch fibers (type I), which are rich in mitochondria, produce higher ZnPP levels at pH 4.75, whereas fast-twitch fibers (type II) exhibit reduced ZnPP formation under the same conditions. These findings underscore the role of mitochondrial activity in ZnPP synthesis (Wakamatsu et al., 2019).

In vitro studies have demonstrated that the source of meat significantly affects ZnPP formation, with horsemeat exhibiting the highest rates under high-pH conditions (approximately pH 5.3–6.0). This was attributed to its elevated zinc chelatase activity and superior heme content and zinc availability compared to other meats (De Maere et al., 2017; De Maere et al., 2018). Furthermore, pork and chicken were found to exhibit lower ZnPP formation rates than horsemeat, highlighting the variability in ZnPP synthesis across different meat sources (De Maere et al., 2018).

Wakamatsu et al. (2006) investigated ZnPP formation across various animal by-products, with porcine liver exhibiting the highest ZnPP-forming capacity. This phenomenon was linked to the mitochondria-rich environment of the hepatocytes, which facilitated ZnPP synthesis without requiring anaerobic conditions. ZnPP distribution studies in Parma ham revealed that intermuscular and subcutaneous fat had significantly higher ZnPP concentrations than lean tissues. This suggests that ZnPP is initially synthesized in lean muscle and subsequently migrates to adipose tissues during curing, where it accumulates due to the lower metabolic activity of fat (Wakamatsu et al., 2006).

Bou et al. (2018) demonstrated a positive correlation between the ZnPP content, proteolysis index, and intramuscular fat content in Parma ham. Proteolysis releases peptides and amino acids that facilitate iron-to-zinc transmetallation, whereas intramuscular fat enhances the retention and distribution of ZnPP. These findings were further corroborated by studies on nitrite-free dried hams, which emphasized the critical roles of marbling and proteolysis in ZnPP formation and their impact on color development (Parolari et al., 2009). These results highlight the significance of meat source, muscle fiber type, and composition in optimizing ZnPP formation, providing valuable insights into nitrite-free, clean-label meat products.

ZnPP formation is a critical factor determining the visual quality and consumer acceptance of nitrite-free meat products and is primarily influenced by the processing conditions, particularly temperature and time. The processing temperature directly modulates the enzymatic activity of FECH at moderate temperatures (10°C–20°C), achieving optimal catalytic efficiency. Both low and high temperatures inhibit enzymatic function, thereby affecting the efficiency and stability of ZnPP synthesis (Abril et al., 2022). Zhai et al. (2022) demonstrated that moderate temperatures not only promote stable ZnPP formation but also ensure the structural integrity of ZnPP-apo-hemoglobin complexes, which are predominant in cured products such as Parma ham. This underscores the dual role of temperature in facilitating pigment formation and ensuring pigment stability in nitrite-free systems.

The enzymatic pathways exhibited significantly reduced activity at lower curing temperatures (3°C–4°C), leading to the predominance of non-enzymatic pathways. However, slower reaction rates increase curing durations by approximately three months compared to conventional methods, posing challenges for industrial scalability and efficiency (Parolari et al., 2016; Zhai et al., 2022). This temperature range, while decelerating the curing process, enhances the stability of non-enzymatically formed ZnPP, particularly in cured products, such as Parma ham, where ZnPP predominantly exists as apo-hemoglobin-bound complexes (Zhai et al., 2022).

The processing duration is equally crucial as it determines the balance between enzymatic and non-enzymatic pathways in ZnPP formation. At moderate curing temperatures, ZnPP accumulation reaches its maximum during the initial and intermediate stages of processing, when the enzymatic activity is robust (Llauger et al., 2023). However, extended durations diminish enzymatic activity due to enzyme degradation, thus reducing ZnPP synthesis (Abril et al., 2022). At low curing temperatures, non-enzymatic mechanisms predominate, necessitating extended processing times to achieve comparable ZnPP levels (Parolari et al., 2009; Parolari et al., 2016).

The combined effects of temperature and time introduce additional complexity into the formation of ZnPP. Llauger et al. (2023) employed a response surface methodology to optimize the parameters for ZnPP synthesis, revealing that anaerobic conditions at 25°C–55°C over 3–30 h yielded high pigment production while maintaining microbiological safety. These findings emphasize the importance of precisely managing processing temperature and time to achieve optimal ZnPP formation in nitrite-free meat products.

In conclusion, the temperature and time are critical determinants of ZnPP formation. Moderate temperatures and shorter durations favor enzymatic pathways, whereas extended periods at lower temperatures promote non-enzymatic mechanisms. Understanding these interactions provides valuable insights into enhancing the quality and visual characteristics of nitrite-free meat products. This knowledge provides a foundation for developing efficient and scalable processing strategies tailored to meet consumer demand for natural clean-label products in the competitive meat industry.

Oxygen availability plays a pivotal role in regulating ZnPP synthesis, directly influencing the balance between enzymatic and non-enzymatic mechanisms. In oxygen-rich environments, ZnPP formation is impeded by oxidation of myoglobin to metmyoglobin, which lacks the capacity to facilitate ZnPP synthesis. For the process to resume, metmyoglobin must be reduced back to its active myoglobin form, a reaction that is inhibited by oxidative stress (Ishikawa et al., 2006; Wakamatsu et al., 2006). Anaerobic conditions are essential for maintaining the enzymatic activities, particularly FECH, a key enzyme in ZnPP synthesis within skeletal muscles (Ishikawa et al., 2006; Wakamatsu et al., 2019).

Porcine skeletal muscle extracts demonstrate significantly higher ZnPP production under low-oxygen conditions because oxygen exposure destabilizes intermediates and oxidizes myoglobin, limiting its capacity to support ZnPP synthesis (Wakamatsu et al., 2004b; Wakamatsu et al., 2019). In contrast, porcine heart tissue exhibits robust ZnPP formation under both aerobic and anaerobic conditions because of its superior enzymatic systems and high mitochondrial activity, which confers resistance to oxidative stress (Ishikawa et al., 2006).

Certain LAB strains, such as L. lactis subsp. cremoris, exhibit the unique ability to facilitate ZnPP synthesis under oxygenated conditions by employing alternative metabolic pathways. This capability renders LAB particularly advantageous in fermented meat products where oxygen exclusion is challenging (Kauser-Ul-Alam et al., 2020; Wakamatsu et al., 2020).

Traditional dry-curing methods, such as those utilized for Parma ham, rely on oxygen-restricted conditions to enhance the accumulation of ZnPP. Fluorescence imaging studies revealed that ZnPP was predominantly localized in the inner layers of Parma ham, where oxygen exposure was minimal. This spatial distribution highlights the critical role of oxygen diffusion in the formation of ZnPP during curing (Wakamatsu et al., 2006).

These findings underscore the significance of oxygen availability in ZnPP synthesis, providing practical insights into nitrite-free meat processing techniques with controlled oxygen levels.

Nitrites and NO significantly impede ZnPP formation via various biochemical pathways (Adamsen et al., 2006; Wakamatsu et al., 2010). Nitrites interact with myoglobin to produce nitrosylmyoglobin, the primary pigment responsible for the characteristic pink color of cured meat products. This reaction reduces the availability of PPIX, which is a precursor required for the synthesis of ZnPP. Studies have demonstrated that nitrites elevate the redox potential of the meat environment, thereby hindering the chelation of zinc into PPIX (Adamsen et al., 2006; Wakamatsu et al., 2010).

In nitrite-free cured products, such as Parma ham, ZnPP forms naturally and contributes to a stable red color. Conversely, nitrite-cured products exhibit negligible ZnPP levels, underscoring the significant inhibitory effects of nitrites (Adamsen et al., 2006; Wakamatsu et al., 2007). Although less reactive than nitrites, nitrates undergo bacterial reduction to form nitrites during the processing. This conversion indirectly inhibits ZnPP formation by acting as a continuous source of NO, albeit at a slower rate than that of direct nitrite addition (Adamsen et al., 2006; Furukawa et al., 1995).

NO inhibits ZnPP synthesis by interacting with the iron-sulfur cluster within FECH, which is a critical enzymatic component responsible for inserting zinc into PPIX. This interaction destabilizes the structure of the enzyme and effectively deactivates its catalytic function (Furukawa et al., 1995). Furthermore, NO reduces PPIX availability, further limiting ZnPP synthesis via enzymatic and non-enzymatic pathways (Wakamatsu et al., 2010).

Innovative nitrite-free curing methods have been developed to counteract the inhibitory effects of nitrite. The use of LAB, including Lactobacillus fermentum, L. lactis, and L. mesenteroides, has demonstrated significant potential for replicating the visual characteristics of nitrosylmyoglobin (Asaduzzaman et al., 2020; Zhang et al., 2007). For instance, L. fermentum enhances ZnPP formation by increasing PPIX availability and facilitating zinc chelation under anaerobic conditions (Asaduzzaman et al., 2020). These findings underscore the potential of LAB as substitutes for nitrites to produce visually appealing and nitrite-free cured meat products.

In conclusion, nitrites and NO inhibit ZnPP formation by reducing PPIX availability and disrupting enzymatic pathways. Nitrite-free curing methods that leverage ZnPP-forming bacteria provide sustainable and health-conscious alternatives for the meat industry. Such methods not only afford the desired coloration of cured products, but also align with the growing consumer demand for clean-label and nitrite-free food options, presenting significant potential for market expansion.

Sodium chloride (NaCl) is a critical factor that influences the formation of ZnPP in nitrite-free meat products. Although moderate salt concentrations enhance ZnPP formation, excessive levels may alter ZnPP synthesis pathways and influence enzymatic activity (Becker et al., 2012; Bou et al., 2020).

Studies have demonstrated that NaCl serves as a catalyst for ZnPP formation by facilitating iron displacement from myoglobin, thereby enabling zinc to bind to PPIX (Becker et al., 2012). In dry-cured ham, moderate salt concentrations have been observed to enhance ZnPP formation by promoting enzymatic reactions and stabilizing ZnPP pigments (Adamsen et al., 2006; Becker et al., 2012). Moreover, research on Parma-like hams indicates that the ZnPP content increases significantly after 40 weeks of processing, with no significant difference between refined salt and sea salt, suggesting that ZnPP formation is primarily influenced by aging duration rather than salt impurities (Wakamatsu et al., 2009).

While moderate salt concentrations facilitate ZnPP formation, excessively high concentrations can inhibit enzymatic activity by reducing water activity and destabilizing ZnPP-related enzymes (Becker et al., 2012). Studies on Serrano ham have demonstrated that ZnPP content is positively correlated with salt content when measured on a dry-weight basis, but inversely correlated when measured on a desalted basis, suggesting that salt content exerts a complex influence on ZnPP formation (Bou et al., 2020). The effect of NaCl on ZnPP is further modulated by pH and enzymatic activity. Research indicates that as the salt concentration increases, the final pH of meat decreases, potentially influencing ZnPP synthesis (Bou et al., 2020).

In addition, ZnPP formation is influenced by microbial activity. Studies have demonstrated that LAB, including Lactobacillus plantarum, L. lactis subsp. cremoris, and Leuconostoc lactis, can sustain ZnPP synthesis even in environments with a 3% salt concentration (Kauser-Ul-Alam et al., 2021). However, at salt concentrations exceeding 5%, ZnPP formation decreased as LAB metabolism and enzymatic activity were inhibited. This observation indicates that while LAB can be beneficial for ZnPP formation, elevated salt concentrations may adversely affect their efficacy (Kauser-Ul-Alam et al., 2021).

The formation of ZnPP in meat products is influenced by the availability of metal ions, particularly Fe and Zn. FECH plays a central role in this process by inserting Fe²⁺ into PPIX to form heme under normal conditions (Becker et al., 2012; Hunter et al., 2008; Taketani and Tokunaga, 1982). However, under certain conditions, FECH can also remove Fe²⁺ and insert Zn²⁺, resulting in ZnPP formation (Becker et al., 2012; Paganelli et al., 2016; Taketani and Tokunaga, 1982).

Although Fe²⁺ is the preferred substrate for FECH, the enzyme exhibits the capacity to insert Zn²⁺, Co²⁺, and other metal ions into PPIX. However, elevated Fe²⁺ concentrations may inhibit ZnPP formation by competitive binding at the FECH active site (Hunter et al., 2008). The efficacy of metal insertion follows the order of Fe>Zn>Co>Ni, suggesting that Zn²⁺ may serve as a substitute for Fe²⁺ under conditions of low Fe availability (Taketani and Tokunaga, 1982).

Zn²⁺ is essential for ZnPP formation and has been demonstrated to significantly enhance ZnPP synthesis in vitro (Becker et al., 2012; Schivazappa et al., 2024). However, excessive Zn²⁺ concentration can inhibit FECH activity by binding to an additional inhibitory site on the enzyme (Hunter et al., 2008). Environmental factors such as phosphate concentration can facilitate ZnPP formation by increasing Zn availability in the system (Becker et al., 2012).

ZnPP formation in meat products is regulated by the interplay between Fe and Zn availability, FECH activity, and environmental conditions. Although Fe²⁺ generally inhibits ZnPP formation by competing with Zn for FECH binding, Zn²⁺ functions as a crucial substrate for ZnPP synthesis when Fe levels are low. Elucidation of these factors can facilitate optimization of ZnPP formation during meat processing, thereby enhancing product quality.

ZnPP has revolutionized nitrite-free meat processing by functioning as a natural pigment that eliminates the need for synthetic additives. It forms naturally through enzymatic and microbial pathways, providing a vibrant red color in nitrite-free products and addressing consumer demand for clean-label options (Bou et al., 2020; Wakamatsu, 2022). ZnPP mitigates the health risks associated with synthetic nitrites, such as N-nitrosamine formation during cooking, while maintaining color vibrancy and stability during extended storage and processing (Wu et al., 2024). As shown in Table 3, its role is particularly evident in traditional products such as Parma ham, where ZnPP forms under low-oxygen conditions and optimal pH levels without the addition of nitrites or nitrates (De Maere et al., 2016; Wakamatsu, 2022; Wakamatsu et al., 2019). In dry-cured fermented sausages, controlled pH, temperature, and processing time facilitate ZnPP synthesis, resulting in a rich and stable red color (De Maere et al., 2016; De Maere et al., 2018). Similarly, ZnPP-rich pork liver homogenates were effective in preserving the color of nitrite-free liver pâtés even under thermal processing conditions, rendering ZnPP a viable solution for clean-label trends, aligning with consumer preferences for minimally processed natural products (Llauger et al., 2024).

Microbial pathways play a crucial role in ZnPP synthesis, with bacteria such as L. lactis, L. mesenteroides, and Enterococcus faecium facilitating ZnPP production in fermented meat products (Asaduzzaman et al., 2020; Wu et al., 2024). These microorganisms ensure vibrant and stable pigmentation, even after thermal processing, rendering ZnPP suitable for diverse product applications.

Technological advancements have significantly enhanced the efficiency and scalability of ZnPP production. Ultrasound-assisted extraction methods increased the FECH activity by up to 33%, reduced processing times by 50%, and achieved higher ZnPP yields (Abril et al., 2021; Abril et al., 2023b). In addition, moderate drying temperatures ranging from 10°C to 20°C, preserved the enzymatic activity and ensured consistent ZnPP yields during processing, while minimizing degradation (Abril et al., 2022). Such technological innovations render large-scale ZnPP production feasible, affording stable meat coloration and extending the shelf life of the product.

Optimized fermentation techniques utilizing engineered Escherichia coli strains, such as HAEM7, have resulted in production yields exceeding 2.2 g/L, minimizing by-products and reducing costs for industrial applications (Choi et al., 2022). Furthermore, system-level metabolic engineering approaches, including feedback regulation elimination and transporter optimization, have further enhanced ZnPP production efficiency, rendering it a sustainable and cost-effective alternative to synthetic curing agents (Choi and Lee, 2023).

Thus, the integration of ZnPP into meat processing is highly promising. However, substantial challenges and limitations persist (Table 3), including variability in ZnPP formation, technological impediments, and incomplete mechanistic understanding, constraining the widespread industrial application of ZnPP.

A major challenge in ZnPP research is the variability in its formation across different muscle types, which complicates standardization of meat processing. The type of muscle fiber significantly influences ZnPP synthesis, with type I fibers requiring acidic pH (approximately 4.75) and type II fibers requiring near-neutral pH. This variability in optimal conditions poses challenges for achieving consistent coloration across different meat matrices (Wakamatsu et al., 2007; Wakamatsu et al., 2019). Moreover, factors such as heme concentration, myoglobin levels, and the presence of metal ions introduce additional complexity, necessitating tailored optimization for each product type (Bou et al., 2020; De Maere et al., 2016).

The commercial adoption of ZnPP-enhancing methodologies has been constrained by technological and economic barriers. Advanced techniques, such as ultrasound-assisted extraction and controlled fermentation, have demonstrated efficacy in enhancing the ZnPP yields. However, these methods require significant investment in specialized equipment and process optimization, limiting their scalability and cost-effectiveness for industrial use (Abril et al., 2021; Abril et al., 2023a; Abril et al., 2023b). For instance, although ultrasound treatment can increase FECH activity and reduce processing time by up to 50%, its high energy requirements and operational costs prohibit its widespread implementation (Abril et al., 2021). Similarly, fermentation technologies involving engineered bacterial strains require precise environmental control, which further increases production costs (Choi et al., 2022).

The limited understanding of the ZnPP formation mechanisms impedes the development of efficient production methods. Although enzymatic and non-enzymatic pathways are involved, the precise interactions among variables, such as pH, temperature, salt concentration, and oxygen levels, remain underexplored. For example, high salt concentrations can either promote or inhibit ZnPP formation depending on other environmental factors (Adamsen et al., 2006; Parolari et al., 2016). Additionally, nitrite and NO, which are commonly used in traditional meat curing, inhibit ZnPP synthesis by interfering with key enzymatic processes (Wakamatsu et al., 2010). These gaps in understanding necessitate further research to optimize production parameters for consistent and scalable ZnPP yields.

ZnPP has not yet received approval from the Food and Drug Administration (FDA) as a food colorant, largely because of the absence of well-defined safety specifications and regulatory frameworks governing natural colorants, which are not subject to the same certification process as synthetic colorants (Simon et al., 2017). This regulatory gap presents significant challenges to the approval process. Therefore, a comprehensive toxicological assessment of novel natural colorants is imperative (de Mejia et al., 2020).

Consumer acceptance of such additives is influenced by factors such as perceived naturalness, ingredient transparency, and generational differences (Maruyama et al., 2021). Notably, providing information about ingredient sources improves acceptance, whereas explaining functionality alone has limited impact. The increasing demand for natural colorants and clean-labelled food products suggests a potential market for ZnPP (de Mejia et al., 2020; Nieto et al., 2024). However, the utilization of advanced technologies such as metabolic engineering and chemical extraction may elicit concerns among regulatory bodies and consumers regarding the naturalness of ZnPP as an additive. Addressing these concerns through transparent labeling and regulatory guidance could be essential for market acceptance.

The natural formation of ZnPP in certain meat products, such as Parma ham, indicates its potential cost-effectiveness (Wakamatsu, 2022). However, natural colorants generally face challenges related to stability and vibration, which must be addressed to ensure their commercial viability (de Mejia et al., 2020). The successful integration of ZnPP as a food additive requires overcoming regulatory hurdles, addressing consumer perceptions, and improving stability through continued research and effective communication strategies.

To address these challenges, a multidisciplinary approach is essential. Future research should focus on elucidating the detailed mechanisms underlying ZnPP formation, optimizing cost-effective production methods, and addressing regulatory and consumer concerns. By addressing these barriers, ZnPP could emerge as a transformative solution for nitrite-free meat production, offering both industrial and consumer benefits.

Despite advancements in the elucidation of ZnPP formation, significant knowledge gaps persist regarding its biochemical pathways. A critical area of inquiry is the mechanism by which FECH preferentially incorporates zinc into PPIX. Previous studies utilizing model systems have demonstrated that zinc substitution can spontaneously occur under anaerobic conditions during curing (Wakamatsu et al., 2004b; Wakamatsu et al., 2007). Further investigation of the molecular factors and environmental conditions influencing this substitution could lead to enhanced ZnPP yields.

The role of non-enzymatic mechanisms in ZnPP formation, particularly in dry-cured meat products with limited enzymatic activity, warrants further investigation. For instance, storage and processing conditions influence the balance between enzymatic and non-enzymatic pathways. Furthermore, studies have shown that both endogenous enzymes and curing conditions contribute to ZnPP formation (Wakamatsu et al., 2009). Elucidating the interaction between these pathways will be crucial for optimizing ZnPP production across diverse meat processing systems and for understanding the role of heme destabilization during proteolysis.

Additionally, exploring the potential health implications of ZnPP is essential. Unlike synthetic nitrites, ZnPP may help reduce the formation of harmful N-nitrosamines, potentially offering a safer alternative to meat products. Although ZnPP has been shown to contribute to color stability in meat products and may influence oxidative stability, its direct antioxidant properties have not been conclusively established and require further investigation (Wakamatsu et al., 2020). In addition, the long-term health effects of ZnPP remain unclear, necessitating systematic research to confirm its safety and potential benefits.

Finally, expanding ZnPP applications to non-meat products, such as plant-based alternatives, seafood, and dairy products, is a promising research direction. Studies on the stability and behavior of ZnPP within diverse food matrices are critical for developing new markets and broadening their industrial applications.

Innovative technologies for enhancing ZnPP formation present promising approaches to address the current production challenges in meat processing. A primary strategy involves bioengineering, including genetic modifications, to enhance the activity of FECH, the enzyme responsible for inserting zinc into PPIX (Choi and Lee, 2023; Choi et al., 2022). Recent studies have demonstrated that overexpression of FECH and optimization of associated metabolic pathways can significantly increase ZnPP synthesis in engineered E. coli strains (Choi et al., 2022). For instance, reducing the concentration of iron in the culture medium and fine-tuning zinc levels has been shown to favor zinc insertion over iron during ZnPP production (Choi et al., 2022; Wakamatsu et al., 2007).

In addition to bioengineering, microbial solutions offer alternative approaches. Specific bacterial strains, such as L. lactis subsp. cremoris, have demonstrated the ability to convert myoglobin into ZnPP both aerobically and anaerobically, rendering them suitable candidates for nitrite-free meat processing (Kauser-Ul-Alam et al., 2020). These bacterial strains not only enhance the red coloration of meat products but also provide a sustainable and scalable solution for industrial applications.

Optimizing biochemical conditions is another critical factor for improving ZnPP production. Studies have demonstrated that manipulating the Zn²⁺/Fe²⁺ ratio in culture media can significantly enhance zinc insertion into PPIX while minimizing competing pathways (Choi et al., 2022; Wakamatsu et al., 2007).

Such advancements in bioengineering and microbial methodologies have the potential to transform ZnPP production, bridging the gap between innovative technologies and the increasing demand of the meat industry for natural nitrite-free solutions.

The increasing demand for natural and clean-label food products has created new market opportunities for ZnPP as a natural meat colorant. Consumers are seeking alternatives to artificial additives and synthetic nitrites due to growing health and environmental concerns (Delgado-Pando et al., 2021; Inguglia et al., 2023). This trend is particularly evident in the processed meat sector, where the demand for nitrite- and nitrate-free products is increasing. ZnPP constitutes a viable alternative, providing vibrant coloration that meets consumer expectations and potentially reduces nitrosamine formation, although further research is necessary to confirm this effect (Wakamatsu, 2022).

ZnPP retains its vibrant red color even under light or heat exposure, rendering it a durable and natural alternative to synthetic additives in meat products (Adamsen et al., 2004; Wakamatsu et al., 2007). Furthermore, high ZnPP-forming food-grade LAB have emerged as promising candidates for nitrite-free meat processing, aligning with consumer demand for clean and sustainable solutions (Kauser-Ul-Alam et al., 2021).

In addition to traditional cured meat products, the food industry and researchers are investigating new natural colorants in emerging categories such as plant-based and alternative foods (Bakhsh et al., 2023; Ryu et al., 2023), and may explore the application of ZnPP. The vibrant red color of ZnPP can effectively replicate the appearance of traditional meat products, rendering it a suitable option for plant-based meat analogs. These factors position ZnPP as a significant innovation in the development of natural meat colorants, addressing consumer preferences for healthier, sustainable, and visually appealing food products.