# Zinc Protoporphyrin-Rich Pork Liver Homogenates as Coloring Ingredients in Nitrite-Free Liver [PERSON]
## 4 Discussion  
The decrease in the protein content with increased liver replacement percentages in the formulations could be due to the higher moisture and lower protein content of ZnPP-P (81.4% and 11.7%, respectively) compared with those of porcine livers (72-75% and 19-20%, respectively) [30; 31]. The higher the concentration of ZnPP-rich ingredients, the higher the final pH. This finding can be attributed to the addition of TSPP to attain a pH of 7.5 in the ZnPP-rich ingredients. The higher ash contents of the 16% ZnPP-H and 16% ZnPP-H/A formulations compared with those of the ZnPP-P formulations could be due to the replacement of water with ZnPP-H containing dissolved salts. However, the pH range (from 6.63 to 6.87) was relatively narrow and similar to that reported in other studies [32; 33]. Figure 3: Total ion chromatogram of the volatile organic compounds found during the elaboration of canned pork liver pâtes containing ingredients rich in Zn-protoporphyrin. The compound number 1 (2-pentanone) was used as an internal standard. Likewise, the proximate compositions of the different gate formulations were within those described in the literature [32; 33]. Thus, the gate reformulation with ZnPP-rich ingredients did not substantially change the proximate compositions of the nitrite-free pates. Conversely, there were important differences in the ZnPP and heme contents, which were mainly explained by the partial replacement of the liver with ZnPP-rich ingredients (Table 3). Nonetheless, in a previous study on liver homogenates, ascorbic acid addition caused a greater decrease in the heme content than the decrease without it, whereas the ZnPP content increased [15]. Hence, it is also possible that ZnPP formation may have occurred, to some extent, during gate elaboration. This could explain not only the ZnPP content in the controls, but also the higher ZnPP content in the 60% ZnPP-P/A formulation compared with that in the 60% ZnPP-P formulation before sterilization. In general, the sterilization treatment caused a heme content decrease, which was significant in all formulations with ZnPP-rich ingredients, whereas the ZnPP decrease with the thermal treatment was only observed in the 40% ZnPP-P/A and 60% ZnPP-P/A formulations. This finding agrees with the reported increased stability of ZnPP extracts at thermal treatments up to 70 \({}^{\circ}\)C when compared with that of extracts containing nitrosyl-heme [34]. The contents of the different porphyrins explain the gate appearances (Figure 1). The pinkish color of the positive control with nitrites was due to the presence of nitrosyl-heme, whereas the dull visual appearance of the negative control could be attributed to protein denaturation and hemichrome formation. Therefore, the red color of the pates containing ZnPP-rich ingredients can be attributed to the presence of ZnPP, which was relatively stable under the thermal treatment (Table 3). The stability of ZnPP during the sterilization treatments is of great interest for obtaining safe, red-colored, and nitrite-free meat products. However, as shown in Figure 1 and Tables 2 and 3, not only the ZnPP content (or level of addition) but also the form of the ZnPP-rich ingredients determined the red color of the product. Interestingly, ZnPP-H addition offered an intense red color (a* value) despite having a lower ZnPP concentration than that in ZnPP-P. The observed instrumental color (L*a*b*) values of the pates formulated with ZnPP-H were similar to those reported in other studies using different ingredients in the formulation of liver pates [26; 33]. The effect of the ZnPP-H-soluble fraction on color was examined by replacing the water in the gate formulation with the ZnPP-H supernatant obtained after centrifugation at pH 4.8 and subsequently adjusted to pH 7.5, causing little effect on the redness of the pates (Supplementary Figure S1). It is unclear whether some soluble compounds in ZnPP-H interacted with other matrix compounds and participated in the appearance of the red color.