3.2. Time–Course of Physicochemical Parameters
The physicochemical characteristics of the 3D-printed products were monitored for 21 days. Time–course changes in pH and water content during storage for each of the two food forms and each MAP condition are reported in
Table 4.
Globally, for form C, pH increased by 0.11 to 0.12 pH units between D0 and D21 (p < 0.05) without a significant difference between the two MAP conditions. For form F, pH remained stable and then decreased very slightly (0.05–0.06 pH units) but significantly (p < 0.05) after day 14 for both storage conditions. As CO2 can dissolve in water and thus decrease pH, the equal concentration of CO2 in both conditioning modes may explain the lack of difference.. Although pH was initially lower for form C than for form F (6.28 vs. 6.41 at day 0; p < 0.05), there was no longer any significant difference between the two forms by day 7. This initial pH difference can be explained by the different compositions, as form C contained a higher proportion of the animal-source mixture, which had a more acidic pH (6.07) due to the presence of raspberry vinegar.
aw values showed no significant changes (p > 0.05), peaking at 0.98 throughout storage, regardless of product form and packaging method (data not shown). This suggests that MAP and time have no impact on this physicochemical parameter in the 3D-printed food. As aw is high, the environment remained favorable to the development of microorganisms, which makes it necessary to store these products at 4 °C to slow down potential microorganism growth.
Form C demonstrated no significant change in water content with time in storage or type of packaging: there was a significant difference at day 7 between N2-MAP and O2-MAP (1.59 kg water/kg dry matter in N2-MAP versus 1.66 kg water/kg dry matter in O2-MAP, p < 0.05), but this difference disappeared afterwards. Once again, the differences observed between the two product forms (regardless of the time in storage, form F always contained less water than form C) are the result of their different initial animal-source/plant-source ratios.
3.3. Time–Course of Iron Content
The total iron concentration measured in raw chicken liver (
Table 5) was two to three times higher than values reported in the literature, such as in Kongkachuichai et al. [
28] (9.9 ± 0.8 mg/100 g) and in the CIQUAL database (9.0 mg/100 g on average) [
27]. The total iron concentration in the raw pork liver was also higher than the value reported by Kongkachuichai et al. [
28] (12.3 ± 3.5 mg/100 g), mainly due to a higher NHI content (9.4 ± 2.4 mg/100 g), but close to the values found in the CIQUAL database (18.4 mg/100 g on average) [
27] and in Tomović et al. [
29] (19.5–23.9 mg/100 g). This variability can be explained by various factors such as genetics, breeding, slaughter practices, feed or geographical location. Total iron concentration of the animal-source mixture is consistent with the proportions of liver used, i.e., 70% poultry liver and 30% pork liver.
For the plant part, the cooked lentil mixture had a total iron concentration of 8.76 ± 0.19 mg/100 g, which is much higher than that which was measured in the CIQUAL [
27] for boiled red lentils (2.2 mg/100 g) and by Quinteros et al. [
5] (2.01–2.25 mg/100 g depending on the cooking method). This discrepancy can be attributed to soaking the lentils, which leads to a decrease in NHI but was not performed here, and to the different cooking conditions. The plant-source mixture had a slightly higher iron content than the lentil mixture alone, because the lupin flour provides additional iron, as it has an iron content of 2.5–11.4 mg/100 g according to Eberl et al. [
30].
Regarding the distribution of the iron forms, NHI was mainly observed in the liver-based preparations according to Bogunjoko et al. [
31], whereas the plant mixtures did not contain HI but mainly free NHI (9.14 ± 0.18 mg NHI/100 g).
For the 3D-printed hybrid products, forms C and F contained 16.40 mg total iron/100 g and 15.77 mg total iron/100 g, respectively, on the day of fabrication (D0), i.e., a ratio of 94% NHI to 6% HI (
Table 6 and
Table 7). This initial composition reflects the proportions of the animal-source and plant-source mixtures incorporated in the recipes. After 21 days in storage, these 3D-printed products still had a total iron content of about 12–14 mg/100 g, regardless of product form (C or F) and packaging method (O
2-MAP or N
2-MAP), which would confer the right to use the nutrition claim “high in iron” as defined in the Annex to EU Directive 90/496/EEC on nutrition labeling for foodstuffs.
This total iron content is naturally lower than that of some fortifying mixtures used as an ingredient based on pork liver (23.8 mg/100 g) [
3] or beef lung powder (59.3 to 61.7 mg/100 g) [
32], but higher than that of a conventional minced steak. These foods thus provide valuable fiber and polyunsaturated fatty acids (
Table 1), but they are also well suited to the daily iron requirements of women and can thus help reduce the incidence of iron deficiency, as the RDAs for iron provided by the Institute of Medicine (IOM) [
33] are 15 mg and 18 mg for women aged 14–18 and 19–50 years, respectively, increasingly to 27 mg in pregnancy.
In the context of a complex hybrid food, it is difficult at this stage to predict the bioavailability of iron in our products without further in vivo or in vitro studies. Rewashdeh et al. [
34] studied the effects of dietary iron sources such as liver, lentils and liver plus lentil mixtures on iron bioavailability and concluded that liver-based diets can improve the bioavailability of plant-based iron, particularly when combined with lentils. Similarly, a recent study showed that some lentil proteins have the ability to bind to iron, which would decrease the free form of iron (Fe (II) and Fe (III)) prior to absorption and thus increase its bioavailability [
35].
HI content decreased significantly during storage for both product forms independently of MAP, but a predominant effect of O
2-MAP was observed. Indeed, HI content (5–10% of total iron) decreased more strongly when the products were stored under O
2-MAP: between days 0 and 7, HI decreased by 0.65 mg/100 g under O
2-MAP against 0.06 mg/100 g under N
2-MAP. Regarding NHI, MAP conditions had no impact on NHI content, which decreased progressively over time for both product forms, although form F showed a significant difference between D0 and D21 (
Table 7).
In contrast to what is presented here, some studies have shown that the NHI content of liver increases during storage in parallel with the decrease in HI content, due to degradation of the heme molecule that leads to the release of iron. However, Estévez et al. [
36] showed that this increase occurred in the final stages of storage after 60 days. It is therefore possible that a 21-day follow-up is not sufficient to observe this conversion and the resulting increase in NHI. Furthermore, the amount of NHI is vastly higher than the amount of HI (about 15 to 43 times higher); the conversion of HI could possibly be masked by the natural decrease in NHI.
3.4. Evolution of Textural Properties
The TPA test is widely used to characterize the textural properties of food products. The double compression allows for the simulation of food chewing and thus brings the perceived texture of the food closer to its real acceptability by consumers [
37].
Table 8 shows a significant effect of MAP conditions on product textural properties, especially hardness, gumminess and chewiness. The 3D-printed products had a significantly harder texture when kept in an oxygen-rich atmosphere, and this change in texture was detected from day 7 onwards and maintained until day 21. Textural changes appeared to have occurred between day 0 and day 7 mainly in the animal-source part, until a plateau was reached. Estévez et al. [
38] observed a similar increase in the hardness of liver pâtés during refrigerated storage. Furthermore,
Figure 3 shows that products stored under N
2-MAP were more friable, crumbling more easily (
Figure 3a), due to a marked dissociation of the animal and plant-based layers, in contrast to (
Figure 3b) the denser and more cohesive products under O
2-MAP that held together better. Even though they are slightly more brittle, ‘N
2-MAP’ products were closer to the structure of the original food. These results are in agreement with Verma et al. [
39], who found that pork loaves stored under N
2-MAP showed significantly lower hardness than products kept under aerobic packaging throughout storage. This textural difference cannot be attributed to a variation in the water content of the product, as composition of the C form remained similar from D0 to D21 regardless of the packaging method. The main explanation of this textural change could be protein oxidations and interactions with other compositional components. Many studies converge to show that variation in the textural properties of meat products is related to changes in protein functionality and, in particular, interactions between the proteins, the continuous aqueous phase and dispersed fat.
3.5. Lipid Oxidation
The TBARs method was used to determine lipid oxidation in the samples during storage under the two MAP conditions. As shown in
Figure 4, the TBARs values of the 3D-printed products under O
2-MAP increased significantly (
p < 0.05) from 0.23 to 1.28 mg MDA/kg product for form C and from 0.09 to 0.67 mg MDA/kg product for form F between days 0 and 7, whereas TBARs values under N
2-MAP remained globally constant at around 0.20 mg MDA/kg for both product forms during the entire 21-day period. Although there is no established threshold value in the literature, it is generally accepted that a product is oxidized if it contains more than 1 mg MDA/kg product [
40]. This was the case for 3D-printed form-C products from day 7 onwards. After day 7, the TBARs values stabilized or decreased slightly. However, this does not mean that oxidation processes were no longer evolving. Indeed, studies have shown that TBARs values are a good indicator of the early stages of oxidation of a product, but that other methods, such as the determination of hydrosoluble Schiff base (HSB) values, are sometimes more appropriate for tracking lipid oxidation over time [
41]. The values obtained here were close to those of Wang et al. [
42] on chicken legs and Estévez et al. [
36] on pork liver pâtés. Moreover, Del Olmo et al. [
43] also observed that the N
2/CO
2 mixture limited the oxidation of pork meat products.
Lipid oxidation is known to cause a deterioration in product quality by affecting sensory properties such as color, flavor and texture (see
Section 3.4). Lipid oxidation involves the degradation of polyunsaturated fatty acids and generates free radicals that lead to the deterioration of proteins and the oxidation of blood pigments [
25]. Many studies have reported that lipid oxidation promotes the formation of metmyoglobin as a result of myoglobin oxidation and makes liver turn brownish grey during storage [
36,
44]. This change in liver color was also visible here in products stored under O
2-MAP compared to N
2-MAP (
Figure 3b). In addition, the heme-protein oxidation caused by lipid oxidation also explained the decrease in measured HI content (
Table 6 and
Table 7). The difference in TBARs values between the two printed forms was mainly due to their different compositions and chiefly the ratio between the animal-source and plant-source parts. TBARs values were higher for form C, which contained more liver mixture and particularly chicken liver, which is rich in unsaturated fatty acids that are more prone to oxidation than saturated fatty acids. The animal part was also rich in NHI, which can further promote oxidation processes. The higher proportion of plant-source mixture in the F form could have helped to curb oxidation, as it contained antioxidant-rich ingredients such as curry powder [
45].
3.6. Interactions between Physicochemical, Nutritional and Textural Parameters
Figure 5 shows the results of PCA performed to map the 3D-printed hybrid form C products to the main variables (physicochemical, nutritional and textural parameters) and explanatory variables (form, storage time and MAP). The additional variables correspond to N
2, CO
2 and O
2 levels measured in the storage trays. These variables were not considered in the determination of the axes, but they serve to visualize the relationship of the variable with all the active variables in order to enrich the analysis. The results for the form F were not presented in the manuscript, because they are similar to those of the form C and provided less information (no texture parameter measurements).
This PCA specifically summarizes the link between the textural properties of the 3D-printed products and the physicochemical phenomena observed. Principal components 1 and 2 explained 69.9% of the total variance. The first component (about 51%) was more related to HI content, TBARs values and textural parameters as a function of MAP composition and thus explains the biochemical and textural changes related to level of product oxidation. Samples with low HI content were characterized by a harder, gummier and chewier texture and higher TBARs values. These samples correspond to those stored under O
2-MAP. Component 2 explains less of the variance (about 19%) and is related to NHI content (the majority of the total iron content) and the adhesiveness of the product. Furthermore, physicochemical parameters such as pH, a
w and water content were fairly orthogonal to all the main PCA variables and were therefore not well represented on these axes, which is consistent with the non-significant results presented earlier. Nevertheless, the PCA map highlights the logical linkage between CO
2 concentration in the tray and pH of the 3D-printed product, which confirms the hypothesis stated in
Section 3.2. Similar results were observed with the biplot of principal components 1 and 3. Principal component 3 explained 11.1% of the total variance and was also related to NHI content, pH and adhesiveness of the product, but variables were poorly represented on the axis.
These results thus show that MAP composition has a major impact on the textural and physicochemical properties of the 3D-printed products and can separate the two groups of samples (O2-MAP and N2-MAP) (p < 0.05). Moreover, biochemical changes occurred mainly between D0 and D7, after which a plateau set in (non-significant differences between days 7, 14 and 21).