3.1. Headspace Gas Composition
The O2 and CO2 concentrations in the headspace of the packages were analysed, except for the vacuum-packed samples.
In the air treatment, the O
2 concentration decreased eight times in the first fourteen storage days. From day 21 onwards, a significant difference was observed (
p ≤ 0.01) as its presence was only 1.32% (
Table 1;
Figure 1). This may be due to the cheese microbiota, since the aerobic microorganisms could have been able to consume the O
2 inside the package and convert it into CO
2. It is less likely that this decrease in O
2 happened due to gas permeation through the packaging material, given the low level of permeability (<3 cm
3/m
2 × bar × 24 h versus a value of ≤80 cm
3/m
2 × bar × 24 h of a conventional PA/PE 20/70 material).
A decrease in the O
2 level from 19.98% to 0.16% was detected in previous experiences when dealing with Domiati cheese in cold air storage, despite using a very high oxygen barrier packaging film [
37].
For both MAP1 and MAP2 treatments, the average O
2 value was 0.12%, and it remained unchanged throughout the storage time. This residual amount of oxygen is indicative of the absence of failures in the container [
38]. There were no significant differences in the O
2 content among the MAP treatments, from 20 to 100% CO
2-packaged cheeses (Samso and San Simón da Costa) [
39,
40], and the mean values were between 0.17 and 0.2%. Similar data (±0.4%) have recently been reported by Albisu et al. [
23] in Idiazabal cheese wedges packaged in four different atmospheres, using a conventional non-recyclable material.
The air treatment presented a seven-fold increase in the first fourteen days and remained stable until the end of storage (
Table 2;
Figure 1). Considering the low CO
2 permeability of this material (<15 cm
3/m
2 × bar × 24 h compared to a value of ≤174 for a conventional PA/PE 20/70 material), the progressive increase throughout the study could mainly be due to the microbial activities of the cheese [
22].
In MAP conditions, unlike air packaging, there was a 17.62% average decrease in CO
2 during storage. In both cases, after 14 days, MAP1 and MAP2 lost 10.23 and 13.21%, respectively. In the study with the PA/PE (20/70) non-recyclable material [
23], a progressive decrease in the CO
2 concentration was observed in the 20, 50, and 80% CO
2 treatments, reaching 39% after eight weeks of storage. This decrease might be attributed to gas dissolution in the cheese matrix, its consumption by anaerobic microorganisms, or by CO
2 loss through the barrier film [
22,
41]. In a similar study by Solomakos et al. [
42] with sheep cheese packaged under 50/50% CO
2/N
2 conditions, after 10 storage days at 4 °C, the CO
2 concentration decreased by 17.15% and remained stable thereafter.
In the first 21 storage days, the O
2 was reduced by 98.68% and remained at a ratio of 0.27% until the end of the period. In a previous study using PA/PE (20/70) as the packaging material, this gas equilibrium was reached at a slower and more progressive rate in almost double the time (42 days) [
23]. In addition to microbial growth, these results could be explained by the higher O
2 and CO
2 permeability of the PA/PE material, whereas the Next flex material used in this study presents a lower gas permeability. In low-permeability materials, a possible accumulation of water on the surface of the product could promote microbial growth [
43,
44]. For highly respiring products, a combination of low oxygen permeability and a high respiration rate easily leads to anaerobic conditions inside the package [
45]. Therefore, film properties do influence the physicochemical cheese characteristics, and, consequently, the cheese microbiota. Related to this fact, Florit et al. [
46] studied the effect of micro-perforation packaging material on the headspace atmosphere evolution.
3.2. Sensory Analysis
Concerning texture (
Table 3), only a slight quality decrease (
p ≤ 0.05) was observed across the two storage months for cheeses packaged in air and vacuum, but they were above the acceptance limit at all times (score out of 4). No significant differences in flavour (
Table 3) were found for vacuum and MAPs, while those kept in air showed significant differences (
p ≤ 0.001) at 14 days, and they were considered unfit from 21 days onwards. These samples displayed off-flavours, such as rancid, mouldy, or an undefined type. Although the oxygen present in the air packaging was low, it may have caused the mouldy and rancid off-flavours by favouring mould proliferation to a certain extend.
The paste appearance changed significantly in all packaging treatments across time, except for MAP2, which remained stable (
Table 4). For air-packed cheeses, the only difference (
p ≤ 0.05) was detected on day 56, where some assessors indicated slight greenish spots. This defect was caused by the proliferation of moulds and was related to the mouldy off-flavours. Vacuum-packaged cheeses had gradually decreasing scores, lowering 2.7 points from the beginning of the storage to the end. This decrease was the result of a plastic and shiny appearance, together with packaging marks and a non-homogeneous colour, which provided an anomalous appearance from day 14 onwards. This packaging treatment showed significantly lower scores (
p ≤ 0.001) with respect to the rest of the treatments. The wedges packaged in MAP1 presented slight changes in the colour of the paste, which could be attributed to the cheese variability itself, and not to the packaging condition.
Regarding holes, significant differences (p ≤ 0.001) over time were only revealed for vacuum-packed cheeses. On day 14, holes started to occlude, and, at the end of the storage, cheeses presented cracks and sinkholes where holes were initially located. These defects were produced by the pressure necessarily exerted in vacuum packing, which produces anomalous colouring and holes.
In summary, vacuum packaging showed the worst results for paste appearance and holes. Air-packaged cheeses were the lowest rated for flavour and presented defects in the paste appearance at the end. Indeed, it has been reported that CO
2 concentrations below 20% do not inhibit the growth of moulds [
21,
47]. Short storage times have been described for cheeses kept in air on account of mould growth [
22,
37,
48]. MAP1 and MAP2 showed no significant changes for texture, flavour, and paste holes during the 56 storage days. In Parmigiano Reggiano cheese, increases in texture and flavour were detected, and, in MAP treatments, moisture and solubility were reduced, but no significant changes in flavour were recorded, except for vacuum-packed cheeses, in which bitterness increased.
Similar results were found in a previous study with Idiazabal PDO cheese using a non-recycled plastic material PA/PE (20/70), where the best-rated cheeses were packaged with 50/50 and 80/20% CO
2/N
2 [
22]. Several authors pointed out that an atmosphere close to 50/50% CO
2/N
2 is the best for preserving the cheese flavour [
32,
48,
49,
50].
3.3. Physicochemical Analysis
Cheeses had mean values of 67.54 ± 1.11% for dry matter, 22.68 ± 0.59% for protein and 39.27 ± 0.90% for fat; the pH was 5.10 ± 0.05, and 0.11 ± 0.07% was quantified for the weight loss.
Dry matter and fat did not show significant differences (
p ≤ 0.05), neither for packaging treatment or storage time, nor for the interactions between them. Protein showed some differences (
p ≤ 0.05) due to packaging conditions and storage time, as well as for their interactions. In a previous study with Idiazabal PDO cheese using PA/PE (20/70) as the packaging material [
23], none of the physicochemical parameters measured in wedges showed significant differences (
p ≤ 0.05), neither for packaging treatment or conservation time, nor for the interactions between them. Kirkin et al. [
51] and Solomakos et al. [
42] did not observe significant changes in the composition of cheese samples throughout the study. The main protein content and total solids remained almost constant for six storage months in vacuum-packaged San Simon da Costa cheeses. The fat content remained constant, although fat migration was observed during storage [
52].
pH levels showed some differences throughout the storage time, mainly between packing and 14 storage of days (an increase of 0.04 units for vacuum packaging and an average increase of 0.14 units for air and MAP treatments). The minimum and maximum values measured were 4.96 and 5.18, respectively. Regardless of the atmosphere composition, the pH value remained stable throughout the storage in a previous study concerning Idiazabal cheese [
23]. Similar pH stability during refrigerated storage has also been reported in other MAP-packaged cheeses [
51]. In vacuum-packaged cheeses, the pH remained constant for six storage months, with no significant differences in non-packaged samples [
52]. It has been explained by lactose metabolization and lactic acid formation in the early stages of the processing, and by the low level of proteolysis.
Weight loss differences were observed for packaging treatments and conservation times (
p ≤ 0.05) (
Table 5). During storage, weight percentage losses remained constant for air and vacuum, whereas MAP1 and MAP2 increased their weight losses from day 42 onwards.
However, there were no statistically significant differences between the four treatments when comparing the percentage of total losses at the end of storage (mean value 0.13% ± 0.02), whereas losses of 0.15% were described for Provolone cheese [
25]. After eight storage weeks of the Idiazabal cheese wedges, weight loss percentages of 0.39 ± 0.43% were reported without differences between the packaging treatments (air, vacuum, and MAP treatments) [
23]. This is consistent with previous experiences where different gas mixtures did not significantly influence weight losses in MAP-packaged ripened cheeses [
22,
50,
53]. The plastic material used prevented dehydration and weight loss [
40].
3.4. Instrumental Colour Parameters
L* and Zi showed differences in terms of the storage time, a* and Yi for the packaging treatment, and b* for both (treatment and time) (
Table 6). L*, a*, and Zi did not show any differences throughout the storage time for any of the treatments. Mean values ± standard deviation for L*, a* and b* colour parameters of the cheese wedges stored for 56 days at different treatments can be downloaded as
Supplementary Materials (Table S1).
Air packaging (a* mean value −3.50) differentiated (p ≤ 0.01) from the rest of the treatments (a* mean value −2.35). This difference could be due to the presence of oxygen in the air treatment, which was able to initiate the colour change perceived via the instrumental measurement.
Throughout the storage time, b* remained stable only in air-packed cheeses (mean value of 13.44). In vacuum, MAP1, and MAP2, b* changed markedly during the first 14 days, decreasing from a mean value of 13.79 on day 0 to 11.60 on day 14, and remained almost stable until the end for all treatments.
Yi did not show significant differences throughout storage time in air- (mean value of 25.20) and MAP1-packed (mean value of 21.71) conditions. However, vacuum and MAP2 did show significant differences for Yi, which presented a 25.64 value on day 0 and a mean value of 21.45 on day 56 for both conditions.
In a previous study on Idiazabal cheese, a* and b* parameters were those with the highest discriminant weight between the preservation conditions. It should be highlighted that a* was able to statistically distinguish air-packaged cheeses from vacuum and MAP cheeses. In the same way, for b* and Yi, air and vacuum packaging treatments were similar, and different from MAP treatments, which were also like each other [
23]. As in the present study, Yi and Zi did not seem to discriminate more than L*, a*, and b*.
In Provolone cheese packaged at different CO2 concentrations, no differences were detected in colour (Yi) [
25], and Di Marzo et al. [
54] suggested that both vacuum and protective atmospheres might stabilize the colour of cheese during storage. Kirkin et al. [
51] did not observe significant changes for L*, while, in Parmiggiano Reggiano cheese packaged with 50%CO
2/50%N
2, a decrease in L* was observed [
32].
3.5. Instrumental Texture Analyses
In the storage time, there were generalized statistical differences (
p < 0.05) in hardness, slope, chewiness, cohesiveness, and resilience for all atmospheres. Air samples remained stable in terms of cohesiveness and resilience.
Figure 2 shows changes in the overall texture profile when comparing the initial sampling days with the last ones.
For all the treatments, the hardness decreased by 75% from the t0 values (33.413 N) to the end (
p ≤ 0.001) after increasing to higher values in the intermediate weeks (38.267 N–40.677 N). Chewiness registered a 78% drop from t0 (19.337 N) to 56 days (
p ≤ 0.001), with the middle values being (23.061 N–23.898 N). The adhesiveness increased significantly (
p ≤ 0.001) by the end of the storage for MAP1 and air. The same tendency was identified in results from the uniaxial compression test, with the lowest values for the parameters measured in the last weeks, especially for the maximum load (
Table 7). Uniaxial compression had an initial increasing behaviour in the three parameters measured, which generally registered the highest values in days 28 or 35. After that, values tended to decrease, nearing values of the initial days. This behaviour was also described in our previous contributions from Albisu et al. [
23] when working with the same kind of cheese, but with a non-recyclable plastic packaging. Texture changes happened more slowly with the current recyclable material when compared to our previous work, as well as to some others, such as Atallah et al. [
37] and Costa el al. [
22], in which the maximum hardness and cohesiveness values were achieved 20–30 days after storage and then decreased, agreeing with gas mixture changes in the modified atmosphere. The current observation is probably due to the stabilization effect offered by MAPs with the CO
2 content above 50% [
23,
49].
Data for the texture profile analysis (TPA) revealed few statistically significant differences (
p ≤ 0.05) among the atmosphere conditions. Air and vacuum packaging were responsible for most of the significant changes identified, and the majority of these happened in the early stages of storage, after around 14 days of packaged refrigerated storage. At that time, the vacuum treatment had a significantly lower hardness (34.920 ± 7.46 N) when compared to other conditions. In the last day, vacuum samples showed significantly higher resilience values (0.464 ± 0.022), as compared to all of the other treatments (0.445–0.447), and it had the highest cohesiveness values (0.744 ± 0.02), which were statistically different when compared to air and MAP1, which were not statistically different and had a mean cohesiveness value ± SD 0.732 ± 0.025. It is usual to find collapsed packaging in vacuum-packed cheeses [
55]. At 14 days of refrigerated storage, the treatment was statistically different in paste appearance and holes. It had the lowest mean values for cohesiveness and resilience, being 0.736 ± 0.218 and 0.436 ± 0.022, respectively. As suggested by sensory observations, a rapid growth of anaerobic bacteria and mould due to quick gas mixture changes could be responsible for these changes in the air-packed samples [
22].
Focusing on uniaxial compression (
Table 7), vacuum-packed samples displayed a behaviour which was slightly different to the others. The maximum load was the most outstanding parameter, as it showed a general tendency towards higher values in these samples, mostly from day 35 onwards. This tendency showed statistical significance (
p ≤ 0.05) at day 42 (comparing to 50/50 CO
2/N
2 samples) and day 56, compared to both MAP1 and MAP2. The maximum load represents the highest force needed to cause the fractures registered during the compression test, thus, this should not be interpreted as hardness or firmness, as it would be the maximum peak force in TPA. The need for higher forces to fracture the samples, with the higher cohesiveness described for vacuum cheese samples in the double compression assay, is significant.
3.6. Discriminant Analysis
Air-packaged samples were clearly differentiated from vacuum-packaged samples, and both types of packaging were also differentiated from MAPs (
Figure 3). The results showed that 92.2% of the samples were correctly classified into their corresponding treatment group, taking 50% and 80% CO
2 MAP as a single treatment group (
Figure 3).
The cross-validation method used for sample classification reported that 93.8%, 87.5%, and 81.3% of the MAP, air-packaged, and vacuum-packaged samples were correctly assigned.
The discriminant variables with higher correlations with canonical functions in the structure matrix were paste appearance and holes, flavour, and yellowness (b*) and yellow index (Yi) colour parameters.