3.1. Microbiological Analyses
Microbial counts of casings and salami during the ripening period are displayed in
Table 1.
Other authors have already deepened the bacteria biodiversity of natural and synthetic casings [
23] and highlighted their role in salami microbiological communities. In agreement with Comi et al. [
24] and Pisacane et al. [
23], in our study, casings were characterized by a Total Aerobic Bacteria at 30 °C of about 5.62 Log (CFU/g). However, if mesophilic bacteria and LAB [
23] appear to overlap literature data, the presence of Total Coliforms did not reflect what was reported by other authors especially because of the high contamination (
Table 1). For this reason, issues regarding the hygienic state of the casings could be hypothesized.
Regarding microbiological measurements carried out on salami during ripening stages, growth trends of each bacterial population were approximately superimposable among products, showing no significant differences. The mesophilic total bacteria count at the beginning of the trial was high in all samples, ranged between 4.96 and 5.78 Log (CFU/g), and increased until the sixteenth day of fermentation. Afterwards, the TBA count remained stable throughout the last ripening period according to Madonia et al. [
2] and to Pisacane et al. [
23]. However, S75s made an exception because they were characterized by a gradual TBA increase during the whole ripening period. In addition, our data showed a close relationship between TBA and LAB counts, whose trend defined them as predominant populations. In addition, despite the absence of starter cultures, the high level of lactic acid bacteria could suggest the positive burden of casings and swine meat on the microbial community of final products, as proposed by Bedia et al. [
25].
Although devoid of significative correlations (data not shown), microbial counts of Total Coliforms suggested a relevant role of casing on microbial communities. Indeed, it is worth noting that for all salami, coliforms had initial high values, which decrease significantly (
p < 0.01) throughout the ripening period. This result could be explained because of the susceptibility of coliforms to the acidification effect of LAB [
26,
27]. Such finding is in agreement with the Pearson’s correlation coefficients calculated (
R2: −0.446;
p < 0.01).
Regarding Pseudomonas spp., microbiological counts significantly differed among salami and sampling time. Nevertheless, these bacteria followed pH variations showing a significant decrease (p < 0.01) at 30 days of maturations, when matrices acidification were highest.
Significant differences among salami were detected for yeasts and molds, which were unusually found in casings. At the beginning of the process, these populations appeared higher in S75 than the others (
p < 0.01). On the contrary, during the ripening period values came closer. Yeasts and molds had similar growth trends with characteristic bell-shaped. Indeed, since the sixteenth day, their number decreased and approached the initial values. As reported by several authors [
27,
28], the presence of yeasts is desirable because they contribute to providing salami sausages with their peculiar flavors and surface appearance, because of their esterification and proteolytic activities [
29].
To monitor the changes in microbial communities after ripening, it was interesting to highlight the behavior of the investigated population. In particular, the ripening process and manufactories adopted might be considered able to control the hygienic state of products, influencing the survival of microorganisms such as Coliforms.
3.2. pH and aw Measurements
The average weight loss of all salami was 40% compared to the initial weight. No significant differences were found among the types of salami. During ripening, pH showed a similar trend among the different salami with a strong decrease until the first month and a slight increase from T1 to T3, as observed in other studies [
22,
30]. Nevertheless, all products showed pH values higher than 5.2 which was considered the stability point of meat products by Ambrosiadis et al. [
31]. Variation in the bacterial population during salami fermentation appeared to influence the meat sourness, as demonstrated by several authors (i.e., [
32]). At the beginning of the study (day 1), the pH was 5.89 ± 0.12 in all samples, reaching values of 5.13 ± 0.04 due to acidification activity of Lactic Acid Bacteria. Instead, a negative and significant correlation was found between LAB concentration and pH value, along whole storage time and in each sample (
R2: −0.510;
p < 0.01). The progressive increase in pH was probably due to proteolytic activities induced by typical meat bacteria with the release of the amino groups [
33,
34], responsible for pH neutralization [
34]. Moreover, Mendonça et al. [
35] hypothesized that it could be influenced by the degradation of lactic acid by molds and internal yeasts.
Significant differences (
p < 0.01) were observed between RS and S75 along the whole storage time (
Table 1), with the lowest values in S75 samples. In spite of the content of Apulo-Calabrese meat, the pH values of S50 salami did not appear influenced by indigenous Italian pig breed and almost overlapped to RS. Aboagye et al. [
36] compared meat quality traits of Apulo-Calabrese with respect to crossbreeds [Duroc × (Landrace × Large White)] and observed that pH values were constantly higher in autochthonous breed meat, albeit slightly.
Water activity measured at the start of the drying process (1 day) was approximately 0.977 ± 0.001 in all samples and decreased during ripening. Throughout the first three months, each sample showed similar aw levels, underlining the same downward trend. Slight differences have been detected at 120 days: white pork salami (RS) reached a value of 0.794 ± 0.008, lower than the others. This may be related to the possible differences in salt concentration among the three formulations.
3.3. Fatty Acids Analysis
Fatty acid composition results measured for each FAME sample in three formulations (SR, S50 and S75) during ripening are shown in
Table 2.
Gas chromatography (GC) analysis showed that in all formulations the dominant fatty acids found were palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1n-9), and linoleic acid (C18:2n-6). As a consequence of the different content of black pig meat, significant differences among the three types of salami were highlighted. The average values of total saturated FA throughout the ripening time were 42.27% in SR, 39.02% in S50, and 36.56% in S75. For monounsaturated short- and medium-chain fatty acids, oleic acid was the most abundant (46.62% in SR, 49.52% in S50, and 51.66% in S75, expressed as average values of all sampling times). A higher amount of SFAs were detected in SR samples (
p < 0.01) than S75 ones, due to greater content in C16:0 and C18:0. According to several authors [
2,
3,
4], Italian native pigs such as
Cinta Senese,
Casertana, and
Nero Siciliano showed a high predisposition to MUFAs depot, mainly oleic, detected in final products as fresh subcutaneous fat and salami products. The high content of C18:1n-9 in salami with Apulo-Calabrese meat (S50 and S75) could be due to the rearing system of the local breeds (almost always outdoors) and the typical dietary regimen of these pigs, which are fed on acorns and chestnuts. Although comparisons with similar studies are difficult due to differences in rearing and management conditions (factors affecting the fatty acids composition of tissues). The high content of oleic acid in meat products from pigs fed on acorns has been reported both by Pugliese et al. [
37] in
Cinta Senese and by Pérez-Palacios et al. [
38] in Iberian pigs. Local pig breeds generally exhibit a higher percentage of MUFAs than crossbreeds [
2,
3,
4,
5], which are characterized by a greater amount of SFAs was found [
2]. This implication is very important from a nutritional viewpoint because stearic and palmitic acids are the predominant SFAs in animal fats whose consumption in the human diet is one of the factors causing cardiovascular disease [
39].
The high PUFAs content may have detrimental effects on the sensory and technological quality [
40] and acceptability of meat products [
41]. The results showed that the lowest PUFAs content was found in SR as the linoleic acid content was significantly lower (
p < 0.01) than in S50 and S75 from the first to the 60th day of ripening. Yu et al. [
42] described similar differences among different genotypes in
longissimus dorsi muscle, recording significantly more PUFAs in Lantang (IMF 2.46%) than in Landrase (IMF 1.43%) breeds. Nevrkla et al. [
5] found the same difference in backfat samples of Prestice Black-Pied breed (IMF 2.89%) than in pigs of hybrid combination [Large White × Landrace sows × Duroc × Pietrain] (IMF 1.99%). In contrast, Aboagye et al. [
17] comparing the fatty acid profile of Apulo-Calabrese and crossbreed pigs reared indoors and fed the same commercial diet, observed significantly lower PUFAs content in the Italian local breed. However, it is difficult to compare studies because the difference in PUFAs content may be due to the different growth performance and adipogenic potential that characterize the different genetic types considered.
In long-aged salami, the PUFAs level should not exceed 12% of the total fatty acids [
43]. At the start of ripening, the percentage of PUFAs was approximately 11–12 in all samples. During ripening, the PUFAs content is more susceptible to oxidation reactions than saturated and monounsaturated fatty acids [
44]. PUFAs decreased in all samples except in SR. SFAs content gradually decreased during the first 60 days and then increased at the end of ripening. In contrast, MUFAs percentage increased during the first 60 days and then suffered a fall at the end of ripening. Regarding P/S, a value greater than 0.4 is recommended for healthy foods and diets [
45,
46] in order to prevent both an excess of SFAs, having a negative effect on the LDL cholesterol plasmatic level, and of PUFAs, some of which are precursors of powerful clotting agents and are involved in the etiology of some cancers [
47].
The PUFA/SFA ratio (P/S), the MUFA/SFA ratio (M/S), the hypocholesterolemic/Hypercholesterolemic fatty acids ratio (h/H), Atherogenic index (AI), and Thrombogenic index (TI) were determined (
Table 3).
The P/S ratio was below the recommended value and ranged on average from 0.26 in SR to 0.31 in S75. The high percentages of MUFAs and C18:1n-9 in S75 samples indicate their suitability for healthier diets, as diets rich in MUFAs (and PUFAs) reduce blood cholesterol levels and are related to a low incidence of cardiovascular diseases [
11]. The M/S ratio was the highest in S75 on the 60th day due to the highest content of oleic acid (47.8%). On the contrary, SR showed the lowest ratio M/S at the end of ripening caused by the lowest percentage of MUFAs, characterized by 41.7% oleic acid.
Among SFAs, C18:0 has poor atherogenic characteristics because it is rapidly desaturated in oleic acid, unlike myristic acid (C14:0) which is considered the main atherogenic fatty acid as it has a hypercholesterolemic power four times higher than C16:0 [
48]. Ulbricht and Southgate [
18] proposed equations for atherogenic and thrombogenic indices. Therefore, the atherogenic index, as well as the thrombogenic index, were calculated in order to evaluate the risk of atherosclerosis, and the potential aggregation of blood platelets, respectively. TI and AI ranged from 1.38 to 1.08 and from 0.55 to 0.44 (mean values) in SR and S75, respectively. The average values for S75 were consistent with the results by Franci et al. [
3] for Cinta Senese. AI and TI values for SR were similar to the values found by Del Nobile et al. [
49] for commercial salami. Our results allow us to consider salami made with black pigs healthier than the salami SR.
These observations agree with the values of the h/H ratio, which is also used to evaluate the nutritive quality of the fat as it is related to cholesterol metabolism [
19]. From a nutritional standpoint, higher HH values are considered more beneficial for human health. The percentage of fatty acids considered as hypocholesterolemic was significantly higher in the S75 (>63%) than in the SR (<58%), while the amount of Hypercholesterolemic fatty acids showed an opposite behavior (<24% in the S75 vs. >26% in the SR). As a result, the mean value of the h/H ratio of the S75 was significantly favorable (>2.6) in particular on the 60th day of ripening (>2.7).
3.5. Thiobarbituric Acid-Reactive Substances (TBARS)
Lipid oxidation is one of the main factors affecting the features of meat products during the storage period. The development of oxidation products in salami is displayed in
Table 7 as TBARS values.
Oxidation significantly increased along the ripening process for all samples (
p < 0.01), except for S75 in the last sampling time. It is worth noting that since the day after manufactories salami SR showed the lowest concentration of malondialdehyde (MDA—0.042 mg/Kg), almost half of the other two formulations (
p < 0.01). Therefore, the presence of Apulo-Calabrese meat seems to play a role in the oxidation process, especially considering MDA differences among S50 and S75 which were clearly influenced by the percentage of indigenous meat present. Other authors demonstrated that lipid oxidation depends on polyunsaturated fatty acids contents, probably because of the lower energy necessary to remove hydrogen from a double carbon bond than methyl carbon, especially when the carbon is between two double bonds [
44,
56]. Nonetheless, PUFAs contents were higher in S75 than others, no significant correlations (data not shown) were found between PUFAs contents and TBARS values. Differences among products remained almost similar until the 16th day of the ripening process, while at the fourth-month disparities disappeared and a significant increase in the MDA values was observed in SR and S50. In particular, the lowest TBARS value was measured in S75 (0.264 mg/kg), probably due to the transformation of oxidation indicators into other decomposition products, afterword achieving its peak [
57]. Furthermore, Pavlović et al. [
58] stated that lipid oxidation slowdown is due to interactions between MDA and additives and organics molecules, as carbohydrates and amino acids.
Table 5 shows the Pearson’s correlation coefficients between TBARS vs. sensory features. Since lipid oxidation was liked to decrease in redness, due to its role as a promoter of myoglobin oxidation, no significant correlation was found. Surprisingly, the value of brightness was negatively correlated (
p < 0.01) with the oxidation lipid index. Though positive correlation was found among TBARS level and hardness (
p < 0.01), because of the common link to high fat content.