3.1. Effect on Physicochemical and Microbial Changes during the Manufacturing Process
Table 1 shows the values of the proximate composition of the sausage batches along the manufacturing process. The evolution of the a
w values is summarized in
Figure 1B. The trends in moisture loss and the a
w decrease during the manufacture of the three sausage groups are very similar than those reported in the literature for other similar dry-fermented sausages [
51,
52,
53] and are basically determined by the size of the sausages and by the environmental conditions (temperature and relative humidity) during the process. The protein, fat, ash and NaCl contents expressed as percentage of the total solids are within the wide range of values reported for similar sausage types and reflect the proportions of lean and fat used and the quantities of salt added in the mix preparation. None of these compositional parameters were significantly affected by the addition of starter cultures. The titratable acidity increased significantly from values of 0.15, 0.17 and 0.15 g of lactic acid/100 g of TS in the mix for the CNT, EQU and SAP batches, respectively, to values of 0.52, 0.69 and 0.71 g of lactic acid/100 g of TS (CNT, EQU and SAP batches, respectively) after 9 days of ripening, and then decreasing until reaching final values of 0.32, 0.59 and 0.67 g of lactic acid/100 g of TS, respectively. Significant differences (
p < 0.001) were observed between the non-inoculated and inoculated batches. These values of titratable acidity are quite low, reflecting a moderate acidification during the manufacture of this sausage type. Information on the evolution of this parameter throughout the maturation of raw-cured sausages is not abundant in the literature, nor is there any discussion of the phenomena involved in such an evolution. The values in the present study are in accordance with that reported in previous works for similar sausages [
52,
53] and the trends in this parameter also agree with those indicated by Salgado et al. [
53] in another variety of chorizo sausage. The decrease after day 14 of ripening is probably due to the consumption of organic acids by the microorganisms present, above all, moulds and yeasts. The increase in titratable acidity during the first 9 days of manufacturing and also the values reached were significantly (
p < 0.001) higher in the batches manufactured using starter cultures than in the control, reflecting the acidifying capacity of the strain of
L. sakei added.
The evolution of the pH values is shown in
Figure 1A. Initial acidification plays an important role in the microbiological, biochemical and sensory characteristics of the fermented foods. In the case of the fermented sausages, the acidification until the pI of the muscle proteins causes denaturation of these proteins and determines the cohesiveness of the mass, the firmness and the sliceability of the final products [
54]. The pH values reached, in addition, modulate the activity of the meat enzymes responsible for the ripening and flavour generation [
55], and avoids the survival and growth of undesirable spoiling and pathogenic microorganisms.
In the present study, the decrease in pH values during the fermentation phase was very moderate (mean values from 6.02, 5.95 and 6.03 in the mix before stuffing to 5.69, 5.51 and 5.54 after 9 days of manufacturing, for the CNT, EQU and SAP batches, respectively). The pH decrease was significantly (
p < 0.01) higher in batches of sausages inoculated with starter cultures, not observing significant differences between the two starter cultures used. From day 9 of manufacture, a slight and constant increase, more marked in the control sausages, was observed, reaching final average values of 5.81, 5.60 and 5.59 for the CNT, EQU and SAP batches, respectively. This pH increase in the last stages of the ripening process was already described by other authors in different sausages [
15,
52] and seems to be due to an increase of basic nitrogen compounds as a result of the proteolytic processes and also to the consumption of lactic acid by the microorganisms.
The pH decrease during fermentation is highly variable in the different sausages and depends on the quantity of fermentable sugars in the mix, the environmental temperature and the activity of the lactic acid bacteria present in the sausages. Therefore, the final pH values of the ripened sausages show a high variability, ranging along the values reported in the literature, from 4.15 [
29] to 6.52 [
56]. In agreement with some other previous observations [
14], the use of starter cultures in the present study decreased the pH values during manufacturing by only a little. This contrasts with the great decrease observed in other studies where
Lactobacillus strains with a greater acidifying capacity were used as starter cultures [
29].
Colour is a very important sensory attribute in this type of food and colour deficiencies are likely to cause rejection even if sausages have a good taste and texture [
57,
58].
Table 2 shows the evolution of the colour parameters during the manufacture of the three batches of sausage. The percentage of conversion of pigments (from heme to nitrosyl-heme) had initial values of 35.16%, 39.63% and 37.56% for CNT, EQU and SAP sausages, respectively, with no significant (
p > 0.05) differences between the three batches. As indicated in
Table 2, the pigment transformation percentages showed a significant (
p < 0.001) upward trend throughout the entire ripening process, reaching final values of 81,88% in the CNT batch and 82.02% and 86.26% in the EQU and SAP batches, respectively. The final value in the SAP batch was significantly (
p < 0.05) higher than in the control batch. The values of percentage of transformation to nitrosyl-heme pigments in the present study are within the wide range of values reported in the literature [
59] and reflect a high transformation of pigments. According to Zaika et al. [
36], the percentage of pigment conversion is as high as the pH value is low, since the low pH values favour the formation of NO from nitrates that then reacts with myoglobin to form nitrosyl-myoglobin. Results in the present work seem to corroborate this appreciation, since in the present case the inoculated batches having lower pH values show higher conversion percentages. The nitrate reductase activity of the staphylococci strains added as starter cultures in the present work could also have some responsibility in the higher percentage of transformation in the inoculated batches.
Regarding the changes in the CIELAB colour coordinates (L*, a* and b*) throughout the drying–ripening process, the use of starter cultures did not have a significant effect on the luminosity (L*) of the sausages that decreased significantly (
p < 0.05) during the whole drying–ripening process (values from 46–49 to 31–32), both in the control and in the inoculated batches. The decrease of this parameter during ripening seems to occur as a result of moisture loss [
60,
61,
62], thus becoming a darker product. The evolution of the a* parameter (red coloration) is in line with the data described by Gómez et al. [
58], with an increase up to 5 days of maturation, reaching a maximum value of 31.94 in the SAP batch, then decreasing to the end of the process with final values of 17.47, 20.70 and 20.25 in CNT, EQU and SAP, respectively. Significant differences were observed in this parameter both during ripening (
p < 0.001) and due to the use of starter cultures (
p < 0.05). The initial increase could be due to the formation of nitrosyl-myoglobin [
63]. The initial redness is also influenced by the use of paprika in the mix formulation with a high colouring power, just as the oxidation of the carotenoids present in this ingredient contribute to the loss of coloration [
58]. Finally, the parameter b * undergoes an evolution similar to that observed for redness. The initial increase in this parameter could be related to lipid oxidation processes. Again, we observed significant differences both during ripening (
p < 0.001) and due to the use of starter cultures (
p < 0.05).
Despite the fact that the correct implantation of these two starter cultures and their dominance over the indigenous microbiota was already observed and demonstrated using molecular methods [
22], the main microbial groups were counted along the manufacture of the three sausage batches using classic culture-dependent procedures. Microbial counts are shown in
Table 3. Counts of the microbial groups (5.48–6.57 log CFU/g for the total aerobic mesophilic bacteria, 4.82–6.42 log CFU/g for the total staphylococci, 4.12–5.47 log CFU/g for the lactic acid bacteria and 2.97–3.09 log CFU/g for the
Enterobacteriaceae in the mix before stuffing) increased until the day 14 of ripening and then remained relatively constant or decreased in the case of the
Enterobacteriaceae until the end of production. Counts and trends of the different microbial groups basically agree with previous data reported in the literature for similar sausages [
12,
64,
65]. In each sampling time, counts of total aerobic bacteria, staphylococci, and lactic acid bacteria were always significantly (
p < 0.001) higher in the inoculated batches than in the control batch, which confirmed the correct implantation of the starter cultures added. Higher counts of lactic acid bacteria and staphylococci in inoculated compared to control batches were also reported by Essid and Hassouna [
15], using
Staphylococcus xylosus and
Lactobacillus plantarum as the starter cultures. Also, from day 14 of ripening, counts of the
Enterobacteriaceae were significantly (
p < 0.01) lower in the inoculated than in control sausages. This phenomenon seems to be due to the lower pH values reached in the inoculated sausages, taking into account the acid-sensitivity of the enterobacteria. In all three batches (CNT, EQU and SAP), the enterobacteria did not completely disappear at the end of maturation. In this sense, there is some discrepancies among the data reported in the literature. While some authors reported the total disappearance of the enterobacteria at the end of the ripening process in inoculated sausages having high pH values (5.63–5.76) [
14], some others reported the survival of this microbial group in sausages reaching very low pH values (4.15) [
15]. The different sources of contamination and the different nature and acid-resistance of the enterobacteria species present in the different sausage types could explain this discrepancy.
3.2. Effect on Proteolytic Changes during the Manufacturing Process
Hydrolysis of the meat proteins, both sarcoplasmic and myofibrillar, is considered one of the main degrading processes during the ripening of meat products playing a determinant role not only in the development of their final aroma and taste but also of the texture properties. Despite the fact that the electrophoretic methods, both the SDS-PAGE techniques [
12,
66,
67] and miniaturized procedures [
68], are the most exhaustive way of study the changes undergone by the proteins during the maturation processes, the quantification of the classical nitrogen fractions is a very satisfactory alternative.
Table 4 shows the values of these nitrogen fractions during the manufacture of the three batches of sausages. The non-protein nitrogen (126.23–133 mg/100 g of TS in the mix before stuffing) significantly increased (
p < 0.001) during the manufacturing process, reaching final values of 167.45, 176.21 and 217.55 mg/100 g TS for the CNT, EQU and SAP sausages, respectively. At the end of the manufacture, values in the SAP batch were significantly (
p < 0.001) higher than in the CNT and EQU batches. The increase in NPN is a common event that takes place in all the ripened sausage types during manufacturing, although in different rates and in unequal proportion during the different steps of the process, as indicated and discussed by Salgado et al. [
53]. The low NPN contents in the mix before stuffing and the moderate increase observed in the present study (from 1.32 fold in CNT and EQU batches to 1.68 fold in the SAP batch) when compared to other dry-fermented sausages [
53] indicate that proteolysis is only moderate in this type of sausage.
The α-aminoacidic nitrogen also increased significantly (
p < 0.001) during the manufacturing, from 31.91, 44.48, and 42.71 to 109.73, 131.97 and 166.92 mg/100 g of TS for the CNT, EQU and SAP batches, respectively. Regarding the total basic volatile nitrogen, the increase was again significant (
p < 0.001) from 13 mg/100 g of TS in the mix before stuffing to 83.05, 91.81 and 95.17 mg/100 g of TS for the CNT, EQU and SAP batches, respectively, at the end of the process. The values observed in the present study for these two nitrogen fractions are in the range of those reported in the literature for other similar sausage types [
53,
59].
Differences in NPN content among batches were not significant during the first 14 days of manufacture and only in the two last sampling times (21 and 30 days) the contents in the SAP batch were significantly (p < 0.001) higher than in the CNT and EQU batches. However, the contents in α-aminoacidic nitrogen were significantly (p < 0.001) higher in the inoculated than in the control batches in all the sampling times, and the same occurred for the total basic volatile nitrogen contents from day 2 of manufacture.
Although there is not a total consensus regarding the importance of the tissue and microbial enzymes in the protein degradation during sausage ripening, it seems evident that proteolysis takes place in two different phases. The initial protein degradation seems to be the responsibility of the muscle calpains and cathepsins, and at a later stage the bacterial enzymes further degrade the protein fragments and polypeptides initially formed [
55]. The fact that in the present study the use of starter cultures had a greater effect on the α-aminoacidic and total basic volatile nitrogen fractions than on the non-protein nitrogen content seems to corroborate this hypothesis.
Release of free amino acids during sausage ripening is a very important event since some amino acids have a particular taste and some others are precursors of taste and odour compounds when degraded following several well-known biochemical pathways [
61]. The contents of the free amino acids in the CNT, EQU and SAP sausage batches during manufacturing are shown in
Table 5. Free amino acids in the mix before stuffing ranged from 428.12 in CNT batch to 444.53 mg/100 g TS in the SAP batch and no significant differences (
p > 0.05) were observed among batches. A significant increase (
p < 0.001) during manufacturing was observed in the three sausage groups reaching final values of 1381.66, 1450.02 and 1593.07 mg/100 g TS for the CNT, EQU and SAP batches, respectively. Significant differences (
p < 0.05) among batches were observed in the final total free amino acid content. Therefore, from the initial and final values, an increase in the free amino acid content of 3.22, 3.33 and 3.58 times for the CNT, EQU and SAP batches can be observed, respectively. Similar increases were observed by other authors during the ripening of other sausage types [
26,
66]. Increases during ripening described in the literature are highly variable (around 1.2 times [
12,
27,
69], around 1.5 times [
65], around 2.5 times [
23,
61,
70] or even more than 4 times [
71]). There are, however, studies in which no increase [
23] or a small reduction [
12] was observed in control batches prepared in studies to determine the effect of the addition of starter cultures. As occurred in the present study, several authors reported that the addition of starter cultures always increased the release of free amino acids during the sausage ripening [
14,
15,
23,
26,
71], which undoubtedly proves the participation of peptidases of microbial origin in the release of amino acids during the maturation of sausages.
The free amino acid profile in the mix before stuffing hardly varied among the batches. Arg was the most abundant FAA (73.21, 73.37 and 72.88 mg/100 g TS for the CNT, EQU and SAP batches, respectively), followed, in a decreasing order, by Tau, Ala, Pro, Lys and Leu, the sum of these six amino acids accounting for 61.09, 60.64 and 60.51% of the total FAA in the CNT, EQU and SAP batches, respectively. The free amino acid profile of the sausage mixes widely varies according to the information reported in the literature [
12,
23,
61,
65,
66,
72,
73], reflecting the diversity of the operating microorganisms, environmental conditions and ingredients. However, in agreement with our observations, some other works [
12,
23,
61,
65,
66,
72,
74] pointed out the abundance of Arg, Tau and Ala in the mix of the sausages. Indeed, Glu, which was reported as the main free amino acid in some studies [
26,
61,
71], was the seventh or even the eighth free amino acid of quantitative importance in the mix before stuffing in the present study.
The individual FAA increased with a different intensity along the manufacturing process (from 2 times in the case of Tau, Arg or Ala to 6 times of Trp, or even 8–10 times of Cys). The most abundant FAA after 30 days of ripening was again Arg (151.09, 152.07 and 167.92 mg/100 g TS for the CNT, EQU and SAP batches, respectively), followed, in a decreasing order, by Ala, Tau, Glu, Pro and Leu. These six FAA accounted for 50.14, 49.65 and 50.13% of the total FAA in 30-day-old sausages in the CNT, EQU and SAP batches, respectively. The FAA profile observed in the mix basically remains in the ripened sausages, with the exception of Glu that increased its abundance and the Lys content that decreased. Again, the FAA profile of the ripened sausages is very variable in the literature [
12,
14,
23,
61,
66,
70,
72]. However, in agreement with our results, the predominance of the Arg [
61,
74] and the abundance of Arg, Ala and Tau [
12,
23,
65,
66] was reported in other works.
The taste of some amino acids and their sensory thresholds were well stablished [
75,
76]. The amino acids Ala, Gly, Thr, Ser and Pro have a sweet taste. Leu, Val, Ile, Met and Phe are bitter. Glu, Asp and His have an acid taste and, in addition, Glu and Asp cause a pleasantly fresh sensation. Moreover, Asp, Tyr and Lys have been considered as responsible for an “aged” taste in the ripened meat products. On the basis of this knowledge, the FAA were grouped according their tastes (
Table 5). In the ripened sausages (30 days) and when compared to the contents observed in mixes before stuffing, sweet FAA increased 3.33, 3.66 and 3.69 times in the CNT, EQU and SAP batches, respectively. Bitter FAA increased 3.70, 3.60 and 3.81 times in the CNT, EQU and SAP batches, respectively. Acid FAA experienced a more marked increase (4.79, 5.16 and 5.44 times, respectively), while “aged” FAA increased 3.66, 3.41 and 3.76 times in the CNT, EQU and SAP batches, respectively. In view of these data, it can be concluded that the use of
Lactobacillus sakei and
Staphylococcus equorum as starter cultures increased the sweet and acid tastes and decreased the bitter taste in the final product when compared with the non-inoculated control sausages. In the same way, the use of
Lactobacillus sakei and
Staphylococcus saprophyticus as starter cultures increased the four tastes when compared with the non-inoculated control sausages. Taking into account that, according to the information reported by Zhu [
75], all these FAA are in the final sausages in concentrations higher than their respective sensory thresholds, the use of the starter cultures assayed in the present work could have some effect on the taste of the manufactured sausages.
Due to their importance for consumer health, derived from their physiological activities, and also due to their effects on the food quality, the biogenic amines are the products of the proteolysis in sausages that demanded more attention in the literature in the last two decades. Fermented sausages offer very favourable conditions for biogenic amine formation because of the high microbial activity during the fermentation process, the high presence of free amino acids (the biogenic amine precursors) as products of the proteolytic processes and the low acidic conditions that favour amino acid decarboxylation [
77].
Table 6 shows the evolution of the main biogenic amines during the manufacture of the three Galician chorizo batches. In the present study, tyramine and spermine were the main biogenic amines in the mix, followed by tryptamine, 2-phenylethilamine, putrescine and cadaverine, with spermidine and histamine being the less abundant ones. The contents of most of these biogenic amines significantly (
p < 0.001) increased during production but in an unequal way, with putrescine (that increased 4.79, 4.72 and 5.24 times in the CNT, EQU and SAP batches, respectively), histamine (4.46, 3.99 and 3.98 times) and cadaverine (4.07, 2.33 and 3.42 times) being the biogenic amines that underwent the greatest increases. Conversely, spermine (whose content did not experience a significant increase), spermidine (that increased 1.71, 1.76 and 1.63 times in the CNT, EQU and SAP batches, respectively) and 2-phenylethylamine (2.06, 1.81 and 1.97 times) were the amines that experienced the lowest increases. As a consequence of the individual amine increase, the total biogenic amine content increased 2.97, 2.48 and 2.53 times in the CNT, EQU and SAP batches, respectively, until reaching final total biogenic amine contents of 289.71, 241.47 and 235.32 mg/kg of TS in the CNT, EQU and SAP batches, respectively. These final content of total biogenic amines are in line with the contents described by other authors [
13,
68,
74], although very much lower (61.71 mg/kg; [
14]) and very much higher (1962.1 mg/kg; [
72]) contents have been reported in the literature. In the final sausages, the main biogenic amine was putrescine, followed by tyramine, tryptamine and cadaverine. In general, tyramine, putrescine and cadaverine were reported as the main biogenic amines in meat products [
24,
78,
79,
80,
81], with the concentration of cadaverine being the most variable [
81]. Spermine and spermidine are the only biogenic amines present at significant levels in fresh meat [
82]. According to the information reviewed by Suzzi and Gardini [
79], several authors reported that strains of the genus
Lactococcus,
Leuconostc and
Lactobacillus are able to produce tyramine, and therefore the generation of tyramine in sausages could be attributable to the decarboxylase activity of the lactic acid bacteria. Cadaverine and putrescine are associated with the activity of
Enterobacteriaceae [
79,
83], and high quantities of these amines indicate poor hygienic practices and high microbial contamination of the raw materials [
84]. In the present study, significant correlation coefficients (
r = 0.692;
p < 0.01) were observed between the counts in MRS agar and the tyramine contents, which seems to corroborate the responsibility of the lactic acid bacteria in the production of this amine. In the same way, a significant positive correlation (
r = 0.727;
p < 0.01) was observed between the counts in MRS agar and the tryptamine contents. The high significant correlations we observed between the counts in VRBGA and cadaverine (
r = 0.635;
p < 0.01) and putrescine (
r + 0.560;
p < 0.01) contents also suggests some implication of the
Enterobacteriaceae in the generation of these two biogenic amines.
In the present study, the final content of total biogenic amines was significantly (
p < 0.001) lower in the inoculated batches than in the control batch, and no significant differences (
p > 0.05) were observed between the two inoculated batches. The use of starter cultures significantly reduced the total biogenic amine content, with the percentage of reduction being 16.65% in the EQU batch and 18.77% in the SAP batch. Reductions were unequal for the different biogenic amines, being cadaverine (45.03% and 36.26% of reduction for the EQU and SAP batches, respectively), tyramine (12.64% and 21.27%), 2-phenylethylamine (16.57% and 19.23%) and putrescine (12.47% and 17.51%) the amines that underwent the major reductions. In accordance with results of other previous studies [
13], the spermine contents remained practically unaltered during the manufacturing process in the three sausage batches.
As indicated by Lorenzo et al. [
81], several studies have demonstrated that the use of starter cultures reduce the biogenic amine formation during the sausage fermentation and ripening due to their inhibiting effect on the spoilage bacteria via acidification. However, some authors reported an increase in the biogenic amine content in the ripened sausages when some starter cultures were added [
14,
85]. This undesirable effect could be due to the increase of proteolysis and subsequent generation of free amino acids (precursors of the biogenic amines), although this possibility was questioned by the results of some studies [
14,
86]. Rather, it could be that the starter cultures favour the production of biogenic amines via a slight acidification that facilitates the decarboxylation reactions or by the direct production of biogenic amines by the strains integrating the starter cultures. In this sense, recent reports [
87] indicated that the species
Staphylococcus xylosus, largely used as starter culture in fermented sausages, is an effective producer of tyramine. In any case, some authors have indicated that a reduction in the formation of biogenic amines through acidification is only real for some biogenic amines, such as putrescine [
85], and that a significant decrease in pH is necessary for this effect to occur. These authors indicated that the acidifying activity of the starter cultures did not reduce the tyramine production. However, our results regarding the tyramine reductions, as well as the results of some other authors concerning the reduction of this biogenic amine by using starter cultures [
88,
89], disagree with this statement. It seems, therefore, that more research is necessary to elucidate these discrepancies. It could be that other inhibitory mechanisms in addition to the pH reduction were involved in reducing the production of biogenic amines and that the strains that produce biogenic amines have a different sensitivity to these inhibitory mechanisms.
For additional information, the biogenic amine index (BAI) and the total vasoactive biogenic amines (TVBA) were calculated. Regarding the BAI, it was first developed and used by Mietz and Karmas [
90], with the aim of assessing the freshness (bacterial quality) of tuna. The initial formula proposed by Mietz and Karmas does not take tyramine into account. In the present study, we used the formula for the BAI calculation proposed by Veciana-Nogués et al. [
91], which does take into account this biogenic amine. Basically, a BAI quantifies the amines that come from microbial metabolism, and their evaluation is of great interest in foods in which any microbial growth is undesirable and indicates spoilage. In fermented foodstuffs (foods and beverages), there is a desirable and normal development of microorganisms during manufacturing. Therefore, these indices do not have an absolutely direct relationship with the microbiological quality of food. In the case of sausages, therefore, this index remains as an indicator of the degree of activity of the decarboxylating microorganisms in the product. The vasoactive amines (tyramine, histamine, tryptamine and 2-phenylethylamine) possess vasoactive and psychoactive properties and therefore indicate a food poisoning hazard. The use of starter cultures significantly reduced the BAI (19.66% in the EQU batch and 20.81% in the SAP batch) and the TVBA (12.12% and 16.23% in the EQU and SAP batches, respectively) in the final ripened sausages. This indicates that the use of these two starter cultures also improves the hygienic quality and safety of Galician chorizo sausage.
3.3. Effect on Lipolytic Changes during the Manufacturing Process
Lipolysis and fat oxidation are major sources of volatiles generated during the ripening of meat products. In order to investigate the effect of the addition of starter cultures on these chemical processes, we firstly analysed some parameters that indicate fat degradation. The results of these analyses are shown in
Table 7. The acidity values that indicate the free fatty acid content increased significantly (
p < 0.001) from 1.38, 1.32 and 1.47 mg KOH/g fat to 10.19, 10.05 and 13.39 mg KOH/g of fat in the CNT, EQU and SAP batches, respectively. At the end of the manufacturing process, the values were significantly (
p < 0.001) higher in the SAP batch than in the CNT and EQU batches. This parameter therefore increased during the manufacture; specifically, 7.38, 7.61 and 9.10 times in the CNT, EQU and SAP batches, respectively. Increases of the acidity value reported in the literature in dry-fermented sausages are very variable. Similar increases than ours were observed by Salgado et al. [
53] (10.42 times), but lower increases were reported by Lizaso et al. [
64] (4.32 times), Franco et al. [
52] (4 times) and Fernández-Fernández et al. [
4] (1.7 times). The final values of fat acidity largely vary in the ripened sausages, as discussed by Franco et al. [
52]. The final values in the present study are similar to those reported by Fernández-Fernández et al. [
4] for the same sausage type, as well as to those observed by other authors in other ripened sausages [
92,
93]. These values indicate that this sausage undergoes during ripening a considerable lipolysis and that the use of the SAP starter enhances this process.
Peroxide values also increased significantly (
p < 0.001), from 0.96, 1.10 and 1.18 to 6.02, 6.30 and 5.93 meq O
2/kg fat in the CNT, EQU and SAP batches, respectively. At the end of ripening, no significant (
p > 0.05) differences were observed among the batches. Usually, an increase in this parameter is noticed during sausage ripening [
4,
24,
29,
53]. However, a decrease was observed in other cases [
94]; in some others, after an initial increase, a decline was reported in the last stages of ripening [
29,
52]. In the present study, the initial values of this parameter were low and, despite the fact that the peroxides increased during the manufacturing, the final values were lower than most of those reported in the literature for other similar sausages [
24,
29,
52,
53,
64,
92,
95]. The reasons for the low values of this parameter in the present study could be the high quality of the fat used in the manufacture, coming from pigs slaughtered 48–72 h before manufacturing and adequately stored under refrigeration, and also the fact that the mincing, mixing and stuffing were carried out under vacuum, thus avoiding air (and therefore oxygen) incorporation during these processes.
The TBA value is a measure of the secondary oxidation processes and quantifies the malondialdehyde, one of the most representative compounds of those coming from the hydroperoxide decomposition. In the present study, the TBA value significantly (
p < 0.001) increased from 0.16–0.22 to 0.54, 0.74 and 0.77 mg malondialdehyde/kg. The use of starter cultures significantly (
p < 0.05) increased the value of this parameter in the final product and no differences (
p > 0.05) were observed between the two inoculated batches. The evolution of the TBA value during the sausage ripening is very variable. As in the present study, Franco et al. [
52] observed a progressive increase during the ripening process. However, in other cases after an initial increase, the malondialdehyde decreases in the final steps of the ripening [
24,
53,
61,
94]. Usually the concentration of malondialdehyde at the end of the manufacture of this type of sausages is around or under 1 mg/kg [
25,
52,
53,
61,
94,
95]. However higher values were frequently reported in the literature [
24,
92,
95]. Domínguez Fernández and Zumalacárregui Rodríguez [
96] reported values of 2.21 mg malondialdehyde/kg in “Chorizo” sausage after 35 days of ripening and indicated that this concentration is insufficient for the sensorial perception of rancidity.
Free fatty acid release during ripening of meat products is an important phenomenon for the sensory characteristics of the final products since most of the volatiles come from fatty acid degradation, mainly via oxidation processes. The evolution of the free fatty acids (FFA) during the manufacture of the batches of sausage is shown in
Table 8. No significant differences (
p > 0.05) were observed in the total FFA content among the batches in the mix. Total FFA contents in the mixes before stuffing (253.62, 247.11 and 263.64 mg/100 g of fat for the CNT, EQU and SAP batches, respectively) are in agreement with those reported in the literature for the mix of other sausages [
12,
26,
61,
70], although higher initial values were reported at the beginning of the manufacture of some other sausages [
14,
29,
67]. Regarding the FFA profile of the mixes in the present work, the oleic acid (C18:1) was the main fatty acid, followed, in decreasing order of abundance, by linoleic (C18:2), palmitic (C16), and stearic (C18), these four FFA accounting for 87.40%, 87.07% and 87.32% of the total FFA of the mix in the CNT, EQU and SAP batches, respectively. The FFA profile in the mixes slightly differed among the batches. In the CNT batch, the fifth, sixth, seventh and eighth most important FFAs were the palmitoleic (C16:1), linolenic (C18:3), myristic (C14) and arachidonic (C20:4) fatty acids, while these same places were occupied by the linolenic C18:3), palmitoleic (C16:1), myristic (C14) and docosadienoic (C22:2) FA in the EQU batch, and by the linolenic (C18:3), palmitoleic (C16:1), arachidonic (C20:4) and myristic (C14) FA in the SAP batch. This FFA acid profile is quite constant in sausages manufactured from pig fat. Regarding the main fatty acids, profiles only differ in the FFA that occupy the second and third place. In some cases, palmitic (C16) is more abundant than linoleic (C18:2) [
26,
64], and in some other, as in the present work, linoleic (C18:2) dominates over palmitic (C16) [
12,
23,
70]. In any case, very small differences between the palmitic (C16) and linoleic (C18:2) fatty acid concentrations have been consistently reported.
The total FFA content increased to final values of 1814.41, 1805.65 and 2295.46 mg/100 g of fat, for the CNT, EQU and SAP batches, respectively. In agreement with the observations made in the acidity values of the fat (
Table 7), the final total FFA content was significantly (
p < 0.001) higher in the SAP than in the EQU and CNT batches, and no significant (
p > 0.05) differences were observed between the CNT and EQU batches. The increases in FFA content (7.15, 7.30 and 8.70 times for the CNT, EQU and SAP batches, respectively) are in agreement with the increases reported by other authors (6–8 times [
12], 6–7 times [
29] and 6 times [
26]). However, lower [
61,
67,
70] and higher [
12] increases have been reported in other works. Usually, the use of starter cultures increases the FFA content during the ripening process. However, in some cases lower FFA contents were reported when commercial starter cultures were added [
14].
In the present study, during the ripening process the different free fatty acids increased at a different rate (e.g., eicosatrienoic acid (C20:3n3) increased 10.88, 21.34 and 25.13 times in the CNT, EQU and SAP batches, respectively, while lauric acid (C12) only increased 3.14, 3.45, and 5.36 times in the CNT, EQU and SAP batches, respectively). The final values of total FFA content in the present work are in agreement with the values observed in other studies [
12,
67]. However, as occurred for the mixes, lower [
26,
61,
70] and higher [
14,
29] values than ours have been reported.
The FFA profile in the ripened sausages hardly varied with respect to the profile in the mixes and in the different batches. The main FFA was again oleic acid (18:1), followed by linoleic (C18:2), palmitic (C16) and stearic (C18) acid, these four fatty acids accounting for 86.48, 83.56 and 87.53% of the total FFA in the CNT, EQU and SAP batches, respectively. The other FFA, in descending order of quantitative importance, were palmitoleic (C16:1), arachidonic (C20:4), docosadienoic (C22:2), linolenic (C18:3) and myristic (C14). The FFA profile in the ripened sausages basically agreed with that reported by other authors in other dry-fermented sausages [
12,
23,
26,
64,
70,
92] and hardly varies between the different authors and works when pork and pig fat are used in the sausage manufacture. This profile notably varies, however, when other fats such as tallow [
25] or hump fat [
28] are used. The FFA acid profile in the ripened sausages basically reflects the fatty acid composition of the fat of the raw materials and the nature of the lipases acting during the ripening process. Monounsaturated fatty acids always dominate in the FFA fraction of the sausages made from pork and pig fat since monounsaturated fatty acids dominate in the triacylglycerols that are the main fraction in the fat used for the manufacture. Some authors [
97] reported that the majority of the FFA derives from the triacylglycerols. In this same line, Muriel et al. [
98] also indicated that, in ripened meat products, the lipolytic processes affecting neutral lipids have a higher incidence in the FFA fraction than those affecting the polar lipids.