Expressed as ppm; § Percentage of transformation into nitrosylheme pigments. CNT: Non-inoculated control batches; EQU: Batches inoculated with *L. sakei* + *S. equorum*; SAP: Batches inoculated with *L. sakei* + *S. saprophyticus*. a–g Means in the same row and sausage group (CNT, EQU or SAP) not followed by a common letter differ significantly (*p* < 0.05) (differences associated to the ripening time). 1–3 Means in the same row and ripening time not followed by a common number differ significantly (*p* < 0.05) (differences associated to the use of starter cultures). SEM: standard error of the mean.

**Table 3.** Evolution of the plate counts (log CFU/g) of total aerobic mesophilic bacteria (SPCA), staphylococci (MSA), lactic acid bacteria (MRS) and *Enterobacteriaceae* (VRBGA) along the manufacturing process of Galician chorizo made without and with starter cultures (means of three replicates in each sausage group).


CNT: Non-inoculated control batches; EQU: Batches inoculated with *L. sakei* + *S. equorum*; SAP: Batches inoculated with *L. sakei* + *S. saprophyticus*. a–g Means in the same row and sausage group (CNT, EQU or SAP) not followed by a common letter differ significantly (*p* < 0.05) (differences associated to the ripening time). 1–3 Means in the same row and ripening time not followed by a common number differ significantly (*p* < 0.05) (differences associated to the use of starter cultures). SEM: standard error of the mean.

**Table 4.** Evolution of the nitrogen fractions (expressed as mg N/100 g TS) along the manufacturing process of Galician chorizo made without and with starter cultures (means of three replicates in each sausage group).


CNT: Non-inoculated control batches; EQU: Batches inoculated with *L. sakei* + *S. equorum*; SAP: Batches inoculated with *L. sakei* + *S. saprophyticus*. a–g Means in the same row and sausage group (CNT, EQU or SAP) not followed by a common letter differ significantly (*p* < 0.05) (differences associated to the ripening time). 1–3 Means in the same row and ripening time not followed by a common number differ significantly (*p* < 0.05) (differences associated to the use of starter cultures). SEM: standard error of the mean.

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.

**Table 5.** Evolution of the free amino acids (mg/100g of TS) along the manufacturing process of Galician chorizo made without and with starter cultures (means of three replicates in each sausage group).



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 [24,78–81], 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. E*ff*ect 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 O2/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.

**Table 6.** Evolution of the biogenic amines (mg/kg of TS) along the manufacturing process of Galician chorizo made without and with starter cultures (means of three replicates in each sausage group).


TBA: Total biogenic amines; BAI: Biogenic amine index (sum of putrescine + cadaverine + histamine+ tyramine); TVBA: sum of the vasoactive amines (tyramine + histamine + tryptamine + 2 phenylethylamine). CNT: Non-inoculated control batches; EQU: Batches inoculated with *L. sakei* + *S. equorum*; SAP: Batches inoculated with *L. sakei* + *S. saprophyticus*. a–g Means in the same row and sausage group (CNT, EQU or SAP) not followed by a common letter differ significantly (*p* < 0.05) (differences associated to the ripening time). 1–3 Means in the same row and ripening time not followed by a common number differ significantly (*p* < 0.05) (differences associated to the use of starter cultures). SEM: standard error of the mean.

**Table 7.** Evolution of the fat parameters along the manufacturing process of Galician chorizo made without and with starter cultures (means of three replicates in each sausage group).

