Expressed as mg KOH/g fat; \* Expressed as meq de O2/kg fat; § Expressed as mg malondialdehyde/kg of sample; 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.

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.

**Table 8.** Evolution of the free fatty acids (mg FFA/100g of fat) along the manufacturing process of Galician chorizo made without and with starter cultures (mean values of three batches in each sausage group).


SFA: sum of saturated fatty acids; UFA: sum of unsaturated fatty acids; MUFA: sum of monounsaturated fatty acids; PUFA: sum of polyunsaturated fatty acids. 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.

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.

#### **4. Conclusions**

The use of autochthonous starter cultures integrated by *Lactobacillus sakei* LS131 + *Staphylococcus equorum* SA25 (starter EQU), or by *L. sakei* LS131 + *Staphylococcus saprophyticus* SB12 (starter SAP) in the manufacture of Galician chorizo slightly but significantly reduced the pH values during the fermentation and improved the colour by increasing the percentage of transformation to nitrosyl-heme pigments as well as the a\* and b\* values in the final products. The two starters also significantly decreased the *Enterobacteriaceae* counts in the final product, but without completely eliminating this microbial group.

Both starter cultures significantly increased the α-amino acidic nitrogen and the total basic volatile nitrogen fractions during manufacturing, also increasing the free amino acid content. Moreover, the two cultures reduced the total biogenic amine content by 20%, also reducing in the same proportion the total vasoactive biogenic amine content. The SAP starter enhanced the lipolytic processes, increasing the content in free fatty acids without modifying the FFA profile.

Due to their performances, these two starter cultures seem to be suitable for increasing the quality and safety of the Galician chorizo sausage.

**Author Contributions:** Conceptualization, J.C.; methodology, J.C. and J.A.C.; formal analysis M.R.-G., S.F. and J.A.C.; data curation, M.R.-G. and J.A.C.; writing—original draft preparation J.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external specific funding.

**Acknowledgments:** This work was financially supported by the Xunta de Galicia (The Regional Government), Spain, through the CITACA Strategic Partnership ED431E 2018/07.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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