**Biochemical Changes during the Manufacture of Galician Chorizo Sausage as A**ff**ected by the Addition of Autochthonous Starter Cultures**

**Miriam Rodríguez-González 1,2 , Sonia Fonseca <sup>1</sup> , Juan A. Centeno 1,2 and Javier Carballo 1,2,\***


Received: 30 October 2020; Accepted: 1 December 2020; Published: 7 December 2020

**Abstract:** In this work, the effect of the use of two autochthonous starter cultures (*Lactobacillus sakei* LS131 + *Staphylococcus equorum* SA25 (EQU), or *L. sakei* LS131 + *Staphylococcus saprophyticus* SB12 (SAP)) on the physicochemical, microbiological, proteolytic and lipolytic changes taking place during the manufacture of Galician chorizo, a traditional Spanish sausage, was studied. Three different batches (control (CNT), EQU and SAP) were manufactured in triplicate and analysed during the manufacturing process (samples were taken and analysed at 0, 2, 5, 9, 14, 21 and 30 days of ripening) for proximate composition, pH, aw, colour parameters, nitrogen fractions, free amino acids, biogenic amines, fat parameters and free fatty acids. The use of either of these two starter cultures slightly but significantly reduced the pH values during the fermentation and increased the percentage of transformation to nitrosyl-heme pigments as well as the a\* and b\* values in the final products. The two starters significantly decreased the *Enterobacteriaceae* counts in the final product, but without this microbial group completely disappearing. 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 and reducing the total biogenic amine content by approximately 20%. The SAP starter enhanced the lipolytic processes, increasing the free fatty acid content. Due to their performances, these two starter cultures seem to be suitable for increasing the quality and safety of the Galician chorizo sausage.

**Keywords:** Galician chorizo; starter cultures; *Staphylococcus equorum*; *Staphylococcus saprophyticus*; *Lactobacillus sakei*; physicochemical characteristics; free amino acids; free fatty acids; biogenic amines

#### **1. Introduction**

In 2018, 1,429,000 Mt of meat products were manufactured in Spain [1]. Cooked sausages were the major products (430,000 Mt), followed by dry-cured hams and forelegs (306,000 Mt), dry-fermented sausages (214,000 Mt), fresh and marinated products (200,000 Mt), cooked hams and forelegs (175,000 Mt) and pre-prepared dishes (104,000 Mt). Despite the strong domestic demand, 63,103 Mt of dry-fermented sausages and 49,138 Mt of dry-cured hams were exported to the international market in 2019. The dry-fermented sausages were the meat products whose production experienced a greater increase in the last five years (from 186,000 Mt in 2013 to 214,000 Mt in 2018), and also the product that registered the greatest increase in exports (from 40,218 Mt in 2013 to 63,103 Mt in 2019) [1]. Spanish dry-fermented sausages are very diverse in size, appearance and organoleptic and nutritional

characteristics, reflecting the traditions as well as the diversity of preferences and climatic conditions in the different regions and areas [2].

Galician chorizo is the typical traditional sausage of Galicia (NW of Spain), the one that enjoys the greatest acceptance and the most widely produced and consumed in this region, being also abundantly consumed in other regions of Spain and foreign countries. Its physicochemical [3–6] and microbiological [7,8] characteristics have been reasonably studied and described. It is both artisanal and industrially produced. Industries have adapted the traditional manufacturing procedures, incorporating the modern technologies in distinct steps of the production process. However, although the manufacturing method is nowadays perfectly standardized, batches of Galician chorizo present in the markets are very diverse in organoleptic characteristics, reflecting, above all, the diversity of the quality of the raw materials used. This variability diminishes the acceptance by the consumers, limits its demand and hinders its expansion in national and international markets.

It is widely known that the organoleptic characteristics of dry-fermented sausages are the result of a series of biochemical changes that take place during maturation [9], promoted by the meat and fat's autochthonous enzymes and by those from the microorganisms that grow during the fermentation and maturation processes [10,11]. Both enzymatic activities are modulated by the rest of the ingredients (salt, spices, additives, etc.) and by the environmental conditions (temperature and relative humidity) present during the production process. Therefore, the diversity of the organoleptic characteristics of the dry-fermented sausages mainly reflects the diversity of the microorganisms acting during the maturation process.

Taking into account all these considerations, the use of an appropriate starter culture seems to be the most feasible solution for the heterogeneity of the organoleptic characteristics of Galician chorizo. The use of starter cultures, generally composed by a lactic acid bacteria and a coagulase-negative staphylococci (CNS), is a common and effective practice in the manufacture of fermented sausages in order to improve the colour and flavour development, ensure safety and extend their shelf-life [12–16]. However, the use of a commercial non-autochthonous starter culture could have a negative impact on the sensory characteristics of the sausages, resulting in losses of the desirable particular organoleptic properties that characterize each type of sausage [17,18].

With the aim of developing a specific and appropriate starter culture for the Galician chorizo sausage, bacterial strains isolated from Galician traditional artisanal sausages were isolated and adequately characterized regarding their technological and safety properties [19–21]. The most suitable lactic acid bacteria and *Staphylococcaceae* strains were then selected, and mixed cultures of *Lactobacillus sakei* and diverse species of *Staphylococcus* were developed and monitored throughout the ripening of this sausage using molecular methods [22]. In this last work, it was possible to verify that these cultures are capable of being implanted in the sausages, dominating the spontaneous flora.

As one of the final steps of this whole work, the aim of the present study was to evaluate the effect of two of these autochthonous starter cultures, consisting of a combination of one strain of *Lactobacillus sakei* and a strain of *Staphylococcus equorum* or *Staphylococcus saprophyticus*, on the biochemical changes that take place during the sausage ripening and that were responsible for the organoleptic characteristics of the final product.

Apart from the novelty of developing and testing a specific starter culture for an economically relevant dry-fermented sausage, the main novelty of the present work is the use of *Staphylococcus equorum* and *Staphylococcus saprophyticus* as starter cultures. *Staphylococcus xylosus* and *S. carnosus* are the only *Staphylococcus* species assayed as starter culture until now [12,14,15,23–26]. Regarding the lactic acid bacteria, the use of *Lactobacillus sakei* as a starter culture is common [13,24,25,27,28], although *Lb. plantarum* [10,11,15,26], *Lactobacillus curvatus* [12,23,29] and *Pediococcus pentosaceus* [14,24] were in many other cases preferred.

#### **2. Materials and Methods**

#### *2.1. Preparation of the Starter Cultures*

In order to inoculate the Galician chorizo batches, one *Lactobacillus* strain (*L. sakei* LS131-CECT 8335-) and two *Staphylococcus* strains (*S. equorum* SA25-CECT 8337- and *S. saprophyticus* SB12-CECT 8336-) were used as starter cultures. These strains were previously isolated from artisanal Androlla and Botillo, two traditional sausages made in Galicia (NW of Spain), and appropriately identified in our laboratory. These strains were chosen from a large set of isolates after testing their suitable technological and safety properties [19–21]. In brief, the strain *Lactobacillus sakei* LS 131 has a mild acidifying activity. The strain *Staphylococcus equorum* SA25 is slightly lipolytic; it has a medium proteolytic activity on the sarcoplasmic proteins and a lack of hydrolytic activity on the myofibrillar proteins. The strain *Staphylococcus saprophyticus* SB12 has high lipolytic activity, high proteolytic activity on the myofibrillar proteins and a lack of activity on the sarcoplasmic proteins [21,30]. The *Lactobacillus* strain was subcultured on MRS broth (Oxoid Ltd., Basingstoke, UK) to a final volume of 500 mL with a concentration of 10<sup>8</sup> CFU/mL, whereas the *Staphylococcus* strains were subcultured on BHI broth (Oxoid) to a final volume of 1000 mL with a concentration of 10<sup>8</sup> CFU/mL. The cell concentration was assessed by interpolation into the correspondent growth curve of the values of absorbance measured at 600 nm. Next, cells were obtained (centrifugation at 4000× *g* for 5 min at 4 ◦C) and washed (with 0.85% NaCl sterile solution). The pellets of cells were finally resuspended in 40 mL of sterile distilled water before addition to the sausage batches.

#### *2.2. Production of Sausages and Sampling*

Following the traditional procedure, three different batches of Galician chorizo were manufactured in triplicate. Batches were designed according to the starter culture added: CNT batch (control not inoculated), EQU batch (inoculated with *L. sakei* CECT 8335 + *S. equorum* CECT 8337) and SAP batch (inoculated with *L. sakei* CECT 8335 + *S. saprophyticus* CECT 8336). *L. sakei* CECT 8335 was inoculated in the sausage mix in a concentration of 106 CFU/g, while *S. equorum* CECT 8337 and *S. saprophyticus* CECT 8336 were added in an amount of 107 CFU/g. The mix of sausages was formulated according to traditional procedures, including lean pork shoulder (80%), pork back fat (20%), sweet paprika (22 g/kg)), spicy paprika (1 g/kg)), garlic (4 g/kg)), salt (15 g/kg)) and water (40 mL/kg). Lean and back fat were firstly ground using a 10-mm diameter mincing plate and next mixed together with the other ingredients for 3 min under vacuum. The resulting mix was allowed to stand at 4 ◦C for 24 h and then stuffed into porcine gut of 36–38 mm in diameter. Sausages were initially fermented for 9 days (6 ◦C and 80% relative humidity) and then dry-ripened for another 21 days (12 ◦C and 75% RH). From each replicate of each batch, samples were taken for subsequent analysis at 0 (mix before stuffing), 2, 5, 9, 14, 21 and 30 days of ripening.

#### *2.3. Microbial Analysis*

Ten grams of sample were taken in triplicate from the mix before stuffing or from the inner of the sausages at the different sampling times. Samples were aseptically added to 40 mL of a sterile solution containing 0.1% peptone (Oxoid), 0.85% NaCl (Oxoid) and 1% Tween 80 (Panreac Química SLU, Barcelona, Spain), and then homogenized in a Masticator Classic blender (IUL Instruments, Barcelona, Spain) for 2 min at room temperature. Serial decimal dilutions in sterile peptone water (0.1% (*w*/*v*)) were prepared and poured or spread in the corresponding agar media. Total mesophilic aerobic bacteria were enumerated in standard plate count agar (SPCA) (Oxoid) after incubation at 30 ◦C for 72 h; staphylococci on mannitol salt agar (MSA) (Oxoid) incubated at 30 ◦C for 48 h; lactic acid bacteria (LAB) in pH 5.7 de Man, Rogosa, Sharpe (MRS) agar (Merck GmbH, Darmstadt, Germany), overlaid and incubated at 30 ◦C for 5 days; and *Enterobacteriaceae* in violet red bile glucose agar (VRBGA) (Oxoid), overlaid and incubated at 37 ◦C for 24 h. Counts were expressed as log CFU/g.

#### *2.4. Determination of the Proximate Composition and Physico-Chemical Parameters*

Moisture, fat, protein (Kjeldahl nitrogen × 6.25), ash and NaCl contents were assessed following the standards ISO 1442:1997 [31], ISO 1443:1973 [32], ISO 937:1978 [33], ISO 936:1998 [34] and ISO 1841-1:1996 [35], respectively. Water activity was measured using a Fast-lab device (GBX, Bourg-de-Péage, France). The pH values were measured with a pH meter GLP21 (Crison Instruments, S.A., Barcelona, Spain) after mixing 10 g of sample with 90 mL of distilled water. Titratable acidity, nitrosyl-heme pigments, total heme pigments and percentage of conversion to cured meat pigments were determined according to the procedures described by Zaika et al. [36]. Colour parameters were measured using a portable CR-400 colorimeter (Konica Minolta Sensing Inc., Osaka, Japan). The results were expressed in the CIELAB space [37] as lightness (L\*), redness (a\*) and yellowness (b\*).

#### *2.5. Determination of the Nitrogen Fractions, Free Amino Acids and Biogenic Amines*

The total non-protein nitrogen (NPN), α-amino acidic nitrogen (NH2-N) and total basic volatile nitrogen (TBVN) were quantified following the methods of Johnson [38], Moore and Stein [39] and Pearson [40], respectively, after precipitation of the proteins with 0.6 N HClO4, according to the procedure described by De Ketelaere et al. [41].

The extraction of free amino acids was performed as described by Alonso et al. [42]. The identification and quantification were carried out by HPLC techniques, using the conditions described by Alonso et al. [42], with some minor modifications. The liquid chromatography equipment consisted of a SpectraSystem module (Thermo Finnigan, San José, CA, USA) equipped with a SCM1000 vacuum membrane degasser, a P4000 pump, an AS3000 automatic injector, a UV6000LP photodiode array detector and ChromQuest Chromatography Workstation software. Separation was made in a reversed phase C18 Ultrasphere 5-ODS, 4.6 mm × 250 mm column (Hichrom Ltd., Theale, Berkshire, UK). The temperature of the column was maintained at 50 ± 1 ◦C with a column heater (SpectraSystem 3000) and the wavelength of the detector was at 254 nm. The standards of the 22 amino acids were supplied by Sigma Chemical Co. (St Louis, MO, USA). All the samples and standards were injected at least in duplicate. Repeatability tests were carried out by injecting a sample and a standard six times consecutively in a day. Reproducibility tests were also performed by injecting the sample and the standard two times per day during three consecutive days under the same experimental conditions. No significant differences (*p* < 0.05) were observed among the results obtained in these tests. Data were expressed as mg/100 g of total solids (TS).

The extraction of the biogenic amines was performed following the method described by Eerola et al. [43]. The separation, identification and quantification were carried out by HPLC techniques also following the procedure described by Eerola et al. [43], using the HPLC equipment already described. The separation was carried out in a reversed phase C18 mod. Kromasil 100 column (25 cm, 4 mm ID) (Teknokroma S. Coop. C. Ltda., San Cugat del Vallés, Barcelona, Spain). The temperature of the column was set at 40 ± 1 ◦C and the wavelength of the detector at 254 nm. The chromatographic conditions used were those described by Lorenzo et al. [44]. A standard containing appropriate amounts of histamine, tyramine, tryptamine, 2-phenylethylamine, putrescine, cadaverine, spermidine, spermine and 1,7-diaminoheptane (acting this later as internal standard) was used for identification and quantification. All the samples and standards were injected at least in duplicate. Repeatability and reproducibility tests were also carried out as indicated for the free amino acid analysis and significant differences (*p* < 0.05) were also not found between the results obtained in these tests. The contents of each biogenic amine were expressed as mg/kg of TS. From the values of the individual biogenic amines, the biogenic amine index (BAI) and the total vasoactive biogenic amine content (TVBA) were calculated as indicated in the foot of the table that shows the amine content in the results section.

#### *2.6. Determination of Fat Indexes and Free Fatty Acids*

After the fat extraction following the procedure of Folch et al. [45], the fat acidity and the peroxide values were determined following the Spanish Official Standards UNE 50.011 and UNE 55.023, respectively [46]. The TBA (thiobarbituric acid) value was measured following the method of Tarladgis et al. [47], with some modifications. All parameters were measured at least in duplicate in each fat sample.

The separation of the free fatty acids from the total fat was carried out in NH2-aminopropyl mini-columns, according to the method described by Kaluzny et al. [48]. The fatty acid methylation was carried out following the procedure described by Shehata et al. [49], with some modifications. The separation, identification and quantification of the fatty acid methyl esters were performed by gas chromatography techniques in a Trace GC chromatograph (Thermo Finnigan, Austin, TX, USA) equipped with a split/splitless, an AI 3000 autoinjector and a flame ionisation detector. The samples were injected in split mode. The separation of the different fatty acids was carried out on an Innowax column (length 30 m, ID25 mm, film thickness 0.25 mm) (Agilent Technologies, Santa Clara, CA, USA). The temperature of the detector was set at 250 ◦C and that of the injector at 230 ◦C. The gases used were hydrogen (35 mL/min), air (350 mL/min) and helium (carrier gas) (30 mL/min). The chromatographic conditions and the procedures for identification and quantification of the individual free fatty acids were those described by Méndez-Cid et al. [50]. All samples and standards were injected at least in duplicate. Repeatability and reproducibility tests were also carried out as previously indicated for the free amino acid determination. The free fatty acid contents were expressed as mg/100 g of fat.

#### *2.7. Statistical Analysis*

In order to analyse significant differences among batches and ripening times in the parameters studied, an analysis of variance (ANOVA) was performed using the General Linear Model (GLM) procedure of the SPSS package, version 23.0 (IBM SPSS, Chicago, IL, USA). The analysis of each parameter and significance was given as *p* < 0.05, *p* < 0.01 and *p* < 0.001. To determine the correlations between variables, Pearson's linear coefficient was used, employing the same SPSS package.

#### **3. Results and Discussion**

#### *3.1. E*ff*ect 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 aw values is summarized in Figure 1B. The trends in moisture loss and the aw decrease during the manufacture of the three sausage groups are very similar than those reported in the literature for other similar dry-fermented sausages [51–53] 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.

**Figure 1.** Evolution of the pH (**A**) and aw (**B**) values along the manufacturing process of Galician sausage made without and with additives (plotted values are means ± standard deviations 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*.

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 [60–62], 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. E*ff*ect 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.

**Table 1.** Evolution of the proximate composition and titratable acidity along the manufacturing process of Galician chorizo made without and with starter cultures (means of three replicates in each sausage group).

