2.2. Cured Meat Product: Spanish Chorizo

Eight different batches (10 samples per batch) of Spanish chorizo were manufactured using the recipe shown in Table 1. Minced meat was purchased in a local supermarket, Hipercor, S.A. (Murcia, Spain). Dextrose, meat protein, and the commercial mix of additives and spices composed of spices, salt, dextrose, lactose, milk protein, emulsifiers (triphosphates E-451, diphosphate E-450), flavour enhancer (monosodium glutamate E-621), preservative (sodium nitrate E-251), antioxidant (sodium ascorbate E-301), and colouring (carminic acid E-120), was used as the control sample (C). A comercial starter culture composed of Pediococcus (50 g per 100 g culture), Staphylococcus xylosus (25 g per 100 g culture), and Staphylococcus carnosus (25 g per 100 g culture). The lyophilised cultures were rehydrated (50 g in 750 mL of chlorinated-free water) for 8 h and then sown in the mass at a rate of 6 × 10⁷ CFU/g. Traditional ingredients of Spanish chorizo (paprika, garlic powder, and oregano) were purchased in a local supermarket, Hipercor, S. A. (Murcia, Spain).

Formulation of Spanish chorizo samples.

Commercial mix^®: composed of spices, salt, dextrose, lactose, milk protein, emulsifiers (triphosphates E-451, diphosphate E-450), flavour enhancer (monosodium glutamate E-621), preservative (sodium nitrate E-251), antioxidant (sodium ascorbate E-301), and colouring (carminic acid E-120). Ct: Citric; R: Rosemary; Ac: Acerola; LAW: Lettuce + arugula + watercress; SCe: Spinach + celery; ChB: Chard + beet.

The meat was then mixed with the starter cultures, additives, spices, and natural extracts. The paste was stuffed into swine casing, slightly curved, 40 to 43 mm caliber, and 300 to 400 mm in length, using an automatic stuffer (Silvercrest^® kitchen tools, Barcelona, Spain). The casing was supplied by Tripas De Murcia, S.L. (Alhama de Murcia, Murcia, Spain) and was previously desalted and washed with chlorinated-free water. The Spanish chorizo samples were labelled and placed in an air-drying chamber, Binder 115 redLine RI (Tuttlingen, Germany), set at 22 ± 1 °C and 90 ± 5% R.H. for two days. After that, the temperature and humidity were adjusted to 14 ± 1 °C and 70 ± 5% R.H. for 20 days. Analysis were carried out at 0 and 50 days. After the curation process, to study the shelf life, the Spanish chorizo samples were vacuum packaged and then stored at 5 ± 1 °C and 65 ± 5% R.H. for 125 days. Analyses were carried out at days 0, 25, 50, 75, and 125 from elaboration. Microbiological growth was determined at day 50, while volatile compounds were measured at days 0, 25, 75, and 125.

Lipid oxidation was related to the concentration of volatile compounds, following the method described by Lorenzo, Bedia, and Bañón [22] with some modifications. For that, 5 g of Spanish chorizo sample were placed in glass vials. Volatile compounds were extracted using solid-phase microextraction (SPME). An SPME device (Supelco, Bellefonte, PA, USA) containing a fused-silica fibre (10 mm length) (polydimethylsiloxane/divinylbenzene (PDMS/DVB)) was used. Extraction was performed at 35 °C for 30 min. Once sampling was finished, the fibre was withdrawn into the needle and transferred to the injection port of the gas chromatograph-mass spectrometer (GC-MS) system. Volatile compounds were analysed in duplicate in all samples at days 0, 25, 75, and 125 from elaboration. Analyses were performed on a Hewlett-Packard 6890 N Series GC gas chromatograph fitted with an HP 5973 mass spectrometer and an MSD Chemstation (Hewlett–Packard, Palo Alto, CA, USA). A split injection port was used to thermally desorb the volatiles from the SPME fibre onto the front of the DB-624 capillary column (J&W scientific: 30 m × 0.25 mm id, 1.4 µm film thickness). Helium was used as a carrier gas with a linear velocity of 36 cm/s. The temperature programme was 40 °C for 2 min and then raised to 100 °C at 3 °C/min, then from 100 to 180 °C at 5 °C/min, and a total run time of 50.8 min. The volatile compounds analyzed were propan-2-ol, octen-2-ol, hexanal, and nonanal. The mass spectra were obtained using a mass selective detector working in the electronic impact at 70 eV, with a multiplier voltage of 1953 V and collecting data at a rate of 6.34 scans/s over the range, m/z, 40 to 300. Compounds were identified by comparing their mass spectra with those contained in the NIST05 (National Institute of Standards and Technology, Gaithersburg, Maryland, USA) library. Volatile compounds were quantified as the area percentage under the chromatograme curve.

Microbiological growth of the total vial count (TVC), total coliform count (TCC), and Clostridium perfringens was determined at 50 days from elaboration. Mass seeding was performed on Rapid E. Coli (to determine TCC), PCA (TVC), and Rapid L. mono (L. monocytogenes). All media were sterilized at 121 °C for 20 min according to product indications. Peptone water (OXOID, Ltd. CM0087 Basingstoke, Hampshire, United Kingdom) was used to make the dilutions. A laminar flow hood (Telstar, BIO-II-A, Madrid, Spain) was used to carry out the analysis. Finally, plates were incubated for 24 h at 37 °C for TCC, 48 h at 37 °C for TVC, and 48 h at 37 °C for C. perfringens. Results were obtained in triplicate and are expressed in cfu/g.