*2.3. Physicochemical Analysis*

#### 2.3.1. Determination of Quality Parameters: pH, Color, and Chemical Composition

The pH value was measured using a portable pH-meter (Hanna Instruments, Eibar, Spain), equipped with a penetration probe. The color determination of sausages was assessed from fresh cut cross-sections in three different points with a portable colorimeter (Konica Minolta CM-600d, Osaka, Japan), according to CIELAB space [21]: Lightness, (L\*); redness, (a\*); yellowness (b\*), with the next settings machine (pulsed xenon arc lamp, angle of 0◦ viewing angle geometry, and aperture size of 8 mm). Hue (Hab) and chroma (C\*) were calculated from the a\* and b\* values according to expressions:

$$C^\* = \sqrt{{(a^\*)}^2 + {(b^\*)}^2} \text{ and } h\_{ab} = acr \tan\frac{b^\*}{a^\*} \tag{1}$$

Moisture, protein, and ash were assessed following the International Organization for Standardization (ISO) recommended standards [22–24]. Total fat was extracted according to the American Oil Chemists' Society (AOCS) official procedure [25], while carbohydrate contents were estimated by the difference. The quantification of total cholesterol, saponification, extraction, and identification was carried out with high performance liquid chromatography (HPLC) in the normal phase, according to Dominguez et al. [26].

#### 2.3.2. Fatty Acid Profile

The total fat was extracted from 12.5 grams of the sample and 50 milligrams were utilized to determine the FA profile. Total FAs were transesterified, following Dominguez et al. [26]. The separation and quantification of the fatty acid methyl esters (FAMEs) was performed using a gas chromatograph (GC-Agilent 6890 N; Agilent Technologies Spain, S.L., Madrid, Spain) equipped with a flame ionization detector following the chromatographic conditions described by Dominguez et al. [26]. Data of FAME profiles were expressed in grams per 100 grams of fat.

To assess the nutritional properties of sausages, the ratios polyunsaturated fatty acid (PUFA)/SFA, *n*-6/*n*-3, hypocholesterolemic/Hypercholesterolemic (h/H) ratios, the nutritional value (NV), the indexes of atherogenicity (AI), and thrombogenicity (TI) were determined. The hypocholesterolemic/Hypercholesterolemic (h/H) ratio was calculated according to Santos–Silva et al. [27]: h/H = ((sum of C18:1*n*9c, C18:2*n*6, C18:3*n*6, C18:3*n*3, C20:3*n*6, C20:4*n*6, C20:5*n*3, and

C22:5*n*3)/(sum of C14:0 and C16:0)). The atherogenic index (AI) and thrombogenic index (TI) were calculated according to Ulbricht and Sauthgate [28]:

$$\text{AI} = [(4 \times \text{C14:0}) + \text{C16:0}] \{ (\Sigma \text{PUFA}) + (\Sigma \text{MUFA}) \}; \tag{2}$$

TI = [C14:0 + C16:0 + C18:0]/[(0.5 × ΣMUFA) + (0.5 × *n*6) + (3 × *n*3) + (*n*3/*n*6)].

#### 2.3.3. Texture Profile Analysis

Sausage pieces of 1 × 1 × 2.5 cm (height × width × length) were compressed at a crosshead speed of 3.33 mm/s in a texture analyzer (TA.XTplus, Stable Micro Systems, Vienna Court, UK). The following parameters were recorded: Hardness, cohesiveness, springiness, gumminess, chewiness, and adhesiveness by compressing to 80%, using a compression probe with 19.85 cm<sup>2</sup> of surface contact.
