*2.3. Chemical Composition*

Moisture, ash and protein were quantified according to the International Organisation for Standardisation (ISO) recommended standards 1442:1997 [28], 936:1998 [29] and 937:1978 [30], respectively. Crude fat was extracted using an Ankom XT10 (Ankom Technology Corp., Macedon, NY, USA), according to the American Oil Chemists' Society (AOCS) Official Procedure Am 5-04 [31]. Protein content was estimated by Kjeldahl method (N × 6.25). Dietary fiber contents (total (TDF), insoluble (IDF) and soluble (SDF) fiber) were determined with the dietary fiber assay kit TDF-100A (Sigma Aldrich, St. Louis, Missouri, U.S.A.) according to the AOAC procedures [32]. The total content of carbohydrate was calculated by difference. Protein and dietary fiber were also measured in the mushroom flours.

Na content was determined from the ashes of 5 g-aliquots of each sample dissolved in 10 mL of 1 M HNO3, and analyzed by inductively coupled plasma (ICP)-optical emission spectroscopy (Thermo-Fisher, Cambridge, UK) equipped with a radio frequency source of 27.12 MHz, a peristaltic pump, a spraying chamber and a concentric spray nebuliser, and controlled by the ICP software. Standard solutions (50, 100, 150, 200 mg/L) were prepared from a stock solution of Na (1000 mg/L; SCP-SCIENCE, Courtaboeuf, France) in 4% HNO3 (*v*/*v*). The results were expressed as milligrams per 100 g sausage.

#### *2.4. Amino Acid Profile*

Amino acid composition (g/100 g of sample) of frankfurters was determined using the methodology described by Marti-Quijal et al. [33] by derivatization of amino acids with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (Waters AccQ-Fluor reagen<sup>t</sup> kit) and

reversed-phase high-performance liquid chromatography analysis (RP-HPLC) (Waters 2695 Separations Module + Waters 2475 Multi Fluorescence Detector + WatersAccQ-Tag amino acids analysis column). Empower 2 TM advanced software (Waters, Milford, MA, USA) was used to control system operation and results management. Amino acids were identified by retention time using an amino acid standard (Amino Acid Standard H, Thermo, Rockford, IL, USA).

#### *2.5. Lipid Oxidation Analysis*

The thiobarbituric acid-reactive substances (TBARs) assay was carried to evaluate lipid stability according to the methodology of Vyncke [34]. For this purpose, 2 g of sample and 10 mL of trichloroacetic acid (5%) were homogenised using an Ultra-Turrax (IKA T25 basic, Staufen, Germany) for 2 min. The homogenate was kept at −10 ◦C for 10 min and centrifuged at 2360 *g* for 10 min. The supernatant was filtered through a Whatman No. 1 (Sigma Aldrich, St. Louis, MO, USA) filter paper. The extract (5 mL) was mixed with a 0.02 M thiobarbituric acid solution (5 mL) and incubated in a water bath at 97 ◦C for 40 min. The absorbance was measured at 532 nm. For quantification a standard curve of malondialdehyde (MDA) was designed, and the results were expressed as milligrams of MDA per kilogram of sample.

#### *2.6. pH and Microbial Analysis*

The pH from three frankfurters of each sample was measured with a digital pH-meter (HI 99163, Hanna Instruments, Eibar, Spain) equipped with a glass probe for penetration.

Total viable counts (TVC) and lactic acid bacteria were determined using TEMPO system (TEMPO Filler, TEMPO Reader, BioMérieuxs, Marcy l´Etoile, France), based on the most likely number technology. Both microorganisms were incubated at 30 ◦C for 24 and 48 h respectively (detection limit of 0.3 CFU/g of sample). *Pseudomonas* spp. counts were enumerated on Pseudomonas agar base with selective supplement for this microbial group (CFC) (Merck, Darmstadt, Germany) after incubation at 25 ◦C for 48 h. Psychrotrophic aerobic bacteria were enumerated on plate count agar (PCA; Oxoid, Unipath Ltd., Basingstoke, UK) following incubation at 7 ◦C for 10 days. Detection limits were 2 log CFU/g for pseudomonads and psychrotrophic bacteria. The microbial results were expressed as log CFU/g.

#### *2.7. Color and Texture Profile*

CIELAB parameters (L\*: lightness; a\*: redness and b\*: yellowness) were measured in slices of 2 cm thickness using a portable colorimeter (Konica Minolta CM-600d, Osaka, Japan) under D65 illuminant and 10◦ observer with an 8 mm aperture.

A texturometer (TA-XTplus, Stable Micro Systems, Surrey, UK) equipped with Texture Exponent 32 software (version 1.0.0.68) was used to measure the texture profile analysis (TPA). The samples were cut in pieces of 2.5 cm height × 2 cm diameter and hardness (N), springiness (mm), chewiness (N/mm), gumminess (N) and cohesiveness (mm/mm) were determined (Bourne, 1978). Textural parameters were measured by compressing to 50% with a double compression cycle test using an aluminum cylinder probe P50 (50 mm diameter) at speed of 20 mm/s and a distance of 30 mm with a 50 Kg load cell. Three slices of each sample were measured.
