## 2 Materials and Methods
### Analysis
#### 2.3.2 Proteolytic Changes and Biogenic Amine Content  
Free amino groups were evaluated in water and in phosphotungstic acid [25]. Water-soluble nitrogen (WSN) was determined in the water-soluble fraction (WSF) of the sample. The contents of free amino-acid groups in water (WSF) and the phosphotungstic acid (PTA)-soluble fractions were measured using a 2,4,6-trinitrobenzenesulffonic acid (TNBS) (Sigma-Aldrich, Saint Louis, MI, USA). A phosphotungstic acid soluble fraction of meat sample was prepared from WSF. The WNS fraction was used to determine the free amino acids as follows: 5 mL of the water-soluble protein fraction was added to 5 mL of 40% trichloroacetic acid (TCA) and stirred on a Vortex RS-va10 (Phoenix Instrument, Garbsen, Germany) for 10 min, before centrifuging at 12,000\(\times\)_g_ for 10 min in a precooled centrifuge MPW-352R, REF (MPW Med. Instruments, Warszawa, Poland) at 4 \({}^{\circ}\)C. Then, 2 mL of solution was removed and dried under nitrogen. The obtained residue was dissolved in 1 mL of 20 mM HCl. The chromatographic separation (HPLC) of free amino acids was performed with Dionex UltiMate 3000 (Thermo Scientific, Waltham, MA, USA). Separation was carried in a Nova-Pak reverse-phase C18 column, 4 um particle size, 150 \(\times\) 3.9 mm (Waters, Milford, MA, USA), thermostated at 37 \({}^{\circ}\)C. The two solvent reservoirs contained the following eleunets: (A) acetate-phosphate buffer pH = 5.2 and (B) a mixture of acetonitrile and water (60:40, \(v/v\)) according to the Waters procedure. The elution program consisted of a gradient system with a flow rate of 1 mL-min\({}^{-1}\) (100% A for 0.5 min, 98% A and 2% B for 14.5 min, 93% A and 7% B to 19 min, 90% A and 10% B to 32 min, 67% A and 33% B to 33 min, 100% B to 37 min, and 100% A to 64 min). Fluorometric detection was carried out using excitation and emission wavelengths of 250 nm and 395 nm, respectively. Analytical kit ACCQ Tag (Waters, USA) was used as a reference. The biogenic amine analysis was performed using HPLC according to the following method: a 10 g sample of meat was mixed with 15 mL of 6% trichloroacetic acid (TCA), homogenized for 2 min (1000 rpm, Vortex), and centrifuged at 14,000\(\times\)_g_ for 20 min at 4 \({}^{\circ}\)C (MPW Med. Instruments, Warszawa, Poland). The obtained supernatant was collected and filtered through Whatman No. 1 filter paper, and the residue was reextracted with 15 mL of fresh of 6% TCA. The volume of the combined supernatants was completed to 50 mL by 6% TCA. The derivatization process was performed by mixing 1 mL of extract with 1 mL of dansyl chloride acetone solution (10 mg-mL\({}^{-1}\)) and 0.5 mL of saturated NaHCO\({}_{3}\) solution. Incubation was carried out at 40 \({}^{\circ}\)C for 60 min with occasional shaking in a thermoblock (Dry Block Heater, IKA, Staufen, Germany). The biogenic amines were extracted twice using 1 mL of diethyl ether for 10 min (22 \({}^{\circ}\)C). The extracts were dried in a stream of nitrogen, and the residue was dissolved in 1 mL of acetonitrile. The solution standards of biogenic amines were prepared by mixing 1 mL of each free base standard solution, containing 0.1 mg-mL\({}^{-1}\) of each biogenic amine with 5 mL of dansyl chloride acetone solution. The derivatization and extraction procedure was performed in the same manner as the samples. The chromatographic separation of free amino acids was performed using Dionex UltiMate 3000 HPLC apparatus (Thermo Scientific, Waltham, MA, USA). The separation was carried in a Nova-Pak reverse-phase C18 column, 4 um particle size, 150 \(\times\) 3.9 mm (Waters, Milford, MA, USA), thermostated at 30 \({}^{\circ}\)C. The two solvent reservoirs contained the following eleunets: (A) acetonitrile and (B) HPLC-grade water at a flow rate of 0.8 mL-min\({}^{-1}\). The elution program was: 65% A and 35% B for 1 min, increasing to 80% A and 20% B for 9 min, increasing to 90% A and 10% for 2 min, increasing to 95% A and 5% B for 4 min, holding for 7 min, and returning to 65% A and 35% B, before holding for 5 min.