Sterile buffered peptone water

The samples (1 mL) were collected and diluted with 9 mL of sterile buffered peptone water (Merck, Darmstad, Germany), and serial dilutions were prepared [12 (link)]. Lactic acid bacteria counts were determined on MRS (de Man, Rogosa and Sharpe) medium (Merck, Darmstad, Germany) after incubation at 37 °C under anaerobic conditions for 72 h, whereas Acetobacter bacteria were assayed on Acetobacter Agar (HiMedia, Mumbai, India), after incubation at 37 °C under aerobic conditions for 48 h. Yeast counts were determined on Rose Bengal Agar at 25 °C for 72 h [13 (link)]. The enumeration of microorganisms was performed in triplicate (by counting plates with 30–300 colonies) and the viable cell counts were expressed as CFU/mL of the samples.

The representative sample cuts (crumb with crust—10 g) were collected and aseptically introduced into a sterile stomacher bag, then diluted with 90 mL of sterile physiological saline (0.9%). The samples were homogenized in a Bag Mixer (Interscience, Saint-Nom-la-Brèteche, France) for one minute and appropriate decimal dilutions were prepared in sterile buffered peptone water (Merck, Darmstad, Germany). Total mesophilic microbial counts were enumerated on Plate Count Agar (Merck, Darmstad Germany), whereas coliforms were determined on Violet Red Bile Glucose Agar (Merck, Darmstad, Germany), both after incubation at 37 °C under aerobic conditions for 48 h. The presence of Bacillus sp. was determined on Mannitol Yolk Polymyxin B Agar (Merck, Darmstad, Germany). Fungal counts were determined on Sabouraud Agar amended with chloramphenicol (0.005%) (Merck, Darmstad, Germany) at 25 °C for 72 h under aerobic conditions. The enumeration of microorganisms was performed in triplicate (by counting plates with 30–300 colonies) and the viable cell counts were expressed as CFU/g of the samples.
A pH meter (CP-411, Elmetron, Zabrze, Poland) was used to determine the pH of the samples by immersing the device’s probe (at 25 °C) directly into the samples homogenized in saline.

Muscle pieces (~ 10 g, without skin) were aseptically excised from the anterior part of the epaxial muscle and transferred to a sterile stomacher bag diluted with 1:10 sterile buffered peptone water (Merck, Germany). The mixture was blended using a Smasher (AES Laboratorie, bioMérieux Industry, USA) for 120 s. Homogenates were further diluted to appropriate concentrations. For raw fillets, total psychotropic counts (TPC) were quantified using long and hammer (L&H) agar, while total mesophilic counts (TMC) and H₂S producing bacteria (HSPB) were quantified using iron agar supplemented with 0.04% l-cysteine (Sigma-Aldrich, Norway) according to the NMKL method No. 184¹⁸ . 49.2 µl of each homogenate was transferred to L&H agar using an Eddy Jet 2 W Spiral Plater (IUL micro, Spain) while 1 ml was transferred to iron agar. L&H agar plates were incubated at 15 °C for 5 days, while iron agar at 25 °C for 72 ± 6 h. HSPB was quantified by the black colonies produced. For smoked samples, microbial analysis was done on the last sampling day using L&H and MRS (de man, Rogosa, Sharpe, Oxoid, UK) agar with Amphotericin B to quantify for TPC and lactic acid bacteria (LAB), respectively. MRS plates were incubated in anaerobic conditions at 25 °C for 5 days according to the NMKL method No. 140¹⁹ . The end of shelf life was defined as the point where microbial counts exceeded 10⁶ cfu g⁻¹.