Violet red bile dextrose agar vrbd

Microbiological analysis of the sausages was performed after 1, 4, 8, 11, 15, 22, and 29 days of refrigerated storage at 4 °C. The samples were prepared according to ISO 6887-2-2017. The total psychrotrophic count (TPC) was determined on Plate Count Agar (PCA) (Merck, Darmstadt, Germany) following incubation at 6 °C for 6 days. The lactic acid bacteria (LAB) were counted on Man Rogosa Sharpe Agar (MRS) (Merck, Darmstadt, Germany) following incubation for 72 h at 30 °C. The quantity of Enterobacteriaceae was determined on Violet Red Bile Dextrose Agar (VRBD) (Merck, Darmstadt, Germany) following incubation at 30 °C for 24 h. The lowest detection limit of the applied enumeration techniques is 10 CFU/g.

For microbiological enumeration, a 10 g sample was transferred to a sterile stomacher bag with 90 mL of sterilized Ringer solution (Merck, Darmstadt, Germany) and was homogenized for 60 s. Samples (0.1 mL) of tenfold serial dilutions of fish homogenates were spread onto the surface of the appropriate media in Petri dishes for enumeration of different spoilage bacteria^{27 (link)}. Total aerobic count was enumerated on Plate Count Agar (PCA, Merck, Darmstadt, Germany) after incubation at 25 °C for 72 h. Pseudomonas spp. were enumerated on Cetrimide Agar (CFC, Merck, Darmstadt, Germany) after incubation at 25 °C for 48 h. For Enterobacteriaceae spp. enumeration the pour-plate method was used, using the Violet Red Bile Dextrose Agar (VRBD, Merck, Darmstadt, Germany), incubated at 25 °C for 48 h. Two replicates of at least three appropriate dilutions were enumerated. The microbial growth was modelled using the Baranyi Growth Model^{28 (link)}. For curve fitting the in-house program DMfit (Inst. Food Research, Reading, U.K.) was used, and kinetic parameters such as the rate (k) of the microbial growth were estimated. Total volatile basic nitrogen analysis was conducted on a single TCA extraction by distillation in a Kjeldhal rapid distillation unit and titration with hydrochloric acid²⁹ .

Bacterial isolation was performed after preparing a ten-fold serial dilution in buffered peptone water (BPW) (Thermo Fisher Scientific Inc., Oxoid Ltd., Basingstoke, UK) up to dilution 10-3. The dilutions were plated in duplicates on Trypticase Soy Agar (TSA) (Merck Millipore, Burlington, MA, USA), Violet Red Bile Dextrose agar (VRBD) (Merck Millipore, Burlington, MA, USA), Reasoner’s 2A agar (R2A agar) (Merck Millipore, Burlington, MA, USA), and Yeast Extract Agar (Merck Millipore, Burlington, MA, USA) plates by spread plate method. VRBD plates were incubated at 37 °C for 24–48 h while plates with the other culture media were incubated at 30 °C for 24–48 h.