Peptone

The commercial starters (BOX-57—Carnobacterium divergens, C. maltoaromaticum and L. sakei; LAK-23—L. sakei; and FP-50—C. divergens, C. maltoaromaticum) were freeze-dried in a foil pouch. F-106 (Lacticaseibacillus casei) was derived from the Collection of the Department of Agricultural Food, Environmental, and Animal Sciences of the University of Udine and was grown in de Man, Rogosa and Sharpe (MRS) broth (Oxoid, Italy). After its growth, the strain was harvested by centrifugation at 8000× g for 10 min and then diluted in peptone water. The commercial starters were thawed, homogenized, and diluted in sterile peptone water (NaCl 0.6%, peptone, Oxoid, 0.1%, and distilled water 1 L) at the time of use. To evaluate the load of each starter, dilutions were performed in sterile peptone water, and 0.1 mL of each dilution was inoculated in the de Man, Rogosa and Sharpe (MRS) medium (Oxoid, Italy) by the double layer method for the LAB and in TSM agar (tryptic soy medium with 5% glucose, 2% NaCl, and pH 8, Oxoid, Italy) in a jar prepared for anaerobic reaction with a gas-packing anaerobic system (BBL, Becton Dickinson, USA) for the carnobacteria. The plates were incubated at 37 °C for 48–72 h, and the grown colonies were counted. Each suspension contained, on average, approximately 11 Log CFU/g.

The chosen starter was sold freeze-dried in a foil pouch. At the time of use, the starter was thawed, homogenized, and diluted in sterile peptone water (NaCl 0.6%; peptone, Oxoid, 0.1%, distilled water 1 L). To evaluate its load, dilutions were performed in sterile peptone water, and 0.1 mL of each dilution was inoculated in Petri dishes, to which de Man, Rogosa, and Sharpe (MRS) medium (Oxoid, Italy) was subsequently added by the double layer method. The plates were incubated at 37 °C for 48–72 h, and the grown colonies were counted. Each suspension contained on average approximately 11 log CFU/g. Then, the starter culture was diluted in natural water used to wash the fish before packaging at a level of approximately 7 log CFU/mL.

Ten grams of each salmon piece was mixed with 90 g of peptone water (1 g/l of peptone (Oxoid) and 8.5 g/l of NaCl (VWR, Belgium), and homogenised in a stomacher (IUL Masticator, Spain), and serially diluted (10-fold) before plating. Lyngby's Iron agar (Oxoid, Norway) supplemented with 0,04% l-cysteine (Sigma-Aldrich) was used for quantification of total aerobic counts and H₂S -producing bacteria, and incubated at 22 °C for 72 ± 6 h [57 ], while MRS and M17 agar were used for quantification of LAB using anaerobic incubation at 25 °C for 2–5 d.