2.5.3. Determination of Total Volatile Basic Nitrogen (TVB-N)

The TVB-N concentration of the fish sample was determined by the reference procedure described in the European Commission’s Decision 95/149/EC [28]. A 10.0 g portion of the fish sample was homogenized in 90 mL of 6% (w/v) perchloric acid solution at 20,000 rpm for 2 min using a laboratory dispenser (T 18 digital Ultra-Turrax, IKA-Werke GmbH & Co. KG, Staufen, Germany). The mixture was then filtered and the obtained extract kept in the refrigerator until use.

A 50 mL aliquot of the extract was mixed with a few drops of silicone antifoaming agent and 6.5 mL of 20% (w/v) sodium hydroxide solution. The mixture was subjected to steam distillation using a Kjeldahl distillation unit (UDK 140, VELP Scientifica, Milan, Italy) till 100 mL of distillate had been produced. The distillate was collected in a flask containing 100 mL of 3% (w/v) boric acid solution and 3-5 drops of Tashiro mixed indicator (0.2 g methyl red and 0.1 g methylene blue dissolved in 100 mL 95% ethanol). Next, the receiver solution was titrated with a 0.01 N hydrochloric acid standardized solution until the endpoint (pH 5.0 ± 0.1) was reached. A blank sample was prepared with 50 mL of 6% (w/v) perchloric acid solution instead of extract and treated identically to the fish sample. The TVB-N concentration was calculated with the following Equation (2):

where V1 is the volume of 0.01 N hydrochloric acid solution consumed for the fish sample at endpoint (mL), V0 is the volume of 0.01 N hydrochloric acid solution consumed for the blank at endpoint (mL), and wfs is the weight of the fish sample (g). Three replicates were run for each fish sample.