Digestor2006

The protein contents of the experimental diets and of fish muscle were estimated by the Kjedahl method, following AOAC (940.25, 2016) [26 ], using a conversion factor of 6.25. Briefly, the samples (250 mg or 150 mg of diets and muscle, respectively) were digested (Digestor 2006; Foss, Denmark) with a catalyst tablet and 25 mL of H₂SO₄ (97%) at 220 °C for 30 min and 400 °C for 90 min. Deionized water (70 mL) was added to cold digested samples that were distillated (Kjeltec 2100, Foss, Denmark) with 100 mL of NaOH (40% m/v). The distillate was collected in 30 mL of H₃BO₃ (4% m/v) with methyl red and bromocresol green as pH indicators for further titration with standardized HCl 0.1 M. Blank assay was prepared at the same conditions as samples. The results were calculated according to Equation (1) and expressed as % FW: $$\mathrm{Protein}(\%\mathrm{FW})=\frac{\left[\mathrm{HCl}\right]\times ({\mathrm{V}}_{\mathrm{S}}-{\mathrm{V}}_{\mathrm{B}})\times 14\times 6.25}{\mathrm{m}}$$
where [HCl]—chloride acid concentration (M); V_s and V_B—volume of HCl spent for samples and blank titration, respectively (mL); m—sample mass (mg).

The total protein assay was performed according to Portuguese Standard NP 4488 (2009 ), applying the Kjeldahl method. Briefly, 0.5 g of sample (or distilled water in the blank assay) were mixed with 2 Kjeldahl tablets (Panreac) and 25 ml of H₂SO₄ (Honeywell, Fluka, MI) in digestion tubes. Samples were further digested in a Kjeldahl digestor (Foss, Digestor2006) at 400°C for 90 min. After samples were cooled down to room temperature, 80 ml of distilled water were added and the ammonia formed was distilled into 30 ml of a 4% H₃BO₃ (Panreac) solution containing bromocresol green (Alfa aesar) and methyl red (Panreac), under alkaline conditions (distillation with 40% NaOH, Fisher scientific), using a Kjeldahl distiller (Foss, Kjeltec2100). The distilled samples were titrated with HCl 0.5 M (VWR).
The crude protein content, represented by the sample's nitrogen content, is expressed as g/100 g of FW, and was calculated using the following equation: $$\text{Protein content}=\frac{6.25\times 0.014\left(V_{\mathrm{a}}-V_{\mathrm{b}}\right)\times N}{W}\times 100$$
where 6.25 is the nitrogen‐to‐protein conversion factor (Jones, 1941 ), V_a is the volume of HCl spent on sample titration, V_b is the volume of HCl spent on blank assay, N is the HCl normality, and W is the sample weight.

The total protein content was determined by the Kjeldahl method [49 ] and was estimated using a conversion factor of 5 specific to red seaweeds [50 (link)]. Seaweed samples (0.5 g) were digested with 25 mL of 97% sulfuric acid and two catalyst tablets (VWR CHEMICALS, Pennsylvania, USA) for 30 min at 200 °C followed by 90 min at 400 °C in a digestor system (Digestor2006, Foss, Hillerød, Denmark). The samples were cooled at room temperature and 80 mL of water was added. The samples were then distilled under alkaline conditions (Kjeltec2100, Foss, Hillerød, Denmark). The resulting distillate was collected in 4% boric acid solution and ammonia was quantified by titration with 0.1M hydrogen chloride using a mixture of methyl red and bromocresol green as an indicator. The protein content was calculated according to Equation (1).
$$\mathrm{Total}\mathrm{protein}(\%)=\frac{\left({\mathrm{V}}_s-{\mathrm{V}}_b\right)\times \mathrm{n}\times 5\times 0.014}{\mathrm{m}}\times 100$$
where V_s represents the volume of HCL (mL) used in the titration of the sample, V_b represents the volume of HCL (mL) used in the titration of the blank solution (prepared in absence of algae biomass), n corresponds to the concentration of the HCL solution used in the titration (M), and m corresponds to the initial mass (g) of the dried seaweed sample.