Digestor 2040

Crude protein was determined by weighing out ~0.25 g of sample and adding 5 mL sulphuric acid (analytical reagent grade, Fisher Scientific, Loughborough, UK) together with 2 copper catalyst tablets (Fisher Scientific, Loughborough, UK), before digesting at 400 o C for 1 h (Foss Digestor 2040; Foss Analytical AB, Högnäs, Sweden). Total nitrogen levels were measured by Kjeldahl (Foss Kjeltec™ 2300, Foss Analytical AB, Högnäs, Sweden) and the crude protein level calculated as N  6.25.

The proximate composition of samples was determined using standard methods (AOAC, 1990) . Moisture content was determined by weighing ∼1 g of homogenised wet sample and placing into a drying oven for 20 h at 105 • C. Ash content was determined by weighing ∼1 g of dried sample and placing into a muffle furnace overnight set at 600 • C. Crude protein was measured by weighing ∼0.25 g dried sample before addition of copper catalyst tablets and 5 ml sulphuric acid (analytical reagent grade, Fisher Scientific, Loughborough, UK). Samples were digested at 400 • C for 1 h (Foss Digestor 2040, Foss Analytical AB Högnäs, Sweden). Total nitrogen levels were measured by Kjeldahl (Foss Kjeltec TM 2300, Foss Analytical AB, Högnäs, Sweden) and the crude protein level calculated as N × 6.25. The moisture content was used to convert results to a wet weight basis. Total lipid was extracted from homogenised wet samples using 20 vol of ice-cold chloroform/methanol (2:1, v/v) using an Ultra-Turrax tissue disruptor (Fisher Scientific, Loughborough, UK) according to (Folch et al., 1957) (link). Non-lipid impurities were isolated by washing with 0.88% KCl and the lipid weight determined gravimetrically after evaporation of solvent using oxygen-free nitrogen and desiccation in vacuo before making up to a known concentration and storing at -20 • C.