In vitro pH-Stat protein hydrolysis of feed ingredients for Atlantic cod, Gadus morhua. 1. Development of the method

Abstract The method described here involves the extraction and partial purification of an enzyme fraction from the dissected pyloric caeca of commercially farmed Atlantic cod, Gadus morhua (1 kg fish) and the development of a pH-Stat method to predict protein digestibility. The various extraction and partial purification steps successfully concentrated the alkaline serine protease enzymes, trypsin (> 4-fold) and chymotrypsin (> 12-fold). It was found that the enzyme fractions produced in the manner described in this study were completely stable for up to 8 months when stored at − 20 °C and at least 10 months when stored − 80 °C after which significant loss of enzyme activity can occur, although the degree of protein hydrolysis (DH) of casein was unaffected after 12 months. It is recommended that enzyme fractions produced in a similar manner should be stored at − 80 °C and used within 8–10 months. The most suitable substrate concentration [S] to use for closed-system in vitro pH-Stat DH assays was established using a standard purified protein source (vitamin-free casein) with four [S] (0.25, 0.5, 0.75 and 1 mg N/mL protein suspension solution). No significant differences (P > 0.05) were found in the DH values between the [S] tested. The DH curve for casein at a [S] of 0.5 mg N/mL showed a rapid increase initially before leveling off at maximum DH (26%) which was achieved within a moderate duration of the assay (5–6 h). The closed-system pH-Stat assay with a [S] of 0.5 mg N/mL and minimum assay duration of 8 h is recommended for further investigation of conventional and novel feed ingredients for gadoid diets.