Improving and standardizing protocols for alkaline protease quantification in fish

Abstract Assessing the activity of alkaline protease is a common procedure in many biological, physiological and nutritional studies, including the maturation and digestive capacity of the digestive tract, in gastric and non‐gastric fish species. After reviewing the available literature on fish digestive alkaline proteases published during a time span of ten years (2008–2018), it has been detected some possible methodological and/or interpretative inconsistencies in this kind of studies when quantifying alkaline protease activity and concluded that special attention should be paid on: (i) the proper preparation of intestine and pyloric caeca extracts, (ii) the use of the proper reaction conditions at the normal range of physiological pH values and temperature and (iii) the proper quantification of the number of peptide bonds hydrolysed for alkaline protease unit calculation. The implementation of these recommendations and the proposal of more suitable (previously tested) methodological steps will promote the standardization of feasible alkaline protease analytical procedures, as well as improve the reliability of comparative studies between different fish species of aquacultural importance, dealing with the alkaline proteolytic enzymes.