The importance of pepsin‐like acid protease quantification in aquaculture studies: a revision of available procedures and presentation of a new protocol for its assessment

Abstract Assessing the activity of acid pepsin‐like protease is a common procedure in many biological, physiological and nutritional studies in gastric fish species. After reviewing the available literature on fish digestive acid proteases (stomach acid pepsin‐like proteases), especially those species of relevance for aquaculture purposes, published during a time span of ten years (2008–2018), authors detected some possible methodological and/or interpretative inconsistencies in this kind of studies when quantifying pepsin‐like enzymes. Thus, we concluded that special attention should be paid on the following issues when assessing digestive acid protease activity: (i) the proper preparation of stomach extracts, (ii) the use of the proper reaction conditions at the normal range of physiological pH and temperature values, and (iii) the proper quantification of the number of peptide bonds hydrolyzed for acid protease unit calculation. The implementation of these recommendations, and the proposal of more suitable (previously tested) methodological steps, will promote the standardization of acid pepsin‐like protease analytical procedures, as well as improve the reliability of comparative studies dealing with this proteolytic enzyme between different fish species of aquacultural importance.