Artificial digestive
solution based on
pepsin is essential for collecting metacercariae from fish. To promote the enzymatic reactivity of
pepsin, the pH of the
solution has to be adjusted to pH 1.0-2.0. Hydrochloride (HCl) is usually used for this purpose, but the use of HCl raises safety concerns. The aim of this work was to address the usefulness of
citric acid as an alternative for HCl for the acidification of
pepsin solution, and to examine its potential to damage metacercariae during in vitro digestion as compared with HCl. Changes in pH after adding 1-9% of
citric acid (m/v) to
pepsin solution were compared to a 1% HCl (v/v) addition. Digestion of fish muscle was evaluated by measuring released
protein concentrations by spectrophotometry. In addition, survival rates of metacercariae in
pepsin solution were determined at different
citric acid concentrations and were compared that of with 1% HCl. The present study shows that addition of
citric acid reduced the pH of
pepsin solutions to the required level. Addition of more than 5% of
citric acid resulted in the effective digestion of fish muscle over 3h in vitro, and 5%
citric acid was less lethal to metacercariae than 1% HCl in
pepsin solution.
Pepsin solution containing 5%
citric acid had digestive capacity superior to
pepsin solution containing 1% HCl after 3h incubation with released
protein concentrations of 12.0 ng/ml for 5%
citric acid and 9.6 ng/ml for 1% HCl. Accordingly, the present study suggests that the addition of 5%
citric acid to
pepsin solution is a good alternative to 1% HCl in
infection studies because
citric acid is a stable at room temperature and has a good safety profile. In addition, we suggest that the use of
citric acid enables the preparation of commercial digestive solutions for the detection of microorganisms in fish and other vertebrate muscle tissue.