The concentration of pepsin (MP Biomedicals, Ohio, USA) used in the present study was 1% (w/v) and the concentration of HCl was 1% (v/v). Citric acid was added at 8 concentrations from 1% to 20% (w/v) (1, 3, 5, 7, 9, 13, 15, and 20%). The digestive capacity

of ADSs containing varying concentrations click here of citric acid were compared with ADS containing 1% HCl. The fish meat used in the study (mullet) were purchased from a market (Noryangjin, Seoul). Tissue samples were prepared by slicing the fish meat to produce 2 cm2 (about 20 g), and these were placed in 50 ml conical tubes with 40 ml of the ADS samples. The tubes were incubated at 37 °C in a shaking incubator for 1–2 h. To determine digestive capacity of each solution, the concentrations of proteins released from digested samples were measured using a Nanodrop 2000 spectrometer (Thermo Scientific, Wilmington, Delaware, USA) at 280 nm. To investigate the effect of citric acid on parasite survival, M. yokogawai, a fish-borne intestinal trematode

parasite, SP600125 chemical structure was collected from sweetfish, Plecoglossus altivelis, captured from a stream in an endemic area in Gyeongsangbuk-do ( Pyo et al., 2013). The sweetfish were finely ground, mixed with ADSs, incubated at 37 °C for 1–2 h, filtered through a mesh (pore size 1 mm × 1 mm), and washed with 0.85% saline repeatedly until clear. The sediment was carefully observed

under a stereomicroscope. Metagonimus metacercariae were identified morphologically and collected ( Pyo et al., 2013). The metacercariae of M. yokogawai were examined for the effects of ADSs on parasite survival. Metacercariae were incubated at 37 °C with each ADS, and surviving metacercariae were counted under an optical microscope (CHS-213E; Olympus, Tokyo) at 2 h intervals during the 8 h incubation period. The survivability of metacercariae was confirmed by their morphological characteristics. A metacercaria was considered dead if it did not move for 5 min at 25–37 °C with loss of the body wall integrity and faintness of the excretory bladder with few excretory granules. The conventional composition of ADS contains 0.5–1% Adenosine of pepsin and 0.8–1% of HCl with a pH of 1.5–2.0. The addition of citric acid instead of HCl acidified the pepsin solution (Fig. 1). The pH of the pepsin solution itself was 3.76, and the addition of 1% HCl decreased the pH to less than 2.0. The addition of 1% citric acid decreased the pH to 2.44, and the addition of citric acid at concentrations greater than 5% decreased the pH to less than 2.0. ADSs containing citric acid between 5% and 20% resulted in pH values between 1.5 and 2.0 (Fig. 1). These findings show that citric acid concentration needs to be greater than 5% to achieve maximal pepsin activity.