Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine and Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul 110-799, South Korea.
Vet Parasitol. 2013 Nov 15;198(1-2):111-5. doi: 10.1016/j.vetpar.2013.08.004. Epub 2013 Aug 14.
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.
基于胃蛋白酶的人工消化溶液对于从鱼类中收集囊蚴至关重要。为了提高胃蛋白酶的酶促反应性，必须将溶液的 pH 值调整至 1.0-2.0。为此，通常使用盐酸 (HCl)，但 HCl 的使用存在安全隐患。本研究旨在探讨柠檬酸替代 HCl 酸化胃蛋白酶溶液的实用性，并比较其在体外消化过程中对囊蚴的潜在损伤与 HCl 的差异。比较了向胃蛋白酶溶液中添加 1-9%柠檬酸（m/v）与添加 1% HCl（v/v）后 pH 的变化。通过分光光度法测量释放的蛋白质浓度来评估鱼肌肉的消化情况。此外，还测定了不同柠檬酸浓度下囊蚴在胃蛋白酶溶液中的存活率，并与 1% HCl 进行了比较。本研究表明，添加柠檬酸可将胃蛋白酶溶液的 pH 值降低至所需水平。添加超过 5%的柠檬酸可导致鱼肌肉在体外 3 小时内有效消化，并且 5%的柠檬酸对胃蛋白酶溶液中的囊蚴的致死性低于 1%HCl。在 3 小时孵育后，含有 5%柠檬酸的胃蛋白酶溶液的消化能力优于含有 1%HCl 的胃蛋白酶溶液，释放的蛋白质浓度分别为 12.0ng/ml 和 9.6ng/ml。因此，本研究表明，在感染研究中，向胃蛋白酶溶液中添加 5%柠檬酸是替代 1%HCl 的一种很好的选择，因为柠檬酸在室温下稳定且具有良好的安全性。此外，我们建议使用柠檬酸可以制备用于检测鱼类和其他脊椎动物肌肉组织中微生物的商业消化溶液。
