1Department of Food Science and Nutrition, Zhejiang University, Hanzhou Zhejiang 310029, China, 2Grudate School of Fisheries Science, Hokkaido University, Hakodate, Hokkaido 041-8611, 3Hokkaido Research Organization, Fisheries Research Department, Yoichi, Hokkaido 046-8555, Japan
A quick, simplified, and quantitative method for preparing ATP and its derivatives (Nuc) from fish meat was developed. Two steps were modified. The first step was the extraction of Nuc from fish meat. Nuc was extracted from 1 g of meat by smashing with a glass rod in 10 volumes of 5% perchloric acid (PCA). A single extraction procedure was sufficient for a full recovery of Nuc. The next step was neutralization of PCA used for the extraction of Nuc with KOH. To simplify the step, neutralization with 1 M KOH was done without removing denatured protein from the mixture. Adjustment of the pH of the mixture to 7 released proteins from the aggregates, while no protein was found when the pH was adjusted to 2-3.5. The volume of the solution was filled up to 20 mL, and the supernatant was obtained by filtering the mixture through a membrane with 0.45-μm pore size to remove small floating aggregates. The filtrate was further neutralized by adding 20 mM phosphate buffer (pH 7.5) for storage of the sample.