A Simplified Method For Extraction Of ATP Related Compounds In Fish Meat And Its Application

Freshness is an important quality of aquatic products, among the various tests for elucidating the freshness of fish, K value has been widely used by many investigators. However, little improvement is achieved in the preparative method for K value measurement. In this study, a simplified method was established. Based on this method, Japanese plaice stored at different temperatures was used to study the change of K value and ATP related compounds as preliminary study on the prediction of freshness. A freshness investigation of several fishes sold in Hangzhou supermarket was conducted. The main results of the thesis were presented as follows:1. A quick, simplified, and quantitative preparative method of ATP and its derivatives from fish meat was developed. Two steps were modified. The first step was the extraction of ATP related compounds from fish meat. ATP related compounds was extracted from1g of meat by smashing with glass rod in10volumes of5%perchrolic acid (PCA). A single extraction procedure was sufficient for a full recovery of ATP related compounds. The next step was neutralization of PCA used for the extraction of ATP related compounds with KOH. To simplify the step, neutralization with1M KOH was done without removal of denatured protein in the mixture. Adjustment of the pH of the mixture to7released proteins from the aggregates, while no protein was found when pH was adjusted to2-3.5. The volume of the solution was filled up to20ml, and the supernatant obtained by standing of the mixture was filtered through a membrane with0.45μm pore size to remove small aggregates floating. The filtrate was further neutralized by adding100mM phosphate buffer (pH7.5) for the storage of the sample. The amount of ATP and its derivatives obtained by the simplified method was proved to be almost the same as that of the conventional method.2. The stability of nucleotides, the ATP related compounds, in acidic condition was measured. The0.4mM ATP stored at1%PCA, room temperature overnight was hydrolyzed to ADP without further hydrolysis. Totally,15%ATP was decomposed. The decomposition rate of0.4mM ATP solutions stored at pH3and pH7overnight were almost showed the same decomposition rate of about2%, indicating that ATP related compounds are stable for one night at room temperature. A standard mixture solution in acidic condition overnight room temperature confirmed the above result. ATP is stable with5%PCA at ice-water condition for about8hours.3. Application of this method to determine the K value of Japanese plaice showed the freshness lowering rate is a first order reaction in the early phase. The K value of the samples that stored under the temperature shifts from0℃to10℃and5℃to10℃showed the tendency more like that of the constant10℃The K value of seven fish species commonly sold in Hangzhou, China, was measured. The results showed the freshness of the tested fishes has big variations. Based on the information obtained from published data, the K value could be used to estimate the fish temperature history.