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The global food waste problem, especially aquatic product spoilage, stimulates the accurate freshness analysis of food products. However, it still remains a great challenge to realize in-field determination of fish freshness at the time of use. In the present study, a colorimetric enzyme biosensor was developed for one-step detection of hypoxanthine (Hx), which is an important intermediate of adenosine triphosphate decomposition during fish storage. We demonstrate that xanthine oxidase grade I ammonium sulfate suspension (XOD-ASS) possesses peroxidase activity. It can oxidize different peroxidase substrates, including 3,3′,5,5′-tetramethylbenzidine, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and o-phenylenediamine in the presence of H 2 O 2 , producing visible color reactions. Further experiments indicate that XOD-ASS displayed effective peroxidase activity and could be used for H 2 O 2  detection. Based on this, a one-step Hx detection method was established using only XOD-ASS as the catalyst. The method displays a good linear relationship in the range from 20 to 100 μM with a detection limit of 6.93 μM. Additionally, we successfully applied this method in testing Hx accumulation in sea bass fish samples of different storage times. The recovery values range from 97.44 to 102.56%. It is exciting to note that, compared with other methods, our proposed method provides a robust advantage on the economic reaction system, ease of preparation, short time consumption, and moderate reaction temperature. We believe that this method shows good application prospects for on-site fish freshness determination.

frontiers in microbiology

One-Step and Colorimetric Detection of Fish Freshness Indicator Hypoxanthine Based on the Peroxidase Activity of Xanthine Oxidase Grade I Ammonium Sulfate Suspension