Development of a Novel Hygiene Monitoring System Based on the Detection of Total Adenylate

Introduction: ATP is the universal energy molecule found in animals, plants, and microorganisms. ATP rapid hygiene monitoring tests have been employed in food industry to ensure adequate cleanliness is being maintained. However, since ATP is hydrolyzed to ADP and AMP by metabolic processes, heat treatment, or in acidic/alkaline conditions, the components of total adenylate (ATP+ADP+AMP; A3) could be more reliable indicators of lack of cleanliness derived from food residues, which may cause bacterial growth and allergen contamination.

Purpose: In this study, a novel enzymatic method to measure A3 was developed. Furthermore, its significance was verified by comparing the amount of A3 and ATP in various food samples.

Methods: ATP was measured by luciferin-luciferase assay, which produces luminescence and AMP. A3 measurement was based on luciferin-luciferase assay with pyruvate kinase and pyruvate phosphate dikinase, which can convert ADP into ATP and recycle AMP into ATP in the presence of phosphoenolpyruvic acid, respectively. Food samples were blended with water and then processed by centrifugation. The supernatants were diluted with water and applied to the assay. The assays were performed in triplicate. A3 and ATP were quantified by measuring luminescence calibrated against known amounts of standards.

Results: The newly developed A3 assay system afforded equivalent linear calibration curves between relative light units and the amounts of ATP, ADP, and AMP, respectively. The amounts of A3 were more than two orders of magnitude greater than that of ATP alone in various foods, such as meat, seafood, whole egg, dairy, beans and nuts. For example, A3 and ATP (pmol/g) in raw chicken were 2.6×10⁴ (3% CV) and 3.2×10¹(10% CV), respectively.

Significance: The A3 assay was developed and appeared to be more suitable for detection of food residues, which potentially cause bacterial growth and allergen contamination, than the conventional ATP assay.