Evaluation of Automating a Novel Biochemical Freshness Assay for Quantitative Measurement of ATP Degradation Products as a Potential Preventative Control of Fish Intended for the Human Food Market

Introduction: Post-mortemATP (adenosine triphosphate) degradation products in muscle tissue are mainly produced due to autolytic enzymes rather than microbial flora, and can therefore be used as a criterion for tissue freshness measurement and storage age determination, before microbial spoilage starts and the corresponding traditional techniques for spoilage measurement become relevant. Based on the principle that higher freshness is an indicator of lower risk of microbial spoilage, a novel assay has been developed that quantifies freshness of fish and meat tissue.

Purpose: Evaluate potential workflow and throughput solutions of automating a biochemical assay for quantification of inosine monophosphate (IMP), inosine (Ino) and hypoxanthine (Hx) as an indication of freshness in fish samples.

Methods: The assay was evaluated in 96-well microplate format for automated and non-automated modes validated over multiple runs using 3 dilutions of 3 enzymes and the assay blank. Then, a predetermined 7-fold dilution of 5 to 10 grams of fish tissue from 5 commercially available product matrices for the same species were subjected to 4 different storage conditions and analyzed over time in duplicate for all 3 co-factors. Detection of enzymatic conversion to NADH was read at absorbance 340 nm using a monochromator equipped microplate reader. Freshness was quantified based on K (Saito et al. 1959) and K_i (Karube et al.1984) values calculated from corresponding absorbance of each fish extract.

Results: Data demonstrates that lower % IMP (n=10) and higher % Hx (n=10) are indicative of decreasing sample freshness and can be matrix dependent, concluding that freshness calibration indices should be individualized per biologic material and test environment. Assay results are comparative for automated and manual modes using several sample prep options, with inter-assay CV slightly favoring automated results by a margin of 2.15%. Enzyme dilutions produced inter assay average CV of 5.13% regardless of method, with none higher than 7.33%.

Significance: Applications that could benefit from a preventative control for rapid, easy quantification of fish tissue freshness may find this assay useful, such as HACCP management on fish products intended for the human food market.