A Freshness Assay for Seafood

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A denosine triphosphate (ATP) is the principal energy source for cellular processes. When cellular activity ceases, ATP is naturally depleted through a combination of autolytic enzyme and bacterial action that sequentially degrade ATP into smaller nucleoside and purine end products starting with adensoine diphosphate (ADP) and following progressively through adenosine monophosphate (AMP), inosine monophosphate (IMP), Inosine (Ino), and hypoxanthine (Hx) respectively (Figure 1).

Because these catabolites are produced early post-mortem, their measurement in animal tissue can determine food QC parameters such as muscle age and storage time, important indicators of freshness. The quantity of these nucleotides in tissue products can also influence sensory attributes, such as taste and texture.

(BIOTEK INSTRUMENTS)
Figure 1: ATP degradation pathway to Hx following the arrest of cellular respiration post-mortem in muscle tissue.

The freshness of fish tissue was first quantified by a Japanese team of food scientists more than 50 years ago using a six parameter equation that solved to a value K based on all of the freshness pathway products shown in Figure 1. Another team of Japanese scientists later abbreviated this to a three parameter equation solving for a Ki (%) value by using the latter stage catabolites of IMP, Ino, and Hx (Figure 2). The %IMP and %Hx can be expressed as an equivalent freshness index by their relationship in Ki such that the higher the %IMP the fresher the fish, whereas higher %Hx values indicate a transition from freshness to spoilage in muscle tissue.

(click for larger image) (BIOTEK INSTRUMENTS)
Figure 2: NovoCIB Precice Freshness assay principle and workflow. The assay works as three assays in one on up to 11 individual samples in a fixed 96-well microplate format with column 12 reserved for pre-filled standards (top left). Relative content of (%) IMP, (%) Hx, and (%) Ino is calculated following enzymatic conversion of nucleotides to NADH, and detection of NADH at 340 nm (lower left, and right).

Recently, the Precice Freshness assay has been developed by NovoCIB that quantifies the levels of these three latter catabolites in a microplate format by enzymatic conversion of each catabolite to nicotinamide adenine dinucleotide (NADH), which can be measured colorimetrically at 340 nanometers using a microplate reader. Figure 2 illustrates the methodology used in the microplate assay.

This assay involves multiple microplate additions that can be facilitated by inexpensive automation, such as an automated pipetting station. BioTek Instruments has collaborated with NovoCIB to develop an automated cost-effective platform for routine Precice Freshness assays. This involves the BioTek Precision Pipetting System, Epoch 2 Microplate Spectrophotometer, and Gen5 Data Analysis Software. This laboratory instrumentation and software provide automation for the reagent addition steps, NADH detection, and subsequent calculation of %IMP, %Ino, and %Hx in the workflow of the Precice Freshness assay as shown in Figure 2. This automated workflow has been used to assess the freshness profile of a number of commercial products, among them raw, frozen shrimp (Figure 3).

(BIOTEK INSTRUMENTS)
Figure 3: Quantification of freshness pathway (% IMP, % Ino, % Hx) over three months for raw, whole, peeled, imported, farmed, frozen shrimp stored at -20°C.

The parabolic shift of Ino reflects a decomposition dynamic as IMP is depleted in the tissue over time and Hx is formed. This data suggests that between 78 and 82 days higher levels of %IMP (more fresh) begin to cross over to higher levels of %Hx (declining freshness).

Goodrich is an applications scientist at BioTek Instruments. Reach her at goodrichw@biotek.com. Dr. Balakireva is the president and chief scientific officer at NovoCIB. Reach her at lbalakireva@novocib.com.