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Affichez la page en français PRECICE® Freshness Kits Kit version Size Price* PRECICE® % IMP (K-Value) 30 analysis € 369.00 PRECICE® % Hx 30 analysis € 369.00 PRECICE® % IMP & % Hx 20 analysis € 499.00 * Excl. VAT. Pricing updated on November 29th, 2011. Prices are subject to change without notice. Shipping charges are not included. Range of PRECICE® Freshness Assay Kits: Determination of Fish and Meat freshness through a rapid quantification of ATP degradation products: IMP (or K-Value), Inosine and Hypoxanthine ATP degradation products are well recognized markers for freshness level of Fish and Meat products: for instance, IMP content, expressed as a percentage of all ATP degradation products, is an excellent indicator of high freshness level, whereas hypoxanthine (Hx) content indicates the beginning of spoilage. PRECICE® K-Freshness Assay Kits are the first enzymatic assays where IMP, Hx and Inosine are measured directly by using a spectrophotometer or a plate reader. After a very simple extraction boiling procedure, the extracts are incubated for 1h with enzymatic mixtures, allowing the complete conversion of IMP, inosine and hypoxanthine to NADH which is then easily measurable by absorbance at 340nm. Three versions of PRECICE® Freshness Assay Kits are available, depending on the level of freshness that is required to be checked out: measurement of % IMP (K-Value), measurement of % Hx or measurement of both % IMP and % Hx. Each PRECICE® Freshness Assay Kit: • is an ideal cost-efficient and rapid tool for a routine control of freshness to replace chromatographic methods (BA, TVBN), • measures up to 30 samples at once (20 for %IMP and %Hx measurement) in 60 min using a standard microplate reader, • is very easy to use ("add and measure" format), • has a high precision, independent on sample size (3-4 g are sufficient) • conveniently presents the results as a % of IMP and/or HX over the total amount of ATP-degradation nucleotides
Download: Product Information Sheet  User Manual	Click to get further information or to order: % IMP measurement, % Hx measurement, % IMP & % Hx measurement
Freshness and ATP degradation products   Case Study   Kit Content

How do ATP degradation products indicate the freshness level of flesh?
What is K value?
ATP (adenosine triphosphate) is the major energy currency in cell. Its content is particularly high in muscle tissue where ATP is used to power contraction. As soon as an animal dies, cell respiration stops as well as ATP formation. Once the rigor mortis stage is reached, within minutes to few hours after the animal has been slaughtered, ATP pool starts being progressively and sequentially depleted through a succession of reactions, as shown on the following figure.

Post-mortem ATP depletion is mainly due to autolytic reactions and controlled by endogenous enzymes, even if the microbial flora, when developed enough, may contribute to the process. A high content of inosine monophosphate (IMP) in the flesh indicates a high level of freshness whereas a high content of hypoxanthine (Hx) indicates that spoilage phase will soon start, if not started yet. Measurement of ATP degradation products is an excellent tool for fish freshness assessment, before the traditional techniques (BA, TVBN) regarding bacterial spoilage become relevant.

The K value concept
In the late 1950's, a Japanese research team (Saito et al.) proposed a new concept, called "K value", for the indication of the freshness of fish flesh. The K value is based on ATP breakdown and the subsequent formation of its by-products, namely adenosine diphosphate (ADP) and monophosphate (AMP), inosine monophosphate (IMP), inosine (Ino) and, at a later stage, hypoxanthine (Hx). The K-value measures how far ATP degradation has progressed within the tissue. It is expressed as a percentage of the content of the last two final compounds resulting from ATP catabolic pathway, i.e. Ino and Hx, over the total content of ATP and its degradation by-products: ATP, ADP, AMP, IMP, Ino and Hx.
However, because ATP decomposes very quickly to IMP in most animals, a simplified K value (generally called K_i value) was soon proposed by Karube et al. (1984) and is currently considered as equivalent to the original 6-parameter equation for K-value.

K (%) =	Ino + Hx	The K value is recognized for several decades as the most effective and objective indicator of the freshness of fish and seafood products, as well as of meat (beef, pork, lamb and poultry).
	IMP + Ino + Hx

Since IMP content, when expressed as a percentage of the three ATP degradation products (namely IMP, Ino and Hx) is directly linked to the K-value formula (%IMP = 100% - K-Value), it may appear more convenient to consider %IMP as an equivalent freshness marker for which, the higher % IMP, the fresher the fish.
Measurement of ATP degradation products offers an additional key-advantage over other freshness criteria: Since IMP, Ino and Hx are stable to heating or freezing, their content can be measured on fresh, frozen or even cooked fish products.

(^Top) Why and when measuring ATP degradation products (and K value)?
Freshness is the most important quality criteria regarding fish and seafood products. If freshness can be quite easily checked on entire crude and chilled finfish by sensory analysis, freshness control remains puzzling when considering transformed fish, for instance.
Current CQ analytic tools, such as TVBN, BA or TMA, are routinely used but they are based on the biochemical changes that appear when spoilage has started, and can be relevant for a medium- or a late-stage of the process. However, since ATP depletion is the very first biochemical process that occurs once a fish has died, long before spoilage starts, K-value changes concern the early stage of fish transformation and storage and allows professionals to anticipate the beginning of spoilage (e.g. TVBN formation) and to better control the "high" freshness level of the product.
In other words, K-value and complementary %Hx measurements are the choice freshness markers at the early stage of the process.
Most of Japanese researchers consider that raw fish can be eaten as sashimi when the K-value is below 20%.

References (with links) A. Rodriguez et al. (2009): Chemical changes during farmed coho salmon (Oncorhynchus kisutch) canning: Effect of a preliminary chilled storage Food Chem. 112(2), 362–368 F. Özogul et al. (2008): Nucleotide degradation and biogenic amine formation of wild white grouper (Epinephelus aeneus) stored in ice and at chill temperature (4 °C) Food Chem. 108(3), 933–941 R. Pacheco-Aguilar et al. (Jan. 2008): Postmortem changes in the adductor muscle of Pacific lions-paw scallop (Nodipecten subnodosus) during ice storage Food Chem. 106(1), 253–259 K. Sasaki et al. (2007): Changes in the amounts of water-soluble umami-related substances in porcine longissimus and biceps femoris muscles during moist heat cooking Meat Science 77(2), 167–172 K. Saito et al. (2007): Effects of a humidity-stabilizing sheet on the color and K value of beef stored at cold temperatures Meat Science 75(2), 265–272 V. Losada et al. (2005): Inhibition of chemical changes related to freshness loss during storage of horse mackerel (Trachurus trachurus) in slurry ice Food Chem. 93(4), 619–625 N. Hamada-Sato et al. (2005): Quality assurance of raw fish based on HACCP concept Food Control 16(4), 301–307 M. Dondero et al. (2004): Changes in quality of vacuum-packed cold-smoked salmon (Salmo salar) as a function of storage temperature Food Chem. 87(4), 543–550 V.P. Lougovois et al. (2003): Comparison of selected methods of assessing freshness quality and remaining storage life of iced gilthead sea bream (Sparus aurata) Food Resear. Intern. 36(6), 551–560 C. Alasalvar et al. (2001): Freshness assessment of cultured sea bream (Sparus aurata) by chemical, physical and sensory methods Food Chem. 72(1), 33–40 R. Mendes et al. (2001): Changes in baseline levels of nucleotides during ice storage of fish and crustaceans from the Portuguese coast Eur. Food Res. Technol. 212(2), 141–146

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Case Study

Changes in content of ATP-breakdown products, namely IMP and Hypoxanthine, were measured using PRECICE^® %IMP & %Hx Freshness Assay Kit on several samples of fish, stored on ice from slaughter until 10-18 days, depending on the fish species. ≈ 3g of fish muscle were collected and boiled for 20min. Fish extracts were recovered and diluted with 1 volume of water. 4-10µL of the diluted extract were put into 6 wells on a 96-well plate before adding 200µL of Reaction buffer (for Blank control), Enzyme mix I (for IMP quantification), Enzyme mix II (for IMP + Ino + Hx quantification) or Enzyme mix III (for Hx quantification), all in duplicate. After 1h of incubation at 37°C, absorbance was measured at 340nm using an iEMS Reader MF (Labsystems) microplate reader. Absorbance data were used for the determination of nucleotide contents. The evolution of IMP and Hx contents over time are shown for Trout (Figure A), Sole (Figure B), Cod (Figure C) and Sea Bass (Figure D). IMP and Hx are expressed as a percentage of the total content of ATP degradation products (IMP + Ino + Hx).

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K-Value is directly determined form %IMP: K-Value = 100% - %IMP
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Kit Content
Three different PRECICE^® K-Freshness Assay Kits are available.

	% IMP (K value) 30 analysis	% Hx 30 analysis	% IMP & % Hx 20 analysis
• "Enzyme mix I", lyophilized, for IMP quantification	V	X	V
• "Enzyme mix II", lyophilized, for IMP + Ino + Hx quantification	V	V	V
• "Enzyme mix III", lyophilized, for Hx quantification	X	V	V
• Reaction buffer    • vials containing cofactors (powder)    • a transparent microplate (round-bottom 96-well plate Corning, Costar®, ref. 3797)	V	V	V