WO2006064488A1

WO2006064488A1 - A kit for colorimetric assays of food and beverage analytes

Info

Publication number: WO2006064488A1
Application number: PCT/IE2004/000171
Authority: WO
Inventors: Barry V. Mccleary; Simon Charnock
Original assignee: Megazyme Ip Limited
Priority date: 2004-12-17
Filing date: 2004-12-17
Publication date: 2006-06-22

Abstract

The invention relates to a kit for measuring the amount of analyte in a sample, comprising a tablet comprising a tetrazolium salt, diaphorase and NAD+, and at least one enzyme active on the analyte to be measured is described. The kit according to the invention may be used for conducting colorimetric assays and in particular may find use in the wine industry. The invention also provides a tablet for use in a diagnostic kit comprising a tetrazolium salt, diaphorase and NAD+

Description

Title

A kit for colorimetric assays of food and beverage analytes

Field of the Invention

The present invention relates to a kit for the colorimetric assay of analytes in food and beverages. The invention also relates to a reagent composition in tablet form for colorimetric assays for use in the wine industry. In particular, the assay is any assay in which NADH is formed.

Background to the Invention

It is well known in the food and beverage industry that samples have to be taken periodically during production to ensure optimum quality of the end products. For example in the wine industry, samples have to be taken periodically to assess the quality of the wine. In particular samples are tested at several stages during the alcoholic fermentation process to test for the presence of key analytes such as reducing sugars (D-glucose plus D-fructose) and ethanol, and during the malolactic fermentation process to test for the presence of key analytes such as L-malic acid and L-lactic acid.

Traditionally, a range of analytes of importance in defining food and beverage quality have been analysed in coupled reactions in which NADH or NADPH is formed. In these reactions, measurement of the increase in absorbance at 340 nm (ultraviolet range) due to production of NADH or NADPH gives a direct measurement of the amount of the analyte in the test sample. However, in some instances, such as reactions involved in the measurement of L-glutamate with glutamate dehydrogenase or D-sorbitol with sorbitol dehydrogenase, the equilibrium of the reaction is in favour of the analyte. In such cases, a second reaction involving diaphorase and iodonitrotetrazolium chloride (INT) has been included to utilize the NADH product and thus "pull" the reaction in the desired direction towards the products. This diaphorase/TNT reaction has only been employed when it has been necessary to ensure complete reaction of the analyte. In the measurement of L-malic acid, D-glucose, D-fructose, L-Lactic acid etc. several enzymic reactions are linked with the ultimate conversion OfNAD⁺ to NADH or NADP⁺ to NADPH. The amount of NADH or NADPH formed in these coupled reactions is stoichoimetric with the amount of L-malic acid, D-glucose, D-fructose, L- lactic acid etc. present in the test sample. As stated above, in some reactions, the equilibrium of coupled reactions lies in favour of the NAD⁺ or NADP⁺, so a further reaction must be included to "pull" the reaction towards formation of NADH or NADPH. One such reaction is the conversion of L-glutamic acid to 2-oxoglutarate by glutamate dehydrogenase (GlDH), shown below:

(GlDH)

(1) L-glutamic acid + NAD⁺ + H₂O > 2-oxoglutarate + NADH + NH₄ ⁺

Since the equilibrium of this deamination reaction lies markedly in the favour of the reactants, a further reaction catalysed by diaphorase is required, in which

NADH reduces iodonitrotetrazolium chloride (INT) to yield an INT-formazan product (2), leading to a rapid and quantitiative conversion of L-glutamic acid to 2-oxoglutarate.

(diaphorase)

(2) INT + NADH + H⁺ > NAD⁺ + INT-formazan.

The amount of INT-formazan formed in this reaction is stoichiometric with the amount of L-glutamic acid. It is the INT-formazan which is measured by this increase in absorbance at 492 nm.

This diaphorase/INT reaction is well known. However it has not been widely used for several reasons. For example, in cases where an Ultraviolet (UV) spectrophotometer is readily available, there is no need to incorporate these extra reagents and analytical steps. In addition, some sample mixtures contain reducing substances, such as L- ascorbic acid in fruit juices, or sulphur dioxide in jam, which interfere with the assay as they react with INT causing a "creep" reaction. Such samples require pre-treatment with alkaline hydrogen peroxide before assay. A major disadvantage of adding the diaphorase/INT reaction to assay formulations is the need for the extra steps to be carried out in the assay. In addition the light sensitivity/instability of INT in solution is disadvantageous.

In certain industrial and production situations, a need exists for an on-the-spot determination of key analytes e.g. L-malic acid and reducing sugars (D-glucose + D- fructose). However, the particular facility may not be able to justify expensive analytical equipment such as a UV spectrophotometer. Using an external analytical laboratory, which is the current practice, requires rapid delivery of the sample to the laboratory for assay and analysing the results. For example in the wine industry, at harvest time there may be a backlog of samples requiring analysis, so there may be a time delay in getting the results. Even having to wait just 1-2 days is a major disadvantage to the winemaker. This situation is not ideal. There is thus a need for a more efficient, cost-effective method for assaying key analytes, allowing the close monitoring of the process.

In certain facilities, for example, in small wineries, the production of a colored end product, rather than one that is visible in the UV range, is clearly desirable and advantageous. This allows measurement of reaction products with a cheap colorimeter rather than an expensive UV spectrophotometer. Furthermore, in such situations, it is essential that the assay reagents are compact, robust and simple to employ. Moreover it is desirable that the assay format employs ready-to-use reagents in the simplest possible format.

Object of the Invention

It is thus an object of the invention to provide an assay kit which allows accurate and convenient determination of the amount of analyte in a sample.

It is a further object of the invention to provide a cost-effective and time-saving method for colorimetric assay of analytes. A further object is to provide an assay which does not require expensive equipment for determination of a result.

A still further object is to provide an assay which can be conducted "in-house" without the need to send samples to a laboratory for determination. Summary of the Invention

Accordingly, the invention provides a kit for measuring the amount of analyte in a sample, comprising a tablet comprising (i) a tetrazolium salt (ii) diaphorase and (iii) NAD⁺ , and at least one enzyme active on the analyte to be measured.

The kit enables the convenient and accurate determination of the amount of analytes affecting food or beverage quality present in a sample to be carried out on the production floor without the need for expensive analytical equipment, such as for example, a UV spectrophotometer.

In an alternative embodiment of the invention, the tablet of the kit further comprises ATP. ATP is required for the determination of the presence of certain analytes, for example D-glucose and D-fructose.

Suitably the tablet further comprises a flow agent, which may be water soluble. Preferably the flow agent comprises sodium benzoate. However, it will be appreciated by those skilled in the art that other flow agents may be used.

Preferably the tablet may comprise a bulking agent. The bulking agent may be selected from the group consisting of lactose, mannitol, maltose or sorbitol.

Further preferably, the tablet may comprise a disintegration agent. The disintegration agent may comprise a mixture of sodium bicarbonate and an organic acid such as citric acid, L-malic acid, D-malic acid, succinic acid, for example. Suitably the kit further comprises a buffer solution. Suitably, the pH and composition of the buffer solution are selected to facilitate optimum activity of the included enzyme(s), as would be well known to those of skill in the art.

In a preferred embodiment, the buffer comprises surfactant selected from the group consisting of Triton X-IOO, polyoxyethylene ether, polyoxyethylene sorbitan or other non-ionic detergent at a concentration of approximately 0.5% (v/v). The buffer provides and maintains the correct pH for the assay. The pH will be selected depending on the active enzymes being used. It will be appreciated by those skilled in the art that if the pH changes the enzyme(s) will be less active or unstable, either of which will impair the test method. Likewise different enzymes are most active at different optimum pHs, and accordingly the pH should be adjusted to achieve optimum activity for the enzyme used.

Suitably the analyte is selected from the group consisting of D-glucose, D-fructose, D- galactose, L-malic acid, D-malic acid, L-lactic acid, hydroxybutyric acid, D-mannitol, D-sorbitol, L-arabitol, xylitol, acetaldehyde , ethanol or L-glutamate. Other analytes wherein prior coupled reactions produce NADH may also be assayed using the kit according to the invention.

The enzyme(s) may be selected from the group consisting of hexokinase, glucose 6- phosphate dehydrogenase, phosphoglucose isomerase, L-malate dehydrogenase, D- malate dehydrogenase, L-lactate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, D-mannitol dehydrogenase, D-sorbitol dehydrogenase, 3- hydroxybutyrate dehydrogenase, galactose dehydrogenase and glutamate dehydrogenase. It will be appreciated by those skilled in the art that other enzymes could also be used whereby the enzyme is selected according to the analyte to be assayed.

Suitably the enzymes are supplied as a suspension in ammonium sulphate or another salt solution such as lithium sulphate or dipotassium phosphate, as a solution in glycerol at approximately 50% (v/v) or as a freeze-dried (lyophilised) powder. The tablet of the kit is used in combination with an enzyme or enzymes that in combination with NAD⁺ catalyse the production of NADH in a linked reaction related to the concentration of the specific analyte.

Suitably the tetrazolium salt is of the type to be reduced by NADH in the presence of diaphorase and is present in an amount sufficient to allow quantitative detection of specific analytes in a test sample.

In a preferred embodiment the tetrazolium salt comprises iodonitrotetrazolium chloride, INT. It will be appreciated by the person skilled in the art that other tetrazolium salts may also be employed.

Suitably the diaphorase is of the correct type and has an activity level to catalyse the indicator-forming reaction involving the enzyme, NADH and a tetrazolium compound.

The invention also provides a tablet for the determination of an analyte in a food or beverage sample, the tablet comprising: (i) a tetrazolium salt (ii) diaphorase and ⁽ⁱⁱⁱ⁾ NAD⁺.

In an alternative embodiment the tablet further comprises ATP.

Suitably the tablet further comprises a buffer and/or surfactant in powder form. Preferably, the buffer, when dissolved in water, or water containing surfactant, yields the correct pH and concentration of required components.

The invention also provides a method for measuring the amount of analyte in a test sample comprising the steps of

(i) adding test sample to a buffer solution; (ii) adding a tablet to said solution;

(iii) taking an initial absorbance reading;

(iv) selecting an enzyme according to the analyte to be detected and adding same to the solution; and (v) taking a final absorbance reading.

Suitably the method includes spectrophotometry measurement of the INT-formazan compound at about 400-5 IOnm.

Brief Description of the Drawings

Figure 1 shows the time course of colour formation in the determination of L-malic acid using the procedure as outlined in Example 1. A. Reaction mixture containing 6.5 μg of L-malic acid. B. Reaction mixture containing no L-malic acid.

Reaction performed at room temperature (approximately 22⁰C) under standard assay conditions as detailed in Example 1.

Detailed Description of the Drawings

The diaphorase/INT reaction used in the assay kit involves quantative utilization of NADH with stoichiometric formation of the INT-formazan complex. This reaction could potentially be applied to all assay situations where NADH is formed, offering the advantages of producing a red coloured (INT-formazan) complex which can be measured with a simple colorimeter and also, of potentially increasing the speed of the reaction by removal of NADH.

The invention will now be described in more detail by way of the following examples. The reagent tablet was used to assay for the presence of a variety of key analytes as outlined in the following examples.

Examples Example 1 L-Malic Acid Determination Reagent

Step 1. A buffer stock solution was prepared by dissolving 13.2 g of glycylglycine (Sigma G- 1002), 14.7 g of L-glutamate (Sigma G- 1251) and 10 mL of Triton X-100 in 1.5 litres of distilled water. The pH was adjusted to 10.0 with 4 M NaOH and the volume to 2 litres.

Step 2. Tablets for the measurement of L-Malic acid were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD⁺ and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.0 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added, allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (Ai) was taken and recorded. An aliquot (20 μL) of L-malate dehydrogenase (7500 U/mL) was added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx 5 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of L-malic acid was calculated.

Figure 1 shows the time course of colour formation in the determination of L-malic acid using the procedure outlined above. In this particular example, a test tablet as described in example 1 was added to 3.0 mL of buffer solution as described in example 1 and allowed to dissolve completely. An aliquot (0.1 mL) of sample solution containing 6.5 μg of L-malic acid was added and the initial absorbance reading was taken. An aliquot (20 μL) of L-malate dehydrogenase (7500 Units/mL) in 3.2 M ammonium sulphate was then added and the increase in colour was monitored in a recording spectrophotometer. A Unit as mentioned here and throughout the patent specification, refers to an International Unit of enzyme activity.

Example 2

D-Glucose and D-Fructose Determination Reagent

Step 1. A buffer stock solution was prepared by dissolving 34 g of imidazole (Sigma 1-1025), 2.4 g anhydrous magnesium chloride and 25 mL of Triton X-IOO in 4 litres of distilled water. The pH was adjusted to 7.6 and the volume to 5 litres.

Step 2. Tablets for the measurement of D-glucose and D-fructose were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD⁺, 6 g ATP and flow, bulking and disintegration agents such as sodium benzoate, lactose or mannitol, sodium bicarbonate and citric acid to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.0 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added and allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (Ai) was taken and recorded. An aliquot (20 μL) of a mixture of hexokinase (425 Units/mL), glucose 6-phosphate dehydrogenase (212 Units/mL) and phosphoglucose isomerase (1000 Units/mL) was added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx 10 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of D-glucose + D- fructose was calculated.

Alternatively, an aliquot (20 μL) of just hexokinase (425 Units/mL) and glucose 6- phosphate dehydrogenase (212 Units/mL) was added and the absorbance increase after approx 10 min (A₂) was recorded; subsequently, an aliquot (20 μL) of phosphoglucose isomerase (1000 Units/mL) was added and the absorbance increase was again recorded after 10 min (A₃). D-Glucose was calculated from the absorbance difference A₂-Ai and D-fructose was calculated from the absorbance difference A₃-A₂.

Example 3

D-Malic Acid Determination Reagent

Step 1. A buffer stock solution was prepared by dissolving 13.2 g of glycylglycine (Sigma G-1002), 7.5 g potassium chloride, 20 g of magnesium chloride.6 H₂O and 1OmL of Triton X-IOO in 1.5 litres of distilled water. The pH was adjusted to 8.0 with 4 M NaOH and the volume to 2 litres.

Step 2. Tablets for the measurement of D-Malic acid were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD⁺ and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.0 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added, allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (Aj) was taken and recorded. An aliquot (20 μL) of D-malate dehydrogenase (220 Units/mL) was added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx 30 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of D-malic acid was calculated.

Example 4 L-Lactic Acid Determination Reagent

Step 1. A buffer stock solution was prepared by dissolving 26.4 g of glycylglycine, 29.4 g of D-glutamate and 10 mL of Triton X-100 in 1.5 litres of distilled water. The pH was adjusted to 10.0 with 4 M NaOH and the volume to 2 litres.

Step 2. Tablets for the measurement of L-Lactic acid were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD⁺ and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.0 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added, allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (A|) was taken and recorded. An aliquot (20 μL) of L-lactate dehydrogenase (2000 Units/mL) was added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx 10 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of L-lactic acid was calculated.

Example 5

Ethanol Determination Reagent

Step 1. A buffer stock solution was prepared by dissolving 79 g of tetrapotassium pyrophosphate (anhydrous Sigma p-8260) and 10 mL of Triton X-100 in 1.2L of distilled water. The pH was adjusted to 9.0 with 8 M HCl and the volume to 2 litres.

Step 2. Tablets for the measurement of ethanol were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD⁺ and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.0 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added and allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (Ai) was taken and recorded. An aliquot (20 μL) of alcohol dehydrogenase (167 Units/mL) was added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx 5 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of ethanol was calculated.

Example 6 Acetaldehyde Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 79 g of tetrapotassium pyrophosphate (anhydrous Sigma p-8260) and 10 mL of Triton X-IOO in 1.2L of distilled water. The pH was adjusted to 9.0 with 8 M HCl and the volume to 2 litres.

Step 2. Tablets for the measurement of acetaldehyde were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD⁺ and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.0 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added and allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (Ai) was taken and recorded. An aliquot (20 μL) of aldehyde dehydrogenase (7.9 Units/mL) was then added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx 5 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of acetaldehyde was calculated.

Example 7

D-Mannitol Determination Reagent

Step 1. A buffer stock solution was prepared by dissolving 24.2 g of Trizma base, 1 ,5 g of bovine serum albumin and 10 mL of Triton X-100 in 1.5 litres distilled water. The pH was adjusted to 9.0 with 8 M HCl and the volume to 2 L.

Step 2. Tablets for the measurement of D-mannitol were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD⁺ and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.0 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added and allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (Ai) was taken and recorded. An aliquot (20 μL) of D- mannitol dehydrogenase (300 Units/mL) was then added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx. 4 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of D-mannitol was calculated.

Example 8 D-Sorbitol Determination Reagent

Step 1. A buffer stock solution was prepared by dissolving 17.8 g of TEA, 1.8 g KCl and 10 mL of Triton X-100 in 1.5 litres of water. The pH was adjusted to 8.6 and the volume to 2 litres.

Step 2. Tablets for the measurement of D-sorbitol were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD⁺ and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.0 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added, allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (Ai) was taken and recorded. An aliquot (50 μL) of D- sorbitol dehydrogenase (40 Units/mL) was then added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx. 15 min (until there was no significant increase in absorbance over a 2 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of D-sorbitol was calculated.

Example 9

Hydroxybutyric Acid Determination Reagent Step 1. A buffer stock solution was prepared by dissolving 74.2 g of TEA, 8.7 g dipotassium hydrogen phosphate and 25 mL of Triton X-IOO in 4 litres of water. The pH was adjusted to 8.6 and the volume to 5 litres.

Step 2. Tablets for the measurement of hydroxybutyric acid were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD⁺ and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.5 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added and allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (Ai) was taken and recorded. An aliquot (20 μL) of 3- hydroxybutyrate dehydrogenase (270 Units/mL) was then added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx. 6 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of hydroxybutyric acid was calculated.

Example 10

D-Galactose Determination Reagent

Step 1. A buffer stock solution was prepared by dissolving 121.4 g of Trizma base (BDH cat. no. 271195Y), plus 7.4 g of EDTA (Sigma cat. no. ED2SS) in 4.0 litres of distilled water. The pH was adjusted to 8.6 and the volume to 5 litres.

Step 2. Tablets for the measurement of D-galactose were prepared by mixing 4500 Units of diaphorase, 176 mg of INT, 5 g NAD⁺ and flow, bulking and disintegration agents to a weight of 100 grams. After thorough mixing, this was compacted into tablets of approx. 60 mg using a Manesty tablet press.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.0 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added and allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (Ai) was taken and recorded. An aliquot (20 μL) of galactose dehydrogenase (100 Units/mL) was then added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx. 30 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of galactose was calculated.

Example 11 L-Glutamic Acid Determination Reagent

Step 1. A buffer stock solution was prepared by dissolving 74.2 g of TEA, 8.7 g dipotassium hydrogen phosphate and 25 mL of Triton X-100 in 4 litres of distilled water. The pH was adjusted to 8.6 and the volume to 5 litres.

Step 3. Assays were performed by adding 0.1 mL of test sample to 3.0 mL of buffer solution in a special test tube designed to fit directly into a colorimeter. A test tablet was added, allowed to disintegrate over 1-2 min in the dark with occasional agitation. An initial absorbance reading (Ai) was taken and recorded. An aliquot (50 μL) of glutamate dehydrogenase (200 Units/mL) was then added and the reaction allowed to proceed at room temperature (preferably above 22⁰C) for approx. 10 min (until there was no significant increase in absorbance over a 1 min period). This absorbance reading (A₂) was recorded. From these absorbance values, the concentration of L-glutamate was calculated.

The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Claims

1. A kit for measuring the amount of analyte in a sample, comprising a tablet comprising (i) a tetrazolium salt

(ii) diaphorase and

(iii) NAD⁺ , and at least one enzyme active on the analyte to be measured.

2. A kit according to claim 1 wherein the tablet further comprises ATP.

3. A kit according to claim 1 or 2 wherein the tablet further comprises a flow agent.

4. A kit according to Claim 3 wherein the flow agent is water soluble.

5. A kit according to any one of claims 1 to 4 wherein the tablet further comprises a bulking agent

6. A kit according to claim 5 wherein the tablet further comprises a disintegration agent.

7. A kit according to any preceding claim further comprising a buffer solution.

8. A kit according to any one of claims 1 to 7 wherein the buffer further comprises surfactant selected from the group consisting of Triton X-100, polyoxyethylene ether, polyoxyethylene sorbitan or other non-ionic detergent at a concentration of approximately 0.5% (v/v).

9. A kit according to any preceding claim where the analyte is selected from the group consisting of D-glucose, D-fructose, D-galactose, L-malic acid, D-malic acid, L-lactic acid, hydroxybutyric acid, D-mannitol, D-sorbitol, L-arabitol, xylitol, glycerol, acetaldehyde , ethanol or L-glutamate.

10. A kit according to claim 9 wherein the enzyme is selected from the group consisting of hexokinase, glucose 6-phosphate dehydrogenase, phosphoglucose isomerase, L- malate dehydrogenase, D-malate dehydrogenase, L-lactate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, D-mannitol dehydrogenase, D-sorbitol dehydrogenase, 3 -hydroxy butyrate dehydrogenase, galactose dehydrogenase and glutamate dehydrogenase.

11. A kit according to any preceding claim wherein the tetrazolium salt is iodonitrotetrazolium chloride (INT).

12. A kit according to any one of claims 3 to 11 wherein the flow agent is sodium benzoate.

13. A kit according to any one of claims 5 to 12 wherein the bulking agent is selected from the group consisting of lactose, mannitol, sorbitol or maltose.

14. A kit according to any one of claims 6 to 13 wherein the disintegration agent is selected from the group consisting of sodium bicarbonate and an organic acid such as citric acid, malic acid and succinic acid.

15. A tablet for the determination of an analyte in a food or beverage sample, the tablet comprising:

(i) a tetrazolium salt (ii) diaphorase and (iii) NAD⁺.

16. A tablet according to claim 15 further comprising ATP.

17. A tablet according to claim 15 or 16 further comprising a buffer.

18. A tablet according to any of claims 15 to 17 wherein the tetrazolium salt is iodonitrotetrazolium chloride (INT).

19. A method for measuring the amount of analyte in a test sample comprising the steps of:

(i) adding sample to a buffer solution; (ii) adding a tablet to said solution; said tablet comprising:

(i) a tetrazolium salt (ii) diaphorase and (iii) NAD⁺

(iii) taking an initial absorbance reading;

(iv) selecting an enzyme according to the analyte to be detected, adding same to the solution and allowing reaction to proceed; (v) taking a final absorbance reading.

20. A kit substantially as described herein with reference to the examples and/or the accompanying drawings.

21. A tablet substantially as described herein with reference to the examples and/or the accompanying drawings.