Malic acid assay

The analyte

Malic acid occurs in two chiral molecules, the D- and L- form. L-malic acid is a naturally occurring organic acid that is used in the body to derive ATP from food (citric acid cycle), and exists in vegetables and fruits. D-malic acid almost does not occur in nature, and is prepared chemically.
L-malic acid has a sour taste of apples and gives a fresh acidic taste in food. In the food processing industry, L-malic acid is used as souring agent and additive in yogurt, drinks, wine-fruit juice mixtures and jellies. In the pharmaceutical industry, L-malic acid is used in production of preparations, tablets and syrups, or added into amino acid injection to increase the use factor of amino acid.
Besides, acidic taste of malic acid remains longer in the mouth than citric acid, therefore, combined use with artificial sweetener such as aspartame or steviocide and can offset the bad aftertaste in food. L-malic acid concentration plays an important role in the production of wine. On one hand insufficient wine acidity can affect wine quality and stability. Otherwise the production of well-balanced wines requires the reduction of excess acidity, especially in the colder regions of the world.
The yeast Saccharomyces cerevisiae, responsible for alcoholic fermentation, metabolise only small and insignificant amounts of L-malic acid. Therefore most red wines and some white wines are subjected to the process of bacterial malolactic fermentation (MLF), during or soon after alcoholic fermentation.
Lactic acid bacteria are added to the wine. The result is the decarboxylation of L-malic acid to L-lactic acid and carbon dioxide. Malolactic fermentation reduces the levels of L-malic acid, increases the pH and L-lactic acid concentration, and leads to enhanced microbial stability of the wine. The MLF could be controlled by the determination of L-malic and L-lactic acid.


Enzymatic analysis of L-Malic acid

For years enzymatic methods have an important place in food analysis. Enzymatic methods are contained in several method books of national and international associations in industry, as well as in governmental locations (EN, OIV, IFU, AOAC, national regulations).
Due to the high specificity of the enzymes L-Malic acid assay has enabled the analysis of wine, fruit juices or any other sample matrices without the need of complicated sample preparation techniques. This makes L-Malcic acid assay by enzymatic reaction a common method for manufacturers of food products, trade companies and in the governmental food inspection.


The benefits of DiaSys L-Malic acid assay (and all other DiaSys reagents!)

· Ease of use:
No errors and no time wasted in reconstitution by using the liquid and ready to use reagents. Pipetting steps are minimized by the use of 3 reagents per assay. 2 can be used premixed. The pipetting order is user friendly by standardization on 4+1+1 format (2000 ml + 500 ml + 500 ml).
· Online monitoring:
Endpoint in less than 5 min at 37 °C. Combination from simplified assay procedure and fast reaction allows online monitoring of production processes.
· Cost-efficient by automation:
The assay can be adapted easily for use on fully automated systems (applications on request).
· Environmental friendly:
The reagents are not hazardous. Due to the liquid and stable form the reagents are always available when needed and can be used up to the very last drop.


Assay principle of Malic acid assay

3-component, ready to use reagent for photometric determination of L-Malic acid in food stuffs and other sample material

In the presence of NAD+ L-Malic acid is oxidized by the enzyme L-malate dehydrogenase (L-MDH) to oxaloacetate. The formed NADH is measured spectrophotometrically at 334, 340 or 365 nm (UV measurement) as a measurement of L-malate. In the balance L-malate is greatly favoured. For quantitative turnover of L-malate, oxaloacetate is removed in a further reaction. There oxaloacetate is convert into L-aspartate in the presence of L-glutamate by glutamate-oxaloacetate transaminase (GOT).

L-Malate + NAD+ ß L-MDH à Oxaloacetate + NADH + H+

Oxaloacetate + L-Glutamate ß GOT à L-Aspartate + 2-Oxoglutarate

Performance characteristics
Reagent preparation: The reagents are ready to use
Measuring range (at 340 nm): 7 – 500 mg/l
Specifity: The determination is specific for L-malic acid. D-malic acid does not react. No interferences were observed.
Sensitivity (at 340 nm): 2.5 mg/l
Applications for analyzers: Vitalab Selectra; Konelab, Hitachi, Respons

Also available as liquid assay:
Glucose, Fructose, Sucrose, L-Lactic acid, D-Lactic acid, Glycerol and others
 

More information about Malic acid - foodanalysis@diasys.de