Brief Description:
The enzymatic analysis of malate is a relatively simple procedure that can be performed in many basic laboratories. It is a spectrophotometric analysis of an enzymatic assay. The readings are taken at 340nm. The reaction employs the reduction of NAD+ to NADH by malic acid. This reduction is able to occur in the presence of malic dehyrodgenase enzyme. The enzyme allows the reaction to be quicker, more specific and more sensitive. The reduction of NAD+ creates a product with a UV/Vis active chromophore at the 340nm wavelength. The absorbance of the analyte is read after a reaction is run. The value is then entered into Beer’s Law (A=εbc) calculation or can be compared to a standard curve to calculate the concentration of malate in the initial sample. This test is run at a pH of 9.5. Because the malate reaction is in favor of the substrates additional enzymes or substrates may be employed to increase the reaction rate. This procedure is fairly rapid, easy, sensitive and specific which allows a convienent way to test for specific analytes such as malate. However, there are precautions when using this method including enzyme impurities, requirement of optimal pH and temperature conditions, and error occurring from improper standards or incorrect analyte dilutions that can interfere with the absorbance readings.
Application in Wine Microbiology:
This analysis is an important and frequently used procedure within the wine industry. Malate is a cause of tartness in grapes, so its depletion is at times important in lowering the acidic nature of a wine. This is achieved in wine fermentations by the malolactic conversion that occurs in the presence of lactic bacteria. Malolactic fermentations can prove beneficial in adjusting the sensory attributes of wine but also in the increasing the stability of the wine. The most efficient way to ensure that malolactic fermentation is occurring is by measuring the amount of malate present. This is true because other detection characteristics of malolactic fermentations such as TA, pH or lactic acid levels can be attributed to other variables in the wine. Though it is possible to use paper chromatography to the indicate presence of malic acid; this test does not quantify the amount of malate present in the wine. Enzymatic analysis is used to quantify the amount of malic acid. The materials for this test are available in kits, making the process even more streamlined and effective. Because of its overall efficiency, enzyme analysis is a convenient way to test the fermentations of lactic acid bacteria.
References:
- Boulton, Roger B., Singleton, Vernon L., Bisson, Linda F., Kunkee, Ralph E. Principles and Practices of Winemaking. New York: Springer, 1998.
- McCloskey, L. Enzymatic Assay for Malic Acid and Malo-lactic fermentations. American Journal of Enology and Vitculture. Vol 31. 1980.
- Segundo M., Rangel A. Kinetic determination of l(−)malic acid in wines using
- sequential injection analysis. Analytical Chemistry. 499:2003 (99-106).