Determination of organic acids by means of reversed phase chromatography/ion chromatography
This method is suitable for wine, fruit juice and other non-alcoholic beverages.
The organic acids are separated using two combined columns, reversed-phase HPLC and an ion exchange column and are then determined using a UV detector.
Determination of L-malic acid by enzymatic means
This method is suitable for malt, wort, beer and beer-based beverages non-alcoholic beverages.
L-Malic acid (L-malate) is oxidized by nicotinamide adenine dinucleotide (NAD) in the presence of L-malate dehydrogenase (L-MDH) to oxaloacetic acid:
L-Malic acid + NAD+ \(^{\underrightarrow{L-MDH}}\) oxaloacetic acid + NADH + H+
The equilibrium of this reaction disproportionately favors malic acid. However, the oxaloacetic acid can be captured with the help of a downstream reaction involving the enzyme glutamate-oxaloacetate-transaminase (GOT) in the presence of L-glutamic acid, shifting the reaction in favor of oxaloacetic acid and NADH:
Oxaloacetic acid + L-glutamate \(^{\underleftrightarrow{GOT}}\)L-aspartate + 2-oxoglutarate
The amount of NADH produced during the reaction is equivalent to the quantity of L-malic acid and its absorption is determined photometrically at 334, 340 or 365 nm.
The method is suitable for the determination of water vapor volatile aroma compounds in beer.
Volatile aroma compounds are driven out of the sample through steam distillation. The ethanolic distillate is saturated with NaCl. Potassium hydrogen sulfite is added to separate carbonyl groups that might interfere with the analysis. The extraction of the aroma compounds is performed by shaking out with dichloromethane and the phases separated by centrifuging.