Determination of citric acid by enzymatic means
This analysis is suitable for malt, wort, beer, beer-based beverages and soft drinks
Fruit juices:
The acid spectrum typical of certain types of fruit are used, along with other criteria, as a basis for recognizing unadulterated fruit juices. Tartaric acid, citric acid and L-malic acid are recorded here, which, with a few exceptions, determine the total acidity of the fruit.
Citric acid occurs as the primary acid in citrus juices and other juices. Orange juice usually contains 3–17 g/l citric acid (AIJN).
In citrus juices, an addition of citric acid can be detected via the citric acid/D-isocitric acid ratio, as this lies within relatively narrow limits. In orange juice, values below 130 are found.
D-isocitric acid is partly present in fruit products as a lactone. The lactone must first be saponified prior to enzymatic determination in order to detect the total D-isocitric acid content.
Malt, wort and beer:
Citric acid is an organic acid and is present in malt and wort and is also produced during fermentation.
Citric acid (citrate) is converted to oxaloacetic acid and acetic acid catalyzed by the enzyme citrate lyase (CL):
Citrate oxaloacetic \(^{\underrightarrow{CL}}\) acid + acetate
In the presence of the enzymes malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), oxaloacetic acid and its decarboxylation product pyruvic acid are reduced to L-malic acid and L-lactic acid, respectively, by reduced nicotinamide adenine dinucleotide (NADH):
Oxaloacetate + NADH + H+ \(^{\underrightarrow{L-MDH}}\) L-malate + NAD+
Pyruvate + NADH + H+ \(^{\underrightarrow{L-LDH}}\)L-lactate + NAD+
The sum of the quantity of NADH consumed during the reaction is equivalent to the quantity of citric acid. The absorbance is determined photometrically at 334, 340 or 365 nm.
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 D-isocitric acid by enzymatic means
This analysis is suitable for non-alcoholic and beer-based beverages.
Fruit juices:
The acid spectrum typical of certain types of fruit are used, along with other criteria, as a basis for recognizing unadulterated fruit juices. Tartaric acid, citric acid and L-malic acid are recorded here, which, with a few exceptions, determine the total acidity of the fruit.
Citric acid occurs as the primary acid in citrus juices and other juices. Orange juice usually contains 3–17 g/l citric acid (AIJN).
In citrus juices, an addition of citric acid can be detected via the citric acid/D-isocitric acid ratio, as this lies within relatively narrow limits. In orange juice, values below 130 are found.
D-isocitric acid is partly present in fruit products as a lactone. The lactone must first be saponified prior to enzymatic determination in order to detect the total D-isocitric acid content.
D-Isocitrate in the sample is isolated as a barium salt through precipitation. The concentration of D-isocitrate is determined enzymatically. The enzyme isocitrate dehydrogenase (ICDH) catalyzes the oxidative decarboxylation of D-isocitrate to α-ketoglutarate with nicotinamide adenine dinucleotide phosphate (NADP):
D-Isocitrate + NADP+ \(^{\underrightarrow{ICDH}}\)α-ketoglutarate + NADPH + CO2 + H+
The amount of NADPH formed is proportional to the amount of D-isocitrate and is measured photometrically through the increase in absorbance.