B-590.30.112 [2020-10] Citric Acid (Citrate) – Enzymatic Method

Citric acid is the primary acid in brightly colored juices and citrus juices.

Orange juice generally contains 3–17 g/l citric acid (AIJN).

The addition of citric acid in citrus fruit juice can be detected through the ratio of citric acid to D-isocitric acid, as this value falls within a relatively narrow range. Values for orange juice are below 130.

The positive effects of fermented beverages on human health have been known for hundreds for years. For this reason, the cult drinks kvass (Russia) and kombucha (Asia) can be traced over a long history and have been consumed for their healing powers through time. Microorganisms such as lactic acid and acetic acid bacteria are used for non-alcoholic fermentation. The products of fermentation include organic acids such as lactic acid and gluconic acid, which facilitate digestion and metabolic processes. Fermented beverages are especially favored by consumers as healthy, natural refreshments due to their slightly sour flavor.

In addition to other criteria, the acid spectrum typical for certain types of fruit serves as the tool for evaluating whether a fruit juice is pure, and its composition has not been altered. Generally, tartaric acid, citric acid and L-malic acid are analyzed. These are the primary acids, with a few exceptions, that are responsible for the total acid in fruits.

Application/Purpose

Determination of citric acid by enzymatic means

Scope of Application

This analysis is suitable for malt, wort, beer, beer-based beverages and soft drinks

Principle

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.

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