The ethanol/alcohol content is determined directly (B-590.10.024 Stammwürze, Extrakt und Alkohol - destillativ (Amtliche Methode)) or indirectly (B-590.10.007 Stammwürze, Extrakt und Alkohol - Katalytische Verbrennung (SCABA)).
The methods B-590.10.070 Stammwürze, Extrakt und Alkohol - refraktiometrisch, B-590.10.026 Stammwürze, Extrakt und Alkohol - Biegeschwinger und Schallmessung, B-590.10.181 Stammwürze, Extrakt und Alkohol – Biegeschwinger und NIR und B-590.10.025 Stammwürze, Extrakt und Alkohol – thermoanalytisch are employed to determine the ethanol/alcohol content indirectly.
For beers containing negligible amounts of alcohol, indirect measurements and methods employing distillation can lead to inaccurate results. In such cases, the enzymatic method for determining ethanol is therefore recommended [1, 2]. The enzymatic method has been adopted in the Official Collection of Analysis Methods according to § 64 of the LFGB (formerly § 35 of the LMBG), chapter 36.00/12 (German regulations governing foods).
Determination of ethanol by enzymatic means
Suitable for beers, non-alcoholic beers, reduced-alcohol beers, beer-based drinks, NAB, juice, beverages.
Ethanol is oxidized by nicotinamide adenine dinucleotide (NAD) in the presence of the enzyme alcohol dehydrogenase (ADH) to acetaldehyde:
Ethanol + NAD+ \(^\underleftrightarrow{Al-DH} \) acetaldehyde + NADH + H+
The equilibrium of this reaction favors the side with ethanol and NAD. In an alkaline medium and through removal of the acetaldehyde produced, the equilibrium can be shifted to favor the substances on the right side of the equation. Acetaldehyde is quantitatively oxidized to acetic acid in the presence of aldehyde dehydrogenase (Al-DH):
Acetaldehyde + NAD+ + H2O \(^\underleftrightarrow{Al-DH} \) acetic acid + NADH + H+
The amount of NADH produced in the reaction is equivalent to half of the amount of ethanol and is measured photometrically due to its absorption at wavelengths of 334, 340 or 365 nm.
Specificity of the determination [1]
The influence of aldehydes and ketones is eliminated by the order of reagent addition during the test. Methanol is not converted due to unfavorable KM values (Michaelis-Menten constant) of the enzymes used. n-propanol and n-butanol are quantitatively converted under test conditions, higher primary alcohols lead to sample-dependent creep reactions. Secondary, tertiary and aromatic alcohols do not react. Glycerin does not interfere with the test even at higher concentrations.