Hop products with isomerized or reduced iso α-acids intended for use in beer brewing or elsewhere in the food industry
Hop products with isomerized or reduced iso α-acids are dissolved with methanol. The bitter acids are separated through reverse phase high-pressure liquid chromatography (RP-HPLC) and isocratic elution. They are then measured at a wavelength of 270 nm.
Simultaneous determination of iso-a-acids and reduced iso-a-acids (rho, tetra, hexa) in beer and beer-based beverages using HPLC
This method is not suitable for beer and beer-based beverages containing a mix of hexa- and iso-products (co-elution).
This method separates iso-α-acids and reduced iso-α-acids (rho, tetra, hexa) chromatographically [2].
The separated compounds are detected spectrophotometrically at 270 nm. Quantification is performed using International Calibration Standards (ICS).
These standards do not contain all of the isomers of a certain type of iso-α-acids. Currently, ICS-I (DCHA-Iso) contains only the trans-isomeric complexes with dicyclohexylamine (DCHA). ICS-R (DCHA-rho) only has the cis-isomers in a complex with DCHA. ICS-T (tetra) comprises both cis- and trans-isomer complexes with DCHA, and ICS-H (DCHA-hexa) includes only cis-isomers with DCHA.
Commercially available hop products contain a greater number of isomers than the standards.
Thus, there are more peaks in the chromatogram than in the corresponding international calibration standards, in particular, with the cis-iso-cohumulone as well as the cis-iso-n-humulone and cis-iso-ad-humulone. The application of this method to these kinds of beers should be conducted with this in mind, since it is not entirely known whether these additional peaks are actually isomers of iso-α-acids.
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.