Determination of glycerin by enzymatic means
This method is suitable for wort, beer, beer-based beverages and non-alcoholic beverages.
Glycerol is a by-product of fermentation. Glycerol is phosphorylated to form L-glycerol-3-phosphate in a reaction with adenosine 5'-triphosphate (ATP) and is catalyzed by glycerol kinase (GK):
Glycerol + ATP \(^{\underrightarrow{\text{GK}}}\)L-glycerol-3-phosphate + ADP
The resultant adenosine 5'-diphosphate (ADP) is converted by pyruvate kinase (PK) back into ATP, and as part of this reaction, phosphoenolpyruvate (PEP) loses its phosphate group to become pyruvate:
ADP + PEP \(^{\underrightarrow{\text{PK}}}\) ATP + pyruvate
Pyruvate is hydrogenated to L-lactate through the action of reduced nicotinamide adenine dinucleotide (NADH) in the presence of the enzyme L-lactate dehydrogenase (L-LDH); in the process NADH is oxidized to NAD:
Pyruvate + NADH + H+ \(^{\underrightarrow{\text{L-LDH}}}\) L-lactate + NAD
The amount of NADH formed during the reaction is equivalent to the amount of glycerol. NADH is the parameter measured, and thus it is determined based upon its absorbance at 334, 340 or 365 nm.