Applicable for all (laboratory) wort
The Congress wort is heated in order to inactivate the amylolytic enzymes, and afterwards added to an Erlenmeyer flask along with 7.5 g/100 ml yeast where it is allowed to completely ferment out under constant agitation. The difference in the extract before and after fermentation is measured in order to calculate the limit of attenuation.
Determination of maltose and maltotriose by enzymatic means
Suitable for wort, beer, malt beverages, nutrient beer, mixed beer beverages, NAB, juices and beverages.
Maltose is the main component of beer wort or wort extract.
Maltose and sucrose are cleaved by the enzyme α-glucosidase (maltase) at pH 6.6 into two molecules of D-glucose and D-fructose, respectively:
\(\text{Maltose}+H_2O \hspace{0.8em} \xrightarrow{α–glucosidase} \hspace{0.8em} {2 \hspace{0.2em} \text{D–glucose}}\)
\(\text{Sucrose}+H_2O \hspace{0.8em} \xrightarrow{α–glucosidase} \hspace{0.8em} {\text{D–glucose}+\text{D–fructose}}\)
The D-glucose formed is phosphorylated by the enzyme hexokinase (HK) and adenosine 5'-triphosphate (ATP) to glucose 6-phosphate (G-6-P):
\(\text{Glucose}+\text{ATP} \hspace{0.8em} \xrightarrow{HK} \hspace{0.8em} \text{G-6-P} \hspace{0.2em} + \hspace{0.2em} \text{ADP}\)
In the presence of the enzyme glucose-6-phosphate dehydrogenase (G6P-DH), G-6-P is oxidized from nicotinamide adenine dinucleotide phosphate (NADP) to gluconate-6-phosphate. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is formed:
\(\text{G-6-P} \hspace{0.2em} + \hspace{0.2em} \text{NADP}^+ \hspace{0.8em} \xrightarrow{G6P-DH} \hspace{0.8em} \text{gluconate-6-phosphate} \hspace{0.2em} + \hspace{0.2em} \text{NADP}^+ \hspace{0.2em} + \hspace{0.2em} \text{H}^+\)
The amount of NADPH formed during the reaction is equivalent to the amount of glucose. NADPH is measurand and is determined based on its absorbance at 334, 340 or 365 nm.
The enzyme α-glucosidase is group specific, i.e., the specificity is directed to the type of glycosidic bond.
Only α-1,4 bonds, i.e., in addition to maltose, sucrose and maltotriose, but not maltotetraose, are cleaved under the given conditions. Therefore, the sucrose content must be taken into account in the maltose calculation (the maltose approach records the glucose formed from maltose and sucrose and the free glucose, the sucrose approach records the glucose formed from sucrose and the free glucose).