Determination of glucose and fructose by enzymatic means
Glucose and fructose are phosphorylated by the enzyme hexokinase (HK) and adenosine 5'-triphosphate (ATP) to glucose 6-phosphate (G-6-P) and fructose 6-phosphate (F-6-P):
Glucose + ATP \(^{\underrightarrow{HK}}\) G-6-P + ADP
Fructose + ATP \(^{\underrightarrow{HK}}\) F-6-P + 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:
\(\text{G-6-P}\hspace{0.2em}+\hspace{0.2em}\text{NADP}\hspace{0.8em}^{\underrightarrow{\text{G6P–DH}}}\hspace{0.8em} \text{glucanate-6-phosphate} + \text{NADP}+\text{H}^+\)
The amount of NADPH formed during the reaction is equivalent to the amount of glucose. NADPH is a measurand and is determined based on its absorbance at 334, 340 or 365 nm.
After the reaction is complete, F-6-P is converted to G-6-P by phosphoglucose isomerase (PGI):
F-6-P \(^{\underrightarrow{PGI}}\) G-6-P
G-6-P reacts in turn with NADP to form gluconate-6-phosphate and NADPH. The additional amount of NADPH formed is equivalent to the amount of fructose and is determined photometrically based on its absorption at 334, 340 or 365 nm.
Determination of glucose, fructose, sucrose by enzymatic means
Suitable for wort, beer, malt beverages, nutritive beer, beer-based beverages, NAB, juices and beverages
The D-glucose content is determined before and after enzymatic hydrolysis of sucrose. D-fructose is measured following D-glucose determination.
D-glucose determination before inversion:
Glucose is phosphorylated by the enzyme hexokinase (HK) and adenosine 5'-triphosphate (ATP) to glucose 6-phosphate (G-6-P):
\(\text{Glucose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{G-6-P + ADP}\)
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:
\(\text{G-6-P + NADP} \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{gluconate-6-phosphate + NADP + H}^+\)
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.
D-fructose determination:
Hexokinase catalyzes the phosphorylation of D-fructose with ATP to D-fructose-6-phosphate.
\(\text{Fructose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{F-6-P + ADP}\)
After the reaction is complete, F-6-P is converted to G-6-P by phosphoglucose isomerase (PGI):
\(\text{F-6-P} \space ^{\underrightarrow{\text{PGI}}} \space \text{G-6-P}\)
G-6-P reacts in turn with NADP to form gluconate-6-phosphate and NADPH. The additional amount of NADPH formed is equivalent to the amount of fructose and is determined photometrically based on its absorbance at 334, 340 or 365 nm.
Enzymatic inversion:
Sucrose is hydrolyzed to glucose and fructose by the enzyme β-fructosidase (invertase) at pH 4.6:
\(\text{Sucrose}+H_2O\hspace{0.8em} ^{\underrightarrow{\text{B-fructosidase}}} \hspace{0.8em} \text{glucose + fructose}\)
The D-glucose determination after inversion (total D-glucose) is carried out as described above.
The sucrose content is calculated from the difference between the glucose concentration before and after enzymatic inversion.