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 sucrose by enzymatic means
Suitable for wort, beer, malt beverages, nutrient beer, mixed beer beverages, NAB, juices and beverages
Sucrose is important as a fermentable sugar for the technology of wort and beer production. Sucrose also plays a role in the evaluation and assessment of malt beverages and nutritional beers.
D-glucose content is determined before and after enzymatic hydrolysis of sucrose.
Sucrose is hydrolyzed by the enzyme β-fructosidase (invertase) at pH 4.6 to glucose and fructose:
\(\text{Sucrose + } H_2O \space {\xrightarrow{β-fructosidase}} \space \text{D-glucose + D-fructose}\)
Glucose is phosphorylated by the enzyme hexokinase (HK) and adenosine 5'-triphosphate (ATP) to glucose 6-phosphate (G-6-P):
\(\text{Glucose}+\text{ATP} \space \xrightarrow{HK} \space \text{G-6-P + ADP}\)
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:
\(\text{G-6-P + NADP} \hspace{0.8em} \xrightarrow{G6P-DH} \hspace{0.8em} \text{gluconate-6-phosphate + NADP + H}^+\)
The amount of NADPH formed during the reaction is equivalent to the amount of glucose. NADPH is a measurand and is determined on the basis of its absorbance at 334, 340 or 365 nm.
The sucrose content is calculated from the difference between the glucose concentration before and after enzymatic inversion.
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).
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.
Determination of the concentration of dissolved carbon dioxide in carbonated beverages in bottles and cans
The total gas pressure in beer is measured after the beer has been forcefully shaken. The carbon dioxide is then bound through the addition of potassium hydroxide. The amount of air in the beer contributes the remaining volume of gas. Once the value for the total pressure has been corrected by subtracting the quantity of air present in the beer, the carbon dioxide can be measured [1].
Determination of the total air in bottles and cans
Determination of the total air in containers for beer, beer-based beverages and carbonated beverages
Through heating and shaking, the gases contained in beer are collected in a burette filled with potassium hydroxide. The carbon dioxide is bound by potassium hydroxide, and the remaining volume of gas, consisting of oxygen and nitrogen, is measured [1].