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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):

\(\text{Glucose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{G-6-P + ADP}\)

\(\text{Fructose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{F-6-P + ADP}\)

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 (NADP + H⁺) is formed:

\(\text{G-6-P + NADP}^+ \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{Gluconate-6-phosphate + NADP + H}^+\)

The amount of NADP + H⁺ formed during the reaction is equivalent to the amount of glucose. NADP + H⁺ is a measurand and is determined based on its absorbance at 340 nm.

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 NADP + H⁺. The additional amount of NADP + H⁺ formed is equivalent to the amount of fructose and is determined photometrically based on its absorption at 340 nm.

Note:

Alternatively, NAD⁺/NAD + H⁺ can be used instead of NADP⁺/NADP + H⁺:

\(\text{G-6-P + NAD}^+ \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{Gluconate-6-Phosphate + NAD + H}^+\)

The D-glucose content is determined before and after enzymatic hydrolysis of sucrose. D-fructose is measured following D-glucose determination.

D-glucose/D-fructose determination before inversion:

Glucose and fructose are phosphorylated by the enzyme hexokinase (HK) and adenosine-5'-triphosphate (ATP) to glucose-6-phosphate (G-6-P):

\(\text{Glucose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{G-6-P + ADP}\)

\(\text{Fructose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{F-6-P + ADP}\)

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 (NADP + H⁺) is formed:

\(\text{G-6-P + NADP}^+ \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{Gluconate-6-phosphate + NADP + H}^+\)

The amount of NADP + H⁺ formed during the reaction is equivalent to the amount of glucose. NADP + H⁺ is measurand and is determined based on its absorbance at 340 nm.

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 NADP + H⁺. The additional amount of NADP + H⁺ formed is equivalent to the amount of fructose and is determined photometrically based on its absorbance at 340 nm.

Enzymatic inversion:

Sucrose is hydrolyzed to glucose and fructose by the enzyme β-fructosidase (invertase) at pH 4.6:

\(\text{Saccharose + H}{_2}\text{O} \space ^{\underrightarrow{\text{β-Fructosidase}}} \space \text{Glucose + Fructose}\)

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.

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.

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).

Separation by ion chromatography followed by conductivity detection.

Ethanol is oxidized by nicotinamide adenine dinucleotide (NAD) in the presence of the enzyme alcohol dehydrogenase (ADH) to acetaldehyde:

\(\text{Ethanol + NAD}^+ \space ^{\underrightarrow{\text{ADH}}} \space \text{Acetaldehyd + NADH + H}^+\)

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):

\(\text{Acetaldehyde + NAD}^+ \space ^{\underrightarrow{\text{Al-DH}}} \space \text{acetic acid + NADH + H}^+\)

The amount of NADH+H⁺ produced in the reaction is equivalent to half of the amount of ethanol and is measured photometrically due to its absorption at wavelengths of 340 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.