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Hop constituents are distributed between an aqueous acidic methanolic phase and diethyl ether. Hop bitter substances extracted with ether are subsequently separated according to their different solubility properties in cold methanol and hexane into fractions: total resins, soft resins and hard resins. The soft resins are further separated according to their capacity to form complexes with lead salts into α-acids (conductometer value) and a β-fraction.

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 by nicotinamide adenine dinucleotide phosphate (NADP⁺) to gluconate-6-phosphate. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is formed:

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

The amount of NADPH+H⁺ formed during the reaction is equivalent to the amount of glucose. NADPH+H⁺ is determined based upon its absorbance at 340 nm.

Note:

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

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

Volatile aroma compounds are driven out of the sample through steam distillation. The ethanolic distillate is saturated with NaCl. Potassium hydrogen sulfite is added to separate carbonyl groups that might interfere with the analysis. The extraction of the aroma compounds is performed by shaking out with dichloromethane and the phases separated by centrifuging.

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:

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

The amount of NADP + H⁺ formed during the reaction is equivalent to the amount of glucose. NADPH + 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):

\(\text{F-6-P} \space ^{\underrightarrow{\text{PGI}}} \space \text{G-6-P}\)

The amount of NADPH + H⁺ formed during the reaction is equivalent to the amount of fructose. NADPH + H⁺ is the parameter being measured and is determined on the basis of its absorption at 340 nm.

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⁺/NADPH + 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.