/ WORT, BEER AND BEER-BASED BEVERAGES / Beverages in General / Carbohydrates

B-590.17.112 [2020-10] Glucose and Fructose – Enzymatic Method

The extract content of normal beer wort is typically composed of around 90 % carbohydrates, which occupies the position of highest importance for beer production.

The starch reserves originally found in the barley kernel are converted to some extent into soluble carbohydrates by degradation during malting; however, the majority of soluble carbohydrates are released during mashing. The products of starch degradation can be classified into three groups:

sugars fermented by brewing yeast (maltose, maltotriose, sucrose, glucose and fructose)
unfermentable oligosaccharides consisting of 4–20 glucose units, exhibiting a negative reaction when exposed to iodine
dextrins consisting of more than 20 glucose units, showing a positive reaction when exposed to iodine

Monosaccharides, disaccharides and trisaccharides are present in different amounts and affect the initial phases of fermentation, primary fermentation and the ultimate degree of attenuation of wort and beer. A deficit of monosaccharides has a negative impact at the beginning of fermentation, while an insufficient amount of disaccharides slows fermentation and reduces the final degree of attenuation.

The concentration of sugars in the wort and their subsequent fermentation by the yeast has a substantial influence on the quality of the beer. Oligosaccharides represent a non-fermentable fraction for the brewing yeast. Nonetheless, this fraction is important in beer as a component of chill haze. These substances also play a role as carrier substances for aromas in the beer and as dispersion agents. Moreover, based on the composition and concentration of this group of substances in the wort, certain conclusions can be drawn regarding the progression of amylolysis during the mashing process.

Higher molecular weight degradation products (dextrins) belonging to the last group are rarely present in wort and beer.

The determination of sugar is important for defining the nutritional value of a food. Glucose, fructose and sucrose occur most frequently. Maltose, maltotriose and dextrins are found in functional sports drinks as an energy source. D-sorbitol is present in pome and stone fruits.

Determination of sugars can be performed by means of HPLC and refractometric detection, ion chromatography and pulsed amperometric detection or through enzymatic analysis.

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The determination of glucose and fructose are of particular importance in the analysis of...

...highly attenuated beers, in which the primary goal is reduction of the carbohydrate content. For the analysis and evaluation of non-alcoholic malt-based beverages, the concentration of glucose and fructose is a definitive factor, one among several defining factors. Yeast can easily metabolize glucose and fructose; therefore, they may affect the fermentation process.

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The concentrations of the coenzyme pairs NAD/NADH⁺ or NADP/NADPH⁺ are typically measured spectrophotometrically at a wavelength of 340 nm.

It is also possible to detect the coenzyme pairs photometrically at the wavelengths Hg 334 or Hg 365 nm.
It must be taken into account that the molar extinction coefficient (ε) changes with the wavelength.

Wavelength	ε (Molar extinction coefficient)
340 nm	= 6.30 in l × mmol^-1 × cm^-1
Hg 365 nm	= 3.50 in l × mmol^-1 × cm^-1
Hg 334 nm	= 6.18 in l × mmol^-1 × cm^-1

Application/Purpose

Determination of glucose and fructose by enzymatic means.

Scope of Application

Suitable for beers, mixed beer beverages, malt beverages, non-alcoholic soft drinks, NAB, juices and drinks.

Principle

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⁺:

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