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B-590.21.112 [2020-10] Starch – 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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Principle

Starch, the main carbohydrate in malt, is hydrolyzed during the mashing process partly to fermentable sugars and partly to (iodine-normal) dextrins. The determination of starch is therefore of interest for brewing technology.

In the beverage industry, maltodextrins are used in sports and energy drinks.

Starch is broken down into glucose by the enzyme amyloglucosidase (AGS) at pH 4.6:

\(\text{Starch + (n-1) H}_{2} \text{O} \space ^{\underrightarrow{\text{Amyloglucosidase (AGS)}}} \space \text{n D-Glucose}\)

The D-glucose formed is phosphorylated by the enzyme hexokinase (HK) and adenosine 5'-triphosphate (ATP) to glucose 6-phosphate (G-6-P):

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

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 (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. NADP+H⁺ is a measurand and is determined on the basis of its absorption at 340 nm.

Note:
The enzyme AGS hydrolyses α-1,4- and α-1,6-glucosidic bonds regardless of molecular weight (amylose, amylopectin, glycogen, dextrin, maltose, maltotriose, etc.)

It is not possible to distinguish enzymatically between high-polymer and low-molecular starch.