Determination of the content of steviol glycosides and stevia products in beverages.
All mixed beer drinks and beverages in general.
The decarbonated and diluted sample is chromatographically separated on a polar-endcapped amino-HILIC phase and detected using a mass spectrometer with ESI source. The quantitative evaluation is carried out by external calibration.
The method is suitable for beers of all original gravities and of any alcohol content.
The gas chromatography headspace method is used to determine the higher alcohols and esters present in beer, i.e., the volatile compounds are transferred from the headspace in the sample vial into the GC system for analysis. The following substances are measured in this analysis:
Acetaldehyde
Propanol-1
Ethyl acetate
2-Methylpropanol
3-Methylbutanol
2-Methylbutanol
2-Methylpropylacetate
Butyric acid ethyl ester
3-Methylbutyl acetate
2-Methylbutyl acetate
Hexanoic acid ethyl ester
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 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 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].