Determination of the correct Velcorin® dosage
flavored beverages, liquid tea concentrate, fruit wine, non-alcoholic wine
DMDC (Velcorin®) is used for the cold sterilization of non-alcoholic beverages. It essentially kills yeasts, bacteria and molds, with little effect on mold spores or yeast ascospores. Certain species of Lactobacillus possess an elevated resistance to Velcorin®.
There should be fewer than 500 microbes/cm3 at the time the dosage is added.
According to the EU guideline EG 1129/2011 [1], up to 250 mg/l DMDC may be added to flavored non-alcoholic beverages, non-alcoholic wine and liquid tea concentrates.
Dimethyl dicarbonate (Velcorin®) quickly dissociates in aqueous solutions almost completely to carbon dioxide and methanol. In addition, small amounts of ethyl methyl carbonate are formed through the reaction of DMDC with ethanol, which can be detected through GC-MS analysis techniques [2]. The amount of DMDC added to a beverage can be determined by measuring the content of EMC and ethanol. The Velcorin® dosage can be checked by measuring the amount of methanol quantitatively using GC analysis; however, the initial amount of methanol present in the product prior to adding Velcorin® must be determined.
H2O + C4H6O5 (DMDC) → 2CH3OH + CO2
250 mg Velcorin → 119.5 mg methanol + 164.1 mg carbon dioxide
C2H5OH + C4H6O5 (DMDC) → C4H8O3 + CH3OH+ CO2
Determination/calculation of the apparent extract content from the SGA20/20 or the density of a liquid
wort, beer, beer-based beverage, NAB, beverage
Determine the SGA20/20 obtained from pycnometry or the density measured with a precision hydrometer or another device for measuring the density. Using the value from the SGA20/20 measurement or the density from the sugar, alcohol, original gravity and correction table according to GOLDINER/KLEMANN, BLOCK, KÄMPF or a polynomial, determine the apparent extract content of the sample.
Determination of glucose by enzymatic means
Suitable for beers, mixed beer beverages, malt beverages and NAB
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:
\(\text{G-6-P + NADP} \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{gluconate-6-phosphate + NADP + H}^+\)
The amount of NADPH formed during the reaction is equivalent to the amount of glucose. NADPH is determined based upon its absorbance at 334, 340 or 365 nm.
Determination of glucose and fructose by enzymatic means
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):
Glucose + ATP \(^{\underrightarrow{HK}}\) G-6-P + ADP
Fructose + ATP \(^{\underrightarrow{HK}}\) 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 (NADPH) is formed:
\(\text{G-6-P}\hspace{0.2em}+\hspace{0.2em}\text{NADP}\hspace{0.8em}^{\underrightarrow{\text{G6P–DH}}}\hspace{0.8em} \text{glucanate-6-phosphate} + \text{NADP}+\text{H}^+\)
The amount of NADPH formed during the reaction is equivalent to the amount of glucose. NADPH is a measurand and is determined based on its absorbance at 334, 340 or 365 nm.
After the reaction is complete, F-6-P is converted to G-6-P by phosphoglucose isomerase (PGI):
F-6-P \(^{\underrightarrow{PGI}}\) 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 absorption at 334, 340 or 365 nm.
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.