After corn starch is converted through the addition of purified, heat-stable α-amylase during a 15 min boiling process and subsequent Congress mash procedure, the extract content is determined.
Determination of oxalic acid by enzymatic means
Suitable for malt, wort, beer, beer-based beverages and soft drinks
Oxalic acid is primarily derived from malt. By reacting with the calcium ions in the brewing liquor, haze caused by calcium oxalate can form. These crystals also serve as nucleation sites for the spontaneous and rapid release of carbon dioxide (gushing). The precise determination of oxalic acid is therefore of great importance in brewing technology.
Oxalic acid (oxalate) is oxidized to carbon dioxide and hydrogen peroxide by the enzyme oxalate oxidase.
\(\text{ Oxalate} \hspace{0.5em}^{\underrightarrow{oxalatoxidase}}\hspace{0.5em} H_2O_2\hspace{0.3em}{+}\hspace{0.3em}CO_2\)
In the presence of the enzyme peroxidase (POD), hydrogen peroxide reacts with MTBH (3-methyl-2-benzo thiazolinone hydrazone) and DMAB (3-dimethyl amino benzoic acid to form a blue quinone complex.
\(H_2O_2+MTBH+DMAB\hspace{0.8em}^{\underrightarrow{POD}} \hspace{0.8em} \text{quinone complex} \space + \space H_2O\)
The intensity of the color is proportional to the concentration of the oxalate in the sample and is measured at 590 nm.
Determination of ascorbic acid by enzymatic means
This analysis is suitable for wort, beer, beer-based beverages and NAB
L-Ascorbic acid (ascorbate) as well as the reducing substances (X-H2) reduce the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in the presence of PMS (5-methylphenazinium methosulfate), which mediates electron transfer, at a pH of 3.5 to a formazan:
L-Ascorbate (X-H2) + MTT+ \(^\underrightarrow{PMS}\) dehydroascorbate (X) + MTT formazan +H+
Of all the reducing substances present in the blank, only the ascorbic acid portion of the sample is oxidatively removed by ascorbate oxidase (AAO) in the presence of oxygen. Dehydroascorbate is produced in the reaction and does not react with MTT/PMS:
L-Ascorbate (X-H2) + ½ O2 \(^\underrightarrow{AAO}\) dehydroascorbate (X) + H2O
The absorbance of the sample minus the absorbance of the blank is equivalent to the quantity of ascorbic acid in the sample. MTT formazan serves as the measured variable, and its absorption can be determined photometrically in the visible part of the spectrum at 578 nm.
Determination of glycerin by enzymatic means
This method is suitable for wort, beer, beer-based beverages and non-alcoholic beverages.
Glycerol is a by-product of fermentation. Glycerol is phosphorylated to form L-glycerol-3-phosphate in a reaction with adenosine 5'-triphosphate (ATP) and is catalyzed by glycerol kinase (GK):
Glycerol + ATP \(^{\underrightarrow{\text{GK}}}\)L-glycerol-3-phosphate + ADP
The resultant adenosine 5'-diphosphate (ADP) is converted by pyruvate kinase (PK) back into ATP, and as part of this reaction, phosphoenolpyruvate (PEP) loses its phosphate group to become pyruvate:
ADP + PEP \(^{\underrightarrow{\text{PK}}}\) ATP + pyruvate
Pyruvate is hydrogenated to L-lactate through the action of reduced nicotinamide adenine dinucleotide (NADH) in the presence of the enzyme L-lactate dehydrogenase (L-LDH); in the process NADH is oxidized to NAD:
Pyruvate + NADH + H+ \(^{\underrightarrow{\text{L-LDH}}}\) L-lactate + NAD
The amount of NADH formed during the reaction is equivalent to the amount of glycerol. NADH is the parameter measured, and thus it is determined based upon its absorbance at 334, 340 or 365 nm.
Determination of the nitrate content of wort, beer and other beverages as well as that of malt and hops.
This method is suitable for wort, beer and other beverages as well as for malt and hops.
Essentially, the nitrate content of beer originates with the nitrate derived from the brewing liquor and the hops (whole hops, hop powder). Nitrate is reduced to nitrite by yeast has not been found to occur at any stage in the brewing process.
Nitrate is reduced with reduced nicotinamide adenine dinucleotide phosphate (NADPH) to nitrite in the presence of the enzyme nitrate reductase (NR) [1].
Nitrate + NADPH + H+ \(^{\underrightarrow{NR}}\) nitrite + NADP+ + H2O
The quantity of NADPH consumed in this reaction is proportional to the amount of nitrate in the sample. NADPH is measured and the determination is performed at 334, 340 oder 365 nm, due to its absorption at these wavelengths.
Determination of acetic acid by enzymatic means
This analysis is suitable for malt, wort, beer, beer-based beverages and soft drinks
Acetic acid (acetate) is converted to acetyl-CoA in the presence of the enzyme acetyl-CoA synthetase (ACS) by adenosine-5'-triphosphate (ATP) and coenzyme A (CoA).
Acetate + ATP + CoA \(^{\underrightarrow{ACS}}\) Acetyl-CoA + AMP + pyrophosphate
Acetyl-CoA reacts with oxaloacetate in the presence of citrate synthase (CS) to form citrate.
Acetyl-CoA + oxaloacetate + H2O \(^{\underrightarrow{CS}}\) citrate + CoA
The oxaloacetic acid required for reaction (2) is produced from malic acid and nicotinamide adenine dinucleotide (NAD) in the presence of malate dehydrogenase (MDH). In doing so, NAD is reduced to NADH:
Malate + NAD+ \(^{\underleftrightarrow{L-MDH}}\) oxaloacetate + NADH + H+
The formation of NADH forms the basis of this analysis, which is measured as an increase in the absorbance at 340, 334 or 365 nm. Since this concerns a previous indicator reaction, the quantity of NADH is not linearly proportional to the acetic acid concentration.