This method describes how to determine iso-α-acids, α-acids and β-acids in isomerized pellets by means of reverse phase high pressure liquid chromatography (RP-HPLC).
Isomerized pellets intended for use in beer brewing or elsewhere in the food industry
The bitter substances in isomerized hop pellets contain a substantial amount of iso-α-acids; however, in addition to these, non-isomerized α-acids and β-acids are also present. In order to determine their content, a specific method is required.
After milling, the substances in question are extracted from the isomerized pellets using a diethyl ether/methanol mixture and a hydrochloric acid solution. The iso-α-acids, α-acids and β-acids dissolved in the ether phase are separated using reversed-phase high-performance liquid chromatography (RP-HPLC) and an elution gradient. They are then measured spectrophotometrically at wavelengths of 270 nm (iso-α-acids) and 314 nm (α-acids and β-acids).
Hops and hop products intended for use in beer brewing or elsewhere in the food industry
After extraction with toluene, the α-acids and β-acids in the hops and hop pellets are determined using spectrophotometry.
Determination of iso α-acids and α-acids in beer, beer-based beverages and wort
This method is not suitable for beer, beer-based beverages or wort, which contain saccharin, p-hydroxybenzoic acid ester, salicylic acid or sorbic acid.
These bitter substances are extracted from the acidified sample using iso-octane. Certain disruptive substances are eliminated through washing the extract with acidified methanol. The concentration iso-α-acids as well as α-acids is determined by measuring the absorbance in alkaline methanol at 255 nm and 360 nm.
Determination of D-gluconic acid by enzymatic means
This analysis is suitable for non-alcoholic beverages and for those containing alcohol.
Fruit juices
The positive effect of fermented beverages on the human body has been known for centuries. Current beverage trends, like kvass (Russia) and kombucha (Asia), stem from traditions with roots deep in the past. They have always been consumed as healing beverages. Non-alcoholic forms of fermentation employ microorganisms, such as lactic and acetic acid bacteria. They produce organic acids like lactic acid and gluconic acid, which promote digestion and metabolism. Due for the most part to their slightly acidic flavor, these kinds of fermented beverages are popular with consumers as a healthy natural refreshment.
Malt, fruit juice and tea serve as a base for fermented beverages.
As a rule, fermented beverages contain 0.5 – 15 g/l D-gluconic acid.
D-gluconic acid is phosphorylated by adenosine 5'-triphosphate (ATP) in the presence of gluconate kinase to gluconate-6-phosphate
D-Gluconate + ATP \(^{\underrightarrow{\text{gluconate kinase}}}\) D-gluconate-6-P + ADP
The enzyme 6-phosphogluconate dehydrogenase (6-PGDH) catalyzes the oxidation of gluconate-6-phosphate to ribulose-5-phosphate with nicotinamide adenine dinucleotide phosphate (NADP):
D-Gluconate-6-phosphate + NADP+ \(^{\underrightarrow{6-PGDH}}\) ribulose-5-phosphate + NADPH + H+ + CO2
The amount of NADPH formed during the reaction is proportional to the amount of D-gluconic acid.
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.