Search: fruit acids

Titratable acidity represents the sum of the free acids present in a beverage, with the exception of the dissolved carbon dioxide (carbonic acid). In fruit juices and the beverages prepared from them, they usually consist of malic acid, citric acid and tartaric acid.

The titration of the degassed beverage sample (freed from carbonic acid) is carried out potentiometrically using 0.25 mol/l sodium hydroxide solution either to a pH of 7.0 calculated as tartaric acid or to a pH of 8.1 calculated as citric acid.

Hop extracts and isomerized hop extracts are dissolved in methanol. The iso-α-acids, α-acids and β-acids are separated using hig-pressure liquid chromatography (HPLC) with gradient elution and measured spectrophotometrically at a wavelength of 270 nm (iso-α-acids) and 314 nm (α-acids and β-acids).

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).

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.

iso-α-Acids (isohumulones), α-acids (humulones) and β-acids (lupulones) are chromatographically separated using this method.

The bitter substances are chromatographically separated by means of RHPLC. The humulones and lupulones are detected at 314 nm, whereas the iso-α-acids are detected at 270 nm.

Equipment

Analytical balance, ± 0.0001 g

Water bath, 20 °C

Ultrasound bath

Centrifuge, 750 g

Vials, crimp caps, crimping tool

Disposable syringes, 20 ml

Syringe filter, 0.45 µm, PTFE

High performance liquid chromatography set up for gradient operations, column oven 35 °C, UV-VIS detector (270 nm and 314 nm)

High pressure liquid chromatograph and UV-VIS detector, equipped for gradient operation

HPLC column, Machery & Nagel CC 125 / 4 Nucleodur 100 5 C18 EC or a comparable column

Graduated cylinder, 1000 ml, 50 ml

Beaker, 50 ml

Volumetric flasks, 100 ml, 50 ml

Volumetric pipettes, 30 ml, 10 ml

Erlenmeyer flask, 100 ml

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).

\(\text{(1) Acetate + ATP + CoA }\space ^{\underrightarrow{\text{ACS}}} \space \space\text{Acetyl-CoA + AMP + Pyrophosphate}\)

Acetyl-CoA reacts with oxaloacetate in the presence of citrate synthase (CS) to form citrate.

\(\text{(2) Acetyl-CoA + Oxalacetate + H}_2\text{O }\space ^{\underrightarrow{\text{CS}}} \space \space\text{Citrate + CoA}\)

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:

\(\text{(3) L-Malate + NAD}^+ \space ^{\underrightarrow{\text{L-MDH}}} \space \text{Oxalacetate + NADH + H}^+\)

The formation of NADH+H⁺ forms the basis of this analysis, which is measured as an increase in the absorbance at 340 nm. Since this concerns a previous indicator reaction, the quantity of NADH+H⁺ is not linearly proportional to the acetic acid concentration.