12 Automatic Analysers in Oenology 659
Certain instruments are only capable of adding one reagent and this precludes
reactions for which two reagents are required, though of course the reverse is not
the case.
12.3.4 Usual Methods Based in Sequential Analysis
12.3.4.1 Determination of Acetic Acid
According to McCloskey (1980), Doneche and Sanchez, (1985) and Dubernet et al.
(1997).
Principle
Enzymatic Method
In the presence of ATP, acetic acid is transformed into acetyl-phosphate in a reaction
catalysed by acetate kinase:
Acetate+AT P —AcetateKinase−→Acetyl-phosphate+ADP (1)
The ADP formed by this reaction is reconverted into ATP by reaction with phos-
phoenolpyruvate in the presence of pyruvate kinase:
ADP+Phosphoenolpyruvate —PyruvateKinase−→Pyruvate+AT P ( 2 )
Pyruvate is reduced to l-lactate by reduced nicotinamide-adenine-nucleotide
(NADH) in the presence of lactate-deshydrogenase:
Pyruvate+NADH+H+ —Lactatedeshydrogenase−→Lactate+NAD++H 2 O(3)
The quantity of NADH oxidised in reaction (3) is determined by measuring its
absorbance at 340 nm, which is proportional to the concentration of acetic acid in
the wine.
A fourth reaction, the elimination of acetylphosphate is used to force the equilib-
rium of reaction (1) in favour of the products:
Acetyl-phosphate+CoA—Phosphotransacetylase−→ Acetyl-CoA+inorganic phosphate
(4)Polyvinylpyrrolidone (PVP) is added to the reaction medium in order eliminate
interference due to wine phenolic compounds.
Characteristics of the Method
Intra-laboratory reproducibility: 0.04g/L
Inter-laboratory reproducibility: 0.10g/L