A new enzymatic method for determination of sulphite in food

A novel enzymatic method for determining sulphite is described. Using the enzyme sulphite oxidase (EC 1.8.3.1) isolated from chicken liver, sulphite is oxidised to sulphate and hydrogen peroxide is formed. This is allowed to react with reduced nicotinamide-adenine dinucleotide (NADH) in the presence of NADH-peroxidase from microorganisms (EC 1.11.1.1). The decrease of NADH, which is proportional to the concentration of sulphite, is measured photometrically.In spite of doubts about determining sulphite quantitatively according to this principle, the reaction conditions could be optimised: sulphite is oxidised in triethanolamine buffer (TEA) at pH8·0 with 40 mU/ml sulphite oxidase and 47 mU/ml NADH-peroxidase quantitatively within a period of 20 min. The precision of measurement is very high in the range 13–205 μmol sulphite/litre test solution. The coefficient of variation is found to be CV = 0·67% in a solution of 67 μmol SO₂/litre.The accuracy of the enzymatic measurements in pure aqueous sulphite solutions is confirmed by comparison with chemical determination. The correlation with the iodimetric method is R = 0.995.High specificity for sulphite is found when applying the enzymatic test described here. Carbonyl/sulphite addition compounds (e.g. those occurring in wine) are converted to sulphate and the free carbonyl compound. The majority of sulphur-containing compounds do not react in the test. Only glycosides of isothiocyanates (e.g. in mustard) are oxidised like sulphite, though more slowly.The majority of substances occurring in food do not interfere significantly with the test. Ascorbic acid influences the determination if it is present in high concentrations. Therefore, it should be removed from the sample before measuring sulphite.The method has been proven in use for a large number of foodstuffs. By using the method described here, sulphite can be determined in food rapidly and with high reliability.