Enzymatic analysis is a useful tool to determine concentrations of some wine components. The enzyme is used as an analytical reagent to catalyze a specific reaction of the compound to be determined. The substrates, products, and rate of the reaction can be equated to the concentration of the compound. Products and coenzymes are typically measured. Spectrophotometry has been used to measure the increase in cofactors such as NADPH or NADH. The main advantage of enzyme assays is that they are specific. Ideally the product of the enzyme-catalyzed reaction is formed in a stoichiometric ratio to the compound being assayed. Another advantage of enzymatic analysis is the time they take to run. It has been reported that 100 samples can be run in an hour. Enzymatic assays require that standards with known concentrations be run. These standards take into account effects on product formation due to pH, temperature, and matrix effects (Devlin 1959).
The measurement of glucose and fructose are carried out using the enzymatic reactions below (enzymes are parenthetically stated) (McCloskey et. al 1984):
Glucose + ATP®(HK) Glucose-6-Phosphate + ADP
Fructose + ATP®(HK) Fructose-6-Phosphate +ADP
Glucose-6-Phosphate + NADP®(GDH) Gluconate-6-Phosphate +NADPH +H+
Enzymatic analysis is particularly useful in determining low levels of residual reducing sugars in dry table wines. Samples of 1000 mg/L can be assayed after undergoing dilution four or five times dilutions. Samples that are suspected to be higher in concentration need further dilution McCloskey et. al. 1984). In some cases samples can have a dilution factor of twenty. Samples are added to a reagent solution with the proper pH buffer for the enzyme being used. Following an incubation period of 10 minutes, absorbance readings, using a spectrophotometer, are measured. A second reagent mixture is added to the sample and the final absorbance readings are measured. The change in absorbance and the absorbance of a standard can then be used to determine the concentration of the compound of interest (McCloskey 1978). Water blanks are run to determine a correction factor. When calculating the concentration of the compound dilution factors must be taken into account.
Importance in Winemaking:
Determining the glucose and fructose concentration is important in determining the amount of fermentable sugar in wine. Typically enzymatic analysis of fructose and glucose is done to determine residual sugar of a wine. Enzymatic analysis is capable of measuring small amounts of residual sugar and is very specific to the types of sugars present (McCloskey 1978). Residual glucose and fructose serve as substrates for spoilage yeast and bacteria. Bacteria are able to use glucose and convert it to undesirable compounds. Stuck fermentations with residual sugar can cause a wine to be too sweet (Boulton et. al. 1996).
- Boulton R. B., Singleton V.L., Bisson L.F., Kunkee R.E. Principles and Practices of Winemaking. 1996. Chapman and Hall. New York.
- McCloskey L.P. Enzymatic Analysis of Glucose and Fructose. 1978. Amer. J. Enol. Vitic. 29(3):226.
- McCloskey L.P., Painter B., Noyse D., and Draper P. A New Enzymatic Assay for Glucose and Fructose. 1984. Amer. J. Enol. Vitic. 35(4): 257.