A Bacterial Enzymatic Method for Determining Tyrosine in Cheese

Free tyrosine determinations of biological materials on a quantitative scale present a complex problem.Important basic difficulties were early recognized by Folin and Cioealteu (3) in their classical studies on protein hydrolyzates and, later, were more extensively discussed by Mogensen (10).The accurate determination of free tyrosine in cheese requires either prior separation of interfering compounds, such as tryptophan; lower peptides and tyramine, or the introduction of a procedure with specificity for tyrosine.Neither paper chromatography nor microbiological assay has proved entirely satisfactory, as the former method lacks sensitivity for tyrosine and the latter is subject to serious errors (6) from peptide interference.Gale (4) has proposed a method for L-tyrosine with tyrosine decarboxylase at pII 5.5 to catalyze the following reaction: L-tyrosine ~ tyramine -t-C02The C02 evolved is measured in a Warburg apparatus and equated to tyrosine.Optimal concentrations of tyrosine for this assay were between 0.5 and 2.0 rag.This method could not be applied to dairy products by the present authors because the size o~ sample possessing the optimum tyrosine concentration was too large for the Warburg apparatus.In addition, C02 evolution in recovery experiments bore tittle relationship to calculated recoveries, evidently because of the complex gas absorptive nature of cheese components.From the above enzyme reaction a measurement of tyramine instead of CO_might serve equally well to determine tyrosine in dairy products.Kosikowski and Dahlberg (8) have successfully applied a sensitive analytical procedure for extracting tyramine out of cheese, but a time element of 24-48 hours was required.In the present study, an adaptation of this method, involving multiple ether extractions from a stronger alkaline buffer, gave excellent recoveries of tyranfine in a much shorter thne and was used to advantage in subsequent determinations.The proposed method for tyrosine, described in detail here, is based on the following considerations.