The Elimination of Interfering Substances in the Kay–Graham Phosphatase Test when Used for Hard Ripened Cheese

Summary A technic was developed for the selective separation of free phenol from interfering substances in the Kay-Graham phosphatase test when used on hard ripened cheese. Tyrosine and tyramine were important interfering substances. The elimination of the interfering substances was accomplished by using trichloracetic acid as a precipitant for the cheese proteins, and by extracting the free phenol, formed as a result of phosphatase activity in an alkaline substrate, with ether under acid conditions using a Mojonnier-type extractor. The ether containing the phenol was placed in distilled water and then was boiled off. The aqueous solution then was treated with the Folin-Ciocalteu reagent and the amount of phenol determined with a colorimeter. The amount of interfering substances in ripened cheese not removed by using the new trichloracetic acid technic was extremely low, as values averaging 0.004 mg. phenol per 0.5 g. cheese were obtained for 25 cheeses varying in age from 7 months to 10 years. Phenol values obtained on cheese using the new technic showed changes in phosphatase activity which corresponded well with results obtained on the same cheeses with the Sanders-Sager phosphatase method. The phenol values obtained using this new technic on cheese were on the order of those obtained on milk using the standard Kay-Graham method, but no complete data have been obtained yet to establish the final details of the test and to show at what point the new technic would distinguish raw from pasteurized milk cheese.