2-Thiobarbituric Acid as a Reagent for Detecting Milk Fat Oxidation

Abstract

Kohn and Liversedge (3) observed that various animal tissues after incubation under aerobic conditions, produce compound(s) which give a color reaction when heated with thiobarbituric acid (TBA).Although they did not demonstrate the nature of the color producing compound(s), their research established the absorption spectrum of the color complex and certain chemical characteristics of the color reaction.Research by Bernheim et al. (2) revealed that the colorproducing materials in various animal tissues are lipid and, more specifically, that egg lecithin or fatty acids isolated from it, brain lecithin, fatty acids from linseed oil, cephalin and brain proteih-lecithin complex give colors with TBA after aerobic incubation.The absorption spectra of the colors obtained from these materials were shown to be identical with that described by E:ohn and Liversedge and that obtained with linolenic acid.More recently Wilbur et al.(5) have further explored the TBA color reaction with regard to certain sugars and aldehydes, as well as the oxidation products of linolenic and certain other unsaturated fatty acids.These studies revealed no specific compound which gives a color spectrum identical with those obtained from oxidized lipid materials or aerobically incubated animal tissues.A knowledge of the compound(s) which produce the typical color on heating with TBA not only might clarify certain aspects of fat oxidation, but also could give some insight into the biological utilization of unsaturated fatty acids in animal tissues.The above-mentioned studies have indicated that the TBA reagent is particularly sensitive in measuring photo-chemical, biological or autoxidation of lipid materials.The possibility that this reagent might be applied usefully in the measurement of milk fat oxidation therefore is quite evident.This paper is concerned with a fundamental investigation of the TBA test and its application to the measurement of milk fat oxidalion. EXPERIMENTALThe color reaction of TBA with milk fat.It was of primary interest to ascertain whether milk fat would respond to the TBA test.TBA (Eastman) reagent was prepared, as directed by Kohn and Liversedge (3), and the test applied to dry milk fat samples of various ages prepared according to conventional procedures from fresh sweet cream.Quantities of melted fat (1.1 ml.) were placed in 5-in.test tubes and, successively, 2.5 ml. of water, 2 ml. of TBA reagent, 1 ml. of 20 per cent trichloroacetic acid and a boiling chip were added, after which