148 MISC. PUBLICATION 5 4 0, U. S. DEPT. OF AGRICULTURE 



Because of the ease and rapidity with which enzymes can be de- 

 tected, the relation between enzyme destruction and storage life 

 has been studied extensiyely. It should be noted that the methods of 

 testing for enzymes are empirical and that the degree of destruction 

 corresponding to adequate blanching yaries not only with the vege- 

 tables but probably also with the yariety, maturity, and other factors. 

 On the other hand, the amount of blanching (as measured by both 

 time and temperature) required to obtain an adequately blanched 

 product may likewise vary. 



In spite of yariations in results, enzyme tests are the chief means of 

 estimating the amount of blanching treatment that dehydrated vege- 

 tables haye receiyed. With certain exceptions, dehydrated yegetables 

 are tested for either catalase or peroxidase by Goyernment procure- 

 ment agencies. The selection of these enzymes was based chiefly on 

 simplicity of test procedures, wide distribution of the enzymes in 

 yegetables, and their sensitiyity to heat. The procedures for the tests 

 and the tolerances adopted during the early stages of the war were 

 largely a matter of expediency ; they represented an attempt to employ 

 a safe margin of blanching eyen though adequate data on storage 

 quality as related to blanching treatment were not ayailable. Proce- 

 dures are being modified continually in accordance with results of 

 research. 



Care is required in making the tests. Although the obseryations 

 are generally subjectiye and qualitative, it is important to follow 

 instructions carefully, since definite concentrations rather than excesses 

 of reagents are used. For example, a two-fold variation in concen- 

 tration of either hydrogen peroxide or guaiacol, as used in the early 

 peroxidase tests, will cause a twofold yariation in color obtained. 

 Variations of several fold may occur unless (1) the hydrogen peroxide, 

 which is yery unstable in diluted solutions, is prepared fresh each day 

 from full-strength concentrated reagent; (2) the amount of water 

 used to coyer the sample is controlled, and (3) the amounts of reagents 

 added to the reaction mixture are properly measured. If the reaction 

 mixture is excessively cold or warm, the results may vary as much as 

 twofold. Also light, particularly sunlight, causes the color to fade, 

 making it necessary to guard against excessive illumination. 



A number of materials besides peroxidase has been reported to 

 cause color formation in the peroxidase system. These include hemin, 

 heavy metal salts, lignin, and others. The hemin and heavj^ metal 

 salts ordinarily are not present in sufficient amounts to give a false 

 positive test, and lignin, contrary to report, does not react with 

 guaiacol either in the presence or absence of hydrogen peroxide. 

 Benzidine, however, reacts with lignin to give a golden-yellow color, 

 readily distinguishable from the blueblack formed in the oxidation 

 of benzidine by peroxidase. The formation of the golden-yellow 

 color is unaffected by hydrogen peroxide. The greater amount of 

 blanching required to destroy the tendency of skins of corn and lima 

 beans, and other specialized vegetable tissues, to color in the peroxi- 

 dase test may be due to the nature of the peroxidase in these tissues 

 cr to the physical environment in which it occurs. Kegardless of the 

 explanation, it is unnecessary to blanch long enough to destroy the 

 material responsible for this coloration. A positive test due to heat- 

 stable contaminants such as the heavy metal salts can be detected by 



