Babcock and Russell (33) and Freudenreich (34) demonstrated in milk the presence of protein digesting enzymes. The bacteria found in all market milks are in many instances capable of digesting proteins. There is, therefore, abundant opportunity for milk used for the preparation of "protein-free milk" after it has been kept for an interval of 24 hours or more to contain partial digestion products of protein that would not be removable by acidifying or by coagulating. All such compounds would remain in the final product.

Osborne and Mendel felt justified by the work of Munk (35) in assuming that the 0.7 per cent of nitrogen in "protein-free milk" was not a disturbing factor in their experimental studies. Munk had, twenty years previously, sought by coagulation and by precipitation with various reagents, to determine the amount of "non-protein" nitrogen in milk (36). He arrived at the conclusion that one-fifteenth of the total nitrogen in milk is in forms other than protein. Osborne and Mendel accepted this figure as correct and reasoned as follows: A certain amount of milk contains a known amount of nitrogen, a part of which is in the form of proteins and can be removed by the methods employed. On evaporating the resulting whey to dryness the "protein-free milk" which remains contains 0.7 per cent of nitrogen in forms not known. From the weight of "protein-free milk" obtained, the total nitrogen it contained was calculated. From this amount they subtracted one-fifteenth of the total nitrogen contained in the milk with which they started. The remainder was assumed to represent "protein," or nitrogen having a nutritive value. There is no evidence in Munk's data, or in any furnished by Osborne and Mendel to show that the one-fifteenth of the total nitrogen of milk, which remained after the removal of the casein and lactalbumin, and which was disposed of by mathematical treatment of their analytical data, was not a source of amino-acids, and capable, therefore, of enhancing the nutritive value of poor proteins making them appear to have higher biological values than they possessed.

A diet such as was used in their studies, and which contained 18 per cent of added protein in purified form, together with 28 per cent of "protein-free milk," would derive 93 per cent of its total nitrogen from the former and 7 per cent from the latter source. The biological evidence is conclusive that this 7 per cent of the nitrogen of their diets supplemented certain proteins with which it was fed, in a very effective manner and led to entirely erroneous conclusions. This is well illustrated by the way in which it supplements gliadin, one of the principal proteins of wheat, and lactalbumin of milk.