on this particular phase of the subject a matter of considerable importance. During our recent investigations of the Washington milk supply we have made the following observations on the coagulation of slightly sour milk at or below boiling:

It will be seen from our results that of those samples of milk which coagulated on heating, sample 15 C. contained the smallest amount of acid at the time of coagulation, viz, 0.225 per cent. (See observation 21.) This sample curdled in two minutes at 65°-67° C. Other samples containing from 0.306 to 0.315 per cent of acid curdled at temperatures varying from 65°-78.5° C., in from two to ten minutes, and as a general rule those milks are most easily coagulated by heat which have the highest acidity. On the other hand, while one of the samples having an acidity of only 0.225 per cent curdled at 65°-67° C., in two minutes, another sample containing 0.243 per cent of acid did not curdle even on boiling. Thörner (15) found the acidity necessary

to coagulate milk on heating to be 0.207 per cent. On the other hand, Rideal (16) found the amount of acid required to effect the coagulation to be somewhat higher than this. According to him the tendency to coagulate is not very marked even when the acidity is as high as 0.23 per cent. Rideal's observations agree very nearly with

our own.

According to Stokes (3) milk which has an acidity somewhat less than 0.3 per cent of lactic acid will coagulate on boiling. He records the fact, however, that 3 samples of milk containing as much as 0.54 per cent of lactic acid did not coagulate on boiling.

Richmond (2) has been able to confirm Stokes's results almost absolutely. He found fresh milk to have an acidity of 20 degrees, corresponding to 0.18 per cent lactic acid. According to him milk curdles on boiling when it has an acidity of 33 degrees, corresponding to 0.297 per cent of lactic acid.

It is evident therefore from our results and those obtained by other observers that the coagulation of milk is dependent on several factors, among which are: time, temperature, degree of acidity, quantity and solubility of the calcium salts, etc.; and that in order to avoid accidents resulting from curdling in the pasteurization of milk the only safe rule to follow is to determine the effect of heat on small samples of the milk which it is proposed to pasteurize, or better still, to pasteurize the milk as soon as it is drawn from the cow.

Another important change in milk effected by heat is the destruction of the bacteria and other micro-organisms normally present in fresh milk, including of course those pathogenic forms which frequently gain access to milk and cause the spread of infections through this medium or which give rise directly to highly poisonous substances. For obvious reasons therefore this phase of the subject, namely, the pasteurization of milk, has received a great deal of attention during recent years at the hands of dairymen and sanitarians. It is foreign to the immediate scope of this communication, however, to enter upon a discussion of this subject. Suffice it to say in this connection that pasteurization either checks or hinders those changes which occur in milk as the result of the life and growth therein of micro-organisms, and affords more or less adequate protection against the spread of microbic diseases through the medium of milk. According to Pasteur (17) milk can be sterilized by heating it to about 110° C and Duclaux kept milk five years by heating it to 120° C. and preserving it in vessels which had been exhausted of air.

Effect of heat on milk enzymes.-Another less obvious change in milk which is brought about by the action of heat is the destruction of the enzymes normally present in fresh milk. Like all enzymes those contained in fresh milk are destroyed by boiling or by heating

the milk above certain temperatures. On account of the important bearing which the thermal death point of milk ferments may have on the pasteurization of milk the following data relative to this subject are here submitted:

Galactase. According to Babcock and Russell (18), the destruction temperature of galactase, the proteolytic enzyme of milk, is somewhat higher than the thermal death point of trypsin. So far as its proteolytic activity is concerned, these observers found it to be weakened by heating for ten minutes at 76°-80° C. That such is the case may be seen from the following table, in which is shown the percentage of soluble nitrogen which milks which had been heated for ten minutes at the temperature indicated, contained at the end of fifty-three days.

a The soluble nitrogen originally present in the milk used in these experiments was found to be 0.05 per cent.

Similar tests were made upon the proteolytic activity toward gelatin of galactase which had been heated for ten minutes at different temperatures, using Fermi's (19) method. Equal amounts of the galactase solution were poured over the surface of carbolized gelatin contained in test tubes and kept under observation seven weeks. The results of this series of experiments is given in Table II.

TABLE II.

[++=rapid digestion; +=digestion by galactase; --no digestion.]

Hence so far as its power to digest gelatin is concerned the activity of galactase is destroyed in alkaline solution by ten minutes' exposure to temperatures of 65° C. in neutral solutions at 70° C. and in acid solutions at a temperature of 65° C.

Babcock and Russell (18) have also employed the power on the part of galactase to accelerate oxidation by hydrogen peroxide as a test for the presence of the ferment, and have made use of this test as a means of determining the destruction temperature of galactase. This we now know is merely a test and measure of the peroxidase activity of the solution and not a test or measure of the activity of galactase at all. Indeed, as has been shown subsequently to the work of Babcock and Russell on this subject, galactase as prepared from separator slime is not a pure enzyme, but a mixture of enzymes. Thus Wender (20) points out that galactase consists of milk trypsin or galactase proper, milk catalase, and milk peroxidase. According to Wender, the trypsin of milk becomes inactive at 76° C.

The use of V. Storch's tests (see p. 330), viz, with an iodide and starch or p-phenylene-diamine and a few drops of hydrogen peroxide as a means of determining the effect of high temperature on the activity of galactase, as employed by Babcock and Russell, is therefore chiefly interesting as throwing light on the stability of milk peroxidase under the conditions employed in their work. Their results are given in Table III.

Babcock and Russell (18) conclude therefore from their experiments that galactase is more stable in alkaline or neutral solution, and that it shows a close resemblance to trypsin, but is less sensitive to

acids than the latter ferment. The heat boundary of its activity is influenced by the chemical reaction of the solution in which it is present, being lower in acid than in neutral or alkaline solution. When galactase that has been heated to 70° C. for ten minutes is added to milk, the digestion is slowed, and heating to 76° C. for ten minutes entirely destroys its digestive powers. In the Fermi (19) gelatin tests no digestion took place with specimens of the ferment that had been heated to 65° C., whereas with the unheated controls, the gelatin liquefied.

Von Freudenreich (21) has confirmed the observations of Babcock and Russell on galactase. According to this observer a temperature of 75° C. for half an hour causes a falling off in the proteolytic activity of this enzyme. On the other hand, he found an exposure to 60° C. for half an hour to scarcely weaken it to a noticeable degree. According to Hippius (22) the proteolytic ferments of milk can withstand an exposure to 60° C. for one hour and an exposure to 65° C. for half an hour.

Milk amylase, according to this author, can withstand a temperature of 60° for one hour, but is destroyed by heating above 75° C.

Milk lipase. Gillet (23) has found that milk lipase is destroyed at 65° C. According to Hippius (22) the lipase of woman's milk can withstand a temperature of 60° for one hour and a short exposure to 62°, but is weakened at 63° C., and is destroyed at 64° C.

The salol-splitting ferment (Nobécourt and Merklen) is, according to Hippius (22), destroyed above 65°C. However, the existence of this ferment is doubtful. (See p. 338.)

Hougardy (24) has found that the activity of lactokinase is greatly enfeebled by heating for twenty minutes at 75° C. and practically destroyed by heating at 75° C. for half an hour.

THE OXIDIZING FERMENTS OF MILK.

While our knowledge of the soluble ferments is of too recent date for an exact understanding of their significance and powers of resistance, the well-known reactions of the oxidases have furnished us with a useful criterion for distinguishing between living (raw) and heated milk (Zelinski 25).

According to Marfan (see Zelinski (25)) the oxidases of milk are destroyed at 79° C. According to Hippius (22) they can withstand a temperature of 60° C. to 65° C., but are destroyed by a short exposure to 76° C.

Peroxidases. According to Wender (20) the peroxidase of milk becomes inactive at 83° C. According to Schardinger (26), using methylene blue as a reagent, the critical temperature of the milk