ate, which, like the salts of other weak acids, is readily hydrolyzable, yielding a certain amount of free base, namely, calcium hydroxide, and partly on the insoluble phosphates. In the case of milk the alkalinity toward lacmoid depends on these two factors and also on the presence of diphosphates. The greater alkalinity of cow's milk depends partly on the larger quantity of insoluble phosphates present, but principally on the presence of diphosphates. As has been repeatedly shown, human milk is more alkaline than cow's milk. According to Courant, however, it, like cow's milk, is also acid to phenolphthalein and alkaline to lacmoid. In the case of cow's milk he found the ratio of alkalinity to acidity to be =2.1, and in the

case of woman's milk

1.08 0.36

=

4.1

1.95

3. According to this author the rela

tively slight acidity of woman's milk is due to the small quantity of caseinogen which it contains and also in all probability to the fact that it contains its caseinogen in the form of tricalcium caseinogenate. To return for a moment to the subject of the rennin coagulation of milk, it would seem that certain aspects of this change exhibit an analogy to the action of a toxin. It has been shown, for example, by Hammarsten and Roden (43) that normal horse serum contains a substance capable of inhibiting the action of rennin. In other words, it contains an antirennin. Similarly, by repeated injection of small amounts of rennin into the blood of animals, Morgenroth (44) obtained an antirennin. According to Fuld and Spiro (45) the antirennin of normal horse serum prevents the coagulation of milk by binding the calcium ions. Arrhenius is therefore of the opinion that in these reactions rennin corresponds to the toxophorous group, the calcium ions to the haptophorous group of a toxin, and the antirennin to an antitoxin.

Many additional facts concerning the rennin ferment are known. Like other ferments it is affected by heat, and the rate of the rennin coagulation is determined both by the quantity of rennin acting and by the temperature. It has been shown that the ferment can withstand a temperature of 180° C: without injury. At temperatures higher than 44° C. the ferment gradually loses its activity, and exposure to a temperature of 50° to 60° C. for a considerable time has been found to be more harmful than a short exposure to a higher temperature. The effect of temperature is also determined by conditions surrounding the ferment, whether it is moist or dry, and also by the reaction of the solution containing the ferment. In the dry state it can withstand a temperature of 100° to 140° C. Its destruction by heat has been found to follow the law for a monomolecular reaction.

The influence of temperature on the rennin coagulation has been studied by Fuld (46). Some of his results are given in the following

It will be observed that there is a good agreement between the observed and the calculated values up to 40° C. Above this temperature the observed values of k become smaller than the calculated values on account of the gradual destruction of rennin by heat.

In 1870 Segelke and Storch (47) showed that rennin coagulates milk in intervals which are inversely proportional to the concentration of the rennin solution. This conclusion has been confirmed by the later work of a number of observers. Thus Lörcher (48) obtained the following results from his measurements:

Recently Madsen (49) has also investigated the effect of concentration of the rennin on the rennin coagulation, working at a tem

perature of 36.55° C. The following are the results of his measure

The influence of various other factors, such as the reaction of the milk, the action of salts, the effect of ultraviolet rays, and the action of various organic substances on the rennin coagulation of milk has also been the subject of numerous investigations. It is ofttimes a difficult matter to determine whether these various influences are exercised toward the ferment itself or whether they react on the milk or participate only in the second phase of the rennin coagulation. The further consideration of such agents is beyond the scope of this communication. Finally, it should be observed that Hillman (50) has studied the rennin coagulation of milk in its practical aspects. This author has found that the milk of fresh cows is better suited to the rennin coagulation than the milk of cows which are nearly dry. In his opinion this is probably to be explained by the diminution in the calcium content of milk during the period of lactation. He found, further, that the degree of acidity of milk in relation to the calcium content is an important factor. According to this author a high calcium content and high acidity prevail at the beginning of lactation and are usually accompanied by high total albumin and a high caseinogen content, all of which conduce to a large yield of paracasein. He also finds that the time of coagulation and the yield of paracasein are independent of one another; generally, however, a short coagulation time and a large yield of paracasein are associated. Strong dilution of the milk with water tends to diminish the yield of paracasein, whereas the addition of soluble calcium salts tends to increase it.

According to this author the action of rennin consists not only in the splitting of caseinogen into paracasein and whey proteid, but also in the conversion of other milk proteids into more soluble form. He seems to think that under favorable conditions paracasein may be formed from the albumin as well as from the caseinogen.

PART II-(4a) CHEMICAL CHANGES IN MILK PRODUCED BY BACTERIA

AND VARIOUS OTHER MICRO-ORGANISMS.

The more obvious changes in milk with which we are familiar are those that are brought about by bacteria and various other micro-organisms. Among these changes may be mentioned: The ordinary souring and curdling of milk, with the production of lactic acid as the chief product; the production in milk of various odoriferous substances, or highly flavored substances, many of a somewhat disagreeable character, good examples being met with in the ripening of cream and cheese; the production of colored substances which impart to the milk unusual colors, such as the formation of blue milk; the formation of mucilaginous, or mucin-like substances, which serve to impart to the milk a characteristic ropiness, known as ropy milk, and finally we must include under this head those bacterial changes in milk which result in the formation of poisonous substances, such as tyrotoxicon, toxins, etc.

The lactic acid fermentation of milk. The lactic acid fermentation is the commonest and best known of all the many bacterial changes that occur in milk. The fact that on standing at ordinary temperatures milk gradually turns sour and finally curdles has been known ever since milk was first used as a food by man. In early times the acid of milk was supposed to be acetic acid, the same as is present in vinegar, and as has already been pointed out this acid does, according to Béchamp (1), occur in even freshly drawn milk in small quantities. The substance really responsible for the souring of milk, viz, lactic acid, was first discovered in milk by Scheele in 1780. The new acid was also studied by Berzelius and its composition definitely established through the work of Mitscherlich and Liebig in 1832. Its chemical constitution and its relation to other varieties of lactic acid, occurring in nature or the products of chemical synthesis, were first established as the result of the labors of Strecker, Erlenmeyer, and Wislecenus.

In 1847, Blondeau (2) discovered micro-organisms is sour milk, but attached to these no particular significance so far as the souring of milk is concerned. It remained for Pasteur (3), in 1857, to definitely and conclusively show as one of the results of his classic investigations on fermentation that the souring of milk is really a kind of fermentation, 24907-Bull. 41-08- -23

which is accomplished by a peculiar kind of micro-organism, to which he gave the name of levure lactique (lactic yeast). His first communication on this subject was read to the Scientific Society of Lille, August, 1857, and afterwards to the French Academy in November, 1857. Since then our knowledge of the lactic acid fermentation has been considerably extended through the labors of Pasteur's students and still later through the work of other bacteriologists and chemists. For example, Boutroux (4) in 1878, in continuing the investigations of Pasteur on the souring of milk, arrived at the conclusion that the lactic acid ferment and the mycoderma aceti, which is concerned in the transformation of alcohol into acetic acid in vinegar making, are identical, but that these vary in function, depending on their general environment and the composition of the liquid in which they grow. This communication also contains a description of the lactic ferment and an enumeration of its morphological characteristics, which are beyond the scope of the present communication. He observed that the organism grew best in a nutrient medium containing, besides albuminous matter, invert-sugar or glucose. He also found that under these conditions the liquid can attain a maximum acidity of 1.5 per cent lactic acid. Larger amounts of acid than this checked the life and growth of the organism, and hence if it is desired to convert all of the sugar into lactic acid the acid must be neutralized with chalk or zinc carbonate as fast as formed. Under proper conditions the lactic acid organism employed by Boutroux produces lactic acid only.

The lactic acid fermentation of milk sugar was also investigated by Richet (5), who found that when milk is kept at 40° C., it becomes acid and coagulates and finally attains an acidity of 1.6 per cent, which amount it never exceeds. He made the further interesting observation that if gastric juice be added to milk the casein is coagulated and finally dissolved, and in less than twenty-four hours the milk contains a larger quantity of lactic acid than otherwise would have been present in a week, and after four or five days as much as 4 per cent of lactic acid was formed. He observed that while neither a pure solution of lactose nor gastric juice will ferment, if the two be mixed fermentation takes place; and that the casein of milk after it has been dissolved by gastric juice also ferments, yielding lactic and butryic acids, besides other products of fermentation. On the other hand the whey of milk obtained by coagulation with rennin never attains an acidity higher than 1.6 per cent of lactic acid, even after having been kept for six months. He found that the lactic acid fermentation is increased by exposing a large surface of the milk to the air. The activity of the ferment increases up to 44° C., remains constant between 44 and 52° C., and above 52° C. diminishes in activity as the temperature rises. Digestive juices and peptones