Under certain conditions, the addition of alcohol as well as the application of heat may cause coagulation of milk. Milk may be coagulated by the addition of rennin or by bringing the acidity of the milk to the isoelectric point of the casein. When milk is combined with other foods, the salt content of the food or the tannin content of the food may be factors that aid coagulation.

Alcohol coagulation. The addition of 70 per cent alcohol to milk may cause coagulation. As the pH of the milk decreases, it becomes susceptible to alcohol precipitation, though this varies with different milks. Usually the milk is precipitated by alcohol while it is still stable to heat and sterilizing temperatures. Some freshly secreted milk is coagulated by alcohol, but this milk is usually abnormal in some way. About 2 cc. of alcohol are added to an equal quantity of milk for the test. Casein is precipitated by alcohol as calcium caseinate, calcium is not released as by acid coagulation.

Rennet coagulation. Milk may be coagulated by the addition of rennet. Rennet is an extract that is usually obtained from the inner lining of the stomachs of calves and lambs. The rennet contains an enzyme called rennase or rennin.

The clotting of the milk is generally believed to be the direct action of the rennin on the casein. But the manner in which these changes is produced is not fully understood. A very small amount of rennin is capable of coagulating a large amount of milk. At favorable or optimum hydrogen-ion concentrations for clotting, 1 part of the fairly pure enzyme preparation is able to coagulate 3,000,000 or more parts of milk.

Mechanism of clotting. It is usually stated that the casein is changed to paracasein by the action of the rennin. It is also often stated that the clotting is brought about in two steps, the first being the action of rennin on the casein and the second the precipitation of the changed casein. Rogers reviews the many theories of rennin coagulation. Some investigators claim the changes are purely chemical; others maintain the rennin affects only the physical state of the calcium caseinate. However, if the change can be explained on the basis of colloid chemistry, it is probable that absorption and the electric charge play an important role in the process. Rogers states that Hammarsten regards casein in milk as a calcium caseinate-calcium phosphate complex. "As a matter of fact the compound called calcium caseinate is most probably a true calcium phosphocaseinate, if, as seems likely, the second and third hydrogens of the orthophosphoric acid esterfied with certain of the amino acids in the casein molecule react with calcium. The correct conception of the term 'calcium phosphocaseinate,' as it is now commonly employed, is that of a colloidal calcium phosphate (or phosphates) sol protected by a calcium caseinate (or caseinates) sol in a manner as yet imperfectly understood." The stabilization of sols is best explained by the theory of Helmholtz, i.e., each colloidal particle is surrounded by an electrical double layer. "In the case of negatively charged sols, in which class calcium caseinate and calcium paracaseinate evidently fall, the outer layer consists of hydrogen ions. If these are replaced by a sufficient number of positively charged ions of higher charge, e.g., calcium ions carrying two positive charges"; or, in other words, if these ions are more strongly adsorbed than the hydrogen ions, the colloid particle will readily precipitate, the rate of clotting being determined by the rate of replacement.

Richardson and Palmer state that rennin itself may reduce the charge of the calcium caseinate micelle and thus reduce the stability of the casein sol. They found indications that the isoelectric point of rennin is about pH 6.9 to 7.0. Above this pH the rennin is negatively charged and below pH 6.9 it is positively charged. They found that rennin lowered the electro-phoretic velocity of calcium caseinate and calcium phosphocaseinate micelles when the casein sol was negatively charged and the rennin was positively charged, but not when the rennin was negatively charged (above its isoelectric point, pH 6.9 to 7.0). From this evidence and from the fact that paracaseinate micelles are not affected by rennin, which agrees with the fact that casein once coagulated by rennin has lost its sensitiveness to this enzyme, they suggest that rennin acts by sensitizing the casein by a preliminary reduction of the electric charge on the casein micelles.

During the clotting of the milk, aside from the consistency of the milk, there is little change in its physical properties. The hydrogen-ion concentration does not change during the clotting process.

Factors affecting action of rennin. Several factors influence the activity of the rennin in bringing about coagulation. These may be listed as follows: (1) temperature for rennin action; (2) heating the milk before the addition of rennin; (3) hydrogen-ion concentration; (4) concentration of casein, calcium, and phosphate ion; (5) character of cations used for coagulation.

Temperature for rennin action. The optimum coagulation by calf rennin is about 40° to 42°C. Below this temperature coagulation is less rapid and no clotting occurs below 10° to 15°C. Also no clotting occurs above 60° to 65°C. The clot is softer at low temperatures and tougher and stringy at high temperatures. By optimum is meant the temperature at which coagulation takes place most rapidly for a definite concentration of rennin and milk.

Effect of previously boiling the milk upon rennin coagulation. If milk is boiled and then cooled before the rennin is added, the rate of coagulation is retarded and a much softer, more flocculent clot is obtained. Pasteurization also affects the rate of coagulation of the milk and the type of clot formed by rennin but not to the extent that boiling does.

Richardson and Palmer found by electrokinetic evidence that heat increased the electric charge on the casein micelles or the cataphoretic velocity of the casein solution. The fact that rennin does not form as firm a clot with milk that has been previously heated indicates that rennin reduces the charge on the casein particles but not sufficiently to form a firm clot. This offers a colloidal explanation of why the addition of active cations (as calcium chloride) to heated milk causes the rennin to coagulate the milk normally.