Casein is a white amorphous powder, practically insoluble in water. It is an acid and as such readily dissolves in solutions of the hydroxides or the carbonates of alkalies and alkaline earths by forming soluble salts.
Pure casein salt solutions and fresh milk do not coagulate on boiling, but in the presence of free acid coagulation may take place below the boiling temperature. The coagulum formed in the case of milk includes fat and calcium phosphate. The slight pellicle which coats over milk when it is warmed is of the same composition.
49. Use of acid.—A commonly accepted explanation of the precipitation of casein by acids is that the casein is held in solution by chemical union with a base (lime in the case of milk); that added acid removes the base, allowing the insoluble casein to precipitate; and that excess of acid unites with casein, forming a compound which is more or less readily soluble.
50. Robertson's theory.—According to Robertson's conception, in a soluble solution of a protein or its salt, the molecules of the protein unite with each other to a certain extent, in this way forming polymers. The reaction is reversible, and the point of equilibrium between the compound and its polymeric modification varies under the influence of whatever condition affects the concentration of the protein ions. Addition of water, or of acid, alkali or salt, or the application of heat has such an effect, and consequently alters the relative number of heavier molecule-complexes. Robertson's experiments give evidence that one of the effects of increase of temperature on a solution of casein is a shifting of the equilibrium in the direction of the higher complexes. He explains coagulation as being a result of these molecular aggregates becoming so large as to assume the properties of matter in mass and to become practically an unstable suspension and then a precipitate. The acid curd then is casein or some combination of casein with the precipitant acid.
51. Rennet curd.—Rennet extract and pepsin coagulation differs from coagulation by acids, and cannot be looked on as a simple removal of the base from a caseinate. The presence of soluble calcium salts (or other alkaline earth salts) seems to be essential, and the precipitate formed is not casein or a casein salt, but a salt of a slightly different nucleoalbumin called "paracasein." Many writers, following Halliburton, call this modification produced by rennin the "casein" and that from which it is derived, "caseinogen." Foster and a few others have used the term "tyrein" for the rennet clot.
A number of investigations have been made on the conditions essential or favorable to formation of the coagulum, especially with regard to the effects of the degree of acidity and of conditions affecting the amount of calcium present, either as free soluble salt or bound to the casein. Soluble salts of calcium, barium and strontium favor or hasten coagulation, while salts of ammonium, sodium and potassium retard or prevent coagulation.
The bulk of the coagulum from milk is a calcium paracaseinate, but it carries down with it calcium phosphate and fat, both of which bodies have been helped to remain in their state of suspension in milk by the presence of the casein salt. Lindet (1912) has concluded that about one-half of the phosphorus contained in the rennet curd is in the form of phosphate of lime (probably tricalcic), the other half being organically combined phosphoric acid.
52. Hammarsten's theory.—According to Hammarsten (1877, 1896), whose view has been commonly held, the distinctive effect of the ferment is not precipitation but the transformation of casein into paracasein. This is evidenced by the fact that if rennet be allowed to act on solutions free from lime salts no precipitate occurs; but there is an invisible alteration of the casein, for now, even if the ferment be destroyed by boiling the solution, addition of lime salts will cause immediate coagulation. (See also Spiro, 1906.) Hence the process of rennet coagulation is a two-phase process; the first phase is the transformation of casein by rennin, the second is the visible coagulation caused by lime salts.
Furthermore, if the purest casein and the purest rennin were used, Hammarsten always found after coagulation that the filtrate contained very small amounts of a protein. This protein he designated as the "whey protein."
In accordance with these observations, Hammarsten (1911) explains the rennin action "as a cleavage process, in which the chief mass of the casein, sometimes more than 90 per cent, is split off as paracasein, a body closely related to casein, and in the presence of sufficient amounts of lime salts the paracasein-lime precipitates out while the proteose-like substance (whey-protein) remains in solution."