By continued action of rennin on paracasein, a further transformation has been found in several cases (Petry, 1906; Van Herwerden, 1907; Van Dam, 1909), but perhaps due to a contamination of the rennin with pepsin, or to the identity of these two enzymes. The action which forms paracasein and whey-protein takes place in a short time (Hammarsten, 1896; Schmidt-Nielson, 1906). The composition and solubilities of paracasein have received considerable attention. (See Loevenhart, 1904; Kikkoji, 1909; Van Slyke and Bosworth, 1912.) It is more readily digested by pepsin-hydrochloric acid than is casein (Hosl, 1910).
53. Duclaux theory.—Duclaux (1884) and Loevenhart (1904) and others do not accept Hammarsten's theory; but to most workers it seems probable, at least, that the action of the rennin is to cause a cleavage of casein with formation of paracasein. However, the chemical and physical differences observed between casein and paracasein appear to be so slight that Loevenhart and some others think that they are only physical, perhaps differences in the size of the colloid or solution aggregates. Loevenhart conceives of a large part of the work of the rennet (or of the acid, in acid and heat coagulation) as being a freeing of the calcium to make it available for precipitation. Some think that the aggregates of paracasein are larger than those of casein, but there is more evidence of their being smaller, which idea corresponds with the findings of Bosworth, though he looks on the change as a true cleavage.
54. Bang's theory.—Another description of the precipitation is given by Bang (1911), who studied the progress of the coagulation process by means of interruptions at definite intervals. His observations confirm the idea that rennin causes the formation of paracasein, and that the calcium salt serves only for the precipitation of the paracasein; the rennin has to do also with the mobilizing of lime salts. According to Bang, before coagulation occurs, paracaseins with constantly greater affinity for calcium phosphate are produced. These take up increasing amounts of calcium phosphate, until finally the combination formed can no longer remain in solution.
55. Bosworth's theory.—By a very recent work of L. L. Van Slyke and A. W. Bosworth (Van Slyke and Bosworth, 1912, 1913; and Bosworth and Van Slyke, 1913), in which ash-free casein and paracasein were compared as to their elementary composition, and as to the salts they form with bases, and the properties of these salts, it is indicated that the two compounds are alike in percentage composition and in combining equivalent, the paracasein molecule being one-half of the casein molecule. Moreover, Bosworth (1913) has shown that, if the rennin cleavage be carried out under conditions which avoid autohydrolysis, no other protein is formed; also that, if the calcium caseinate present be one containing four equivalents of calcium, the paracaseinate does not precipitate, save in the presence of a soluble calcium salt, while, if the calcium caseinate be one of two equivalents of base, rennin does cause immediate coagulation. Bosworth concludes that the rennin action is a cleavage (probably hydrolytic) of a molecule of caseinate into two molecules of paracaseinate, the coagulation being a secondary effect due to a change in solubilities, dicalcium paracaseinate being soluble in pure water but not in water containing more than a trace of calcium salt, and the monocalcium caseinate being insoluble in water. The alkali paracaseinates, as well as caseinates, are soluble. This explanation seems to promise to harmonize the observations with regard to acidity and the effects of the presence of soluble salts. This theory represents, therefore, many years of continuous work at the New York Experiment Station centered primarily on American Cheddar cheese. Disputed points remain for further study but these workers have contributed much toward a clear description of the chemical constitution of casein as affected by rennet action and bacterial activity.
The investigations of these authors and of Hart with regard to the changes which the paracasein, the calcium and the phosphorus undergo during the ripening of cheese (Van Slyke and Hart, 1902, 1905; Van Slyke and Bosworth, 1907, 1913; Bosworth, 1907) contributed to this interpretation.
Bang, Ivar, Ueber die chemische Vorgang bei der Milchgerinnung durch Lab, Skand. Arch. Physiol. 25, pages 105-144; through Jahresb. u. d. Fortsch. d. Thierchem. 41, pages 221-222, 1911.
Bosworth, A. W., The action of rennin on casein, N. Y. Exp. Sta. Tech. Bul. 31, 1913.
Bosworth, A. W., Chemical studies of Camembert cheese, N. Y. Exp. Sta. Tech. Bul. 5, 1907.
Bosworth, A. W., and L. L. Van Slyke, Preparation and composition of basic calcium caseinate and paracaseinate, Jour. Biol. Chem. Vol. 14, pages 207-210, 1913.