At the present time, two substances are used to coagulate milk for cheese-making,—rennet extract and commercial pepsin.20 Many substances will coagulate milk, such as acids and other chemicals. Enzymes in certain plants will also coagulate it.

The curing or ripening of the cheese seems to depend on the physical and chemical properties of the curd, on the activity of certain organisms and on enzymes produced by them or in the milk. Rennet extract and pepsin are the only known substances which will produce curd of such character as will permit the desired ripening changes to take place. Until recently, rennet extract was principally used to coagulate the milk, but because of the scarcity, pepsin is now being substituted.

44. Ferments.—Many of the common changes taking place in milk are due to fermentations. The souring of milk is one of the most familiar cases of fermentation. The important change taking place is the formation of lactic acid from the milk-sugar. The change is brought about by certain living organisms, namely, the lactic acid-forming bacteria. Another familiar case of fermentation is the coagulation of milk by rennet extract or pepsin. In this case, the change is produced by a chemical substance, not a living organism. Fermentation may be defined as a chemical change of an organic compound through the action of living organisms or of chemical agents.

There are two general classes of ferments: (1) living organisms, or organized ferments; (2) chemical, or unorganized ferments. Organized ferments are living microorganisms, capable, as a result of their growth, of causing the changes. Unorganized ferments are chemical substances or ferments without life, capable of causing marked changes in many complex organic compounds, while the enzymes themselves undergo little or no change. These unorganized ferments are such as rennin, pepsin, trypsin, ptyalin. The rennet and pepsin must, therefore, be very thoroughly mixed into the milk to insure complete and uniform results, because they act by contact, and theoretically, if they could be recovered, might be used over and over again. Practically, the amount used is so small a percentage that recovery would be impractical even if possible.

45. Nature of rennet.—Two enzymes or ferments are found in rennet extract, rennin and pepsin. They are prepared from the secreting areas of living membranes of the stomachs of mammalian young. For rennet-making, these stomachs are most valuable if taken before the young have received any other feed than milk. Rennin at this stage appears to predominate over pepsin which is already secreted to some extent. With the inclusion of other feed, the secretion of pepsin comes to predominate. Rennin has never been separated entirely from pepsin. Both of these enzymes are secreted by digestive glands in the same area, perhaps even by the same glands. They are so closely related that many workers have regarded them as identical. In practical work the effectiveness of rennet preparations has been greatest when stomachs which have digested feed other than milk are excluded. The differences, therefore, however difficult to define, appear to be important in the commercial preparation of rennet.

It was the practice until a few years ago for each cheese-maker to prepare his own rennet extract. Each patron was supposed to supply so many rennets. Now commercial rennet extract and pepsin are on the market; however, some Swiss cheese-makers prefer to make their own rennet extract. For sheep's and goat's milk cheese, some makers hold that rennet made from kid or lamb stomachs is best for handling the milk of the respective species. The objection to the cheese-maker preparing his own rennet extract is that it varies in strength from batch to batch and is liable to spoil quickly. Taints and bad odors and flavors develop in it and so taint the cheese.

46. Preparation of rennet extract.—This extract may be manufactured commercially from digestive stomachs of calves, pigs or sheep. An animal is given a full meal just before slaughtering; this stimulates a large flow of the digestive juices, containing the desired enzymes.

The stomach is taken from the animal, cleaned, commonly inflated and dried. It may be held in the dry condition until needed for use. Such stomachs are usually spoken of as "rennets" in the trade. Such old rennets may be seen to-day hanging from the rafters of some of the older cheese factories. When wanted for use, rennets are placed in oak barrels and covered with water. Before placing them in the barrel, they are cut open so that the water may have easy access. Salt is usually added to the water at the rate of 3 to 5 per cent. They are stirred and pounded in this solution from five to seven days. At the end of this time, they are wrung through a clothes-wringer to remove the liquid. The rennets are put back into a fresh solution of salt and water, the object being to obtain all the digestive juices possible. They are usually soaked from four to six weeks. At the end of this time, most of the digestive juices will have been removed. The liquid portion is passed through a filter made of straw, charcoal and sand. When clean, an excess of salt is added to preserve it.

Such extracts cannot be sterilized by heat because the necessary temperature would destroy the enzyme. Effective disinfectants cannot be used in food products. The extract, therefore, should be kept cool to retard bacterial growth. The extract is kept in wooden barrels, stone jugs or yellow glass bottles to protect it from light, which is able to destroy its activity. Rennet extract should be clear, with a clean salty taste and a distinct rennet flavor. There should be no cloudy appearance and no muddy sediment in properly preserved rennet. Rennet extract is on the market in the form of a liquid and a powder, the former being much more common. The commercial forms of rennet have the advantage in the skill used in their preparation and standardization. The combined product from large numbers of stomachs may not be as effective a preparation as the most skillfully produced sample from the very choicest single stomach, but it gives a uniformity of result which improves the average product greatly.

47. Pepsin.—Pepsin is on the market in several commercial forms, as a liquid, scale pepsin and in a granular form known as spongy pepsin. Some commercial concerns put out a preparation which is a mixture of rennet extract and commercial pepsin.

48. Chemistry of curdling.—The chemistry of casein21 and of curd formation under the influence of acid and rennet extract and pepsin has been the subject of many years' research. While many points remain unsettled, the general considerations together with a large mass of accepted facts may be presented and some of the unsolved problems pointed out as left for future researches.

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."

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.

Duclaux, Émile, Action de la prÉsure sur le lait, Compt. Rend. Acad. Sci. 98, pages 526-528, 1884.

Hammarsten, Olof, Zur Kenntnis des Caseins und der Wirkung des Labfermentes, Nova. Acta Regiae Soc. Sci. Upsaliensis in Memoriam Quattuor Saec. ab Univ., Upsaliensi Peractorum, 1877.

Hammarsten, Olof, Ueber das Verhalten des Paracaseins zu dem Labenzyme, Zeit. physiol. Chem. 22, pages 103-126, 1896.

Hammarsten, Olof, A text book of physiological chemistry, from the author's 7th German edition, 1911.

Hosl, J., Unterschiede in der tryptischen und peptischen Spaltung des Caseins, Paracaseins und des Paracaseinkalkes aus Kuh- und Ziegenmilch, Inaug. Diss. Bern., 31 pp., 1910.

Kikkoji, T., Beitrage zur Kenntniss des Caseins und Paracaseins, Zeit. physiol. Chem. No. 61, pages 130-146, 1909.

Lindet, L., SolubilitÉ des albuminoides du lait dans les ÉlÉments du sÉrum; rÉtrogradation de leur solubilitÉ sous l'influence du chlorure, Bul. Soc. Chim. (ser. 4) 13, pages 929-935.

Lindet, L., Sur les ÉlÉments mineraux contenus dans la caseine du lait, Rep. Eighth Internat. Congr. of Applied Chem. 19, 199-207, 1912.

Loevenhart, A. S., Ueber die Gerinnung der Milch, Zeit. physiol. Chem. 41, pages 177-205, 1904.

Petry, Eugen, Ueber die Einwirkung des Labferments auf Kasein, Beitrage z. Chem. Physiol. u. Path. 8, pages 339-364, 1906.

Robertson, T. Brailsford, On the influence of temperature upon the solubility of casein in alkaline solutions, Jour. Biol. Chem. 5, pages 147-154, 1908.

Schmidt-Nielson, Sigval, Zur Kenntnis des Kaseins und der Labgerinnung, Upsala lÄkaref. FÖrh. (N. F.) No. 11, Suppl.

Hammarsten Festschrift No. XV, 1-26; through Jahresb. u. d. Fortschr. d. Thierchem. No. 36, pages 255-256, 1906.

Spiro, K., Beeinflussung und Natur des Labungsvorganges, Beitrage z. Chem. Physiol. u. Path. 8, pages 365-369, 1906.

Van Dam, W., Ueber die Wirkung des Labs Auf. Paracaseinkalks, Zeit. physiol. Chem. No. 61, pages 147-163, 1909.

Van Herwerden, M., Beitrag zur Kenntnis der Labwirkung auf Casein, Zeit. physiol. Chem. 52, pages 184-206, 1907.

Van Slyke, L. L., and A. W. Bosworth, I. Some of the first chemical changes in Cheddar cheese. II. The acidity of the water extract of Cheddar cheese, N. Y. Exp. Sta. Tech. Bul. 4, 1907.

Van Slyke, L. L., and A. W. Bosworth, Composition and properties of some casein and paracasein compounds and their relations to cheese, N. Y. Exp. Sta. Tech. Bul. 26, 1912.

Van Slyke, L. L., and A. W. Bosworth, Method of preparing ash-free casein and paracasein, Jour. Biol. Chem. Vol. 14, pages 203-206, 1913.

Van Slyke, L. L., and A. W. Bosworth, Preparation and composition of unsaturated or acid caseinates and paracaseinates, Ibid. Vol. 14, pages 211-225, 1913.

Van Slyke, L. L., and A. W. Bosworth, Valency of molecules and molecular weights of casein and paracasein, Ibid. Vol. 14, pages 227-230, 1913.

Van Slyke, L. L., and A. W. Bosworth, Composition and properties of the brine-soluble compounds in cheese, Jour. Biol. Chem. 14, pages 231-236, 1913.

Van Slyke, L. L., and E. B. Hart, A study of some of the salts formed by casein and paracasein with acids; their relations to American Cheddar cheese, N. Y. Exp. Sta. Bul. 214, 1902.

Van Slyke, L. L., and E. B. Hart, Casein and paracasein in some of their relations to bases and acids, American Chem. Jour. 33, pages 461-996, 1905.

Van Slyke, L. L., and E. B. Hart, Some of the relations of casein and paracasein to bases and acids, and their application to Cheddar cheese, N. Y. Exp. Sta. Bul. 261, 1905.