Issued April 22, 1913. ?3={ U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN 166. A. D. MELVIM, CHIEF OK BUREAU. -JEMICAL CHANGES PRODUCED IN COWS 1 MILK BY PASTEURIZAlQfJjajR *^TJ *C of California i Regional ^ Facility BY PHILIP RUPP, M. D., PH. D., Chemist, Dairy Division. AUG 7 ir WASHINGTON: GOVERNMENT PRINTING OFFICE. Issued April 22, 1913. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN 166. A. D. MELV1N, CHIEF OF BUREAU. CHEMICAL CHANGES PRODUCED IN COWS' MILK BY PASTEURIZATION. BY PHILIP RUPP, M. D., PH. D. Chemist, Dairy Division. WASHINGTON: GOVERNMENT* PRINTING OFFICE. 1913. BUREAU OF ANIMAL INDUSTRY. Chief: A. D. MELVIN. Assistant Chief: A. M. FARRINGTON. Chief Clerk: CHARLES C. CARROLL. Animal Husbandry Division: GEORGE M. ROMMEL, chief. Biochemic Division: M. DORSET, chief. Dairy Division: B. H. RAWL, chief. Field Inspection Division: R. A. RAMSAY, chief. Meat Inspection Division: R. P. STEDDOM, chief. Pathological Division: JOHN R. MOHLER, chief. Quarantine Division: RICHARD W. HICKMAN, chief. Zoological Division: B. H. RANSOM, chief. Experiment Station: E. C. SCHROEDER, superintendent. Editor: JAMES M. PICKENS. DAIRY DIVISION. B. H. RAWL, Chief. HELMER RABILD, in charge of Dairy Farming Investigations. S. C. THOMPSON, in charge of Dairy Manufacturing Investigations. L. A. ROGERS, in charge of Research Laboratories. ERNEST KELLY, in charge of Market Milk Investigations. ROBERT McAoAM, in charge of Renovated Butter Inspection. 2 LETTER OF TRANSMITTAL. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY, Washington, I). C., January 30, 1913. SIR: I have the honor to transmit for publication as a bulletin of this bureau the accompanying manuscript, entitled "Chemical Changes Produced in Cows' Milk by Pasteurization," by Dr. Philip Hupp, chemist in the Dairy Division of this bureau. The paper de- scribes a series of experiments made with the object of determining the effect of pasteurization at various temperatures on the chemical composition of cows' milk. The work was undertaken because of certain objections which are made against pasteurization, it having been asserted that the process injures the milk from a digestive and nutritive standpoint, particularly as a food for infants. It is satis- factory to note from the results of the work that, so far as the ordi- nary temperatures used in commercial pasteurization are concerned, these objections are not well founded. The method of filtering the milk necessary for the carrying on of the tests consists of a new application by Dr. Hupp of the clay -cell filtration used by other scientists. This method is fully described and illustrated by a diagram. Respectfully, A. D. MELVIN, Chief of Bureau. Hon. JAMES WILSON, Secretary of Agriculture. CONTENTS. Page. Introduction 5 Method of pasteurization employed 5 Chemical changes in the phosphates 6 Method of filtration 6 Methods of analysis 7 Changes in the composition of the serum during filtration. 8 Chemical changes in the proteins 11 Lactalbumin 11 Methods of analysis 11 Casein 13 Method of coagulation 14 Changes in the acidity 15 Summary and conclusions 15 ILLUSTRATION. Page. Fia. 1. Apparatus for filtering milk 7 4 CHEMICAL CHANGES PRODUCED IN COWS' MILK BY PASTEURIZATION, INTRODUCTION. One of the objections frequently raised against pasteurized milk is that the heating produces changes in the chemical composition which make it unsuitable for infant nourishment. It is claimed that a portion of the soluble phosphate of lime becomes insoluble and that this change produces defective nutrition, while the changes in the lactalbumin and hi the casein render the pasteurized milk more indigestible than raw milk, and therefore inferior as a food. These objections were brought forward at a time when high temperatures were employed in the sterilization of infants' milk, and they are made use of at the present day by the opponents of pasteurization, even though the temperatures used in pasteurizing are considerably lower. The objects of the work herein described were to study the chemical changes produced by the heating of milk at the different temperatures used in commercial pasteurization and to determine whether a tem- perature that destroys all pathogenic germs can be used in pasteuri- zation without having any appreciable effect on the chemical compo- sition of the milk. Tliree points are considered: First, the changes in the phosphates are discussed in order to show to what extent the soluble phosphate of lime becomes insoluble. Second, the proteins are examined to determine to what extent the albumin is precipi- tated and the casein changed in its characteristic property of coagu- lating with rennin. Finally, the acidity of the milk is considered. METHOD OF PASTEURIZATION EMPLOYED. The milk was pasteurized in a constant-temperature bath. The water having reached the required temperature, the cool milk, in an Erlenmeyer flask closed with a valve to prevent evaporation, was placed in the water and occasionally moved about until it had ac- quired the temperature of the water. This usually required from 15 to 20 minutes, according to the height of the temperature. It was then allowed to remain for 30 minutes, after which it was rapidly cooled to about 12 C. (53 F.). The milk used was of very good quality and had a very low bacterial count. The variation in tem- perature during pasteurization was less than 0.5 C. (0.9 F.). 5 6 CHEMICAL CHANGES PRODUCED BY PASTEURIZATION. CHEMICAL CHANGES IN THE PHOSPHATES. The vast majority of the experiments which show a diminution in the quantity of soluble calcium salts in heated milk as compared with raw milk refer to milk which has been heated to the boiling point. But few definite results can be found showing a diminution of the soluble calcium phosphate in pasteurized milk. Diffloth l found a decrease in the soluble phosphates, amounting to a loss of 25.9 per cent, on heating milk to 60 C. (140 F.) for 30 minutes, while Babcock 2 believes that a change in the solubility of the limp, salts is brought about when the milk is pasteurized, and bases his conclusion on the less ready coagulability of the milk by rennet. In order to determine the effect of pasteurization on the solubility of the phosphates in the milk, the serum of both raw and pasteurized milk was examined. METHOD OF FILTRATION. A new application of the clay-cell filtration as used by Soxhlet * was employed. The clay cell of a galvanic element is thoroughly cleansed by boiling in dilute caustic soda and then washed by filter- ing hot water through the cell by suction. The cell is next boiled in dilute hydrochloric acid and again washed by suction with hot dis- tilled water until the filtrate no longer gives a reaction for chlorin. The method of filtration is easily understood by a description of the accompanying diagram (figure 1). An hi verted bell jar (a) (9 inches high and 4 inches in diameter) is covered with a perforated glass plate (6) holding a tube containing a plug of cotton to admit air. Inside the jar is a porous clay cell (c) of a galvanic element (5 inches high, 2J inches in diameter, and the cell wall one-eighth of an inch thick) the opening of which is covered with a piece of rubber dam (d) of medium thickness and supported on a heavy glass plate (e). Both the rubber dam and the glass plate are perforated for the passage of a rubber stopper containing a glass tube with a Geissler stopcock (/). The upper end of the tube is flush with the glass plate e, while the lower end passes into a filtering flask (g) of 750 c. c. capacity. A glass tube with a two-way stopcock (h) is attached to the outlet of the flask, and this in turn is connected with the suction apparatus by means of a heavy rubber tube. In order to obtain airtight joints a little stopcock grease is placed on the glass plate, and the edge of the clay cell is also coated with a thin layer. The cover (6) of the bell jar is held down in the same manner. 1 Diffloth, Paul. Du r&le de quelqucs agents physiques et chimiques dans 1'insolubilisation des phosphates du lait. Bulletin des Sciences Pharmacologiques, vol. 10, p. 273-279. Paris, 1904. Abstract: Zeitschrift ttir Untersuchung der Nahrungs- und Genussmittel, vol. 11, no. 7, p. 455-456. Berlin, Apr. 15, 1906. * Babcock, S. M. The centrifugal separation of casein and insoluble phosphates from milk. Wisconsin Agricultural Experiment Station, Twelfth Annual Report (1895), p. 93-99. Madison, 1896. Soxhlet, Franz. Beitrage zur physiologischen Chemie der Milch. Journal fur Praktische Chemie, new series, vol. 6, no. 1/2, p. 1-52. Leipsic, Aug. 24, 1873. CHEMICAL CHANGES IN THE PHOSPHATES. To begin filtration remove the cover, evacuate the apparatus, and while the suction is still on fill the bell-jar with milk. Then cut off the suction at h, replace the cover, and remove the suction tube. The apparatus is now ready to be placed in the refrigerator. The advantage of this method over those previously employed is that the filtration can be continued indefinitely and the serum removed at any time without interfering with the filtration. Further- more, no concentration of the serum can take place, as evaporation is excluded. The serum can be removed at any time by closing the stopcock /, admitting air by opening 7i, and removing the filtering flask. Then replace the flask, evacuate, close the stop- cock h, and open/, and the apparatus is ready for further filtration. METHODS OF ANALYSIS. Weigh about 40 grams of milk (equiva- lent to about 0.1 gram of phosphoric acid), evaporate to dryness, and ignite at a low red heat until the ash is free from carbon. Dissolve the ash in hot water containing a little hydrochloric acid and dilute to about 100 c. c. Basic acetate precipitation. Almost neutralize the solution of the ash with ammonia, add 2 c. c. of dilute acetic acid and a slight excess of ferric chlorid solu- tion of known strength (7.5 c. c. of a 10 per cent solution). Now add 13 to 14 c. c. of a 10 per cent ammonium acetate solution, dilute to 350 to 400 <. c. and boil for about one-half to one minute, stirring occasionally to prevent bumping. If the quantity of ferric chlorid added was sufficient the precipitate will be brownish- red, and the precipitation will be complete as the boiling point is reached if the acetate was added in sufficient quantity. Filter boil- ing hot through two 12.5 cm. filters and wash immediately with a hot dilute ammonium acetate solution (one-half to one per cent), using a fine jet which is played around the edge of the precipitate, thus cut- ting it free from the paper in order to produce rapid filtration. Do not allow to drain dry. Wash the precipitate in this manner three to four times until free from chlorids, return to the original beaker with hot water, and dissolve by the addition of nitric acid. When FIG. 1. Apparatus for filtning milk. 8 CHEMICAL CHANGES PRODUCED BY PASTEURIZATION. the basic acetate precipitation is carried out as described above the filtration is always rapid and the precipitate never runs through the filter. In washing the precipitate with hot water, some flakes gen- erally appear on concentrating the filtrate and washings. This, however, is not the case if a hot dilute solution of ammonium acetate is used. The phosphoric acid in the basic acetate precipitate was determined by the molybdate method and weighed as magnesium pyrophosphate. The filtrate and washings from the basic acetate precipitation were concentrated and the calcium precipitated with ammonium oxalate. The oxalate was redissolved, reprecipitated, and weighed as calcium oxid. The nitrate and washings from calcium precipitation were evaporated, ammonium salts removed, and the magnesium deter- mined as pyrophosphate. For serum analysis evaporate about 70 grams and ignite at a dull red heat until the ash is free from carbon. In the basic acetate pre- cipitation use 6 c. c. of ferric chlorid solution and 9 to 10 c. c. of ammonium acetate solution. The casein phosphorus was determined by Neumann's method, 1 using, however, only 10 c. c. of concentrated sulphuric acid. The total phosphorus in the milk was also determined by Neumann's method. The specific gravity was taken by means of a picnometer. CHANGES IN THE COMPOSITION OF THE SERUM DURING FILTRATION. De Vries and Boekhout, 2 using a Chamberland filter and pressure, found that the quantity of calcium in the serum did not vary in differ- ent fractions during the filtration, the first 20 c. c. of serum containing the same quantity of calcium as the second and third. In order to determine whether the serum remains of constant composition during filtration and for what length of time milk can be filtered at a tem- perature of 5 to 8 C. (41 to 46. 4 F.) without changing its composi- tion, skim milk was filtered and the serum analyzed during a period of two weeks. Skim milk was used for this experiment because it niters more rapidly than whole milk. The results are shown hi Table 1. i Neumann, Albert. Einfache Veraschungsmethode (Sauregenusch-Veraschung). Zeitschrift ftir Physiologische Chemie, vol. 37, no. 2, p. 116-142. Strasburg, Dec. 20, 1902. * Ott de Vries, J. J., and Boekhout, F. W. J. Beitrag zur Kenntnis der Labgerinnung. Die Land- wirtschaftlichen Versuchs-Stationen, vol. 55, no. 3, p. 221-239. Berlin, Feb. 12, 1901. CHEMICAL CHANGES IN THE PHOSPHATES. 9 TABLE 1. Quantity of soluble phosphorus in the serum on different days during the filtration of raw skim milk, separated at 30 C. (86 3 F.). Quantity. Specific gravity. Acidity.' P S 8 . CaO. MgO. Casein Grams. Per &nt. 0.0638 Per cent. Per cent. Raw skim milk 1.034 17.8 .2352 0. 1671 0.0185 Serum: First day 3 hours 34.3 1.023 .0753 .0450 .0133 First day next 18 hours 81.5 1.025 8.8 .1029 .0554 .0133 Second day .... 109 8 1.025 9.0 . 1054 0581 .0134 Do .1055 .0588 .0133 Third day 109.3 1.025 9.0 .1055 .0578 .0127 Fourth day 112.0 1.025 9.0 .1048 .0585 .0127 Fifth day... . 105 7 1.025 9.0 . 1054 .0589 .0130 Sixth and seventh days. . . 205.0 1.025 9.0 . 1057 .0579 .0129 Do .1053 .0582 .0131 Tenth and eleventh days 155.0 1.025 10.0 .1074 .0609 .0131 Thirteenth and fourteenth davs 158.5 1.025 12.0 . 1082 .0633 .0139 Fifteenth and part of sixteenth day 105.0 14.6 .1110 .0683 .0145 1 Number of cubic centimeters of alkali required to neutralize 100 c. c. of serum. As will be seen from the above table there is an absorption of both phosphoric acid and calcium at the beginning of filtration, while the magnesium passes completely through the cell wall. For this reason the first portion of the serum must be rejected as worthless for making comparisons. The quantity to be rejected varies with the size of the cell used and may amount to from 60 to 90 c. c. The specific gravity of the serum collected during the first 3 hours was 1.023, and it increased to 1.025 during the next 18 hours, after which time it remained constant during the remainder of the experi- ment. The acidity was 8.8 on the first day and 9 on the second day. After the ninth day the acidity began to increase gradually, being 10 on the tenth and eleventh days, while on the fifteenth day it was 14.6, showing that the lactic acid had considerably increased. The phosphoric acid during the first 3 hours amounted to only 0.0753 per cent, and it increased to 0.1029 per cent during the next 18 hours. On the second day it was 0.1054 per cent, and then re- mained constant until the tenth and eleventh days, when it began gradually to increase, amounting to 0. 1 1 1 per cent on the fifteenth day. The calcium content was also low during the first 3 hours of filtra- tion, amounting to 0.045 per cent, and it increased to 0.0554 per cent during the next 18 hours. On the second day it was 0.0581 per cent, and began to increase gradually as the acidity increased, amounting to 0.0683 per cent on the fifteenth day. The magnesium remained constant during the first 11 days and then increased very slowly up to the fifteenth day. The experiment shows, furthermore, that the filtration of a clean milk with low bacterial count can be continued for about one week without any appreciable chemical changes taking place in the serum, provided the temperature be kopt at 6 to 8 C. (42.8 to 46.4 F.). 10 CHEMICAL CHANGES PRODUCED BY PASTEURIZATION. Before proceeding to the filtration of raw milk in the refrigerator, several experiments were made in which formaldehyde had been added to the milk as a preservative. This was done in order to carry on the filtration at room temperature. The results obtained showed that ike serum, after the first day's filtration, remained of constant composition, and led up to carrying out the filtration of a pure milk hi the refrigerator. Having thus shown that the serum remains of constant composi- tion after the first day of filtration, the next step was to determine whether there were any demonstrable changes produced in the soluble phosphates by pasteurization. In order to produce a maxi- mum precipitation the highest temperature, 68.3 C. (155 F.), employed in the holder process was chosen. The results obtained are shown in Table 2. TABLE 2. Quantity of soluble phosphates tr? the serum of raw whole milk and in whole milk pasteurized at 68.3 C. (155 F.)for SO minutes. Specific gravity. Acidity. PjOo. CaO. MgO. Casein Per cent. 0595 Per cent. Percent. Raw whnlfi milk 1.029 18 2219 1749 0203 Raw serum: Second and third days 1 026 9 8 1074 0625 0142 Do .1078 .0624 0142 Fifth and sixth days 1.026 9.6 1082 0631 0143 Pasteurized serum: Second and third days 1.026 9 6 1078 0611 0140 Do 1063 0613 0140 Fifth and sixth days. . 1 026 9 4 1089 0627 0141 It will be seen from the table that the changes produced by pasteurization at 68.3 C. (155 F.) for 30 minutes are trifling. The phosphoric acid content remains the same in both the raw and the pasteurized serum. The quantity of lime salts, while a trifle lower in the pasteurized than in the raw serum, is still within the limit of error, and the magnesia remains the same in both serums. Other experiments made with skim milk showed results agreeing in every particular with this one made with whole milk. All prelim- inary experiments were made with skim milk because, as previously stated, it filters more readily and thus gives a larger quantity of serum for analysis. Comparing Table 1 with Table 2 we find that the variation in the quantity of soluble lime salts between the two samples of milk is far greater than that found between the raw and pasteurized serums. For this reason the objection frequently raised against pasteurized milk as an infant food, on the ground that the soluble lime salts are diminished by the process of pasteurization, is without foundation. CHEMICAL CHANGES IN THE PROTEINS. 11 CHEMICAL CHANGES IN THE PROTEINS. LACT ALBUMIN. Different investigators do not agree as to the exact temperature at which the coagulation of the albumin begins. Steiner l found no change on heating milk for 25 minutes at 60 C. (140 F.), while Solomin 2 says that the change seems to begin at this temperature, and Jensen and Plattner 3 also found some coagulation in milk heated for five hours at 60 C. (140 F.). Babcock 4 could find no change on heating milk for 20 minutes at 65 C. (149 F.), while Woll 5 states that coagulation begins at this temperature. According to Willoughby 6 the change begins at 70 C. (158 F.), while de Freud- enreich 7 found 15 to 20 per cent coagulated on heating milk for 30 minutes at 68 to 69.5 C. (154.4 to 157 F.). A series of experi- ments were therefore undertaken to determine at what temperature the change in the albumin begins, and to what extent it is coagulated at the different temperatures commonly employed in the pasteuriza- tion of milk. METHODS OF ANALYSIS. Casein. To 10 grams of milk were added 50 c. c. of distilled water at 40 C. (104 F.), then 1.5 to 2 c. c. of a saturated alum solution were added gradually. The precipitate was allowed to settle, was then filtered and washed. The precipitate and filter paper wore treated by the Gunning method, adding about 0.2 gram of copper sulphate. Albumin and globulin. The albumin and globulin in the filtrate and washings were precipitated with 10 c. c. of Almen's tannin solu- tion; this was allowed to settle, was then filtered and washed. The precipitate was treated the same as under casein. Tables 3 to 6 show the results of the experiments to determine the lactalbumin precipitated in whole milk pasteurized for 30 minutes at temperatures ranging Jrom G2.S C. (145 F.) to 71.1 C. (160 F.). 1 Steiner, U. Beitriigezur Kenntnisdos Einflussesder Pasteurisiorung auf die Beschaffcnheit dor Milrh und auf den IHittertings I'rozesz. Milch-Zcitung, vol. 30, no. 2(i, p. 401-403, June 29; no. 2S, p. 435, July 13. Leipsic, 1901. 'Solomin, 1'. Uober die boiin Erliit7.cn dor Milrh ausfallenden Eiweissinongen. Archiv fur Hygiene, vol. 2s, no. 1, p. 43-48. Munich and Ixsipsic, ls%. * Jensen, Orla, and Plattner, Ernest. De I 'act ion du chauffage stir lo lait do vae.he. Hevue (Jo^ne'rale du Lait, vol. 4, no. Id, p. 3f>l-3r>8, May 30; no. 17, p. 3SS-397, June 15; no. IS, p. 41!)-424. Lierre, I'.Ml.x 4 Babcock, S. M. Tho centrifugal separation of casein and insoluble phosphates from milk. Wisconsin Agricultural Experiment Station, Twelfth Annual Report (1S95), p. 93-99. Madison, 1S9>. & Woll, F. W. Tho effect of pasteurization and sterilization on the viscosity and fat globules of milk and cream. Wisconsin Agricultural Experiment Station, Twelfth Annual Report (18951, p. 1C4-173. Madison, 18%. Willoughby, Edward V. Milk, its production and uses. Ix>iuion, 1903. See p. 12o-12<'>. T Pe Freudenrcich, Ed. Stir la pasteurisation du lait dans raliiucntatiou de 1'eufanee. Kevue (U'-niTale du Lait, vol. 4, no. 19, p. 433-437. Lierro, July lo, 1905. 12 CHEMICAL CHANGES PRODUCED BY PASTEURIZATION. TABLE 3. Quantity of albumin precipitated in raw whole milk and in milk pasteurized at 62.8 C. (145 F.)for SO minutes. Raw milk. Pasteurized milk. I. II. Average. I. II. Average. Casein nitrogen Per cent. 0.4003 .0996 Per cent. 0.4027 .0998 Per cent. 0.4015 .0997 Per cent. 0.4015 .0996 Per cent. 0.4010 .0997 Per cent. 0.4013 .0997 Albumin+globulin nitrogen PROTEIN- NITROOENX6. 38. Casein 2.554 2.569 2.562 2.561 2.558 2 560 Albumin +globulin .635 .637 636 635 636 636 No albumin precipitated. TABLE 4. Quantity of albumin precipitated in raw whole milk and in milk pasteurized at 65.6 C. (150 F.)for SO minutes. Raw milk. Pasteurized milk. I. II. Average. I. II. Average. Per cent. 0.4260 .0983 Per cent. 0.4218 .0978 Per cent. 0.4239 .0981 Per cent. 0.4320 .0920 Per cent. 0.4328 .0929 Per cent. 0.4324 .0925 Casein nitrogen Albumin+globulin nitrogen PROTEIN=NITROGENX6.38. Casein 2 717 2 691 2 704 2.756 2 762 2 759 Albumin+globulin . .627 .624 .626 .587 593 590 Albumin precipitated=5. 75 per cent. TABLE 5. Quantity of albumin precipitated in raw whole milk and in milk pasteurized at 68.8 C. (155 F.)for 30 minutes. Raw milk. Pasteurized milk. I. II. Average. I. II. Average. Casein nitrogen Per cent. 0.3739 .0963 Per cent. 0.3812 .0981 Per cent. 0.3776 .0972 Per cent. 0.3910 .0863 Per cent. 0.3878 .0833 Per cent. 0.3894 .0848 Albumin+globulin nitrogen PROTEIN=NITROGENX6.38. Casein 2.385 2.432 2.409 2.495 2.474 2.485 Albumin+globulin . .614 .626 .620 .551 .531 .541 Albumin precipitated 12.75 per cent. CHEMICAL CHANGES IN THE PROTEINS. 13 TABLE 6. Quantity of albumin precipitated in raw whole milk and in milk pasteurized at 71.1 C. (160 F.)for SO minutes. Raw milk. Pasteurized milk. I. II. Average. I. II. Average. Casein nitrogen Per cent. 0.3860 .0961 Per cent. 0.3851 .0989 Per cent. 0.3856 .0975 Per cent. 0.4200 .0681 Per cent. 0.4169 .0667 Per cent. 0.4185 .0674 Albumin+gfobulin nitrogen PROTEIN= NITROGENX6.38. Casein 2.462 2.457 2.460 2.679 2.660 2.670 Albumin -(-globulin .613 .631 .622 .434 .426 .430 Albumin procipitated= 30.87 per cent. From an analysis of the above tables it is apparent that no albumin is coagulated on heating milk for 30 minutes at 62.8 C. (145 F.), the temperature most commonly used in the holder process in com- mercial pasteurization. At 65.6 C. (150 F.) the separation of the albumin has begun, 5.71 per cent having become insoluble. At 68.3 C. (155 F.) the quantity has increased to 12.76 per cent, wnile at 71.1 C. (160 F.) 30.87 per cent of the albumin has been coagulated. CASEIN. The condition in which casein exists in milk has not as yet been fully explained. The effect of heat shows itself in two ways. In the first place, the rennin coagulation may be accelerated, retarded, or even inhibited; and in the second place, the coagulum may assume a different character from that obtained from raw milk. The great majority of authors agree that no change in rennin coagulation takes place below 65 C. (149 F.). Steiner l found a retardation in milk heated to 60 C. (140 F.), while Fleischmann and Morgen 2 found the milk less sensitive to rennin when heated for two hours from 60 to 70 C. (140 to 158 F.). According to Van Slyke and Publow 3 milk heated above 65.6 C. (150 F.) for a considerable length of time coagulates less rapidly and the coagulum is highly flocculent. De Freudenreich l finds the coagulum unchanged at 68 to 69.5 C. (154.4 to 157.1 F.). Jensen and Plattncr l state that retardation begins on heating milk for five hours at 70 C. (158 F.), while Mayer 4 places the beginning at 75 C. (167 F.). Stassano and ' IMC. cit. * Fleischmann, W., and Morgen, A. Kiniges iilxr die nach ScherfT's Verfahren conservirte Flaschcn milch. Din Landwirthsohaftlichen Versuch Stationen, vol. 28, p. 321 332. Berlin, 18S3. 'Van Slyke, Lucius L., and Publow, Charles A. The science and practice of cheese-making, p. 310, New York, 1909. Mayer, Adolf. Bestimmungen der Wirksamkeit des Labfermentes unter verschiedenen auszeren Um- standen. Milch-Zcitung, vol. 10.no. 2, p. 17-19, Jan. 12; no. 3, p. 33-38, Jan. 19; no. 4, p. 49-52, Jan. 26; no. 6, p. 81-84, Feb. 9. Bremen, 1881. 14 CHEMICAL CHANGES PRODUCED BY PASTEURIZATION. Talarico l differ from all the preceding in that they observed an accel- eration of rennin coagulation in milk heated from 55 to 65 C. (131 to 149 F.), while at 70 C. (158 F.) the heated milk behaved the same as raw milk, and above 70 C. (158 F.) the retardation began. The following experiments were made with raw milk and milk pasteurized at various temperatures: METHOD OF COAGULATION. The apparatus consisted of a large galvanized-iron box having four cylindrical compartments surrounded by water. The upper openings were of sufficient size to admit a cylindrical percolater (1 foot high and 2| inches in diameter), the lower openings being about one-fourth of an inch in diameter. A small glass tube drawn out into a capillary was inserted into the neck of the percolator by means of a perforated rubber stopper. In making the test, the water in the box was heated to the required temperature, the percolators were then placed in the compartments and allowed to remain until they had acquired the temperature of the bath, the upper openings being covered. Next 200 c. c. of milk were heated, the rennin solution added, and, after mixing, poured into the percolator. The upper opening was then closed with a cover and the time required for the milk to coagulate in the tube noted. The flow through the tube was regulated by breaking off the capillary to such an extent that all tubes dropped at the same rate. For each experiment 200 c. c. of milk were heated in an Erlenmeyer flask to 35 C. (95 F.) and 5 c. c. of rennin solution (0.15: 100 c. c. of water) added. This was taken as the beginning of the coagula- tion time; the end point was when the milk stopped dropping from the capillary tube. The rennin solution was made of such strength that the raw milk coagulated in about 20 minutes. The results are shown in the following table: TABLE 7. Time required for rennin coagulation of raw whole milk and of whole milk pasteurized at different temperatures. Milk 200 c. c., rennin solution (0.15 gram: 100 c. c. of water) 5 c. c. Experiment. Raw milk. Milk pasteurized at 55C. (131 F.). 60 C. (140 F.;. 65 C. (.149 F.). 70 C. (158 F.;. 75 C. (167 F.). I. { M. s. M. 8. M. 9. M. s. M. s. M. s. 18 30 18 08 19 34 19 23 17 28 16 56 17 10 16 53 17 12 17 12 1 II I 20 38 20 25 36 30 37 30 \ 1 Stassano, H., and Talarico, J. De I'influencede lacuisson sur la cas^i float ion du lait par le lab-ferment. Comptes Rendus Hebdomadaires des Seances et Me'moires de la Societo de Biologic, vol. 69 (annee 62, tome 2), no. 28, p. 254-255. Pan ;, Aug. 5, 1910. SUMMARY AND CONCLUSIONS. 15 From the above table it will be seen that milk pasteurized at 55 C. (131 F.), 60 C. (140 F.), and 65 C. (149 F.) aU coagulate more rapidly with rennin than does the raw milk. This result was obtained in all cases in a large number of experiments. At 70 C. (158 F.) the retardation of rennin coagulation has begun, while at 75- C. (167 F.) the time has about doubled and the coagulum is highly flocculent. CHANGES IN THE ACIDITY. Woll l and de Freudenreich l find 110 appreciable change in the acidity of milk pasteurized at 67 to 68 C. (153 to 154.4 F.) for 20 minutes. Hoft 2 and Jensen and Plattncr 1 on the other hand find a diminution in the acidity on heating milk. From a large number of experiments made during the coagulation of milk by rennin, it was found that the acidity of the milk always diminished during pasteurization. While the diminution was slight it amounted to several tenths of a cubic centimeter, and was always sufficient to be determined by titration with tenth-normal caustic- soda solution. The diminution, however, is insignificant and is probably due to a loss of carbonic acid during the heating. Further- more, the diminution varies in different samples of milk. SUMMARY AND CONCLUSIONS. 1. Milk pasteurized by the holder process at 62.8 C. (145 F.) for 30 minutes does not undergo any appreciable chemical change. 2. The soluble phosphates of lime and magnesia do not become insoluble. At 68.3 C. (155 F.) the quantity of phosphoric acid, lime, and magnesia in the serum of both raw and pasteurized milk are practically the same. 3. The albumin does not coagulate at 62.8 C. (145 F.), but at 65.6 C. (150 F.) 5.75 per cent of the albumin is rendered insoluble. As the temperature increases the amount of coagulated albumin increases. At 68.3 C. (155 F.) the quantity increases to 12.75 per cent, and at 71.1 C. (160 F.) it amounts to 30.78 per cent. 4. The time required for coagulating the casein by rennin is slightly less in milk pasteurized at temperatures up to 65 (\ (149 F.) than it is in raw milk. At 70 C. (158 F.) there is a slight retardation, while at 75 C. (1(>7 F.) the time has almost doubled. 5. The acidity as determined by titration is slightly diminished in pasteurized milk. 1 Loo. cit. 3 Hoft, 11. l"elx>r dio VoriinderuiiK dor Aoiditiit dor Milch beim Krhiizen. Miloh-Zeitunp, vol. HO. no. 7. p. 10:i. Leipzig, Feb. Iti, 1901. o University of California SOUTHERN REGIONAL LIBRARY FACILITY 405 Hilgard Avenue, Los Angeles, CA 90024-1388 Return this material to the library from which it was borrowed. JAN 1 3 K _j A 001 083 170 9 Universil South* Libra