. (1 Xovi'mlun- 28, 1908. 
 
 U. S. DEPARTMENT OF AGRICULTURE, 
 
 BUREAU OF ANIMAL INDUSTRY. BULLETIN 109. 
 
 A. D. MELVIN, CHIEF OF BUREAU. 
 
 OTEOLYTIC CHANGES IN THE RIPENING 
 OF CAMEMBERT CHEESE. 
 
 BY 
 
 ARTHUR W. DOX, 
 
 Chemist in Cheese Investigations , Dairy Division. 
 
 WASHINGTON: 
 
 GOVERNMENT PRINTING OFFICE. 
 
 1908.
 
 Issued November 28, 
 
 U. S. DEPARTMENT OF AGRICULTURE, 
 
 BUREAU OF ANIMAL INDUSTRY. BULLETIN 109. 
 
 A. D. MKLVIN, CHIKF OF BUREAU. 
 
 PROTEOLYTIC CHANGES IN THE RIPENING 
 OF CAMEMBERT CHEESE. 
 
 BY 
 
 ARTHUR W. DOX. 
 
 Chemist in Cheese Investigations, Dairy Division. 
 
 WASHINGTON: . 
 GOVERNMENT PRINTING OFFICE. 
 1908.
 
 THE BUREAU OF ANIMAL INDUSTRY. 
 
 Chiff: A. D. MBLVIN. 
 
 Assistant Chief: A. M. FAHRINGTON. 
 
 Chief Clerk: CHARLES C. CARROLL. 
 
 Biochemic Division: M. DORSET, chief; JAMES A. EMERY, assistant chief. 
 
 Dairy Division: En. H. WEBSTER, chief; C. B. LANE, assistant cliief. 
 
 Inspection Division: RICE P. STEDDOM, chief; MORRIS WOODEN, R. A. RAMSAY, 
 and ALBERT E. BEHNKE. associate chiefs. 
 
 Pathological Division: JOHN R. MOHLER, chief; HENRY J. WASHBURN, assist n MI 
 chief. 
 
 Quarantine Division: RICHARD W. HICKMAN, chief. 
 
 Zoological Division: B. H. RANSOM, chief. 
 
 Experiment Station: E. C. SCHROEDER, superintendent; W. E. COTTON, assist am. 
 
 Animal Husbandman: GEORGE M. ROMMEL. 
 
 Editor: JAMES M. PICKENS. 
 
 DAIRY DIVISION. 
 Chief: Ed. H. Webster. 
 Assistant Chief: C. B. Lane. 
 Librarian: Miss C. B. Sherman. 
 
 DAIRY FARMING INVESTIGATIONS. 
 
 Assistant in charge, B. H. Raid; assistant, Duncan Stuart. 
 
 Dairy buildings: J. A. Conover; architect, K. E. Parks; ventilation experiments, 
 C. R. Potteiger. 
 
 Herdbook work: Helmer Rabild and William Hart Dexter. 
 
 Southern dairying: S. E. Barnes, J. E. Dorman, J. T. Eaton, H. P. Lykes, J. H. 
 McClain, A. K. Risser, H. R. Welch, and T. R. Woodward. 
 
 HAIRY PRODUCTS INVESTIGATIONS. 
 
 Assistant in charge, L. A. Rogers. 
 
 Butter investigations, Albert Lea, Minn., and Washington D. C.: Chemist, 
 W. H. Berg; bacteriologist. S. H. Ayers. 
 
 Swiss cheese investigations. Albert Lea, Minn.: In charge, C. F. Doane; assistant, 
 T. W. Issajeff. 
 
 Cheese investigations, Madison, Wis.: Chemist, S. K. Suzuki; bacteriologist, Alfred 
 Larson: cheese maker, J. W. Moore. 
 
 Cheese investigations, Storrs, Conn.: Mycologist, Charles Thorn; chemist, Arthur 
 W. Dox; cheese maker, F. R. Thompson. 
 
 Milk secretion investigations, Columbia, Mo.: Chemist, R. II. Shaw; assistants, 
 J. O. Halverson, A. E. Perkins, and C. C. Payne. 
 
 DAIRY MANUFACTURING INVESTIGATIONS. 
 
 Assistant in charge, B. D. White: assistant, S. C. Thompso 
 Creamery records, Albert Lea, Minn.: Creamery practice, John L. Sherk; assistants 
 
 (collaborators), P. W. Noble and J. D. Burk. 
 Creamery practice investigations: J. C. Joslin, Robert Me Adam, F. L. Odell, J. C. 
 
 Winkjer. and Thomas Corneliuson. 
 Market investigations: New York City, C. W. Fryhofer; Chicago, H. J. Credicott; 
 
 San Francisco, C. L. Mitchell. 
 
 MARKET MILK INVESTIGATIONS. 
 
 Assistant chief of division in charge; assistants, Lee H. P. Maynard, Ivan C. Weld, 
 and <eorge M. Whitaker. 
 
 RENOVATED-BUTTER INSPECTION. 
 
 Chief inspector, M. W. Lang. Chicago: assistant, Levi Wells, New York. 
 2
 
 LETTER OF TRANSMITTAL. 
 
 U. S. DEPARTMENT OF AGRICULTURE, 
 
 BUREAU OF ANIMAL INDUSTRY, 
 Washington, D. C., September 21 , 1908. 
 
 SIR: I have the honor to transmit herewith a manuscript entitled 
 "Proteolytic Changes in the Ripening of Camembert Cheese," by 
 Arthur W. Dox, chemist in cheese investigations, Dairy Division, and 
 recommend that it be published as Bulletin 109 of this Bureau. This 
 paper deals with work carried on at the Storrs (Conn.). Agricultural 
 Experiment Station, by cooperation between that station and the 
 Dairy Division of this Bureau. 
 
 Respectfully, A. D. MELVIN, 
 
 Chief of Bureau. 
 Hon. JAMES WILSON, 
 
 Secretary of Agriculture. 
 
 3
 
 CONTENTS. 
 
 Page. 
 
 Introductory 5 
 
 Processes in the ripening of Cameinbert cheese 6 
 
 Enzymes in the cheese 8 
 
 Proteolysis of casein 10 
 
 Nitrogenous constituents of the cheese 11 
 
 Hydrochloric acid precipitate : 12 
 
 Coagulable proteins 13 
 
 Caseoses 14 
 
 Peptones 15 
 
 Polypeptids 15 
 
 Diamino-acids 16 
 
 Monoamino-acids 18 
 
 Ammonia 21 
 
 Absence of putrefac ivc products 21 
 
 Summary 22 
 
 Acknowledgments 22 
 
 References to literature. . . 23
 
 PROTEOLYTIC CHANGES IN THE RIPENING OF 
 CAMEMBERT CHEESE. 
 
 INTRODUCTORY. 
 
 Until comparatively recent years the changes that take place in the 
 ripening or curing of the different varieties of cheese were but little 
 understood. Although the practice of cheese making has been carried 
 on for centuries, all of our knowledge of the chemical changes involved 
 in the ripening process and the various factors that bring about these 
 changes has come to us within the past fifty years. The earliest 
 record we have of any discussion of this subject from a chemical point 
 of view was published only a century ago. In this paper the French 
 chemist, Chaptal, l a discusses the ripening of Roquefort cheese and 
 advances certain theories to account for the changes in appearance and 
 flavor which this cheese undergoes during its sojourn in the natural 
 ripening caves. Biological factors were, of course, not taken into 
 account in Chaptal's paper, for that phase of the subject was unknown 
 until the time of Pasteur. 
 
 No scientific study of the subject, however, was made until the 
 latter half of the nineteenth century. The attention of chemists was 
 then directed to Roquefort cheese by a paper published by Blondeau 2 
 in 1864. Blondeau analyzed cheeses in different stages of ripening 
 and found that the fat content increased from 1.85 per cent in the 
 fresh cheese to 32.31 per cent in the cheese two months old. This 
 enormous increase in the fat he ascribed to a synthesis of fat from 
 protein by the mold. But a comparison of his figures for the other 
 constituents of the cheese as well as the fat with analyses to be found 
 hi any modern book on dairy products will show their utter im- 
 possibility. This inaccurate work of Blondeau, however, served the 
 purpose of directing the attention of other investigators to the sub- 
 ject, and during the next few years the changes in the fat content of 
 cheese were studied by Brassier, 3 Sieber, 4 Jacobstahl, 5 and Von Niigeli 
 and Loew. B 
 
 By this time the subject of cheese ripening had begun to arouse con- 
 siderable interest among scientific investigators, and their researches 
 were extended to other varieties of cheese. Swiss cheese and Cheddar 
 
 " Th' figun s refer to list of literature at end of bulletin.
 
 6 CHANGES IN RIPENING OF CAMEMBERT CHEESE. 
 
 cheese in particular received considerable attention. Among those 
 who worked with Swiss cheese were Weidmann, 7 Rdse, 8 Schulze, 8 and 
 Winterstein. 9 In our own country the Cheddar type of cheese has 
 been made the subject of thorough chemical investigation, and in this 
 connection reference is made to the work of Babcock and Russell 10 and 
 that of Van Slyke and Hart. 11 The German investigators made a spe- 
 cial study of the products of proteolysis, while those in this country 
 studied more particularly the factors that cause the ripening. 
 
 All this work, however, deals with the "hard" cheeses. In this 
 class of cheeses the ripening factors are the enzymes or unorganized 
 ferments present in the fresh curd and the bacteria which occur in 
 enormous numbers in the cheese. Such cheeses require several months 
 for ripening, for the enzymes are present in very small amount, and the 
 bacteria do not produce rapid proteolytic changes. In contrast to 
 the hard cheeses we have another distinct class known as the soft 
 cheeses. The cheeses belonging to this class differ mainly from those 
 of the former class in that they contain a much higher percentage of 
 water. This higher moisture content is much more favorable to the 
 development of micro-organisms, and the ripening proceeds with 
 greater rapidity. 
 
 PROCESSES IN THE RIPENING OF CAMEMBERT CHEESE. 
 
 The variety of soft cheese which w r e shall consider in this paper is 
 the Camembert type, a soft cheese ripened mainly by a surface growth 
 of mold. Of late years it has attained considerable importance in the 
 cheese market, and the public is now more or less familiar with it. 
 Although it is still imported from France in large quantities, its man- 
 ufacture has been undertaken on a commercial scale in our own coun- 
 try with considerable success. The researches carried on by the 
 Dairy Division of the Bureau of Animal Industry, in cooperation 
 with the Storrs Agricultural Experiment Station, with a view to 
 introducing the manufacture of Camembert cheese into the United 
 States, have given very gratifying results. 12 For a description of the 
 details of its manufacture the reader is referred to a bulletin by T. W. 
 IssajefT. 14 
 
 The biological factors essential to the production of Camembert 
 cheese are the lactic-acid bacteria, which are normally present in 
 milk, and two molds, Penicillium camemberti Thorn 13 and Oidium 
 lactis. Other molds that may be present are generally contamina- 
 tions and often deleterious to. the cheese. The penicillium is the mold 
 that produces the actual ripening or digestion of the curd, while the 
 oidium seems to be connected hi some way with the flavor production. 
 The oidium by itself or in conjunction with the lactic-acid bacteria 
 can not ripen a cheese more than a few millimeters below the surface.
 
 RIPENING OF CAMEMBERT CHEESE. 7 
 
 The ripening of all cheeses being essentially a hydrolysis of the para- 
 casein or cheese curd through the agency of various enzymes, the end 
 products or simple substances from which the complex protein mole- 
 cule is built up are set free in varying amounts during the course of 
 the ripening period. At the same time secondary reactions may 
 occur which involve not only hydrolysis, but also oxidation, reduction, 
 desamidation, and removal of carboxyl groups. The products of 
 simple hydrolysis may thus undergo further changes with the forma- 
 tion of substances differing widely in chemical constitution from the 
 substances from which they were derived. The successive steps of 
 such reactions are often difficult to follow, and it is sometimes impos- 
 sible to ascertain precisely what the mother substance is. In most 
 cases, however, the secondary changes involve but one step, the 
 removal of a certain radical from the original hydrolytic product. 
 These changes are caused for the most part by bacteria, and in a 
 cheese where the ripening is produced almost entirely by other agen- 
 cies they are of minor importance. Where they do occur they result 
 not from the direct action of an enzyme acting outside the cell wall 
 of the organism, but rather from activities dependent upon the life 
 history and metabolism of the organism. For this reason the center 
 of a hard cheese shows the same flora and the same chemical compo- 
 sition as the portion near the rind. With cheeses of the Camembert- 
 Brie type, however, there is a marked difference in this respect. The 
 actual ripening here is caused by the proteolytic enzyme of the mold. 
 This enzyme is secreted by the mold growing on the surface of the 
 cheese and diffuses toward the center, digesting the curd through 
 which it passes until the cheese is ripe. The fact that the ripening 
 begins at the surface and proceeds toward the center indicates that 
 the enzyme is produced in the mycelium of the mold. The progress 
 of the ripening is very easy to follow, for the texture and color of the 
 ripened portion are quite different from those of the unripened curd, 
 and there is always a sharp line of demarcation between the two. 
 The mycelium of the mold does not penetrate more than a few milli- 
 meters into the cheese, forming a sort of rind which is removed when 
 the cheese is eaten. 
 
 The fresh Camembert cheese differs from the fresh curd of other 
 whole-milk cheeses mainly in the greater amount of whey it con- 
 tains. This slightly increases the relative amount of the other pro- 
 teins, lactalbumin, whey protein, and lactoglobulin, as well as that 
 of the nonnitrogenous constituents lactose, citric acid, and inor- 
 ganic salts. The amount of butterfat in the cheese varies merely 
 with the richness of the milk. Paracasein, however, is the only 
 protein present in any considerable amount, and the end products of 
 the ripe cheese may be considered as derived from it. Moreover,
 
 8 CHANGES IN RIPENING OF CAMEMBKi: I CHEESE. 
 
 the other three proteins mentioned above yield the same primary 
 disintegration products, and a distinction as to the origin of the 
 latter can not be made. 
 
 The proteolytic changes which constitute the ripening of Camem- 
 bert cheese consist, therefore, in the changes which this paracasein 
 undergoes through the action of proteolytic enzymes. As has already 
 been mentioned, the principal factor is the enzyme secreted by the 
 Camembert penicillium. Other enzymes are present, however, and 
 a brief discussion of these will follow. No appreciable proteolysis 
 occurs until after the cheese is nearly 2 weeks old. But in the 
 meantime certain changes take place in the character and solubility 
 of the curd. These changes have been studied by Bosworth. 15 
 The}' consist mainly in the liberation of paracasein from combination 
 with calcium, due to the formation of lactic acid by lactic acid bac- 
 teria. At the same time the paracasein is converted into a form 
 completely soluble in 5 per cent salt solution, and later it becomes 
 insoluble again. As these are not, strictly speaking, proteolytic 
 changes, a detailed discussion will not be given here. 
 
 ENZYMES IN THE CHEESE. 
 
 With the exception of the enzyme secreted by the mold and of a 
 smaller variety of bacteria, Camembert contains the same ferments 
 that are present in other cheeses. Like the hard cheeses, it contains 
 the milk enzyme galactase, the rennet enzyme chymosin added in 
 the curdling process, and lactic acid bacteria. In the case of the 
 Cheddar type of cheese the action of these three factors has been 
 studied in detail. Babcock and Russell found that galactase and 
 rennet (pepsin) were important agents in the ripening of this variety 
 of cheese. According to Van Slyke and Hart, 11 the rennet alone is 
 capable of ripening a cheese. In their experiments the galactase 
 was first destroyed by heat and then chloroform added to prevent 
 the development of bacteria, yet the ripening went on, though not 
 as rapidly as in the normal cheese, and the character of the chemical 
 products was somewhat different. Thus there was a predominance 
 of paranuclein, caseoses, and peptones, and an abnormally small 
 amount of amino-acids. The entire absence of ammonia was very 
 striking. The function of the bacteria in this variety of cheese has 
 been studied by Rogers. 18 He came to the conclusion that the 
 enzymes produced by the bacteria were responsible for most of the 
 digestion beyond the peptone stage, and consequently the charac- 
 teristic flavors. 
 
 In the short time required for the ripening of Camembert cheese 
 the rennet, galactase, and lactic acid bacteria produce no appreciable 
 digestion. This conclusion was reached by Bosworth, and the expe- 
 rience of the writer confirms it. Even the hard cheeses which are
 
 ENZYMES IN THE CHEESE. 9 
 
 ripened entirely by these agents undergo very little change during 
 the first month. A Camembert cheese, however, should be ripe at 
 the end of a month, and at the same time should contain a greater 
 amount of primary digestion products. This ripening must be due 
 almost entirely to the mold enzyme, for the interior curd, which has 
 not yet been reached by this enzyme, but contains all of the other 
 ferments, shows little evidence of digestion. If the unripened curd 
 in the center of a Camembert cheese three or four weeks old be sub- 
 jected to chemical analysis it will be found that the paracasein is 
 scarcely altered except for the fact that it is liberated from combi- 
 nation with calcium. The galactase can not play more than a very 
 subordinate role, as is shown by the fact that the cheese ripens 
 normally when made from milk which has been pasteurized at a 
 temperature sufficiently high to impair greatly, if not destroy, the 
 activity of this enzyme. Likewise the rennet can not be of more 
 than minor importance as a ripening factor. Recent investigations 
 have shown that chymosin or rennet enzyme is identical with pepsin. 
 But, as will be seen later, the ripening of Camembert cheese bears 
 no resemblance to a peptic digestion. The rennet should show its 
 greatest activity in the interior curd, which is quite strongly acid. 
 But owing to the short duration of the ripening period and the small 
 amount of rennet present, the proteolytic action of the latter is prac- 
 tically negligible. Aside from the hydrolysis of the casein into para- 
 casein and whey protein, its action is inappreciable. The unripened 
 curd shows no evidence of peptic digestion. 
 
 The lactic acid bacteria which constitute nine-tenths of the bac- 
 terial flora of the cheese serve the purpose of converting the milk 
 sugar into lactic acid, thus producing conditions unfavorable to the 
 development cf other bacteria. They are probably responsible for 
 the peculiar flavor which is characteristic of the acid curd in the 
 interior of a Camembert cheese. Their proteolytic action is other- 
 wise hardly noticeable. Experiments in which sterile curd was 
 inoculated with these organisms show that the amount of diffusible 
 nitrogen increases only very slightly at the end of a month, even in 
 the presence of calcium carbonate, which neutralizes the acid. 
 
 We are safe in assuming, therefore, that these three proteolytic 
 factors the galactase, the rennet, and the lactic acid bacteria 
 have very little to do with the actual ripening of the cheese, this 
 being essentially the work of the enzyme from the mold. 
 
 As has already been pointed out, the mold of Camembert cheese 
 (Penicillium caniemberti) secretes a powerful proteolytic enzyme, 
 which is undoubtedly the most potent factor in the ripening of this 
 cheese. The fact that the ripening begins at the surface and proceeds 
 toward the center indicates that the enzyme is produced in the myce- 
 lium of the mold and diffuses inward. The diffusibility of this enzyme 
 58056 No. 10908 2
 
 10 CHANGES IN RIPEX1XU OF CA.MK.M IJKK T CHEESE. 
 
 is also shown by the fact that synthetic culture media upon which 
 this mold has grown for some time have a marked proteolytic activity. 
 Experiments are now being instituted to determine the exact nature 
 of this enzyme and the extent to which it will hydrolyze certain proteins. 
 The results thus far obtained seem to indicate that it is of the nature 
 of erepsin. It attacks casein and peptone readily, but is without 
 action upon fibrin and coagulated egg albumin. 
 
 Vines 17 lias shown that erepsin is very widely distributed jn the 
 vegetable kingdom. This vegetable "ereptase," as he calls it, differs 
 from animal erepsin in that it is most active in the presence of th'e 
 natural acid of the plant. The addition of other acids, or of an 
 alkali, greatly impairs its activity. As long as the acidity is due 
 entirely to acid phosphates, the activity of this ereptase is very 
 pronounced, but the presence of free acid in the medium is inhibitory. 
 It is readily seen, therefore, that a fresh Camembert cheese offers 
 very favorable conditions for the action of ereptase. In the first 
 place, casein and paracasein are readily attacked by this enzyme. 
 In the second place, the lactic acid produced by the bacteria does not 
 accumulate but combines with the calcium phosphate, forming 
 calcium lactate and mono-calcium phosphate. The acidity of the 
 cheese is due to the presence of the latter salt. 
 
 In this connection, however, it must be remembered that the same 
 results are not necessarily obtained with enzymes in the presence 
 of an antiseptic as with organisms in vivo. In the case of Camembert 
 cheese where the proteolysis is effected by diffusion of the enzyme 
 rather than by diffusion of the substratum, the conditions more 
 nearly approach those met with in an artificial digestion experiment. 
 Nevertheless there are certain striking differences which will be 
 pointed out later. A discussion of the enzymes obtained from a 
 pure culture of this organism will be reserved for a future paper. 
 
 PROTEOLYSIS OF CASEIN. 
 
 When casein or any other protein is boiled with strong acid or 
 alkali, a decomposition takes place with the production of simpler 
 substances. These simple substances resulting from such decomposi- 
 tion have of late years been studied by a number of investigators. A 
 similar decomposition occurs when the protein is acted upon by 
 proteolytic enzymes. These enzymes also have the power of trans- 
 forming casein into bodies of less molecular complexity. The 
 changes are of a hydrolytic nature, the original molecule being 
 broken successively at different places and a molecule of water 
 entering at the point of cleavage. The extent to which the protein 
 is hydrolyzed, and consequently the nature of the resulting products, 
 depends upon the enzyme. Pepsin, obtained from the gastric juice, 
 does not carry the proteolysis beyond the peptone stage, while tryp-
 
 NITROGENOUS CONSTITUENTS OF CHEESE. 11 
 
 sin, obtained from the pancreas, breaks up the protein into crystal- 
 line end products. Erepsin, on the other hand, does not attack 
 native proteins, with the exception of casein, but acts readily upon 
 proteoses and peptones. The enzyme isolated by the writer from 
 Camembert mold resembles erepsin in this respect. 
 
 NITROGENOUS CONSTITUENTS OF THE CHEESE. 
 
 The ripening of Camembert cheese being a proteolysis, certain 
 digestion products may be expected to occur in the ripened cheese. 
 As casein, or rather paracasein, forms the main bulk of the proteins 
 in the curd, it would therefore undergo the same changes that occur 
 when casein is digested artificially with an enzyme, though the 
 proteolysis is never allowed to go en as far in a cheese as is usually 
 done in an artificial digestion experiment. The products may be 
 grouped roughly into the following classes: Caseoses, peptones, 
 polypeptids, amino-acids, and ammonia. Methods for the separation 
 of these groups of substances from cheese have already been elabo- 
 rated by Van Slyke and Hart. 11 They consist in extracting the cheese 
 with water, and determining the nitrogen in the precipitates obtained 
 by the addition of various reagents to aliquot portions of this extract. 
 
 In the subsequent pages of this article the separation of the indi- 
 vidual members of these groups will be discussed. The cheeses used 
 for this work were made at the Storrs Experiment Station, and were 
 pronounced by experts to be equal in texture and appearance to the 
 imported brands. Both texture and flavor showed them to be excel- 
 lent cheeses of the Camembert type. 
 
 The analysis of a ripe cheese by the method of Van Slyke and Hart 
 showed the nitrogenous constituents to be present in the following 
 amounts : 
 
 Per cent 
 N itrogen as of cheese. 
 
 Total nitrogen 2. 47 
 
 Water soluble 1 . 79 
 
 Precipitated by hydrochloric acid 40 
 
 Caseoses 10 
 
 Peptones 
 
 Amino-acids 82 
 
 Salt soluble (paracasein) 15 
 
 Ammonia 19 
 
 The polypeptids are included under the peptones and amino-acids. 
 This represents the analysis of an individual cheese. Some varia- 
 tions occur with different samples, but the differences are only slight 
 with cheeses at the same stage of ripening. As none of the individual 
 members of these groups of digestion products had ever been isolated 
 from Camembert cheese, a determination of some of the more char- 
 acteristic ones was considered advisable. Proximate analyses of the
 
 12 CHANGES IN RIPENING OF CAMEMBERT CHEESE. 
 
 cheese at different stages of ripening will be found in Bosworth's 
 paper. 15 These analyses show merely the rate of formation or destruc- 
 tion of three broad groups of digestion products, and throw little 
 light upon the nature of the ripening from the biochemical stand- 
 point. The writer has attempted to determine, as far as the facilities 
 at his command permitted, the relative amounts of the more impor- 
 tant members occurring in these groups. They will be discussed in 
 the same order as the groups are determined in Van Slyke and 
 Hart's method for the analysis of cheese. 
 
 HYDROCHLORIC ACID PRECIPITATE. 
 
 When an aqueous extract of the cheese is made and acidified with 
 hydrochloric acid, a white curdy precipitate appears, which on warm- 
 ing to 50 C. clots together into a gummy mass. If the digestion 
 were of a peptic nature this precipitate should consist of paranuclein. 
 The chief characteristics of this altered phosphoprotein are its high 
 phosphorus content and the comparative slowness with which it is 
 further acted upon by pepsin. On the other hand, trypsin converts 
 casein directly into caseoses without the formation of paranuclein, 
 and at the same time liberates the phosphorus in the form of phos- 
 phoric acid. Erepsin probably acts in the same way as trypsin in this 
 respect. Again, the precipitate might be a coagulose or protein syn- 
 thesized by the reversed action of a proteolytic enzyme. Such a 
 coagulose is Kurajeff s plastein, formed by the action of rennet on 
 albuminoses. 
 
 A similar precipitate has been obtained from other varieties of 
 cheese and has usually been regarded as paranuclein. As the ripen- 
 ing of Camembert cheese is not a peptic digestion, it seemed unlikely 
 that this precipitate could be paranuclein. In order to determine 
 more precisely the nature of the precipitate, several cheeses were 
 subjected to the following treatment. 
 
 An aqueous extract was made by stirring the macerated cheese 
 with water in a bath maintained at a temperature of 50 C. This 
 extraction was repeated several times, filtering off the liquid at the 
 end of half an hour through cotton and asbestos, and adding fresh 
 quantities of water until the filtrate was practically free from nitroge- 
 nous matter. The extract made in this way was acidified to 0.2 per 
 cent with hydrochloric acid, whereupon a white curdy precipitate 
 resulted. The precipitate was washed thoroughly with acidulated, 
 then with distilled, water, and finally dried in a desiccator and 
 extracted with ether to remove adhering fat. 
 
 Upon testing the solubilities of this precipitate, it was found that 
 a small part of it dissolved in 5 per cent sodium chlorid solution, 
 while the greater part was soluble in 50 per cent alcohol. The 
 smaller fraction was studied first. It dissolved in alkalies, was 
 reprecipitated by acids, excess of which dissolved the precipitate.
 
 HYDROCHLORIC ACID PRECIPITATE. 
 
 13 
 
 The substance was readily attacked by trypsin, dissolving com- 
 pletely in twenty-four hours, and giving a solution from which no 
 precipitate was obtained by saturation with ammonium sulphate. 
 A sample dried at 110C. gave the following analysis, based upon 
 the ash-free substance. Alongside it are given Rose's analysis of 
 paracasein and Chittenden's 1S analysis of paranuclein, with the 
 phosphorus as found by Jackson. 
 
 
 Substance 
 from cheese. 
 
 Paracasein 
 (Rose). 
 
 Paranuclein 
 (Chittenden). 
 
 Carbon .. 
 
 53.63 
 
 53 94 
 
 51 29 
 
 Hydrogen 
 
 7 08 
 
 7 14 
 
 7 2C 
 
 Nitrogen 
 
 15 10 
 
 15 14 
 
 15 23 
 
 Sulphur 
 
 .98 
 
 1 01 
 
 08 
 
 Phosphorus 
 
 50 
 
 
 2 75 
 
 Ash 
 
 82 
 
 
 
 12 43 
 
 
 
 
 
 A comparison of these analyses shows that the substance is too 
 low in phosphorus to be paranuclein. The analysis agrees fairly 
 well with that of paracasein, and it can be regarded as paracasein 
 from which a part of the phosphorus has been liberated by enzyme 
 action. Paracasein probably contains about the same amount of 
 phosphorus as casein itself, which, according to Hammarstein, is 
 0.85 per cent. 
 
 The alcohol-soluble part of the precipitate was dissolved in 50 per 
 cent alcohol, filtered and poured into water. A gummy precipitate 
 was formed. It was dried at 110 C., extracted with ether, and 
 analyzed. On fusing the substance with potash and niter for the 
 sulphur determination, a strong odor of skatol was emitted, stronger 
 than that obtained with casein. This indicates the presence of the 
 tryptophane group. The ready solubility in alcohol and insolu- 
 bility in water are properties characteristic of caseoglutin, a sub- 
 stance discovered in Swiss cheese by Weidmann. The analysis, 
 together with Rose's analysis of caseoglutin, follows: 
 
 
 Substance 
 from cheese. 
 
 Caseoglutin 
 (Rose). 
 
 Carbon 
 
 54.34 
 
 54.4 
 
 
 7.30 
 
 7.34 
 
 Nitrogen . 
 
 15.37 
 
 15.29 
 
 
 .95 
 
 .95 
 
 
 .06 
 
 
 \sh 
 
 .28 
 
 
 
 
 
 Both the analysis and the properties of tills substance agree with 
 those of caseoglutin. 
 
 COAOULABLK PKOTKINS. 
 
 Winterstein found in Swiss cheese a small amount of a substance 
 which was precipitated from acid solution by boiling. This substance 
 he calls tyroalbumin. Its exact nature has not yet been determined.
 
 14 CHANGES IN RIPENING OF CAMEMBERT CHEESE. 
 
 Several attempts were made to find this substance in Camembert 
 <-hee>e. but so far they have resulted in failure. On heating the 
 filtrate from the eascoglutin to boiling, both in acid and in neutral 
 solution, no precipitate was obtained. 
 
 CA8EOSES. 
 
 These, together with the peptones, are the intermediate disintegra- 
 tion of casein by ordinary proteolytic enzymes. They can not be 
 regarded as homogeneous substances, as they represent transition 
 products formed by the loss of varying numbers of ammo-acid mole- 
 cules from the original protein. They can, however, be separated 
 into groups according to their solubilities. A method for the separa- 
 tion of albumoses by fractional precipitation with ammonium sul- 
 phate was elaborated by Pick. 19 If nitrogen determinations are to 
 be made, the ammonium sulphate must be removed by dialysis, a 
 long and tedious operation. To obviate this difficulty Zunz 20 used 
 zinc sulphate and found that the precipitation limits were quite as 
 sharply defined. As the elementary analyses of these groups of 
 caseoses have very little value, and the peptones were to be separated 
 from the filtrate, the method of Pick was used in this work. These 
 saturation limits can not, however, be regarded as reliable indexes of 
 individuality. 
 
 The caseoses of the cheese were separated into the four fractions 
 described by Pick. They are designated as follows: Protocaseose, 
 by half saturation of the neutral solution with ammonium sulphate; 
 deuterocaseose A, by two-thirds saturation; deuterocaseose B, by 
 complete saturation; and deuterocaseose C, by acidifying the filtrate 
 from B. In the early stages of ripening, the protocaseose predomi- 
 nates. In the ripened cheese, however, protocaseose and deutero B 
 are present in about equal amounts, and together form about three- 
 fourths of all the caseoses. A distinction will be noticed here from 
 the albumose formation observed by Zunz in peptic digestion. Ac- 
 cording to Zunz, after deutero B has reached its maximum, deutero 
 A predominates, and finally deutero C. 
 
 In purifying the different fractions, the method of Haslam 21 was 
 followed out, viz, rubbing the precipitate in a mortar with am- 
 monium sulphate solution of the same concentration as the filtrate. 
 Several reprecipitations were made before the product was finally 
 freed from ammonium sulphate by repeated precipitation with alcohol. 
 
 The first fraction should contain, besides protocaseose, heteroal- 
 bumose if tliis substance were present in the cheese. Heteroal- 
 bumose could not be derived from casein. Traces were found, how- 
 ever, but they probably came from albumin. Upon subjecting the 
 carefully purified protocaseose to dialysis, a slight residue was left
 
 PEPTONES AND POLYPEPTIDS. 15 
 
 which would not diffuse. The amount was too small for chemical 
 examination, but it was probably heteroalbumose. Ah 1 the fractions 
 gave the biuret reaction, and all except deutero C gave the Millon 
 reaction. The intensity of the lead sulphid reaction seemed to 
 diminish progressively, until with deutero C it was just perceptible. 
 
 PEPTONES. 
 
 The filtrate from the caseoses was nearly neutralized with ammonia 
 and treated with a saturated solution of ferric ammonium sulphate. 
 A gelatinous brown precipitate resulted. This corresponds to the 
 alpha and beta peptones of Siegfried. 22 The precipitate was filtered 
 off, washed with a saturated solution of iron alum, and decomposed 
 by barium hydrate. After filtering off the ferric hydroxid and ba- 
 rium sulphate, a current of air was drawn through the alkaline solu- 
 tion until the ammonia was expelled. The barium was then removed 
 by sulphuric acid, and the solution concentrated under diminished 
 pressure and poured into a large volume of alcohol. A precipitate 
 was obtained which is analogous to Winter-stem's alpha peptone. 
 The filtrate still contained peptone, as was shown by an intense biuret 
 reaction. Further addition of alcohol gave no precipitate. The solu- 
 tion must therefore contain an alcohol-soluble peptone Winterstein's 
 beta peptone. It was reprecipitated, after distilling off the alcohol, 
 by phosphotungstic acid, the precipitate decomposed by barium hy- 
 droxid, and the barium removed by sulphuric acid, carefully avoid- 
 ing an excess. The solution was then evaporated to dryness. The 
 resulting beta peptone may have contained slight admixtures of poly- 
 peptids, but further purification was not attempted. 
 
 The alpha peptone gave no Millon reaction and a strong furfurol 
 reaction. Beta peptone, on the other hand, gave a strong Millon 
 reaction, but no furfurol reaction. Both gave the characteristic red 
 biuret reaction, xanthoproteic reaction, and a slight lead sulphid reac- 
 tion. Dried at 105 C., alpha gave 15.10 and beta 14.80 per cent 
 nitrogen. In this case the analytical figures have very little value, 
 as the substances are hygroscopic, and on drying continue to lose 
 water until a temperature is reached which causes a slight decompo- 
 sition. The two peptones were present in about equal amount and 
 together comprised about 1.6 per cent of the cheese. They had the 
 bitter taste characteristic of peptones. 
 
 POLYPEPTIDS. 
 
 After removal of the caseoses and peptones by means of lead acetate, 
 carefully avoiding an excess of the reagent, the cheese extract still 
 gave a biuret reaction. The polypeptids are the intermediate prod- 
 ucts between peptones and amino-acids. Some of them give a biuret
 
 16 CHANGES IN RIPENING OF CAMEMBERT CHEESE. 
 
 reaction, and their presence is probably the explanation of this phe- 
 nomenon. Some are precipitated by lead acetate, while others re- 
 main in solution. Fischer and Abderhalden obtained by the tryptic 
 digestion of casein a polypeptid, which on hydrolysis yielded alpha- 
 pyrrolidin-carboxylic acid and phenylalanin. These two acids were 
 not found in the free state, and their absence has b^en regarded as 
 characteristic of tryptic digestion. Whether or not erepsin decom- 
 poses this polypeptid is not yet known. It is very probable that other 
 polypeptids exist, temporarily at least, as transition products from 
 the peptones to the amino-acids. Many of them would be destroyed 
 by the action of the mold, while others would be more resistant. 
 Abderhalden 23 found that Aspergillus niger grows readily on glycyl- 
 glycin and dileucylglycylglycin, two polypeptids that are not attacked 
 by trypsin. Winterstein regards his alpha peptone as similar in many 
 respects to Fischer's polypeptid. This, however, would seem im- 
 probable, in view of the fact that he has demonstrated phenylalanin 
 and prolin in the cheese. No satisfactory method has yet been found 
 for separating the polypeptids as a group, and the amount present in 
 a cheese can only be a matter of conjecture. The polypeptids will 
 be made the subject of future study in this connection. 
 
 DIAMINOACIDS. 
 
 The next group of substances to be studied was the diamino-acids, 
 or hexone bases. The three hexone bases, along with ammonia, can 
 be determined quantitatively, and for that reason they have received 
 more attention from investigators studying the disintegration prod- 
 ucts of the proteins than have the monoamino-acids. They can be 
 expressed in definite figures, whereas the other disintegration products 
 have to be expressed in minimal values. They have already been 
 found in cheese in Swiss cheese by Winterstein and ThOny, and in 
 Cheddar cheese by Van Slyke and Hart. Owing to the softer con- 
 sistency of Camembert cheese a somewhat different method of pro- 
 cedure was adopted in making the extraction. 
 
 Three kilograms of the thoroughly ripened cheese made at the 
 Storrs Experiment Station were ground in a mortar and extracted six 
 times with warm water, according to the usual method of analysis, 
 until the volume of the liquid was about six liters. The greater part 
 of the fat rose to the surface and could easily be skimmed off. It 
 was washed by stirring thoroughly with cold water and the filtrate 
 mixed with the cheese extract. The remainder of the fat was found 
 to be precipitated almost quantitatively with the proteins, and thus 
 the necessity of extracting the original cheese with ether was obviated. 
 The extract was filtered through cotton and through asbestos, then 
 acidified with sulphuric acid and warmed until the caseoglutin had
 
 DIAMINO-ACIDS. 17 
 
 settled out, whereupon the liquid was filtered again. The solution 
 was now concentrated at a low temperature until the volume was 
 about two liters. About three volumes of alcohol were added to 
 precipitate the greater part of the caseoses and peptones. After 
 filtering off this precipitate the alcohol was distilled off under dimin- 
 ished pressure, and tannic acid added to precipitate the rest of the 
 caseoses and peptones. The excess of tannic acid was removed by 
 lead acetate and the lead by sulphuric acid. The resulting solution 
 still gave a biuret reaction. It contained, besides traces of second- 
 ary disintegration products and polypeptids, the hexone bases, 
 ammo-acids, and ammonium salts, together with the sodium chlorid 
 present in the cheese. 
 
 The hexone bases were precipitated by a 50 per cent solution of 
 phosphotungstic acid in the presence of 5 per cent sulphuric acid. A 
 large amount of this reagent had to be added before the precipitation 
 was complete, and a voluminous white precipitate was obtained. 
 After standing several days it was filtered off and washed w r ith 5 per 
 cent sulphuric acid containing a little phosphotungstic acid. The 
 washing was a long and tedious operation. It was found necessary 
 to remove the precipitate each time from the funnel and grind it with 
 the sulphuric acid in a mortar. This was repeated until all of the 
 sodium salts had been removed. 
 
 For the separation of the bases KossePs 24 older method was used, 
 after removal of the phosphotungstic and sulphuric acids by barium 
 hydroxid and passing in a current of air to expel the ammonia. The 
 excess of barium was removed by carbon dioxid, and mercuric 
 chlorid added to precipitate the histidin. This precipitate was 
 allowed to stand several days, then filtered and washed again. It 
 was suspended in water and decomposed by hydrogen sulphid after 
 slightly acidifying with sulphuric acid. The filtrate from the mer- 
 curic sulphid was boiled with charcoal until practically colorless, 
 and precipitated with silver nitrate and ammonia. The arginin was 
 precipitated by saturating the solution with barium hydroxid, and 
 adding silver nitrate until a drop of the solution gave a brown color 
 on the addition of silver nitrate. The arginin silver was decomposed 
 by hydrochloric acid and hydrogen sulphid, filtered, boiled with 
 charcoal, and evaporated to crystallization. The filtrate from the 
 arginin was freed from barium and silver by means of sulphuric acid 
 and hydrogen sulphid, and an alcoholic solution of picric acid added. 
 The lysin picrate did not crystallize readily, but after several crystal- 
 lizations the characteristic yellow needles were obtained. The bases 
 were found in the following amounts: IILstidin, 1.1 grains; arginin, 
 0.6 gram; and lysin, 1.9 grains. Histidin was analyzed in the form 
 of the silver salt, arginin as the chlorid, and lysin as the picrate. The
 
 ( HAXCKS IX RIPKXIXc; OF CAMKMBERT CHEESE. 
 
 free histidin gave an intense red color with diazobenzenesulphanilic 
 acid. The analyses of the bases are given below: 
 
 Histidin silver, C^HjN 3 O 2 Ag 2 II 2 O. 
 
 ( Beta-imidoazol-alpha-aminopropionlc add.) 
 
 Argon turn. 
 Nitrogen.. 
 
 Calculated. 
 
 V..M 
 10.85 
 
 Found. 
 
 55. 00 
 10.78 
 
 Arginin hydrochlorid, C 6 H l4 N 4 O. 2 ltCl. 
 
 ( Delta-guanidinc-alpha-aminovalerianic acid. ) 
 
 Chlorin.. 
 Nitrogen. 
 
 Calculated. 
 
 16.87 
 22. GO 
 
 Found. 
 
 Hi. 70 
 22.50 
 
 Lynn picrate,C<HuN 2 2 CJf 3 N 3 7 . 
 (Diaminocaproic acid.) 
 
 
 Calculated. 
 
 Found. 
 
 Nitrogen 
 
 18. 00 
 
 18.50 
 
 Carlx>n 
 
 38.40 
 
 38.53 
 
 Hydrogen 
 
 4.53 
 
 4.54 
 
 
 
 
 The other bases were present in so small amount (about 0.5 gram) 
 that no attempt was made to isolate them. A noteworthy fact is that 
 arginin, which was not found at all in Swiss cheese, is present here. 
 It is possible that some of it is further hydrolyzed into guanidin and 
 aminovalerianic acid or into urea and ornithin (diaminovalerianic 
 acid). The filtrate from the histidin, arginin, and lysin had a very 
 faint but characteristic odor of tetramethylenediamin. This sub- 
 stance would result from the liberation of carbon dioxid from 
 ornithin, one of the cleavage products of arginin. It could not have 
 been present, however, in more than traces. An attempt was made 
 to separate guanidin in the form of the gold salt, but no crystals could 
 be obtained. The small amount of bases remaining in the lysin frac- 
 tion indicates that the occurrence of secondary reactions in this group 
 is very slight. 
 
 MONOAMINO-ACIDS. ' 
 
 A complete separation of all the amino-acids remaining in solution 
 after removal of the intermediate disintegration products and hexone 
 bases can be accomplished only by Fischer's method of distilling the 
 ethyl esters in vacuo. This necessitates delicate and costly apparatus
 
 MONOAMINO-ACIDS. 
 
 19 
 
 which the writer did not have at his command. Certain of the amino- 
 acids, however, can be separated almost quantitatively from the mix- 
 ture without resorting to the method of esterification. Among these 
 are glutaminic acid, tyrosin, and leucin. 
 
 Another lot of cheese (3 kilograms) was treated in the manner 
 described above to remove the caseoglutin, caseoses, and peptones. 
 The residue was evaporated to a small bulk, saturated with hydro- 
 chloric-acid gas, and kept at zero for several days. Crystals were 
 deposited on the walls of the flask, and a pulverulent precipitate sepa- 
 rated out on the bottom. These were found to consist of glutaminic 
 acid, hydrochlorid, and sodium chlorid. They were transferred to a 
 Buchner funnel and washed with concentrated hydrochloric acid, 
 then dissolved in water. The solution was neutralized with caustic 
 soda and boiled with freshly precipitated copper hydroxid. A blue 
 precipitate was formed. It was filtered and washed, and then sus- 
 pended in water slightly acidified, and decomposed by hydrogen sul- 
 phid. The free acid thus obtained was again saturated with hydro- 
 chloric-acid gas and allowed to crystallize as before. The colorless 
 crystals thus obtained were decomposed by the calculated amount of 
 caustic soda (30 c. c. N-NaOH), and the free acid crystallized out. 
 About 5 grams of crystals were obtained. Analysis gave the follow- 
 ing figures : 
 
 (Hutaminic acid, C 5 H 9 A : O t . 
 (Aminoglutaric acid.) 
 
 
 Calculated. 
 
 Found. 
 
 Carbon 
 
 40.82 
 
 40. 62 
 
 Hydrogen . . . 
 
 6.13 
 
 fi. 15 
 
 Nitrogen 
 
 9.52 
 
 9.53 
 
 
 
 
 The filtrate from the glutaminic acid was treated with lead carbon- 
 ate to remove the bulk of the hydrochloric acid, and the lead remain- 
 ing in solution was removed by sulphuric acid. After filtering and 
 neutralizing, the solution was evaporated to incipient crystallization. 
 The first crop of crystals should contain tyrosin and traces of leucin, 
 and the second leucin with traces of tyrosin. The two constituents 
 of each fraction were separated by treatment with glacial acetic acid. 
 The leucin purified in tliis way gave no color with Millon's reagent. 
 The tyrosin was tested for sulphur by fusing a portion of it with 
 sodium carbonate and adding sodium nitroprussid to the aqueous 
 solution. No coloration was obtained, indicating the absence of 
 cystin. About 8 grams of tyrosin and 14 grams of loucin were 
 obtained. It must be borne in mind that while the greater part of the 
 glutaminic acid and leucin can be isolated in this way, the amounts 
 do not represent strictly quantitative results, for a further yield is
 
 20 
 
 CHANGES IN RIPENING OF CAMEMBEKT CHEESE. 
 
 invariably obtained from the higher boiling fractions of the ethyl 
 esters. Following are the analyses of the tyrosin and leucin: 
 
 Tyrosin, C 9 H n ^O^. 
 (P-hydroxyphenyl-alpha-amlnopropionlc acid.) 
 
 
 Calculated. 
 
 Found. 
 
 Carbon 
 
 59.66 
 
 59.53 
 
 Hydrogen 
 
 6.07 
 
 (i.OO 
 
 Nitrogen 
 
 7.73 
 
 
 
 
 
 Leucin, C 6 // 13 A 7 O 2 . 
 (Alpha-aminoisobutylacetic acidJ 
 
 
 Calculated. 
 
 53.96 
 9.92 
 10.68 
 
 Found. 
 
 First 
 crop. 
 
 Second 
 crop. 
 
 Carlwn 
 
 54.99 
 10.53 
 
 54.83 
 
 9.62 
 10.76 
 
 It v<lrogen 
 
 Nitrogen 
 
 
 The tyrosin gave the characteristic color reactions, viz, a red color 
 and precipitate with Millons reagent, a bright red coloration with 
 diazobenzenesulphanilic acid, and a yellow precipitate of nitro ty- 
 rosin nitrate with nitric acid. Under the microscope it showed the 
 characteristic wavy needles. 
 
 The leucin was also characteristic under the microscope. Heated 
 on a platinum foil it sublimed completely, emitting an odor like that 
 of some of the higher alkylamins. 
 
 On further concentrating the filtrate from the leucin, a few needle- 
 shaped crystals were obtained having a sour taste. They did not 
 melt at 225 C. Not enough of the substance was obtained for 
 analysis, but it was probably aspartic acid. 
 
 As has already been mentioned, the remainder of the amino- 
 acids can not be separated without resorting to Fischer's method of 
 distillation. Phenylalanin and tryptophan, however, can be detected 
 qualitatively. On evaporating the solution to dryness and treating 
 the residue with sulphuric acid and potassium dichromate, the 
 writer felt confident that he detected the odor of phenylacetaldehyde, 
 notwithstanding the presence of other aldehydes formed by the oxida- 
 tion with dichromate. This would indicate the presence of phenyl- 
 alanin, but from this test alone it would be impossible to say with 
 certainty whether phenylalanin was really present or not. 
 
 A striking fact is that none of the cheeses examined responded to 
 the tryptophan reaction with acetic acid and bromin water. Aque-
 
 ABSENCE OF PUTREFACTIVE PRODUCTS. 21 
 
 ous extracts of cheeses in all stages of ripening were examined, but 
 in all cases they failed to give any coloration with this reagent. 
 When the mold was grown upon milk the tryptophan reaction was 
 likewise negative, although the casein was broken down into amino- 
 acids, among which leucin and tyrosin were identified. On the other 
 hand, the enzyme preparation from the same mold readily digests 
 milk in the presence of toluol, and the tryptophan reaction is inva- 
 riably positive. If tryptophan were liberated in the cheese, it might 
 undergo further decomposition as a result of bacterial action. In 
 this case the end products would be indol and skatol characteristic 
 products of putrefaction. Both of these substances would have 
 given a coloration with the tryptophan reagent, and must therefore 
 have been absent. 
 
 AMMONIA. 
 
 X 
 
 A normal cheese contains from 0.20 to 0.25 per cent of ammonia. 
 The greater part of this is in combination with acid radicals, for while 
 the ripened cheese is alkaline to litmus it is still acid to phenolphtha- 
 lein. Some of the more highly flavored specimens, however, have a 
 distinct odor of ammonia near the rind. The formation of ammonia 
 does not begin until after the second week, then the amount steadily 
 increases until the cheese is ripe. As long as the ripened portion 
 does not extend more than a few millimeters below the mycelium of 
 the mold no ammonia, either free or in combination, can be detected. 
 Cheeses with a very strong flavor contain, as a rule, more ammonia 
 than the milder ones. In cheeses that are overripe the presence of 
 free ammonia can usually be detected by its odor. For the deter- 
 mination of ammonia, the writer has found Folin's method more sat- 
 isfactory than the regular method of distilling the tannin filtrate with 
 magnesium oxid. It can be used in the presence of proteins without 
 effecting any decomposition. 
 
 ABSENCE OF PUTREFACTIVE PRODUCTS. 
 
 While the ripened cheese possesses a peculiar odor, which to some 
 people is quite disagreeable, it does not resemble that of the typical 
 putrefactive products, nearly all of which are characterized by a foul 
 odor. Among the substances belonging to this class are indol, skatol, 
 mercaptan, hydrogen sulphid, and phenols. Qualitative tests were 
 made repeatedly for all of these substances on different samples of 
 cheese, but in all cases they were negative, except in those cheeses 
 that had gone past the usual ripening. After the ripening is complete 
 putrefaction may set in if the cheese does not receive proper care. In 
 those cases where some of the putrefactive products were found the 
 cheeses were otherwise unfit for eating, as was evidenced by a very
 
 22 CHANGES IN RIPENING OF CAMEMBERT CHEESE. 
 
 disagreeable odor and taste. Nearly all of these *ubstanc?s are 
 found in Limburg cheese, but they do not occur in good Camembert 
 cheese. The failure to find typical putrefactive products, together 
 with the fact that the lysin fraction of the diamino-acids contained 
 only traces of diamins, indicates that whatever the action of the 
 bacteria in the cheese may be, they do not cause secondary reactions 
 of this nature to any extent. 
 
 SUMMARY. 
 
 The following substances have been isolated from Camembert 
 cheese: Caseoglutin, protocaseose, deuterocaseoses A, B, and C, alpha 
 and beta peptones, histidin, arginin, lysin, glutarninic acid, tyrosin, 
 and leuein. 
 
 Among those substances which the writer failed to find are para- 
 nuclein, tryptophan, indol, skatol, mercaptan, hydrogen sulphid, and 
 phenols. 
 
 The ripening of Cam?mbert cheese can not be a peptic digestion, as 
 is shown by the following facts: 
 
 1. Paranuclein, the characteristic product of peptic digestion of 
 casein, is absent. 
 
 2. The greater part of the phosphorus is liberated and appears as 
 acid calcium phosphate. According to Plimmer and Bayliss, 25 
 pepsin acts slowly and incompletely, only 70 per cent of the phos- 
 phorus being liberated from casein in 149 days., and that mostly in 
 the organic form. 
 
 3. Amino-acids and ammonia are present in considerable amount. 
 The ripening resembles ereptic digestion in many respects, as 
 
 follows: 
 
 1. The reaction of the cheese before ripening, i. e., acidity caused by 
 acid phosphates, is most favorable to the activity of ereptase. 
 
 2. The digestion proceeds beyond the peptone stage, with the for- 
 mation of amino-acids and ammonia. 
 
 3. A separate study of the enzyme from the Camembert mold 
 shows that this enzyme is a vegetable ereptase. 
 
 The absence of tryptophan, which is ordinarily liberated in ereptic 
 digestion, is striking. 
 
 The presence of caseoglutin, the remarkable albumose-like body, is 
 also noteworthy. This substance has not, to the writer's knowledge, 
 been observed in digestions with pure enzymes. 
 
 ACKNOWLEDGMENTS. 
 
 In conclusion, the writer acknowledges his indebtedness to Prof. 
 L. B. Mendel, of Yale University, and to Dr. B. B. Turner, formerly of 
 the Storrs Experiment Station, for many helpful suggestions in carry- 
 ing out this work.
 
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 24 HANCIS IN KII'KMN<; < >1 A M I. M 111 K I QHBEBE. 
 
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