Proteolytic changes in the ripening of Camembert cheese


Material Information

Proteolytic changes in the ripening of Camembert cheese
Series Title:
Bulletin / United States Department of Agriculture, Bureau of Animal Industry ;
Physical Description:
24 p. : ; 23 cm.
Dox, Arthur Wayland, 1882-
United States -- Bureau of Animal Industry
U.S. Dept. of Agriculture, Bureau of Animal Industry
Place of Publication:
Washington, D.C
Publication Date:


Subjects / Keywords:
Camembert cheese   ( lcsh )
federal government publication   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )


Includes bibliographical references (p. 23-24).
Statement of Responsibility:
by Arthur W. Dox.
General Note:
"November 28, 1908."

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 029613537
oclc - 22301286
System ID:

Full Text

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Chemistt ,,t C(-'t w" ]'/;*( slh;,,/ioizs, 1),m i /,,; isiou.

Chip. A. D. MELVIN.
Assislltant (C'hie.f: A. M. FARRIN\GTON.
Biorhten, ic Di, ision. M. DORSET. chief, JAMES A. EMERY, assistant chief.
Dairy Diti.sion: Eu. H. WEBSTER. chief C. B. LANE, assistant chief.
Inspaetin Division.: RICE P. STFEDDOM, chief: MORRIS W\OOOEN, R. A. RAMSAY,
and ALBERT E. BEHNKE. associate chiefs.
Patholuqical Dirisionr JoH.N R. MOHILER. chief. HENRY .1. WASHBL'RN, assistant
Quarantine Dirision: RICHARD W. HICKMAN, chiPf.
Zoological hit ision: B. H. R.ANSOM, chief.
Exeiiuent Stluion: E. C. SCHROE.DER, superintendent, \W. E. 'oTTrroN, assistant.
Animal lhusbandman: GEORGE M. ROlIMEL..
Chief Ed. H11. Websiter.
assistantt l 'hirf: C. B. Lane.
Librarian: M iss (C'. B. Shernma .
.4. si.sanI in charge B H. Raw'l: osi.tantl, DIinaun .Stuart.
Dairy buildings' .. A 'ono\er architeol. K. E. Parks; \ontilation experiments,
C. R. Poiieiger.
Herdbook work: Helmer Ralmild and \William Hart Dexter.
Southern dairying S. E. Barnf... J. E Dorman, J. T. Eat'n. H. P. Lykes, J. H.
MrClain. A. K. Risser II. R Welch. and T R. Woodward.
.Issistoni in churqe, L. .4 Rogers.
Butter investigatiun. Alliert Lea. Minn.. and Washington D. C.: Chemist,
W. H. Berg; Iatteriologist, S. H. Ayer,.
Swiss ( heese in\'etligatii'n-. Albert Lea. Minn.: In charge, tC. F. Doane; assistant,
T. W. lsajeff.
Chee-e investigationh, Mndioi. \\'is.: C'hemist. S. K. Suzuki; bacteriologist, Alfred
Lar.on. cheese maker. J. \V. Moore.
CheeqP investigations. Storrs. Conn.: My ecologist, Charles Thorn; chemist, Arthur
W. Dox. cheese maker. F. R. Thompson
Milk e,'relion investiatIion_,. (C'linmbia. Mo. Chemist. R. H. Shaw; assistants,
J. 0 IIalverson. .\. E. Perkins. and C. C. Paync.
A.I.ssistntl in charqi. B. D. White: assistant, S. C. Thonipso
Creanierv r:-'ords, Albert Lea. Minn.: C'reamery practice, John L. Sherk; assistants
t(ollalir'itorsi, P. \V Noble amId .1 D. Burk.
<'reanmery practice investigations- J. (' Joslin, Robert McAdam, F. L. Odell, J. C.
Winkjt-r. and Thorna: ('orneliuson.
Market investigation!. New York City. C. W. Fryhofer: Chicago, H. J. Credicott;
San Fra'ico. O' I.. Mitchell.
.A\.,istant chief of division in icharge: assistants, Lee H. P. Maynard, Ivan C. Weld,
and George M. Whitaker.
Chief inspe tlor, M. \W. Lang. C 'liica. assistant. Levi Wells, New York.


lash ingltorn, D. C., Seplt'm ber 21, 1908.
SIR: I have the honor to transmit herewith a manuscript entitled
"Proteolvtic Changes in the Ripening of Camembert Cheese," by
Arthur W. Dox, chemist in cheese investigations, Dairy Division, and
recommend that it he 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
Dair- Division of this Bureau.
Respectfully, A. 1). MELVIN,
(_"I f : f', Bu,'t m .
Seerretary Of Aorl'i1iit, i-.


I n lt i i i ury ................ . . . . . . . . . . . . . . . . . . 5
Pr',,'es.,- in rhe ripenin of ('arm i':inlI r li -eiese. . ....................... 6
E n y lin es in thie cheese ..................... .......................... 8
P r' ,,i sis if . .j. . . . . . . . . . . . . . . . . 10
N. ilr rJ e n'i -s V1-.9tl (i t u itS ii til' 'ii i ........................... ........ 11
H ydrochlr' -r i il prev pitil -. ..... ............... ......... 12
( ,ng,.n il e pr,,e.i ite . .. . .. .. .. . .. . . . . . . . . . . . . . . . 13
(C': s e e s . . . . . . . . . . . . . . . . . . . .. . . . . . 1 4
P e p t n e s . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 1 5
P l ypi lt ii Is ............ .............. .. .... .. .... 15
D ia rn in ,o -a c id s ............ .. ........ . ...... .... ... .. ...... 16
M I, ,i ;a n i n -ii i dls . . . . . .. . . . . . . . .. . . . . . . . . . . . . 18
A. m ,', a . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1
.\l,,n, e il pitr-la ix P prdiult. ... .... ................... 21
S ii iu i r . . . . . . . . . . . . . . . . . . . . . . . .. . 2 2
.\ k n i, uI, ii. n i s . . . . . . . . . . . . . . . . .. . . . .. . .. . 2 2
H eie' ren, (' i t ierJitu le ................. . . ..................... 23


Until comparatively recent years the change.. that take place in the
ripening or curing (if tlhe dtiierent varieties of cheese were buit little
understood. Although the practice of cheese making has been carried
on for centuries,, all of our knowledge of thle cheltiical changes involved
in the ripening process and thie various factors that bring about these
changes has come top us within the past fifty years. Thlie earliest
record \we have of any discussion of this subject from a chemical point
of view waas publi.thedl only a centni-y ago. In this paper the French
chemist, ('haptal,' dliscusses the ripening of Roqiefort cheese and
advances certain tILhIories to accouInt for the changes in appearance and
flavor which tliis cheese undergoes during its sojourn in tlie natural
ripening caves. Biological factors were, of course, not taken into
account in Chaptal's paper, for that plihase of the subject was unknown
until the time of Pasteur.
No scientific study of the subject, however, was made until tlie
latter half of thlie nineteenthl century. Tlie attention of chemists was
then directed to Roquefort cheese by a paper publihetd Iby Blonileau
ini 1S64. Blondeau analyzed cheeses in different stages of ripening
and found that tlie fat content increased front I.S. per cent in thlie
fresh cheese to 32.31 per cent in thlie cheese two montlis old. This
enormous increase in thle fat lie ascribedl to a synthliesis of fat from
protein by the mold. But a comparison of his figures for tlie othlier
constituents of thie cheese as well as thlie fat with analyses to be found
in any modern book on dairy products will show their utitter im-
possibility. This inaccurate work of Blondeau, however, served thlie
purpose of directing thlie attention of other investigators to tlie sub-
ject, and during thlie next few years the changes in thlie fat content of
cheese were studied Iy Brassier,3 Sieber,' Jacol)stahlil, and Von Ndgeli
anti Loew.A
By this time the subject of cheese ripening had begun to arouse con-
siderable interest amniong scientific investigators, and their researchies
were extended to other varieties of cheese. Swiss cheese anti Chlieddar
a The figure s refer to list utf literaturte at end of bulletin.


cheese in particular -received considerable attention. Among those
who worked with Swiss cheese were Weidmann,7 Rose,8 Schulze,8 and
Winterstein." In our own country the Cheddar type of cheese has
been minade the subject of thorough chemical investigation, and in this
connect ion reference is made to the work of Babcock and Russell'10 and
that of Van Slvke and Hart." 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.
,ll 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.
The variety of soft cheese which we 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 tlhe 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.
The biological factors essential to the production of Camembert
cheese are the lactic-acid bacteria, which are normally present in
milk, and two molds, Penicilliurn carmemberti Thom'' 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 in 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.


Thile ripening of all cheeses being essentially a li \ drolysis of tlhe 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 carhoxvl groups. The products of
simple hydrolysis may thus undergo further changes with the forma-
Stion of substances dliffering widely in chemical constitution from the
substances from which thev were derived. The successive steps of
such reactions are often difficult to follow, and it is sometimes imIpos-
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 hYdrolvtic product.
These changes are caused for the most part by bacteria, and in a
cheese where theli 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 tlie cell wall
of the organism, but rather from activities dependent upon tlie life
history and metabolism of the organism. For this reason thle center
of a hard chlieese shows tihe same flora and the same chemical comrnpo-
sition as the portion near the rind. With cheeses of the Camembert-
Brie type, however, there is a marked difTerence in this respect. Tlie
actual ripening here is caused by the proteolytic enzyme of the nmold.
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 andi proceeds toward (l 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 thle 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,


the oilither three proteins mentionedI above Yield the same primary
disintegration products, and a distinction as to the origin of the
laitler can not he niade.
The protcolytic changes which constitute the ripening of Camem-
bert cheese con,ist, therefore, in the changes which this paracasein
unlergoe, through the action of proteolytic enzymes. As has already
been mentioned, the principal factor is the enzyme secreted by the
Camemnbert 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 thlie curd. These changes have been -studied by Bosworth.'15
rThey consist mainly in the liberation of paracasein from combination
with calcium, idue to the formation of lactic acid by lactic acid bac-
teria. At the same time the paracasein is converted into a form
completely siluble in 5 per cent salt solution, and later it becomes
insoluble again. As these are not, strictly speaking, proteolytic
changes, a detailed discu-ssion will not be given here.
With thlie exception of thle enzyme secreted by the mold and of a
smaller variety of bacteria, Camembert contains tie same ferments
that are present in other cheeses. Like the hard cheeses, it contains
the milk enzyme galactase, the rennet enzy-me chIymosin added in
thle curdling process, and lactic acid bacteria. In the case of the
Cheddar type of cheese thle action of these three factors has been
studied in detail. Babcock and Russell found that galactase and
rennet (pepsin I were important agents in the ripening of this variety
of cheese. According to Van Slyke and Hart." the rennet alone is
capable of ripening a cheese. In their experiments the galactase
was first destroyed by heat. and then chloroformn added to prevent
thle development of bacteria, yet the ripening went onil, though not.
as rapidly as in the normal cheese, and the character of the chemical
products was somewhat different. Thus there was a predominance
ot paranuclein, caseoses, and peptones, and an abnormally small
amount of amino-acids. The entire absence of ammonia was very
-striking. The function of thle bacteria in this variety of cheese has
been studied by Rogers." lie came to the conclusion that the
enzymes_- produced by tlhe bacteria were responsible for most of the
digestion bey'nid thlie peptone stage, and consequently the charac-
teristic flavors.
In the -short time required for thie ripening of Camembert cheese
the rennet, galactae, and lactic acid bacteria produce no appreciable
digestion. This conclusion was reached by Bosworth, and the expe-
rience of the writer cunlirmnis it. Even the hard cheeses which are i


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 sutb-
jected to chemical analysis it will he 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 r6le, as is shown by the fact that the cheese ripens
normally when made from milk which has been pasteurized at a
temperature suiliciently 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, thie 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 camemberti) 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. 109-08-- 2


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. A!
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'7 has shown that erepsin is very widely distributed in the
vegetable kingdom. This vegetable "ereptase," as he calls it, differs 4
from animal erepsin in that it. is most active in the presence of the
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 TI
calcium lactate and mono-calcium phosphate. The acidity of the
cheese is due to the presence of the latter salt. .1
In this connection, however, it must be remembered that the same i
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.
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-,i:


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.


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 nn 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." 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 thle 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 he 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
Per e lnE
Nitrogen as- ofi cnesr...
T o ta l n itro geii ........................... ...... . . . . 2 .4 7
W a ter- so lu b le ... ...... ........ .. ..... ....... ..... ...... 1. 79
Precipitated by hydrochloric acid .... ... 40
Caseoses ....... .. ... 10
P e p to n e s . .. . .. . .. .. .. .. . .. . . . .. . .. .. 28
A m in o -acid s ................. ...... ... . ..... 82
Salt- soluble paracasein ..... ........ ... . . .. .. ... .. ......... 15
A m m o n ia ... .. .................... ..... .......... .. ... .... .. 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


cheese at different stages of ripening will be found in Bosworth's...
paper.' These analyses show merely the rate of formation or destrue-,:.
tion of three broad groups of digestion products, and throw little l
light upon the nature of the ripening from the biochemical stand- i
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.
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 phosphonrus 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 KRirajeff's plastein, formed by the action of rennet on
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, filterixig 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 precipitatA'.,ii


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. 110 C. gave the following analysis, based upon
the ash-free substance. Alongside it are given Rose's analysis of
paracasein and Chittenden's'8 analysis of paranuclein, with the
phosphorus as found by Jackson.

Substance Plaracas(in Paranuc lcin
from cheese (Rose I. (Chittenden).

C a rbon ............. .. ......................... ... 53. 3 53.94 51. :9
H ydrogen ......... . ...... ...... . 7.O I 7.14 1 2G
N nitrogen. ......... ... ... 15.10 15.14 15.23
S u lp h u r ......... ...... .. ... ...... .. .. .. .. .. 1.0 1 (.8
P hosphoruts .......... ........ .... .. ... .. '2 75
A sh ............ ... .. . . . .... ..... 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 R6se's analysis of caseoglutin, follows:

Substance CaseogluEin
from cheese, L Rbsei.

Carbon ............ ... ...... .. . ..... . .. .... 54. 34 54.4
H ydrogen .............. ... ...... .... .. .. . .. ...... ... .. 7.30 7.34
N itrogen ................................ ... ... 15.37 15.29
S u lph u r .............................................. ... . ..... . 95 .95
Phosphorus ........ .................... ....... .. .. ...... . o ..............
A sh ......... ....... ... .......... 8 ...........

Both the analysis and the properties of this substance agree with
those of caseoglutin.

Winterstein found in Swiss cheese a small amount of a substance
which was precipitated from acid solution by boiling. This substance
he calls tyroalbumrnin. Its exact, nature has not yet, been determined.


Several attempts were made to find this substance in Camembert
cheese, but so far they have resulted in failure. On heating the
filtrate from the caseoglutin to boiling, both in acid and in neutral
solution, no precipitate was obtained.
These, together with the peptones, are the intermediate disintegra-
tion of casein by ordinary proteolytic enzynmes. They can not be
regarded as homogeneous substances, as they represent, transition
products formed by the loss of varying numbers of amino-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 albunmoses by fractional precipitation with ammonium sul-
phate was elaborated by Pick.'19 If nitrogen determinations are to
be made, the ammnionium sulphate must be removed by dialysis, a
long and tedious operation. To obviate this difficulty Zunz20 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
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 protocascose predomi-
nates. In thle ripened cheese, however, protocaseose and deutero B
are present in about equal amounts, and together form about three-
fourtlhs of all thle caseoses. A distinction w-ill 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 Haslam2 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 this substance were present in the cheese. Heteroal-
bumose could not be derived from casein. Traces were found, how-
ever, hut. they probably came from albumin. Upon subjecting the
carefully purified protocaseose to dialysis, a slight residue was left


which would not diffuse. The amount was too small for chemical
examination, but it. was probably heteroalbumose. All 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.
The filtrate from the caseoses was nearly neutralized within 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."' 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 whichli is analogous to Winterstein's alphlia 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 dis-tilling 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.
After removal of thlie 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

reaction, and their presence is probably the explanation of this phe-j
nomenon. Some are precipitated by lead acetate, while others re- ii
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 been 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"3 found that Aspergilus 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, anl 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.
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 casepglutin had

.. ::;i ::::.[i[


settled out, whereupon thile liquid was filtered again. Tile solution
was now concentrated at a low temperature until the volume was
about two liters. About three volumnes 4f alcohol were added to
precipitate the greater part of the caseoses andl peptones. After
filtering off this precipitate thle alcohol was distilled otl under dimin-
ished pressure, and tannic acid added to precipitate the rest of tihe
caseoses and peptones. The excess of tamulilc acid wyas reiMoved by
lead acetate and the lead b1y s-ulphuric acid. ''lThe resulting solutionn
still gave a biuret reaction. It contained, besides traces of sectond-
ary disintegration products and polypeptidls, tlihe liexone bases,
amino-acids, and ammonium salts, together within the sodium chloritd
present in the cheese.
The hexone bases were precipitated by a 5(1 per cent solution of
phosphotungstic acid in the presence otf 5 per cent sulphuric acitd. A
large amount of this reagent hadl to be added befor e tlie precipitation
was complete, and a voluminous white precipitate was obtained.
After standing several days it wa. filtered off ;ind a ashel with 5 per
cent sulphuric acid containing a little phlisplhottunim-tic acid. The
washing was a long and tedious operation. It \wa% found necessary
to remove the precipitate each time from thlie funnel anti 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 Kosel'sd' oler method was used,l
after removal of the phosphotungstic and sulIphuric acids by bariumt
hydroxid and passing in a current of air to expel the ammonia. The
excess of barium was removed by carbon tlioxidl, and mercuric
chlorid added to precipitate the hi.stidin. This precipitate was
allowed to stand several days, then filtered and washed again. It
was suspended in water and decomptosed by hydrogen sulphid after
slightly acidifying with sulphuric acidl. The filtrate from the mier-
curic sulphid was boiled with charcoal until practically colorless,
and precipitated with silver nitrate and ammoinnia. The arginin was
precipitated by saturating the solution with barium hydroxidl, and
adding silver nitrate until a drop of tlie solution gave a brown color
dn the addition of silver nitrate. The arginin silver was decomposed
by hydrochloric acid and hydrogen shilphid, filtered, boiled with
charcoal, and evaporated to crystallization. The filtrate from the
arginin was freed from barium and silver by means of s-ulphuric acidl
and hydrogen sulphid, and an alcoholic solution of picric acitl 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: Histidin, 1.1 gram.s., arginin,
0.6 gram; and lysin, 1.9 grams. Ilistidin was analyzed iII the form
of the silver salt, arginin as the chlorid, anti lysin as the picrate. The


free histidin gave an intense red color with diazobenzenesulphanilic
acid. The analyses of the bases are given below:

Histidin silver, C6; N30,Ag:JiO.
Beta-imidoazol-al pha-amniopropionic acid.)

Calculated. Found.

.\rgnLium ... .. i 55 1 55 .O0
Nitrogen ......... 10.85 M.78

Arginiit hydrochloride, (',7H, 1 O,11OIl.
i [If-lia-guanidine-alpha-aminovalerianic acid 1

I Calculated. Found.

C. hlorn . I, 87 1',. 70
Nilrouon. 2. 60 2.50u

lpsiti pirraf,, ('61,4 NT 0 2 .,(Hj )O.
4 Dia1iinlota.Iiroic ac id. I

t'altuluard. Found

S Nilro '-rn. lI ft, I s.50
C 'arhon . s. 0 33853
HyvdrLpvii.. I 4 541

Thile other bases were present in so sminall 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
aninovalerianic acid or into urea and ornithin (dianminovalerianic
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 ot 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.

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


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 glutamninic 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
Biichner 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-NaOHi, and the free acid crystallized out.
About 5 grams of crystals were obtained. Analysis gave thle follow-
ing figures:
(ilulaminl, t id, ( l,,A' .
I .\ TiogliIthrJiv. u iilt

('jifiils Li''l F Fnrii .

Carl on ..... .42 4 '0 1'2
Hydrngen.. 1M f 15
N it r'pien .. Q 52 ; .. |

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 this way gave no color with Miltin'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 leucin 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


invariably obtained from the higher boiling fractions of the ethyl
esters,. Following are the analyses of the tyrosin and leucin:

Trrsirn. C9H, .\0O
(P-hbdrox.rplhenyl-a Ipha-amimopropionic acid.)

Caliedlated.| Found.

Cn rIon ... ..' 59 u' )59.5.3
ll dr,_gn . 07 f! 00
Nitlrgen .... 7 73 7 9

Lotin. C./ 1,.%0.
,..\ phd-j m In e o bu ly Ila.c.cii. ari~d ,
ne ni sant!yltict isin'J
C'li t ejlarel.
First St-cond
crop crop.

Carnon..... 53 '91 .544.9f 54. -,
lli rogun ... .i 4-2 H W) 9 62
Nitrogen 1.. .. I I 5 aI 10. 7h |
L __-

The ty-rosin gave the characteristic color reactions, viz, a red color
and precipitate with Millon's reagent, a bright reel coloration with
diazobenzenesiilphanilic acid, and a yellow precipitate of nitroty-
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 alkylanins.
On further concentrating the filtrate from the leiucin, a few needle-
shaped cry'stals 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 (lichromnate. 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-


ous extract- otf cheeses in all sitages of ripening were examined, lhuiit
in all cases they failed to give any coloration within this reagent.
When the mold was grown upon milk the tryptophan reaction was
likewise negative, although thlie casein was broken down into amino-
acids, among which leucin and tyrosin were identified. On the other
hand, thlie enzyme preparation from the same mold readily digests
milk in the presence of toluol, and the tryptophan reaction i,; 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-characteri-,tic
products of putrefaction. Both of these sub-tances %mould have
given a coloration with the tryptophan reagzent, andml must therefore
have been absent.
A normal cheese contains from 0.20) to 0.25 per cent (if ammiIonia.
The greater part of this i, in combination with acid radicals, for while
the ripened cheese is alkaline to litmus it is still acidI to p)henolphtha-
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 thlie second week, then the amount .steadily
increases until the cheese is ripe. A, long a.i the ripened portion
does not extend more than a few millimeters below tlhe myiiveli-m of
the mold no ammonia, either free or in combination, can lie detected.
Cheeses with a very strong flavor contain, as a rule, more ammonia
than the milder ones. In cheeses that are overril)e the presence of
free ammonia can usually be detected by its oldor. For the dleter-
muination of ammonia, the writer has found Folin's method more sat-
isfactorv than the regular method f dlist-,tilling the tannin filtrate with
magnesium oxid. It can be usedl in the presence of prot.ins without
effecting any decomposition.


While the ripened cheese possesses a peculiar odor, which to ,ome
people is quite disagreeable, it does not resemble that of thle typical
putrefactive products, nearly all of wliich are characterized by a foul
odor. Among thlie substances belonging to this class are indul, skatol,
mercaptan, hydrogen sulphid, and phenols. Qualitative tests were
made repeatedly for all of these substances on different sample, 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 veiy


disagreeable odor and taste. Nearly all of these ;ubstancs'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
o)f this nature to any extent.
The following substances have been isolated from Camembert
cheese: Caseoglutin, protocaseose, deuterocaseoses A, B, and C, alpha
and beta peptones, histidin, arginin, lysin, glutaminic acid, tyrosin,
and leucin.
Among those substances which the writer failed to find ara para-
nucleit, tryptophan, indol, skatol, mercaptan, hydrogen sulphid, and
phen, ls.
The ripening of Cami mibert 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,"
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
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 (if anino-acids anti 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.
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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13. THo i. CHARLES Fungi in cheese ripening: Camembert and Roquefort. U. S. 'i
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