THE RELATION BETWEEN THE CALCIUM
AND PHDSPHOBUS CONTENT AND
THE CURD TENSION 0F MILK.

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' Ernest Phipps Black
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THE RELATION BETWEEN THE CALCIUM AND
PHOSPHORUS CONTENT AND THE
CURD TENSION OF MILK

THESIS
by

ERNEST PHIPPS BLACK
Hun
Respectfully submitted to the faculty
of the Graduate School of Michigan
State College in partial fulfillment
of the requirements for the degree

of Master of Science.

\\~\ I (E f) .
July first
1953

THE-1515

 

The author of this thesis feels himself

to be highly indebted and sincerely grate—
ful to Professor C. D. Ball for his invalu-
able guidance during the course of the work
described herein, and in the preparation

of this thesis.

The assistance of Professor C. F. Huffman
in procuring the milk samples used and the
kindly loan of the curd tension measuring
apparatus by the Department of Dairy Hus-

bandry are also greatly appreciated.

CONTENTS

Introduction
Historical
Experimental
Tables and Figure
Discussion
Conclusions

Bibliography

23
27a
28
36
37

INTRODUCTION

Within the past several years considerable
interest has been aroused among research workers,
nutrition students, and the dairy industry by ob—_
servations made from time to time in research re-
ports on the physical character of milk curds form-
ed subseouently to the action of enzymes. This phys-
ical character, or curd tension, as it is commonly
called, is subject to wide variation from soft, body-
less, to tough, rubbery consistancies, depending on
various physical and chemical phases in the constit—
ution of milk.

Application of the existence of hard and soft
curds has been made in the manufacture of cheese, in
which a hard curd is advantageous; in infant nutrition,
for which a soft curd is required for greater ease of
digestion, and to facilitate maximal nutritional
advantage; and in the treatment of gastric ulcers,
for which the easily digested soft curd is obviously
to be preferred.

Many of the curd tension regulating influences

have been partially explained or indicated, but the

(1)

natural complexity of the physics and chemistry of

milk invests the studies first, with a difficulty of

approach; and secondly, with an ambiguity of results.
It is with tne hole of making some contribution

toward a clearer understanding of the influences reg-

ulating curd tension that this work was undertaken.

HISTORICAL

In 1916 Alleman and Schmidt (1) published a
report of their investigations of SOLE of the factors
influencing the coagulating properties of cows' milk.
The report deals with some of the most fundamental
aspects of the phenomenon of coagulation, every one
of which must be considered in a discussion of the
subject.

For the purpose of measuring the hardness
of rennin curds these workers invented a method
which was based on the grams of pull measured with
a Spring scale required to draw thrOugh the curd a
tool consisting of concentric rings on a horizontal
plane attached to the end of a centrally placed, per-
pendicular rod. The principles involved seem to have
been adopted for all succeeding methods of measuring
curd tension.

INFLUENCE OF TILE AFTER COAGULATIGN IS
COMPLETE: Alleman and Schmidt (1) found that the
curd tension increased in direct preportion to th
time allowed for the rennin to act until a maximum
of hardness was approached. The period of time elapsing
between the moment of coagulation and the measurement

of curd tension has been frequently referred to in

their report as the "Wartzeit". The longest "Wartzeits"

presented were twelve minutes only. Between the
eleventh and twelfth minutes curd tension was in-
creasing at rates of from one to three grams per
minute. From their data the authors demonstrated
that a constant may be found for any particular
sample of milk by dividing the curd tension by
the "Wartzeit" in minutes. This constant when
calculated for one milk is invalid for other
samples of milk with a different degree of re-
action to coagulation.

DEPENDENCE OF CURB TEESICN ON TEE ALOUNT
OF RENNIN ADDED: Curd tension and speed of co-
agulation increased in direct prooortion to the
amount of rennin added, according to the work of
Alleman and Schmidt.

When a "Iartzeit" equal to one tenth (or
any given fraction) of the time required for co-
agulation was established, it was found that for
any given sample of milk a constant curd tension
develooed, regardless of the amount of rennin used.

INFLUEHCE OF ACIDITY OK COAGULATION: By
coagulating the milk with a constant amount of
rennin and adding equal volumes of varying strengths
of acetic acid, Alleman and Schmidt demonstrated

that increased acidity favored a harder curd.

In this case as before, the curd tension was in direct
proportion to the speed of coagulation. The conclusion
was drawn that the acid itself has no soecific effects,
but only creates conditions which favor coagulation.
This conclusion was more or less supported by the more
recent work of Rona and Gabbe (39) which indicated
that the action of rennin in formation of paracasein
is most efficient at pHs from 6.0 to 6.4; and the
principle, demonstrated by Palmer (34) and Bell (2),
that under conditions of increased acidity calcium
is made more available for the precipitation of cal-
cium paracaseinate.
INFLUENCE OF CALCIUH SALTS ON COAGULATICN:
Curd tension increases prOportionally to the amount of
calcium chloride added. The mathematical relationship
found by Alleman and Schmidt (1) between added calcium
chloride and curd tension indicates that the salt,
like acid, created conditions favorable for coagulation.
The authors stated that the calcium had no specific
effect, but in view of the function of free calcium
recoanized by Palmer and Richardson (33) and Hammarsten
(l?) as the positive radical of an insoluble paracaseinate,
the element seems to assume a more significant position.
Addition of calcium hydroxide has a softening

influence on the curd according to Bosworth and Bowditch

(4).

In connection with the natural calcium con-
tent of milk, it has been stated by Weisberg, EcCollum
and Johnson (50) that soft curd milk contains less of
the element than hard curd milk. Espe and Dye (16)
found that removal of a large part of the colloidal
CaHPO4 by centrifuging has no effects on the curd
tension. The results of Hill (20) verified this view,
showing that either removal or remixing of cream
separator slime, which according to Espe and Dye (16),
contains a large part of the colloidal calcium has no
effect on curd tension. On the contrary, Teisberg,
thollum, and Johnson (50) have made the statement
that the suspensoid phase of milk, including calcium
phosphates, through its concentration and mode of
distribution controls the curd character.

INFLUEECE OF AXHONIA ON COAGULAT ON: Alleman
and Schmidt (1) have shown that the addition of
ammonia slows up coagulation and brings about a de-
crease in curd tension. This effect is, in some de-
gree, only in opposition to that of addition of acid,
but it varies from the latter effect in that the de-
crease is not in strict prooortion to the amount of
ammonia added. The curd tension falls at a rate fast-
er than the increase of ammonia additions, which prob—
ably indicates a decomposition of the casein, rennin,

or the casein-rennin combination which may exist. In

this connection it is also noteworthy to recall the
demonstration by Van Slyke and Bosworth (43) and
Porcher and Brigando (36) that ammonium or sodium
radicals may tend to replace the calcium in the
caseinate or paracaseinate, and that Rimmington and
Kay(37) and Bosworth (6) have shown that weak alkalie
tends to remove phosphorus. Hill (20) has recorded
that the addition of sodium or potassium in a form
not stated decreases curd tension.

INFLUENCE OF TEMPERATURE N OOAGULATION:
Alleman and Schmidt (1) confirmed the well known fact
that coagulation occurs best at about 40 degrees C.
Above or below this point the process becomes slower
as the temperature increases or decreases. The change
in Speed of coagulation is accompanied by a corres-
ponding change of curd tension, but not in the same
proportion; for the increase in Speed of coagulation
decreases per degree as the temperature mounts toward
40 degrees, while the curd tension increases in dir-
ect prooortion to the temperature.

INFLUENCE OF PHYSICAL TREAT E T OF MILK ON
COAGULATION: Muller (32) found that vigorous agitation
of milk reduces its capability for coagulating. The
accuracy of this earlier observation was later born out
by the work of Alleman and Schmidt (1).

Hill (18), (19) and Espe and Dye (16) found

that pasteurizing or boiling milk has a softening

effect on the subsequently formed curd. Weisberg,
McCollum, and Johnson (50) suggested that this effect
is due to the precipitation by heat of colloidal CaHPoé,
but Espe and Dye (16) have refuted this view on the
basis of evidence obtained by the removal of some of
the colloidal calcium salts by centrifuging. The curd
tension was not altered by this treatment. The same
result was obtained by Hill (20) by removal of separ-
ator slime which contains a high prooortion of the
colloidal calcium. Rupp (41) and Bell(2) have shown
that heating at pasteurizing temoeratures has little

or no effect on the amount of soluble calcium in milk
filtrates, but Nagee and Harvey (a7) and Nattick and
Hallett (28) have presented evidence to the contrary.
Working with colloidal solutions of CaHP04 stabilized
with gelatin, Palmer (84) found it very easily pre—
cipitated by heat. To show the effect of calcium in

the two mentioned physical states, Palmer proceeded to
dialyzed milk until it must have been practically free
of all soluble calcium, then tested the effect of rennin
on the dialyzed product. Coagulation absolutely failed
until a Small amount of CaClg was added (one drop of

4 molar) upon which the curd formed instantly. Addition
of two or three drops of dilute hydrochloric acid also

permitted the curd to form. Palmer reached the conclusion

that the effect of heat on the curding properties of
milk was due to a denaturation or decomposition of the
casein, or to a disturbed "conditions which govern what
is regarded as normal clotting of calcium paracaseinate".
Although direct evidence in the matter is Sparse,
the opinion appears to be prevalent that heat causes a
definite alteration of the casein molecule. Lacqueur
and Sackur (23) showed that casein dried at 94 to 100
degrees C. underwent a cleavage yielding an alkalie
soluble fraction that possessed more acid properties
than casein, and showed a higher base binding power.
Zoller (53) found that pasteurizing caused milk to
yield a much softer curd by acid precipitation, and
that the curd so formed contained more moisture as the
temperature of preheating was increased. The temperatures
used were from 50 to 120 degrees C. inclusive. In an-
other study by this worker (52) solutions of casein and
sodium hydroxide were heated to 118-135 degrees in
sealed tubes. In solutions of pH below 6.5 the caseinates
failed to precipitate though held at 135 degrees for
forty minutes. In solutions of pH above 6.5 precipitation
occurred. During the heating period the pH dropped .18
to .54, the drop being more pronounced in the more
alkaline solutions. The coagulum was soluble in acids
and alkalies and resembled curd made from heated or

sterilized milk. Michaelis and Marui (30) demonstrated

10

that the higher the temperature to which pure casein

in alkaline solution was raised the Slower was the sub-
sequent process of coagulation by rennin and calcium
chloride. The assertion was made in their report that
the effect is on the casein itself rather than on the
reaction between the calcium and the casein. Kumatsu
and Okinaka (22) heated casein with water in stoppered
bottles to 116-120 degreesC. and obtained a product,
the weight of which, when freed from water, was greater
than that of the original sample, evidence which was
interpreted as an indication of hydrolysis. It was
found in their work that groups, some of which con—
tained diamino nitrogen were removed from the protein
molecule by this treatment.

Wright (51), on the other hand, was unable to
find any alteration in the optical rotation or racemiz-
ation of solutions of casein that had been heated as
high as 120 degrees C. in an autoclave.

A quantitative conception of the effect of heat
on curd tension is readily gained from the work of
Hill (19). By heating milk samples from a number of
cows to 92 degrees for five minutes and determining
curd tensions before and after heating, he arrived at
the following averages over a period of several days

on which the tests were repeated:

11

Curd tension Curd tension after
of fresh milk heating to 90 degrees
C. for 5 minutes

194 68
57 24
179 56
51 45
167 66
22 6
142 46
78 12
26 4
145 67
51 8
111 34

Furthermore, Hill has followed the changes in
curd tension through the commercial processes of evap-
oration and condensation (2).As a result of this study
he has been able to report that heating to 114 degrees F.
(48 degrees C.) only slightly affects curd character. In
one case the first heating to 180 degrees F. (80 degrees C.)
reduced the curd tension almost as 1dr as the final heating
to 204 degrees F. (95 degrees 0.). The final sterilization
of evaporated milk in cans heated to 234 degrees F. (112
degrees C.) has the most pronounced effect of all. A
series of curd tensions that might be accepted as typical

for mixed herd milk can be compiled as follows:

Treatment Curd tension
Raw milk 63
Evaporated milk 62
Sterilized and evaporated 25
Boiled milk 19

Evaporated milk,diluted 50¢ 12
Sterilized and evaporated
diluted 50b 5

 

I
I

 

 

 

 

 

12

The precipitation of casein by acid is affected
in a way similar to the rennin precipitation, according
to Courtney (50), who found that evaporated milks give
a bulky and fluid curd, milk dried by the roller process
a rather compact and cheesey curd, and fresh milk raw,
pasteurized, and boiled, and milk dried by the spray
process yielded curds with characteristics between those
of the other two groups.

INDIVIDUALITY OF cows: Alleman and Schmidt (1)
and Hill (18) found that curd tensions of milks from
different cows show variations as wide as those well
known in the chemical composition of milks. Alleman and
Schmidt demonstrated that milks from different cows
show no constant ratio between "Wartzeit" and curd ten-
sion. The individuality of each animal seems to be
fairly well established,and to be altered only by the
sex cycle,disease, or temporarily by a drastic change
of diet. Individual cows show slight variations in
the tensions of their milks taken at different periods
of the day.

Experiments of Alleman and Schmidt with mixed
milks show that when a hard curd milk is mixed with a
soft curd milk, the curd formed from the mixture is
from 10 to 20 percent harder than would be calculated

from the tensions of the separate components. This

13

would indicate that whatever influences tend toward a
hard curd extend their action to the softer curd milk
upon being mixed with it. This observation has been
verified in feeding experiments by Bergeim and co-
workers (3).

INFLUENCE OF THE PHYSIOLOCICAL COHDITIQN OF
THE COW: Certain fluctuations in curd tension are
found to correscond to the progress of the sex cycle
of the animal. The results of Alleman and Schmidt (1)
show a decrease in the efficiency of the action of rennin
on milk taken during the heat period, but no convincing
alterations in curd tension. Hill (18), however, ob—
served that after the cow freshens and as soon as the
colostrum is exhausted the milk immediately assumes a
SOmewhat higher curd tension. This condition continues
for from four to six weeks. Hill also stated that to-
ward the end of the lactation period the curd usually
becomes harder, but sometimes the occosite effect is
observed.

Monier and Sommer (31) noted that many low curd
tension cows but no high curd tension animals were
found to have histories of chronic udder infection.
Unpublished results of the work of C. S. Bryan, Mich-
igan State College Department of Bacteriology, have
shown that milk from udders in an advanced state of
mastitis infection failed entirely to coagulate with

rennin.

14

Washburn and Biglow (48) have found no relation
between leucocyte counts of milk and data on coagulation.
EFFECT OF THE COfi'S DIET ON CUPD TENSIDN:
Alleman and Schmidt (1) found no significant variations
in curd tensions resulting from dietary conditions. Hill
(18) wrote that he found no indication of any appreciable
influence of normal diet on curd tension, but that changes
might be brought about by sudden and drastic alterations
in diet, the effect of which. is, however, only temporary.
Very dry diets may cause a harder curd by favoring the
production of a more concentrated milk; or diets con-
taining an abundance of water and moisture dispose to-
ward a softer curd by causing dilution.

INFLUENCE OF PROTEIN OF MILK ON CURD TEKSI N:
Weisberg et al (50) make the sweeping statement that
the concentration of casein is the major factor in de-
termining curd character; a high concentration favoring
a hard curd and vice versa. This idea is considered to
be supported by experiments in coagulation of diluted
milk. ESpe and Dye (18) have observed no difference
in alteration of curd tension when milk was diluted
with distilled water in one case, and with milk whey
in another. Argument is presented to show that in two
samples studied the difference in casein content ac-
counted for only 81 percent of the difference in curd

tension.

 

 

15

Monier and Sommer (51) have indicated that the ratio
of casein to albumin is an influencing factor in coag-
ulation. Albumin apparently favors softness of curd.

Van Slyke and Bosworth (45) have found that acid in milk,
a factor that tends toward a harder curd, reduces the
absorption of albumin by casein. Hill (19) found that

curd tension seems to increase along with total protein at
the beginning and end of lactation, but the curd ten—
sion increases faster than the protein content itself
seems to warrant.

Espe and Dye (18) have stated that there is prob-
ably no direct proportion between the curd tension and the
~"concentration of any one of the constituents of milk,
'although there may be marked correlations.

I INFLUENCE OF FAT: The actual presence of fat
in milk is an influence that softens the curd accord-
ing to Hill (18). However, as a general rule, it is
found that milk of a naturally high fat content yields
a hard curd. teisberg and co-workers (50) included
fatty constituents in the suSpensoid phase which they
claim controls the curd tension.

MILK PHOSPH HUS AND CUHD TENSION: The most
committal statement in regard to the relation of phos—
phorus to curd tension has been made by Honier and

Sommer (31). They record that although natural phos—

16

phorus seems to be higher in hard curd milks, added
phoSphates depress coagulability. Perusual of the
reports of Van Slyke and Bosworth (45), Porcher and
Birgando (38), Piettre (35) and Rossi (40) convinces
one that there is some controversy over the physical
and chemical states of both calcium and phosphorus in
milk. Weisberg, McCollum, and Johnson (50), however,
thought it probaole that the mode of distribution of
these elements in their inorganic forms has some part
to play in the determination of the coagulating prop-
erties. Palmer (34) has contributed striking evidence
that the calcium phOSphates of milk are all present
as CaHPO4, some in the colloidal and some in the

true solution; that in the true solution monopoliz-
ing the functions that this salt performs relative

to coagulation.

' RELATION or BREED: Hill (18) has found that
the median of curd tensions is lower in the Holstein
than in the Jersey breed, and Watson observed (49)
that the former freed, in general, yielded milk of
a lower buffering capacity than the latter.

RELATICES OF KIKOR COESTITUEEFS: Citric
acid has been attributed by Honier and Sommer (31)
with an appreciable softening influence on the milk

curd. Although it has been suggested by Porcher-and

17

Biigandc (36) and Piettre (35) that this effect may
result from a pronensity of citric acid or citrates.
to remove calcium by precipitation, Bosworth (5) has
shown that the soluble calcium of milk is increased by
addition of sodium citrate, and that the less in—
solubles sodium-calcium caseinate is formed.

Work of Loevenhart (28) indicates that the
magnesium precipitates paracasein in a manner that
appears to be quite closely comparable to that of
calcium. Van Slyke and Bosrorth (44) have found mag-
nesium in milk filtrates to the extent of about one
sixth of the weight of the calcium. However, no
ouanitative results have come to light in connection
with the effect of natural magnesium on curd tension.

We are equally in ignorance of the influence
of chlorine, lactose, and natural sodium and potassium.
Loevenhart (28) found that the anions chloride, sul-
fate, and nitrate were without effect on coagulation
of casein.

RELATION OF SUFFERING POWER Oi MILK: Notice
has been given by Watson (49) and Erennemann (13) to
the fact that soft curd milks have allow buffering
power. The lack of direct relation between buffering
power and curd tension is indicated by comparing
the findings of Buchanan and Peterson (8) and Espe

and Dye (16). The former workers found that milk

18

may be diluted thirty percent of its volume without

any change in buffering power, while the latter have
shown that in one case represented the dilution of a '
sample of milk to the extent of twenty five percent of
its volume caused a drop in curd tension of almost forty
percent.

There is some disagreement arising in the work
of Buckley (9), who found that Jersey and Guernsey milks,
which tend toward harder curd than the Holstein and
Ayrshire milks, according to Hill (18), are in general,
more easily precipitated by weaker dilutions of hydro-
chloric acid than the milks of the latter breeds, This
indicates a lower buffering power among the Jersey
animals, and is in direct Opposition to the statement
of Watson (49).

CURD TENSION AND DIS;ST OX: Since it is scarcely
the function of a thesis bearing on this particular
subject to make a thorough review of the very vol-
uminous literature concerned with the digestability of
milk, only some representative works will be considered
here in order to establish a connection with certain
applications of knowledge of curd tension.

There has been considera.le dissention over
the relative merits of boiled and unboiled milk as a
food. The status of the question has been well review-

ed by Lane-Claypon (25) for the period previous to

19

1912, and summed up by conclusions which, as far as
our subject is concerned, have had but little contra-
diction since. Only one phase of the controversy enters
in to our discussion; that is, the effect of boiling
on digestability.

Brennemann (11) has written that for years
European physicians have been feeding milk to basics
without the dire results that were considered in
America to follow such a diet, and, in fact, with less
digestive troubles than were experienced by American
infants nurtured on raw milk. The discovery of this
situation by American nutrition students led to in-
vestigations that have given highly concordant results.
Two esoecially enlightening investigations were carried
out by Brennemann (10) and Bergeim et a1 (3) using
the same technique and arriving at almost identical
results.

These workers were each fortunate in procuring
a human subject who could regurgitate his food at will.
By exploitation of this gift the following conclusion
were reached. Raw cows' milk coagulateé within thirty
seconds after it reaches the stomach. At first small
curds form which grow, coalesce, and harden, to form
chunks as large as a man's thumb, and hard enough to
resist breakage when dropped on the floor, These curds

grow for one to two hours, then gracually disappear,

20

disintegrated by peripheral digestion W41Ch continues
in the stomach for five hours or more.

Boiled milk, on the other hand, forms curds
of a soft, flaky nature, which may achieve a size
equal to that of a small pea. These curds pass from
the stomach in acout three hours. Pasteurized milk gave
curds with qualities between those of raw and boiled
milks, but resembled the raw milk curd most strongly.
Milks modified by sodium citrate, sodium carbonates,
or lime water; condensed milk and buttermilk yielded
soft curds.

In the feeding of raw and boiled milks, very
convincing clinical evidence is offered in favor of
boiled milk for infants as reported by Brennemann,
(43,44,45,48), Variot (47) and Bennett (49). The
clinical pictures presented in these reports are so
true to form that tiers is no need of separate discuss-
ion of each. Although a great many babies are quite
capable of normal and undistressed progress on a
raw milk diet, a considerable number of infants do
hate digestive and nutritional difficulties with
raw milk that can be prOmptly cured by feeding boiled
milk. Symptoms of such difficulties often become
apparent in the form of regurgitations, dyspepsia,

diarrhea, chunks of undigested curd in the stools,

liquid stools, foul stools, and even rickets and gen-
eral malnutrition. A striking number of these afflicted
babies have been returned to normal, undistressed

growth and development by the simple exoedient of
boiling their milk. Variot(48) has written of consistent
success in treating infant eczema with boiled milk.

‘I

It is well known that human milk, cein

espec-

an

ially designed for human babies, has the property of
yielding a curd that is remarkably soft and digestible.
Brennemann (13) has made the comment that all milk
modifications and substitutes have one factor in common:
the curd has been reduced in size and consistancy so
as to approach more closely to the qualities of mothers'
milk. This is clearly shown by the evidence presented
above.

As has been pointed out earlier in the re-
sults of Alleman and Schmidt(l) and Hill (18,19,20),
the milk of all Cows is not the same in curding char-
acteristics. In fact cows milk is to be had that very
nearly approaches human milk in this respect without
being modified.

The first demonstrations of the use of un-
modified cows' milk with much the same effect as
boiled milk has been shown to have were made by Hill
in collaboration with Blood (18, 19). These workers

furnish case reports showing great benefits derived

22

in cases of infant indigestion, malnutrition and
rickets, and in unusually satisfying normal development.

Somewhat more quantitative data are presented
by Espe and Dye(16) Who carried out digestion exper-
iments with dogs, calves, and humans, using for obser-
vation Pavlov pouches, roentgenograms, and flouroscooic
methods respeCL'vely. It was found that an adult dog
will digest 300 cc. of milk with a curd tension of 98
grams in fram 1.7 to 1.8 hours, while milk with a
curd tension of 190 grams regains in the same animal's
stomach for from 2.3 to 2.8 hours. Undoubtedly in-
dividuality would account for differences in these
figures as determined in different animals of the
same Species.

It was also shuwn that although the rate of
gatric secretion seems not to be influenced by the
curd character, the persistance of the secretion
does reapond to curd toughness.

Returning to heat treated milks in the light
of their digestibility, it is interesting that Wallen-
Lawrence and Koch (47) report an increase digestibility
by Trypsin in vitro of evaporated and boiled milks. The
increased ease of digestion seems to be a function of
the temperature to which the milk is heated, and the

length of time it is held there.

23

EXPERILEHTAL

In view of the imoortance that has been attrib-
uted to the role of the inorganic constituents of milk
in the process of enzymic coagulation, these comaounds
seem to offer a promising field for investigation. Cal—
cium and phOSphorus were selected as the outstancing sub-
jects of this research because precedenting investig—
ations seemed to indicate that a study of these constit-
uents would contribute some light toward an under—
standing of the controlling factors of curd tension.

The milk used for this work was obtained from
the fiichigan State College eXperimental herd of Hol-
steins. Holstein milk was considered to be most adapt—
ed to this work because of the wide variations in
curd tension to be found in the breed, and because of
the fairly uniiorm and low fat content. The samples
were all taken from the mixture of the total morning
milk output of each cow. Precautions were taken to
avoid milks from mastitis-infected animals, or from
animals in those periods of the sex cycle in which
the curd tension might be affected.

Determinations were made of curd tension,
total calcium, total phOSphorus, acid soluble phos-
phorus, specific gravity and in some cases total solids

and pH.

24

Curd tension was determined by the use of the
apparatus called the "American Curd-O-Meter" manufact-
ured by the Heusser Instrument Company of Salt Lake City,
Utah, and claimed by the manufacturers to give results
closely comparable to those obtained by the standard
Hill apparatus. Coagulation was carried out as prescrib-
ed by Hill (18) using as a coagulant a mixture of three
parts of .6 percent pepsin solution (l-SOOO dry scale)
to one part of a calcium chloride solttion containing
378 grams of calcium chloride, U.S.P., per liter. A
coagulation period of ten minutes was used. A parallel
series of determinations of curd tension was made,
using three parts of the pepsin solution to one part of
distilled water as the coagulant without calcium chlor-
ide. Preliminary results obtained by these methods are
shown on Table 1.

Calcium and acid soluble phoso orus were
determined on a tricnloracetic acid filtrate after
the procedure of Sanders (42). Sanders found that
after precipitating the protein of milk with four
volumes of ten percent trichloracetic acid all of the
milk calcium (and magnesium) and 18.1 to 31-6 percent
of the total phosohorus were converted to a soluble
form and could be determined quantitatively in the
filtrate with a great saving of time. The results of

these determinations were carefully checked by Sanders

25

against those obtained by ashing methods. The filtrate
was preoared by placing 20 cc of milk in a 100 cc cal-
ibrated volumetric flask and diluting, very slowly at
first, and with rotation of the flask, witn 10 per cent
trichloracetic acid to the mark. This was allowed to
stand with occasional snaking for thirty minutes to
permit a complete precipitation of the proteins and
liberation of the calcium from the colloidal state and
the calcium caseinate. The proteins were then filtered
off. A 50 cc aliquot of the filtrate was placed in a
Kjeldahl flask and ashed wet with 25 cc of a l-l mix-
ture of concentrated H8804 and concentrated HNO3. in
.order to assure complete ashing and subsequently a
ready precipitation of the phosphorus in the filtrate
it was found advisable to boil off an additional 15-20
cc of concentrated nitric acid. The ditestive mix-
ture was carezully mashed into a 400 cc beaker, using
water as required until a piece of blue litmus paper
dropped into the flask remains blue. Ninety-five per-
cent alcohol to a volume five times that of the di-
gest and washings was added. The CaSO4 precipitated

in this way was allowed to settle over night. The
precipitate was filtered off, placed in a crucible,
and ashed for the calcium determination. This ash was

dissolved in concentrated HCl by digesting 45 minutes

at 50—75 degrees, diluted, and examined for calcium
content by the Meigs, Blatherwick, Cary modification of
Abderhalden's and McCrudden's met ods combined (28).

A comparison of this method with a shorter one
desribed in the hethods of Analysis of the Association
of Official Agricultural Chemists, third edition, page
288, was made. The object was to shorten the procedure
by omitting the digestion and precipitating calcium as
the oxalate directly from the trio loracetic acid fil-
trate. The method was slightly mddified in that after
the first precipitation of calcium oxalate from the
filtrate was complete the precipitate was dissolved in
dilute HCl, treated with several drone of concentrated
HKO3 to remove any absorbed organic matter, amd evap-
orated to dryness. This treatment was reoeated once more
before proceeding with the final precioitation. Al-
though the results for some milks were very satisiact-
orv others snowed wide variations from the values ob—
tained by the l,nser method. For this reason the pro—
cedure recommended by Sanders was followed. The re-
sults of the comoarison are shown on Table 2.

Acid soluble ohosohorus was determined on the
filtrate frOm the calcium seocr tion. This alcoholic
filtrate was evaporated on the steam bath; the residual

liquor was treated with concentrated ENC? droo wise
U

27

to oxidize organic matter. neutralized with concen—
trated HH4OH, and used for the determination of phos-
phorus by the gravimetric method, Zethods of Analysis,
A.O.A.C. 3d edition p. 15.

Total phosphorus was determined on a 10 cc
sample of the milk evaporated and ashed with the addi-
tion of magnesium nitrate. The asLe was wetted with
water and dissolved by adding 5—10 cc of concentrated
HNOg, and examining for p.osonorus content by the
same method used for acid soluble phosphorus. The
results of calcium and onosohorus determinations and
their relation to curd tension with and without CaClg
are shown on Tables 3 and 4 and Figure 1.

Total solids determinations were made on weighed
2 cc samples in aluminum dis es. The samples were
evaporated to dryness on the steam bath, and then placed
in an 80 degree oven (C) to dry for several hours, after
which they were cooled in a vacuum dessicator over CaCl8 .
The oven drying was re eated until the sanoles lost less
than .5 milligrams during the final heating.

The pH was determined by use of the ouinhydrone
electrode. The results of total solids and pH deter—
minations are recorded on Table 4.

The specific gravity was measured with a
lactometer which had been tested for accuracy against

pyknometer and Westphall balance determinations.

Cow

TABLE 1

Date

July
n

N
I!

I!
"
II
II

I!
II
II
I!

II
I!
n
M
II

n
I!
I!
u
I!

27a

com-eon (OCDQO) coco-10)

Curd tension

Hill

57
74
51
8O

28
33
41
39

36
60
36
36

73
9O
77

38
31
44
41

37
38
39
38
46

34
33
39
4O
46

4O
48
4O
50
31

Without
0&012
44
38
38
37

Cow

F6
167
611

011
015
D15
D9

D13

Cow

D15
n
u

27b

Curd tension

TABLE 1
(concluded)
Date
Hill
July 6 18
" 7 33
" 8 32
" 9 39
" 18 30
“ 13 30
" 14 3O
" 15 34
" 16 38
Jan.21 50
July 6 16
" 7 39
" 8 28
" 9 30
Jan. 7 20
Feb. 6 55
Mar.15 4O
Apr.11 53
May 16 55
TABLE 2

Calcium by method
of Meigs,Blatherwick,
mg/lOO cc

Cary.

110.4
102.1

95.3
115.0
100.6
128.2
139.4
109.8
126.8

Without

C

aCl
152
30
27
36
23
23
27
:53
38
30

Calcium by modi-
fied A.O.A.C.
method.

105.8
102.8

96.1
114.4

96.8
113.2
119.7
128.6
112.5

mg/lOO co

 

 

TABLE 3
Cow Cfird tension Ca Total Acid sol. Acid insol.
Hill Without mg P mg P mg P
CaCl " Qcc.-l§0.cc.___zr "
G 11 145 0 ¥ 100.6 77.27 60.00 0. 7
G 33 104 43 117.0 103.87 81.95 21.92
" 100 41 139.3 --- --- -—-
" 79 41 108.2 99.92 78.02 21.90
Herd 102 54 167.7 -—- —-- -——
G 12 100 52 140.9 —-— --- ---
G 6 91 53 158.4 93.21 78.59 14.62
G 17 90 39 96.3 84.21 64.49 19.72
" 86 48 134.0 —-- --- ~--
" 85 45 118.2 92.02 74.04 17.98
" 52 30 118.? ~—— —-- ---
G 16: 85 45 126.9 --- --— ——-
" 51 25 78.69. 62.00 16.69
G 31 84 29 139.8 --- --- --—
" 64 32 131.9 93.97 77.12 16.85
" 57 38 142.8 --- -—- ---
" 48 20 110.8 93.14 71.58 21.56
D 15 55 28 144.8 86.43 63.13 23.30
" 55 27 139.5 --- --- ---
" 53 42 141.7 -—- --— -——
" 40 21 149.2 --- --- -—-
0103 41 27 127.6 92.05 76.00 16.05
" 38 16 147.6 95.48 73.54 20.94
167 38 0 @ 102.8 84.80 63.32 21.48
D 9 36 24 109.7 --- --- --—
F 6 34 33 110.4 82.60 66.70 15.70
G 15 29 9 145.6 --- -—— -——
D 13;: 25 22 126.8 67.33 52.14 15.19
# No coagulation in ten minutes.
@ Coagulation but no measureable curd tension in

ten minutes.

270

* Represents one quarter of the udder infected by

streptococci.
Note: Each determination recorded in this table repre—
sents the average of closely checking duplicates.
Note: Specific gravity was used in each case in calcu-
lation of results.

 

 

TABLE 4
Cow erd tension. .pH Total
Hill Without Fresh CoagTd Coag'd

08012 Without Hill solids
CaClZ method percent

G 33 104 43 6.61 --- 5.84 12.75
G 33 100 41 —-- ——- --— 12.55
G 17 95 41 --- --- -—- 10.86
G 6 91 53 —-— --- -—- 12.50

G 17 90 39 -—- 6.52 5.70 -—-
G 17 85 45 -—- 6.52 5.67 11.14
G 31 84 29 --- --- --— 11.63

G 33 79 41 --- 6,67 5.17 --—
G 31 64 32 6.60 6.60 5.87 13.03
D 15 55 28 6.52 6.52 5.92 12.56
D 15 53 42 --- --— ——- 11.49
G 17 52 30 6.52 6.52 5.68 14.06

G 31 48 20 --- 6.35 5.39 -——
0103 41 27 6.65 6.65 5.75 10.75

0103 38 16 -- 6.70 5.84 --

MICHIGAN STATE COLLEGE

 

28

DISCUSSION

CALCIUX: The results arrived at in this work
fail to corroborate those of preceding reporters, wh
in general, assert that the calcium content diminishes
in relation vith tne curd tension by the Hill test. The
contradiction is supported by comparison of milks taken
from the same cow on cifferent days (Table 3). It is
notable that in s0me cases t e samples from an in-
dividual cow that develOp the softest curd prove to
have the highest calcium content.

The lack of apparent relation between total
calcium and curd tension is not in the least surpris-
ing when one considers that it is only the calcium
in true solution that takes part in the process of
coagulation. Van Slyke and Bosvorth (44) working
with milk filtrates obtained by passing milk through
a porous earthenware filter, have found that in tvo
sam les dealt with only 35.16 and 33.33 percent of
the total calcium was present in true solution.

This evidence suggests that the calciun in true sol-
ution would show considerable variation in fifferent
samples, depending uoon pH and the relative pro ortions
of other constituents of the system rather than on
gross calcium content. This argument can apply, how—

ever, only in cases in which milk is coagulated

without the addition of calcium ions. There is an ob-
vious inconsistency in trying to correlate the effect
of naturally occuring calcium with results that are
obtained only after the addition of an excess of these
ions. A comparison of the curd tensions obtained with-

ut and with the added calcium chloride (Tables 1 and
4) convinces one that the treatment is by no neans
equally effective in all cases. The results shown on
Table 3 fail also to Show any relation between curd
tension determined without calcium chloride addition
and natural total calcium.

The relationship in the coagulation process
between the activity of rennin and the increased
availability of calcium ions under conditions of
lower pH would undoubtedly be a moot question at
present, but judging from the hardness of the curd
formed under different conditions, the total pro-
cess seems to take place less readily at pH 6.4- 6.0,
the zone descri ed as the most advantageous for the
conversion of casein to paracasein by rennin, (Rona
and Gabbe!39)) than under conditions of greater acidity.

As has been pointed out above, the effect of
added calcium chloride on curd tension is not ouantita-
tively constant. In some cases observed the tensions
‘measured without the addition of the salt were scarcely

different from those measured with it; while on the

30

opposite extreme were those samples that either failed
to coagulate or yielded a curd that was too soft to
register on the measuring de ice, but which yielded
comparatively hard curds when coagulated with the added
calcium chloride. Most commonly, perhaps, the curd ten-
sion was approximately doubled by the use of calcium
chloride in the prooortions indicated in the Hill test.
No relation was found between the natural calcium
content of milk and the effect of the calcium chloride
addition, although such a relation is suggested in

the cases of tvo milks listed with especially low
calcium figures which failed to coagulate to any
measurable degree in ten minutes without added cale
cium ions.

It seems obvious tvat at lea t a part of the
effect of the calcium Chloride addition lies in the
depression of the pH (Table 4). It has been pointed
out that coa ulation of milk by rennin occurs more
rapidly and results in a harder curd when the pH is
lowered within reasonavle limits. then the acidity is
induced by the addition of an acid, calcium is made
more available for the precipitation of paracasein,
but there is no reason to believe that this is the
only coagulation-favoring reaction that occurs. When

calcium chloride is the cause of the depression of pH,

it would seem probable that the increase of available
calcium would be accomplished even if the pH were not
lowered. The addition of calcium hydroxide, as point—
ed out above, has a depressing effect on curd tension,
which may be the result of a lowering of acidity. Un-
fortunately, no one has added calcium ions to milk
without changing or radically threatening the orig-
inal pH. Again we are reminded of the assertion of
Rona and Gabbe (39) that the change of casein to para—
casein is complete only in the range of pH 6.0-6.4.
This range of pH is somewaat higher than that which

is obtained by the addition of calcium chloride as

in the Hill test.

Some lack of uniror ity may arise among
various workers as a result of using rennin in some
cases and peosin in others for coagulation, for con—
sideration should be given to the fact that pepsin shows
a maximum activity at a lower pH than does rennin.

There is no apfiarent correlation between any
of the pH values recorded in Iaole 4 or the alterations
in pH values and the curd tensions.

Although the discussion on calcium ion addi-
tion and pH alteration has been limited for the sake
of avoiding confusion, such a restricted view is far
from adequate. The picture is considers ly complicated

by variation in casein and fat contents, the effect of

32

which this study is not well adapted to evaluate. In
addition there is the colloidal phase of the ouestion,
which involves variations in stability of casein and
paracasein in solutions of various pH values and con-
ditions of salt equilibrium. Certainly the shift toward
the isoelectric point of casein and paracasein which
results in varying degrees from calcium chloride add—
ition will exert some regulatory influence on the curd
hardness. These physical relations are not at present
understood.

PHOSPHORUS: It is observed that both the acid
soluble and total phosphorus decrease in a general
relation with the curd tension (Table 3 and Figure 1).
So far as can be perceived by these results there is
no appreciable difference between toe activity of total
and of acid soluble phOSphorus in this relation. This
seeming parallelism is affected by the fact that the
total phosphorus represen s the acid soluble plus
the casein phosohorus; and the latter remains fairly
constant: 14.6 to 27.0 percent of the total phosphor-
us. (Sanders: 18.1 to 31.6 percent).

An estimation of the casein content of these
milks is possible based on the analysis of casein by
Eosworth and Van Slyke (7) showing .71 percent phos-
chorus; and the conclusion of Lenstrup (25) that the
acid insoluble phOSphorus of milk is 98.5 percent

casein phosphorus. Casein calculated in this way

I . (VIII. 4

33

appears to have no relation to curd tension.

Nothing has been done to specifically demonstrate
any acti ity of the phosoh te ions in curd formation.
Palmer (34),in fact, produced a ready precipitation of
paracaseinate by the addition of calcium chloride to
rennin treated milks that had been previously dialyzed
until the calcium had been removed, and might therefore
be presumed to be fairly free of phOSphate ions.
Furthermore, Loevenhart(26) found that chloride,
sulphate, and nitrate ions had no effect on coagulation;
evidence which indirectly favors the assumption that
the phosohate radical is impotent, at least in a
strictly Chemical sense. As has been stated above,
it seems probable that the phosohate ion may have some
role in stabilization of casein from the physical view-
point.

Whether or not the principles of calcium
buffering which have been worked out by Kugelmass
(21) enter into this picture is an interesting Question.
If such be the case, it must be effective to only a
limited extent, for in the Hill test calcium is added
in a rather excessive amount:_roughly seven to nine
times the amount find in true solution, and two and
a half to four times the total calcium of milk.

However, the conditions outlined by Kugelmass for
buffering against calcium ions are not exactly

satisfied in milk. These conditions are set up by

34

mixtures of weak acids and their salts, which react to
form inslouble normal calcium salts and soluble inter-
mediates.

Since a large part of the soluble phosphorus
is considered to be present as a calcium phosahate,
it might seem evident that the content of acid soluble
phosphorus might be merely an indication of the amount
of calcium available for precipitation as the para-
caseinate. The case cannot be so simple as that,for
the argument in itself is not explanatory of the re-
lation that the DhOSJhBtBS bear to the effect of added
calsium chloride. Furthermore, the added calsium would
be expected to obliterate the relation between the
phosphorous content and curd tension.

Some of the erratic manifestations of the
results might be explained on the basis that the per-
centage, as well as the weight of the ihOSphorus
present in true solution varies. Van Slyke and Bos-
Aworth (45) found that of the total milk-phosonorus
70.0 and 64.4 percent passed through the earthenware
filter in the respective cases of two cifferent samples
of milk. A curve in which the curd tension were plot-
ted against the ohOSphorus in true solution might

show a more even relation.

55

TOTAL SCLILS: Examination of Table 4 reveals
no relation between total solids and the curd tension.
Reconsideration in respect to curd tensions determined
without the use of calsium chloride is eoually fruit-
less. As in the case of total calsium, this lack of
relationship is not surprising in view of the many
other modifying factors. Total solids contains
colloidal calsium and chosohorus, sodium, potassium,
and magnesium salts, citric acid, lactose, and other
substances; the effect of which on curd tension is nil ,

doubtful, or very little understood.

CONCLUSIZHS

1. Curd tension as measured by the Hill method
is fairly constant for individual cows.

2. The effect of calsium chloride in coagulation
differs markedly with milks from different cows. Al—
though calsium chloride has a pronounced eff ct on the
pH of milk, the effect of added caleium is more deeply
seated than in a mere alteration of pH.

3. There is no indicated correlation between
total solids and euro tension or pH and curd tension.

4. Acid insoluble phosohorus varies slightly
in mi ks of different curd tensions, but there is no
indicated correlation in this respect.

5. Acid soluble phOSphorus varies with curd
tension. In general, milks with a high curd tension

have a higher content of acid soluble pnOSphorus.

57
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