Amerioan Beal-Kap Oorporation, Long Islana Oity, N.: Arnold B. Storrs

A TEST FOR THE PROTEIN STABILITY OF MILK
ARNOLD B. STORRS
Amerioan Beal-Kap Oorporation, Long Islana Oity, N. Y.
The stability of the protein of milk is a matter of great importance in

many phases of the dairy industry. Frequently an excessive loss of protein
stability is the chief factor limiting the extent to which some treatments may
be applied to milk or milk products. The problem is often of concern in the
processing of such products as evaporated milk, ice cream, cream and some'
types of modified fluid milk. .
It is not the purpose of this paper to enter into a lengthy review of pro
tein stability since more adequate treatment of the subject may be found
elsewhere (1, 2). The physical and chemical aspects of the problem will be
mentioned only briefly. Fundamentally, the degree of stability of the milk
proteins is dependent upon the hydration and. the charge of the particles;
These factors are influenced in turn by temperature, reaction, salts, previous
heat treatment, or the action of other dehydrating or denaturing substances.
The controlled application of one or more of these factors has been the basis
for most methods of measuring protein stability.
One of the most widely used methods of studying protein stability has
been that Of subjecting the milk to high temperature under pressure ina
manner simulating the sterilization of evaporated mille The use of pilot
sterilizers by condenseries is a routine operation for determining the degree
of stabllity and the corrective measures needed. The technique of such tests
is described by Hunziker (2). The same general methods have been. applied
by many investigators in studies of heat stability (3,4,5,6,7,8). The use
of a pilot sterilizer or similar equipment makes it possible to duplicate on a
small scale many of the processing operations which affect stability, par
ticularly with respect to the ability of the product in question to withstand
sterilization. While the method is largely of value for the control and study
of evaporated milk and rellJ,ted products, it is not well suited for use with
many of the other dairy products.
Another method of measuring the coagulability of milk is the alcoh()l test
(9), The test was originally devised as a measure of acidity but has been
shown by Sommer and Binney (10) to be of little practical value due to the
influence of the salts and other milk constituents. The test has been used
for the detection or milk lacking stability during sterilization but there is
some disagreement as to its accuracy for this purpose (2).
In 1935 Keith and Freeman (11), to determine the amount of HCI re
quired to produce flocculation, empioyed an acid coagulation test which
consisted of adding varying concentrations of Nj40 HCI in distilled water
to 5 mI.. of ice cream mix.
Reeeived for publieation August 11, 1941.
19
20 ARNOLD B. STORRS
Dahle and Rivers (12), in a study of ice cream, used a modified alcohol
test essentially the same as the acid coagulation test of Keith and Freeman.
The samples were observed for flocculation after the addition to the ice
cream mix of varying concentrations of alcohol in distilled water instead
of HOI.
Another method of estimating protein stability is a determination of the
coagulating time with rennin. Mattick and Hallett (13) and others (6)
have employed this technique in studies of heat stability.
In 1931 Ramsdell, Johnson and Evans (14) proposed a phosphate test
for the detection of milk unstable to heat. After the addition of 0.5 M
mono-basic potassium phosphate to2 m!. of milk and mixing, the tubes were
immersed in boiling water for five minutes, then cooled and examined for
coagulation. A low "phosphate number" indicated low heat stability.
Variolls other methods of studying protein stability have been trieJ
including such general tests as titratable acidity, pH measurements both
with and without the use of coagulating agents, and simple boiling tests.
These methods have been of little value except for some limited and specific
applications, probably because results were too often influenced by factors
having little or no relationship to protein stability.
The writer has been interested in the development of a process for modi
fying milk by the addition of a proteolytic pancreatic enzyme. One of the
problems was a control system which would insure proper treatment of the
milk and at the same time would be a simple and inexpensive routine for
milk plants. Preliminary investigation indicated that the method best
suited for the purpose was one involving a determination of protein stability
and particularly with respect to the ability of the milk to withstand boiling
without coagulation. A number of procedures were attempted before a test
was developed which was both simple and accurate. A description of the
test follows.
METHOD
This test for protein stability includes mixing increasing amounts of

N/I0 HOI with portions of the milk, boiling the mixtures for a specified
length of time and then examining the samples for coagulation. While
originally devised as a control test for the enzymatic treatment of milk, it
need not be limited solely to that application.
Equ1:pment and reagents for the test:
10-m!. volumetric pipette
1-m!. pipette, graduated in 0.1 or 0.05 m!.
Supply of test tubes, 16 x 150 mm. (Pyrex)
Test-tube rack
Water bath
N/I0 hydrochloric acid
A TEST FOR THE PROTEIN' STABILITY OF MILK 21
Procedure:
Arrange and number a series of test tubes as follows, adding NI10 HOI
to each by means of the I-ml. pipette in the amounts shown:
Tube no. NI10HOI

(ml.)
o 0.00
5 0.05
10 0.10
15 0.15
20 0.20
etc., as needed
(Note: HOI in the amounts 0.05, 0.15, 0.25 ml., etc., can be estimated
satisfactorily with a pipette graduated in 0.1 ml.)
The tube numbers correspond to 100 x the ml. of NI10 HOI added to each
tube. This eliminates the decimal point as well as any need for further
interpolation of results.
Add to each tube by means of a volumetric pipette 10 ml. of the milk to
be tested. All tubes are then placed in a water bath maintained at the boil-
ing temperature. After 10 minutes the tubes are removed from the boiling
water and examined for coagulation by tipping . The number of the first
tube in the series which shows coagulation represents the end-point and is
recorded as the stability number of the milk.
RESULTS
In applying this test to a variety of untreated fresh milks a range in

stability of from 40 to 100 has been observed. A frequency table of these
results is shown in table 1. Samples with extremely high or low stability
TABLE 1
Distl'iblttion of stability numb61'S of untreated fresh milk
Stability number No. of times

observed
40-49 . 2
50-59 15
60-69 32
70-79 41
80-89 11
90-99 3
100-109 1
Total observations 105
Ave. nunlber 66.6
numbers have not been found to be common. The average stability number
has been found to be about 60 to 70. Results have been consistent' and
22 .ARNOLD B. STORRS
properly stored milk has maintained the same stability number for several
days.
It was observed in some of the early work with this test that pasteuriza_
tion seemed to increase the stability number of milk. Consequently, an
experiment was run to determine more exactly what effect could be expected.
The samples for this investigation were pasteurized in glass bottles in the
laboratory. Preheating periods of 10, 20 and 30 minutes were used in con-
junction with the usual holding process at 143-145 F. for 30 minutes. The
data from this study are shown in table 2. Pasteurization increased the
TABLE 2
The effeot of the preheating time 1bpon the stability n1bmber of pasteurized milk
After pasteurization
Raw milk Time of preheating
10 min. 20 min. 30 min.
50 60 55 60
70 80 80 85
65 75 75 75
70 85 85 90
60 70 70 70
60 75 80 80
50 60 60 55
70 80 80 80
65 75 75 75
65 75 75 75
Ave. 62.5 73.5 73.5 74.5
stability number of the milk slightly more than 10. In some cases the sta-
bility number increased as the length of the preheating period was increased.
However, in the samples tested the average increase in the stability number
as a result of longer preheating periods was negligible.
There was also an opportunity for a limited study of the effect of copper
contamination upon protein stability. Known amounts of copper; in the
form of copper sulfate, were added to the milk and the effect upon the sta-
bility number observed. As shown in table 3 copper contamination de-
TABLE 3
The effect of oopper upon the stability number of mil10
Copper added, in p.p.ill.

0.5 1.0 2.0
Deerease in stability numher
10 15 20
10 15
5 10
Ave. 5 10 15 20
A TEST FOR THE PROTEIN STABILITY OF MILK 23
creased the stability of the milk, the loss or stability becoming greater as the
mount of copper was increased.
a In the application or this test to the enzymatic treatment of milk it is the
O'eneral practice to control the amount or enzyme activity so that the sta-
bility of the milk is reduced to within the limits of from 20 to 40. This
insures adequate treatment and at the same time provides a satisfactory
margin against coagulation: upon subsequent boiling. ,
With respect to simplicity the method is largely satisfactory. The appa-
ratus and reagents are standard items which can be procured, easiJy and
inexpensively if not already on hand. A minimum of technical knowledge
is necessary for proper performance of the test.
SUMMARy
simple test for protein stability is described which includes mixing

.ell,.
increasing amounts or Njl0 HOI with portions or the milk, boiling the mix-
tures for a specified length of time and examining the samples for coagu-
lation.
The stability number or untreated fresh milk has been round to average
about 60 to 70 as indicated by the test.
Pasteurization tends to increase the stability or milk.
Copper contamination tends to lower the protein stability or milk.
AOKNOWLEDGMENT
The writer wishes to acknowledge the assistance and criticism of Profes-

sor E. O. Anderson of the Department or Dairy Industry, University of
Connecticut, in the preparation of this manuscript.
REFERENCES
(1) SOMMER, H. H. Market Milk and Related Products. Published by the author.
Madison, Wis. 1938.
(2) HUNZIKER, O. F. Condensed Milk and Milk Powder. Published by the author.
La Grange, Ill. 1935.
(3) SOMMER, H. H., AND HART, E. B. Heat Coagulation of Milk. Jour. BioI. Chem.,
40: 137. 1919.. '
(4) SOMMER, H. H., AND HART, E. B. Heat Coagulation of Milk. JOUR. DAIRY SOl., 5:
525. 1922.
(5) ROGERS, L. A., DEYSHER, E. F., AND EVANS, F. R. Relationship of Acidity to the
Coagulating Temperature of Milk. JOUR. DAIRY SCI., 4: 294. 1921.
(6) HOLM, G. E., WEBB, B. H., AND DEYSHER, E. F. Heat Coagulation of Milk. I.
JOUR. DAIRY SCI., 15: 331. 1932.
(7) WEllB, B. H., AND HOLM, G. E. Heat Coagulation of Milk. II. JOUR. DAIRY SCI.,
15: 345. 1932.
(8) WEBB, B. H., AND BELL, R. W'. Effect of Flash Forewarming upon the Heat Stabil
ityof Evaporated Milk. Paper presented at 36th Ann. Meeting Amer. Dairy Sci.
Assoc. Abstract JOUR. DAIRY SCI., 24: 535. 1941.
(9) LABORATORY MANUAL.. p. 236. Compiled and published by the International Asso
ciation of Milk Dealers. Chicago, Ill. 1936.
24 ARNOLD B. STORRS
(10) SOMMER, H. :H., AND BINNEY, T. H. A Study of the Factors that Influence the
Coagulation of Milk in the Alcohol Test. JOUR. DAIRY SCI., 6: 176. 1923.
(11) KEITH, J. I., AND FREEMAN, T. R. Ice Cream Rev., 17: 52, 1933.
(12) DAHLE, C. D., AND RIVERS, P. W. Protein Stability of Ice Cream Mixes and Ita
Effect on Certain Properties. lee Cream Trade Jour. Oetober, 1940.
(13) MATTICE:, E. C. V., AND HALLETT, H. S. Jour. Agr. Sci., 19: 452. 1929.
(14) RAMSDELL, G. A., JOHNSON, WM. T., JR,AND EVANS, F. R. A Test for the Detection
of Milk Unstable to Heat. JoUR. DAIRY SCI., 14: 93. 1931.

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A TEST FOR THE PROTEIN STABILITY OF MILK

The stability of the protein of milk is a matter of great importance in

This test for protein stability includes mixing increasing amounts of

Tube no. NI10HOI

In applying this test to a variety of untreated fresh milks a range in

Stability number No. of times

Copper added, in p.p.ill.

simple test for protein stability is described which includes mixing

The writer wishes to acknowledge the assistance and criticism of Profes-

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