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PRINGPORT, MICHIE

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WO Aen &
A STUDY OF THE ORIGINAL ACID REACTION OF
MIIK TO PHENOLPHTHALEIN.

by

Me Be. Kurtz

Submitted in Partial Fulfillment of the
Requirements for the Degree of

D. Ve. Le

in the

Michigan Agricultural College
1914.
The author takes great pleasure
in thanking L. H. Cooledge, Research
Assistant in Bacteriology at the Michi-
gan Agricultural College, at whose sug-
gestion this investigation was undertaken,
for valuable assistance and encouragement

in the pursuit of the same.

199385
A STUDY OF THE ORIGINAL ACID REACTION OF
MIIX TO PHENOLPHTHALEIN.

Table of Contents.

Introduction . e ° ° ° e ° °
Review of literature . ° e ° ° e
Methods. ° e ° ° ° ° ° °

Acid reaction of different species ° °

Acid reaction of different portions of milking

Acid reaction of all normal samples tested
Influence of breed on acid reaction. °
Influence of age on the acid reaction. °

Influence of lactation on the acid reaction

Effect on the acié reaction caused by standing

Acid reaétion as found in the four quarters
Acid reaction as found from day to day °
Acid reaction caused-by unhealthy cows :
Discussion of results . ° ‘ oe

Summary . ° e ° ° ; . ° °

Page

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16
A STUDY OF THE ORIGINAL ACID REACTION OF
MILK TO PHENOLPHTHALEIN.

Introduction.

Fresh normal milk while amphoteric to neutral
indicators gives an acid reaction when titrated using
phendphthalein. The acidity of milk then, when spoken
of in this thesis will refer to the reaction when phe-
nolphthalein is used as an indicator. Some observations
have been made upon the original acidity and these,
while very limited seem to suggest to the investigators
that the original acid reaction in milk may have some
value in diagnosing inflamatory and gargety condition
of the udder, as well as many other pathological changes
brought about by disease or injury.

As very few observations have been made upon
the original acidity of milk, and as most references
to same are unaccompanied by substantiating evidences,
it is thought that a study of the factors which influence
the reaction of the milk at this period may be of interést
and possibly have some practicable application.

The object of this investigation then will be
to verify the statements already made as to the signifi-
cance of the origina] acid reaction of milk; to detemmine
any possible connection between the original acidity and

pathological changes; to study the relation of the period
of lactation, the effect of standing, care with which

drawn and any other factors that may enter in.
Unfortunately time and facilities will not

permit of making a study of the chemical nature of the

original acid reaction to phenolphthalein in milk.

Review of Literature.

Schoper* in an article "Experimental Studies
on Milk", makes the following statements in regard to
the original acidity of milk: "Milk when drawn from
the healthy udder of the dow has an acidity which is
hardly perceptible. After a very short time the acidity
may be readily detected and determined.

The acidity of fresh milk as determined by
phenolphthalein as indicator is due principally to the
presence of carbonic acid, acid phosphates, ash and
casein. The acidity that develops later is due princi-
pally to the change of lactose into lactic.acid, and
results from bacterial action, this being dependent on
the kinds of bacteria present, their nunber and the
tempature at which the milk is kept. The change known
as coagulation is the result of precipitation of castin-
ogen by the combined action of the acid and calcium
salts. The acid is usually tactic acid and can be
titrated, though coagulation occurs as a result of the
formation of amino aodds and possibly also from other

causes."

*Edwin H. Schorer,- Experimental Studies on Milk,
Journal of Infectious D&seases, Nov. 11. 1912.
- 4 «

The original acidity of milk has been reported
by different observers as varying between .11%5 - .18%
figured as lactic acid. Milk is found to coagulate upon
boiling if the acidity is over .225%. <A sour taste is
first detected at .3% and curdling occurs at .56%.
Shorer goes on to say, “Auzinger states that
fresh milk from one cow has an acidity of .146 to .18%
and that the percentage of acidity of fresh milk may give
valuable information in regard to the cow from which it
was drawn,
If the degree of acidity is less than .11% suspect
mastitis.
If the degree of acidity id between .14 and .20%
suspect first or second week after parturition.
If the degree of acidity is above .20% suspect colos-
trum or pathological milk.

Ruhnn states that milk from a tuberculous udder
has an acidity of less than .16%.

*Hoyberg has devised a method for determining
inflamatory processes in the udder of the cow based on
the alkaline reaction of serum exuded in inflamatory
processes, using rosolic acid in alcoholic solution as
an indicator".

*Metzger studied the value of the alavhol test
in detecting the milk of diseased animals - "The value of

¥Zteschr. f. Fl. v. Mileh-Hyg., 1911, 21, p. 133.

OK. Metzger, Inaug. Disc. Univ. Stuggart, 1912.
Abs. B. 8. R. Vol. XX1X, Wo. 5.

“_
the alcohol test was studied in connection with the acidity
of milk coming from 18 cases of tuberculosis, <6 cases of
indigestion, 17 cases in which the genital organs were af-
fected and 9 causes of miscellaneous diseases. The results
obtained veried, consequently the test is not deemed one
which can be relied upon for general diagnostic purposes.
An exception, however, is supposed to be made in diagnosing
impending abortion. <A negative finding in these cases
points with certainty to the fact that abortion will not
teke place within a certain time.”

An article by *Bordas upon the original acidity
of milk has been abstracted in the Experiment Station Rec-
ord as follows: “The lack of harmony in the various views
concerning the reaction of milk is thought probably due to
interpreting the results on the basis of different indica-
tors. The author believes the best indicato#f to use is
phenolphthaleh. The total acidity of milk is chiefly due
to free casein. Fresh milk never contains a free acid as
lactic acid, citric acid or any acid salt. When there is
an increase in the adidity as a result of decomposition of
lactose, the calcium salt of casein is decomposed before
the lactic acidity is manifest".

Chretien found normal milk to contin .10 to .20%
acidity reported as lactic acid. Colostrum milk had slightly
less. The feeding of a liquid distillery residue did not

*Bordas. (Orig. Commun. 8 Internat. Cong. Appl. Chen.)
Abstract TE. S.R. Vol. XXIX, No. 9.

°Chretien, A. (Hyg. Viande et Lait) 7, (1913) No. 5,
pp. 244-251. Abs. E.S.R. Vol. XXIX, No. 6.
materially affect the acid of milk. The acid content
appeared to increase with the length of lactation period.
Milk from a tuberculous udder shuwéd a decrease in the
acidity while that containing Bacillus mastitis, B. coli,
staphyxzococci, and streptococci showed a greater acid con-
tent than normal milk.

*Bosworth and Prucha, while trying the method of
Blau found fresh normal milk to contain .21% of citric acid.
This they found to gradually disappear during souring.

°Richmond reports the original acidity of milk as
being due to mono- and di-basic phosphates, and partly to

dissolved carbonic acid.

Metnods.

Taking samples. - Samples were taken at the barn
from the whole milk, placed in sterile bottles and brought
to the laboratory where acid determinations were immediately
made. In most cases the sampbes were less than an hour old
wren titrated.

Method of determining acidity.- 40 c.c. of well
mixed milk were placed in a clean white porcelain cup.,

2 c.c. of 1% solution of phenolphthalein was added. Tenth-
normal sodium hydroxide solution was then added from a
graduated burette until the milk had a faint pink color.

*The fermentation of citric acid in milk. Alfred W.
Bosworth and M.J.Prucha. Tech. Bulletin No. 14, Wf.Y. Agric.
ixp't. Station.

°Richmond, H.D. Dairy Chemistry.
The following formula was used in calculating the results:

c.c. of N/1O NaOH used x.009 x 100 = % acidity as
| c.ce of Milk used lactic acid.

Experimental Part.

Wo literature was found snowing the original acid
reaction of milk from different species except from the wow.

In Table I,are given the results of titrations of
fresh milk from different animals. Of the animals examined,
sheep's milx had the highest average with a maximum of .198%
and a minimum of .112. Cow's milk ranged between .114% and

.243%, while mare's milk ranged between .063 and .07?.

 

 

 

Table I.
Animal No. of Samples @% Acid.
_ Examined. 7
Sheep 16 ~ 165
Cow 55 0177
are 3 ~066
Woman L ____ 2951

Table II, shows the difference in acidity of
different portions of the milking. For this experiment a
Sample of the fare milk was taken from the four quarters.
Another sample was taken at the middle of the milking and
a third at the end. The table shows the middle milk to
have the highest average acidity with .159%, strippings

next with .144% and fore milk lowest with .138%.
 

Table II.

 

~_

Acidity to Phenolphthalein

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Cow No. Fore Milk Middle Milk Strippings
1 0179 «207 0193
2 139 ~148 © 135
3 126 ~148 ~1350
4 ~126 0135 e121
5 ~103 0125 107
6 o144 © 157 ~144
? 0154 ~162 -153
8 e112 ~ 148 el21
9 0153 ~198 eL71
190 ~144 0175 0157
11 ~144 ~166 ~162
12 ~ 140 0144 ~135
Average _____ 138 ~159 ~144 _
Table III.
Cow No. l. Cow Nlo. 2.
Portion Acidity Portion Acidity _
1 ~162 1 ~144
2 ~165 2 1180
3 ~175 3 0177
4 - 180 4 ~ 166
5 ~181 5 0157
6 180 6 _ ~ 157
7 2175 Cow No. 3.
8 0175 l ~ LOO
9 0177 2 ~153
10 ~ 166 3 ~ 157
ll ~ 166 4 ~162
12 ~ 166 5 ~ 156
13 - 166 6 0153
14 ~161 7 ~153
15 0155 8 ~ 146
16 o155 9 2144

 

Table III shows the original acidity of different
portions of the milking. As noted in the table, the first

portion has a low acidity, reaching a maximum a little prior
to the middle of the milking, and falling again at the end.
These results correspond closely with those obtained in
fable II.

In fable IV the original acidity of all the nor-
mal samples of milk tested, are tabulated, also a record of

the animals.

 

 

 

_ ; __rable IV. _

Cow. Dairy. Breed. Age. Period of Lactation.% Acid.

1 College Holstein 3 yrs. 9 Months «206

2 " " 4" 6 * 0157

3 " " 4" 8 * ~ 180
4 " “ 4" 2 e211

5 " " 4" 4" ~ 166

6 " " 5 " 35" ~ 166

7 7 " 5" 2" . VEO
8 " " 5 " 4" -202

9 " " 5 ll * ~202
1O " " 6" 6 * ~180
11 " " 7% 13" ~175
12 " " 7” a" 2166
13 " " 8 * 4" 2139
14 " " 8 3” -198
15 " " 10 * 3 ~198
16 ” Guernsey 7 * 7 * 229
1? " " 5 *" 8” e211
18 " " 5 * 4 e211
19 " " 5 * 1 * 0229
20 " “ 7” 9" 0215
21 " Jersey 3 * 4" ~189
22 « « § * 9” ~ 180
23 " « 3°” % ~243
24 . " 2* 1.* 0254:
25 " " 2” 9” o225
26 " " 2* 7” -206
29 0 " 3 0 7” e2il
28 M ” 5 * 7 0 e2il
29 " " 5 ® 7 ® e211
30 " " 5 * 4" ~202
ol . " 6 * 35” e211
o2 " " 7” 6 * 0229
33 " " 8 " 3 ~ 198
34 * Brown Swiss 3 * 8 ~198
35 8 " 5 * 2" 184
56 ._° " 5 * 4 ~184
37 8 " 13 * 19 ® e202
58 Bwpanore Holstein 4.5yrs. 12 * 2169
39 " “ 3.5 ® 10 * -154
40 " " 5.5 " 7 * 0149
 

 

 

 

 

___ Table IV. (cont'd)
Cow. Dairy. Breed. Age. Period of Lactation. % Acid

 

41 Espanore Holstein 4.5yrs. 2 Months £176
42 " " 9 0 12" ~125
43 " " 5 " 12 * ~114
44 ” # 5 a 9 " ~153
— «45 " " 5 " 6 * e145
46 " “ 4 “ 3% e131
4” " " 4 “ 4" 0147
48 " “ 4.5 " 7," e122
49 " " 9 " 10 * 2167
50 " " 11 " 8 ~129
51 " n 7  « 6 * ~ 155
52 ” " 2.5 " ye 0138
53 " " 2.5 " 7 0 171
54 « " 2.5 * 7” ~ 162

 

fable V shows the influence of breed upon the
original acid rmeaction of milk. The Guennsey is found to
be the highest with an average acidity of /219%. The others

ranking as follows; Jersey, Brown Swiss and Holstein.

 

 

Table VY.

Breed No. Tested Highest Lowest Average
Guernsey § 0229 o2il 0219
Jersey 13 02435 «180 e212
Brown Swiss 4 ~202 184 0192
Holstein 32 C* e211 ~114 ~162

 

In Table VI all the normal animals tested are
arranged in four groups according to their age. This table
shows the milk of cows between 2 and 4 years to have an
acid reaction above average for the breed, while cows be-
tween 4 and 10 years have a reaction which is close to the

average for breed. Milk from cows that are over 10 years

has a higher average acidity than the average for breed.
 

Table VI.

 

 

Age No. tested Average for Breed
2-4 yrs. 13 1.8 % above
4-6 * 25 1.2 % below
6 - 10" 13 -71 % above
10 - 15 * - 3 2.5 % above 7

 

Table VII shows the effect of the period of lac-
tation upon the original acid reaction of milk. During the
second to fourth month of the period of lactation, the acid-
ity seems to be unusually high being 6.4% above the average
for the breed. At all othe@ times it is slightly below the

average for the breed.

 

 

_ _ Table Vil.
Period of Lactation. No. tested. Average for Breed.
Under 2 months 5 048% below
z2to4 *" 12 6.4 % above
4to6 8 2.9 % below
10 mohths and over 24 1.7 % below

8 1.3 % below

 

Teble VIII shows the development of acid in samples
of milk which have been carefully drawn and kept at room
temperature. There seems to be no constant rule as to rate
and time of development of acidity, probably depending ujson
the nature and amount of contamination.

Table VIII.

Monday

   
 

-10:00pm.11: 00pm.

0171

1 .099 ~112 -112 ell2.- .112 e111 e112 e112
2 e135 0155 ~170 ~172 1756 ~170 ~170 ~ 165
5 .148 157 ~162 ~153 150 «150 e152 - 150
4 .117 ~112 ~ 126 e121 -124 01235 117 e125
5 .193 ~ 180 °192 0193 ~195 o202 ~193 ~ 185
6 .122 e121 e121 -126 el2l ~125 e121 e121
7 °189 180 ~180 -~ 180 175 0171 0175
 

 

 

-~12-

Table VIII. (cont'd)

Monday
Cow 4:00pm.5:00pm.6:00pm./ ;00pm.8: 00pm .9: 00pm .10:00pm.11:00pm.

 

8 .lei e121 0121 .12i ~126 e121 e120 ~118
9 .1721 e162 ~166 = .171 ol71 e171 ~162 ~157
10 .153 ~153 0153 3 =0.157 ~ 166 ~153 ~152 ~ 157
ll .202 202 207) 3=.202 0202 205 0209 e210
12 .155 ~156 ~-160 .155 ~165 ~ 165 ~ L160 e160
Tuesda Wednesda
Cow 6: d0am.12;:a0pm.5: 50pm. 8:30am.5:50Opm.
1 e110 ~115 e115 e112 441
2 ~L70 ~ 163 ~ 165 ~ 166 -a85
3 e150 ~150 ~148 e150 0357
4 0124 0123 012d e123 ~050
5 e190 e185 0193 ~190 o9l
6 »125 ~126 ~126 126 ~585
7 178 «180 ~185 ~180 499
8 e121 e121 e121 ~118 ~450
9 L171 «160 e175 0171 ~460
10 ~ 160 ~160 e160 ~ 165 ~526
11 0210 e212 216 2207 e511
12 ~163 ~ 159 ~ 160 158 ~489

 

Table IX snows the difference in the original
acidity of milk taken from the four quarters. This table
shows a wide variation in the original acid reaction of the

milk found in the different quarters.

 

 

 

 

Table IX.
Right Rear Right Front Left Rear Left Front
Cow Quarter Quarter Quarter Quarter
1 2 15C e202 -180 e191
2 .157 2126 ~ 150 «150
3 0225 202 162 2252
4 198 229 ~189 258
5 01355 »161 ~ 166 207
6 0135 0152 166 148
? e215 229 -207 211
8 1359 148 0k5? »135
9 0207 202 0215 0202
LO 2157 e157 - 180 e152

 
 

Table X shows the original acid reaction of milk
from day to day after calving. The first two or three days
are seen to be*usually high due to colustrum. It then
reaches 9 point from which it varies slightly either up or

down from dey to day.

 

 

 

Table X.
Oe Lassie College Bertha Aais
Day Elzevere Goda i. Fair
1 «405 0427 «210 e315
2 ~150 2 0290 ~135 ©202
3 137 0279 -162 ~195
4 «103 .270 ~2£10 » 184
5 ~121 .256 ~ 184 «207
6 ~101* ~246 ~ 166 0177
7 ~090* 0216 01756 e162
8 ~101* 202 216 e171
9 ~067* ©186 207 e162
10 0067% e211 ~180 159
11 ~182 ~150
12 *Gargety 0175 © 135
13 - 166
14 0117
15 e178

 

Table XI shows the relation of the health of the
cow to the original acid reaction of mibk. Unfortunately
very few abnormal cows were available for this work. The
Semples obtained show a wide variation due to the different

abnormal conditions.

Table XL.

 

Condition of ¥ilk or Udder % Acid

Sick with indigestion e072
Milk very bloody ~599
Teat injured externally ~ 157
Gargety milk -063

" " -090

" " »050

” " 069

“ 0 ~090

"

~065
 

Discussion of Results.

A study of the original acid reaction of the milk
from the different species shows a wide variation. Of the
species studied, sheep milk was the highest with others
ranking as follows: Cow, mare, woman. The following table
shows an interesting relation between the acid reaction and
the percent of ash and casein present.

Species Acidity Casein Ash

Le

Sheep 2185 5.235 097
Cow 0177 3.00 75
Mare ~066 1484 ~ 30
Woman 031 1.00 220

 

The casein and agh are seen to decrease in exactly
the same manner that the acid reaction does. This bears out
the theory that the original acid reaction of milk is largely
due to casein and ash.

Acid reaction of milk to phenolphthalein shows the
fore milk to have the lowest acid reaction, while the middle
milk is highest and the strippings having a reaction between
the two. The reaction of the fore milk and strippings are
again seen to correspond to the amount of casein and ash
present in the respective portions as noted in relative %
acid reaction to species. The author was unable to find a
table showing the chemical composition of the middle milk.

It is also thought that the presence of alkali producing
organisms in the highly contaminated fore milk might influence

the reaction.
A wide variation in reaction is found for breeds.
There appears to be no eonstant relation between this reac-
tion and the average amounts of casein or ash present.

The average reaction of milk to phenolphthalein
for cows between 2 and 4 years of age is slightly above the
average for the breed, cows between 4 and 10 years are about
average while cows over 10 years ef age have an average re-
action which is 2.5% above that for their breeds. These
differences are so small that this factor isn't thought to
influence the reaction to any appreciable extent.

Between the second and fourth months of the
period of lactation was the only time that the milk had an
acid reaction which was above the arerage for the breeds.
Except for this period the acid reaction gradually increased
during the period of lactation as reported by Chretien.

Milk when very carefully drawn and stored at room
temperature may sometimes be kept as long as 24 hours without
appreciable change in reaction. In some cases,however, the
reaction increases slightly after the first hour. Host
titration in this work were made within one hour after
having been drawn.

The milk from different quarters of the same
cow sometimes has an acid reaction with a wide variation.
This might be accounted for by some pathological condition
of the quarter or by some bacterial invasion of the quarter

in question.
»

« 16 um

Colastrum has an acid reaction which in some cases
was found to be over .4%. Usually this reaction reaches
normal in from three to four days. The high reaction of
the colostrum is probably due to its extremely high percent-
age of casein and ash.

vezy few samples of pathological milk were obtained
for this work. Extreme cawes of gargety milk and milk from
a cow sick with indigestion gave unusually low titrations,
while bloody milk gives a high acid reaction. Judging from
the experiinents the reaction of milk to phenolphthalein is
of no practical importance in diagnosing abnormal conditions
of the milk or udder for the following reasons: Too many
other fadtors enter in and also a condition of the milk or
udder, which could be detected by means of the reaction,

could be much easier and more susely detected by other means.

SUMLIARY .

The acid reaction of fresh milk to phenolphthalein
is of no practical value in diagnosing abnormal conditions
of the milk or general conditions of the cow.

Normal milk was found to have an original reaction
to phenolphthalein varying between .114 and .243 percent as
lactic acid. This according to Auzinger'ts table would include
Old milkers; milk from cows in first or second week after
pabturition, colostrum and pathological milk.

Bad cases of gargety milk and bloody milk were
found to have reactions which were respectively below and

above the limits for normal milk.
- 17 »

These conditions, however, could easily be detected
by other means.

The different breeds were found to have varying
acid reactions. The average reactions being: Guernsey .219%,
Jersey .2124, Brown Swiss .192%, and Holstein .162%.

Middle milk has a higher titration then fore milk
or strippings.

Age of cow apparently has very little influence
upon the reaction.

The acid reaction of the milk Gpparently gradually
increases after the fourth month of the period of lactation.

Considerable variation is sometimes found between
the reaction of the milk from different quarters.

Colostrum has a high ecid reaction.

There seems to be a relation between the acid

reaction and the amount of casein and ash present.
 

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