THE EFFECT OF HYDROGEN MN
C(TrNCENTRATTON ON THE BACTERIAL
CONTENT OF MHJ< BURN“;
PASTEURIZATION

Thesis for the chrec of M. S

Ramon I. Quinet

1928

 

 

 

 

 

 

THE EFFECT OF HYDROGEN ION CONCENTRATION ON THE
BACTERIAL CONTENT OF MILK DURING

BASTEUKIZCTION.

.THE EFFECT OF HYDROGEN ION CONCENTRATION ON THE
BACTERIAL CONTENT OF MILK DURING
PASTEURIZATION.

THESIS

Submitted to the Faculty of the.Michigan State College
in partial fulfillment of the requirements for the

degree of Master of Science.

by
Ramon I. Quinet

June, 1928.

THESTS

 

TABLE OF CONTLNTS

INTRODUCTION

HISTORICAi

EAPERLHENTAL
General Considerations
Collection of Samples
Method of Determining the pH
Pasteurization
Plating and Counting
Tables

DISCOSSION OF DATA

8 Uhhim RY

CONCLUSIONS

ACKNOJLEnGQhNTS

BIBLIOGRAPHY

10332330

Page

12
12
17

21
21
24
26
50
32
53
54

INTRODUCTION

Milk, one of the most indispensable foods in most of
the civilized countries, presents also some very indis-
pensable problems in relation to the health or to the
physical well-being of the consumer. Such problems in-
volve not only the passing of ordinances and laws (10)
concerning its sale but also present various phases of
investigational nature. Prominent among these phases
are those concerning the safeguarding of public health.
The fact that milk is a good food, containing the necessa-
ry elements for growth, makes it also a favorable medium
in which to grow micro-organisms. Improperly handled, it
becomes a source and a disseminator of a number of
diseases and may be, in itself a menace instead of a bene-
fit to the public. For this reason, researches have been
made and have prompted many literary discussions so that
to-day the literature, concerning the various phases of
investigational nature, of milk and its product, is
voluminous.

When it became known that milk was a source and a
carrier of the germs of many diseases such as tubercu-
losis, diphtheria, typhoid fever, septic sore throat,
scarlet fever, dysentery, malta fever, and recently
Alkaligenes abortus (Bang) (This latter only recently
found to be caused through infected milks from infected

cows), the elimination of such diseases became the subject

_ .....-«~

-2-

of much investigation by workers in Public Health Labora-
tories and hospitals, and others interested in safeguard-
ing the health of the public. To-day, except in rare
cases, the public is well assured of a milk supply that is
both sanitary and free from disease producing organisms.
Pasteurization of milk has been the subject of much
investigation and researches have been justified by the
resultant increase in public safety. Any work, therefore,
in this field may be considered as contributing something
in the production of a better and safer commodity. The
following is a contribution to the process of pasteur-
ization showing the effect of H-ion concentration on the

bacterial content of milk during the process.

NISTORICAL

Perhaps there is not a subject in the dairy industry
which has reCeived greater attention than that which deals
with the quality of the milk received and dispensed. The
fact that milk harbors a great number of bacteria which
are either pathogenic or non-pathogenic leads the pioneers
of the dairy industry to subject their product to the
process of pasteurization to eliminate all possible Chances
of infection from the use of their products. The early
beginnings of pasteurization (17) may be traced back to
Spellanzani who in 1765, disproved the theory of spon»
taneous generation by boiling meat extract in sealed flasks
and finding that the contents did not spoil. In 1788,
Scheele advised the use of this process in the preservation
of vinegar by exposing the sealed bottles to boiling water.
Appert, in 1804, used the process in canning, and in 1810,
Durand, taking advantage of the opportunities offered by
this method of preserving foods, obtained a patent for
preserving foods in tin cans and glass jars.

Pasteur, (1860-1864) after revealing to the world the
fact that fermentation was due to living cells and not to
chemical reactions, next focused his attention on the
diseases of wine. He found that spoilage was due to the
presence of certain bacteria and he suggested that this
could be eliminated by heating the grape juice at a tempera-
ture between 122‘ F. and 140° F. Since then this process of

partial sterilization has been called pasteurization.
Pasteurization, however, was not used so extensively
until the medical profession recognized the value of
heated milk in the elimination of certain intestinal
troubles. We find that, in 1886, soxhlet first preposed
the use of heated milk for infant feeding. Other doctors
and pediatricians seeing the possibilities of heated milk
as asafeguard against intestinal troubles of their patients
also adopted its use, and in 1889, Jacobi, a pediatrician,
gave heated milk to his patients. In 1889, Dr. Henry Koplik
was made director of the first pasteurized milk depot in
New York City. The milk he distributed was pastcueized at
185° F. to 195‘ F. The temperature was made high because
he found that milk pasteurized at that temperature kept well
in.a moderately cool place for 24 hours. In 1893, Nathan
Straus, established his first milk depot where he sold milk
pasteurized by the holder method for 20 minutes at 167' F.
From this brief summary of the use of pasteurization
in the dairy industry may be traced the efforts of men who
pioneered in bringing about a modern industry, the products
of which are daily necessities in every man's home. The
bacteriology of this industry is quite extensive and well-
explored and workers in this field have endeavored to help
the industry to maintain the highest quality of product
and to safeguard the public. This present research is only
part of the work that is being carried on to throw more
light on the subject of pasteurization.

-5-

It is well known in bacteriology that bacteria require
a certain amount of acidity or alkalinity in a mediwm to
bring about the best possible growth. It is well known
also that above or below this optimum acidity or alkalinity,
bacteria exhibit certain atypical characteristics or the
so-called dissociations, or may be entirely inhibited in
their growth.

In looking over the literature on this subject, it was
found that the determinations on the acidity of milk have
not been.as extensive as might be expected. Fee (11) was
the first to report the H-ion concentration of cow's milk
which fact he detemmined by the electrometric method. He
found that fresh cow's-milk has a pH value between 6.6886
and 7.6209. Baker and Breed (2) tried to add the indicator
directly to milk contained in test tubes and to compare un-
known samples with those obtained with standard samples of
normal milk. They came to the conclusion that this method
was impracticable due to the lack of definite color
standards and the Opacity of the milk. To these diffi-
culties may be added the fact that some milks contain
higher amounts of phosphates, citrates, and other salts
which increase the buffer action. (Another factor which can
not be very well overlooked in that of the absorption of
the color due to the presence of casein as reported by
Sammie and Santschi (l9) . These investigators (19)
suggested the use of fresh whey as the "whey is said to

have the same H-ion concentration as the milk". However,

they did not state the authority for above conclusion.
Sharp and McInerney (21) indicated that it might or might
not be true depending upon the H-ion concentration of the
milk. They further said, "Also it is known that whey shows
indicator errors....,“ and for this reason was of no value.
These two workers (21) diluted milk 1-20, and using Brmm
Cresol Purple and Phenol Red as indicators, found that at
1-20 dilution, the error was very small. By means of a
correction factor, they were able to transpose the color-
imetric pH into the corresponding pH taken electrometrically,
This factor which they found corresponded to a decrease of
the H-ion concentration equivalent to 0.54 of a pH when the
milk was diluted to 1-20 and compared to the same sample the
pH of which was taken.electrometrically. By using this
correction factor, in the present work, the actual pH or
true pH mentioned was determined.

Frei and Lienhard (12), using colorimetric method by
adding the indicator directly into fresh cow's milk, with
Brom Cresol Purple and Brom Thymol Blue as indicators,
found that fresh, normal cow's milk has a pH range between
5.9 and 6.1.

Tan Slyke and Baker (27) dialized 500 samples of
milk and found that the pH values ranged from 6.4 to 6.7
and that 85 per cent of these have a range between pH 6.5
and 6.7. Schultz and Chandler (23) dialized 1 cc. of
milk against 2 cc. of neutral distilled water for 7 1/2

minutes and determined the H-ion concentration of the

diffusate colorimetrically, using Brom Thymol Blue as

 

indicator and found that the pH values ranged from 6.4 to
6.7. They found that distilled water gave the same results
as physiological salt solution provided equal volumes were
used.

Rice and.Markley (18) diluted milk with water and
found that a dilution of 1-10 did not further reduce the
acidity appreciably when titrated with N/lO NaOH using
phenelphthalein as indicator. They stated, "Dilution
affects the titration values of high acid milk more than
those of low acidity, which would indicate that phosphates
have much to do with the effect." Lisk (14) analized 77
samples of sterile milk diluted 1-10 and found that the
pH value varies from 6.4 to 6.6. She did not state whether
the colorimetric or electrometric method was used. Clark
(4) stated that a well buffered solution may often be
diluted without seriously altering the pH.

The writer found that as long as the pH of the milk
used for analysis did not reach beyond 6.6, diluted 1-80
or even 1-100, the pH value did not change appreciably
provided the diluting water was made neutral. Using
physiological salt solution or neutral distilled water in
1-20 or 1-100 dilution, there was a tendency for the
diluted milk to approach the pH value of the diluting
solution, i.e., instead of having the pH of 7.5 (pH 6.8)
in a dilution of 1-20 or 1-100, the H-ion concentration
decreased and there was an appreciable change toward the

acid side. The same result was also obtained if the milk

was diluted 1-20 or 1-100 and the pH value was brought to

6.5 (pH6.0), it had a tendency to go toward the alkaline
side, even in the 1-20 dilution where there was some buffer
action left to take care of the change. In both cases the
pH determination was not accurate at the above pH ranges.
In lower dilutions, there was buffer effect which could
nottne avoided and in higher dilutions, there was the pH
decrease or increase due to the diluting water. However,
the change effected was so small as to be almost negligible
provided the H-ion concentration of the milk being tested
was not carried far beyond the pH values 6.6 to 6.0 un-
diluted. These results agreed in substance with those of
Baker and Van Slyke (28) who stated that the H-ion concen-
tration.of fresh milk decreased when water was added.

Clark also stated, "Diluting milk with distilled water
lowered the H-ion concentration appreciably”.

Cullen (7) in determining the H-ion concentration of
blood plasma, diluted the serum with 0.9 per cent NaCl and
found it to increase in alkalinity up to dilution of 15 to
20-fold, beyond the 20-fold the change upon further dilution
became so small as to be practically negligible. The writer
also found this to be true in connection with fresh cow's
milk.

Sharp and.flcInerney (22) titrated milk samples and
also determined the pH value electrometrically. The ti-
tration values were expressed in terms of per cent lactic
acid using N/10 NaOH solution. These workers found that
there was a direct relationship between the titrable acidity

and the PHe

Table Ie
The pH of Fresh.uilk and Corresponding Titrable
Acidity, EXpressed as Lactic Acid, Determined from Curve.

~(Fr0m Sharp and McInerney)

 

 

pH Titrable pH Titrable
Acidity Acidity
6.0 ‘ 0.50 6.7 0.145
6.1 0.43 6.8 0.125
6.2 0.56 6.9 0.115
6.5 0.50 7.0 0.105
6.4 0.25 7.1 0.095
6.5 0.21 7.2 0.090
6.6 0.165 7.3 0.085

From the above discussion of the literature, one can
see that in spite of the differences of opinion as to the
methods of determining the pH values colorimetrically,
each investigator believed that his work had advantages
over that of the others.

Dializing the milk, and determining the pH of the
dialysate, eliminates some of the errors due to the ab-
sorption of the color by the casein, and also eliminates
Opacity which by any other method, except by the
electrometric method, is an unavoidable factor of error.
Dilution of the dialysate does not affect the H-ion con-
centration except when carried to a high dilution in

which case the pH value becomes greater and has a tendency

-‘10 -

to go toward the pH of the diluting water or solution. Di-
lution with water causes a sudden change in the pH of milk
but with any subsequent addition of the diluting solution the
pH changes are not as sharp as at first. If these changes
were plotted, there would be at first a sudden drop flattening
out slowly as the dilution increases, arriving finally at the
pH of the diluting solution.

Diluting milk does not change the H-ion concentration
appreciably as indicated by Rice and.Mark1ey (18), Lisk,
(14), Clark (4), Van Slyke and Baker (28), Sharp and
(McInerney (21), so that the pH can be determined to within a
very small degree of error when compared with that taken
electrometrically. Although the determination of pH values
colorimetrically is very susceptible to various factors (20)
which are slightly conducive to error in reading, the small
variations in pH values in the determination.which.deviates
from the true pH may be considered negligible for practical
purposes in the eXperiments that follow.

Some of the pH values of milk as determined by differ-
ent investigators are given in the following table by Erei

and Lindhard (12).

1.
2.
5.
4.
5.
6.

8.

9.

10.

11.

Table II

pH of normal Cow Milk as Reported in Literature

by Various Investigators.
(From Frei and Leinhard.)

Courant (zit. nach Foa)................
D3V1d3°hn 0..O...OOOOOO/OOOOOOOOOOO0.0..

van DamOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoe

AllemanCOOOOOOOOOOO0.000000000000000000

JMilroy'.................................
Clark:..................................
Taylor (zit. nach Schnlte and Chandler..
0bermeiert..............................
Kramer and Green.......................
Slyke and Baker (500 Prob.) ............

About 85 per cent ....................

Schultz and Chandler (160 samples)......

Method - 1 to 8, Electrometric
9 to 11, Dialysis
10, Colorimetric

pH
pH
pH
pH
pH
pH
pH
pH
pH
pH
pH
pH

6.6885 - 7.6209

6.75 1m Mittel

6.74

6.61

6.64 - 6.8
6.6

6.8

6.5 - 6.6

6.5 - 6.7

6.5 - 7.2

6.5 - 6.76
6.4 - 6.7

O

c-
O

.

-
.

‘
.

m
.

4.12-

EXPERIMENTAL

General Considerations.

This work was undertaken to determine if there was
some practical significance of the H-ion concentration
in the pasteurization of milk. It was hoped that it
might aid in answering some of the questions that arise
such as: Does the H-ion concentration of milk have any
effect on the bacterial content during pasteurization?
Does it affect the keeping quality after pasteurization?

Rice and.Mark1ey (18) concluded that normal milk
with a high acidity contained more of all nutrients and
was particularly high in phosphates and casein. They
gound that, "High acid milks under the same conditions of
storage, reached the coagulating point later than.milks of
lower acidity."

Milk, being a highly buffered substance has the prepar-
ty of resisting the changes of pH upon the addition of acid
or alkali up to a certain point, and also, milk has the
property of being amphoteric, i. e., it reacts acid to
blue litmus paper, and alkali to red. It has been.shown
by Rice and markley (18) that milk having a higher acidity
than normal generally shows higher buffer action.than.milk
of low acidity.

Milk heated for sterilization has a tendency to show
decrease in pH. The work of Whittier and Benton (50)
showed that the pH value of heated milk decreased and the
titrable acidity in terms of N/lO lactic acid increased,

a
.
‘ d
l
_.

ml -.

 

this increase being due to lactic acid. The percentage of
increase was proportional to the temperature, time, and the
concentration of lactose present in the milk.

Freshly drawn milk absorbs carbon dioxide from the air,
and this is responsible for the decrease in the pH value
aside from the effect produced from the milk sugar as it is
acted upon by acid-producing bacteria. It is estimated by
Van Slyke and Baker (27), (28), that normal milk contains
about 10 per cent by volume of 002 and that carbon dioxide
absorption is not so marked between the pH values of 6.50
and 6.65, as it is above pH 6.65. Rice and Markley (l8)
concluded that 002 accounted for 0.01 to 0.02 per cent of
the acidity calculated as lactic acid.

Whittier and Benton (29) found that heating milk increased
the acidity equivalent to a concentration of 0.02 mol. and
lactose decreased in amount equivalent to 0.085 mol. In
another experiment these two authors added lactose to the
amount of 5 per cent, and found that the acidity was marked-
ly greater than that of the normal sample. They concluded
that the loss of lactose content in heated milk was more than
sufficient to account for the acid production even on a mol
to mol basis.

The facts, then, are that milk absorbs carbon dioxide
from the air and that heating produces a decrease in the
H-ion concentration due to breaking down of the lactose
molecule and the subsequent oxidation of the glucose forming

into molecules of lactic acid. This acid will cause some

514..

trouble in the adjusted pH values of the samples to be
pasteurized. However, Van Slyke and Baker (28), on their
observations on this point say, "We had noticed that pasteur-
ization, if properly performed, is without observable effect
in changing the H-ion concentration of milk." They also con-
cluded from an experiment as shown in Table III that in.milk
in which carbon dioxide was not removed before heating, the
pH value remained the same before and after heating, even
though the percentage of 002 was decreased by the heating from

10 to 2 per cent.

Table III

Results Showing Effect of Removal of 003 on
Reaction of Pasteurized Milk.
(From Van Slyke and Baker)

 

Before Removal of C02 After Removal of 002

Before Heating : After Heating . Before Heating : After Heating

;

:Per ct. : pH :Per ct. : pH :Per ct.
Value :of 00; :Value :of 002 :Value :of 002
:by Vol. :by Vol. : :by Vol.

’6
p:

pH :Per ct.
Value :of CO:
:by Vol.

L__

.£5. 514:: 1:

0.0 2 6.56

6.54 10 6.60 0.0

ee ee ee we ee
ee ee ee P.
#9 ee ee e

The above results by Van Slyke and Baker (28) were obtained

by heating milk at a temperature of 65° C. for 15 minutes.

- 15 -

Since heating milk without 002 decreases the pH value by only
0.04, it is quite possible that such a small amount of change
is quite negligible considering the H-ion concentration by
colorimetric measurements, inasmuch as the lowest change that
could possibly be detected under ordinary conditions using a
comparator block and blanks, is around 0.1 of a pH. In this
work, it was found that milk under the ordinary method of
laboratory pasteurization using test tubes plugged with
cotton, a majority of the samples tested did not show any
change in H-ion concentration and some showed a decrease in
their pH values. The greatest change in some cases was not
enough so that even approximate values could be designated,
and, therefore, signs were used to express greater than (+)
or less than (-) to designate the change. For experimental
purposes, some of the samples were acidified by adding N/lO
lactic acid until the desired pH was obtained, while in
others N/lO moon was used to make them more alkaline. There
was a tendency for the milk to show a brownish color if ad-
justed to'a concentration higher than pH 7.2 (diluted 100
columes) or equivalent to pH 6.7 in the actual pH using the
correction factors as given by Sharp and Mo Inerney (21).
This borwnish coloration.was probably due to the action of
the alkali upon the protein material in the milk or due to
the alteration of the sugar content. As it was more
frequently found in samples heated in flowing steam, one

might believe that this change was due to the caramelization

of the sugar.

Table IV gives the result of some eXperiments on samples
of milk having known pH values and pasteurized at a temperature
of 62.5“ C. for thirty minutes, and the pH value taken after
cooling to 20° C.

~ Table IV.
The Effect of Pasteurizing Temperature Upon the
H-ion Concentration of Hilk of.Known pH Value.

 

 

Before HeatingiAfter Heating;Before Heating.After Heating

PH 3 PH 3 PH 3 PH
6.8 i 6.8 i 7.2 g 7.1
6.8 i 6.8 i 6.8 g 6.9
6.9 i 6.9 i 6.7 i -6.7
6.9 i 6.9 i 6.6 i 6.6
6.8 i 6.8 i 6.6 : +6.5
5.5 5..

 

The above pH values were obtained by diluting the
milk sample 1-100 both before and after heating. Phenol
red and Brom cresol purple were used as indicators.

As shown by the above data, the change is very
slight and checks closely with that of Table III given

by van Slyke and Baker (27).

.~ 17 -

The Determination of the pH at Which Bacteria
in.Milk are Killed Most Rapidly.

Collection of Samples.

The samples of milk used in this work were obtained
from the college creamery, placed in sterile flasks and
taken to the laboratory and pasteurized in test tubes of fine
same size and thickness. Some of the samples were stored in
the ice-box, the temperature of which varied only from 4‘ C.
to 6° 0. Four days storage was allowed before pasteurizing.
This was done to increase the bacterial count of some of the
samples taken during the winter months. Changes in the H-ion
concentration during storage were also determined. Table V
shows the result of the changes in pH of’ten samples of
fresh milk that were thus stored.

It will be seen in the following table that there were
no appreciable changes in the pH values during the four days
of storage, except in the last sample which was made alkaline
by the addition of N/lO NeoH. This sample showed a change
of 0.2 of a pH after four days storage. It will also be
noted that samples No. 6 and No. 9 showed changes of 0.1 and

less than 0.2 of a pH respectively.

The Effect of'Storage Upon the H-icn

Concentration of Fresh Cow‘s Milk at Ice-Box

 

 

 

Temperature.

Sample Diluted 100 volumes
ATter After
No. Initial 48 hrs. 4 das.

pH pH pH

1 6.8 6.8 6.8

2 6.8 6.8 6.8

5 6.9 6.9 6.9

4 6.9 6.9 6.9

5 6.8 6.8 6.8

6 7.0 7.0 6.9

7 6.8 6.8 6.8

8 6.9 6.9 6.9

9 7.0 7.0 +6.8

*10 7.1 7.1 6.9

 

a

* This sample was made alkaline by the addition
of N/lO Neon.

1...,

 

-19-

Method of pH Determination.

The pH determination was made by diluting 1 cc. of
milk to 100 volumes and the desired amount of indicator
was added to 10 cc. of this dilution. Sharp and Holnerney
(21) used Phenol red and Brom oresol purple as their indi-
cators in a dilution of 1-20 which proved by experimental
evidence that the changes in pH produced by dilution were
gradual. Checking their results by using a 1-100 dilution,
it was found that this latter dilution agreed with their
results from pH 7.2 to pH 6.4. Slight deviations occur
outside these values, especially if Brom thymol blue is
used as an indicator. This deviation was much more
noticeable in 1-20 dilution if Brom thymol blue is used
as an indicator. Table VI shows the relationship between
the three indicators, Phenol red, brom oresol purple, and
Brom thymol blue in 1-20 and 1-100 dilutions.

It will be noted that no values were given for the
undiluted milk having the adjusted pH 7.5 (diluted 1-20)
due to the fact that no correcting factor was given for
that pH. The reason for this was that Sharp and McInerney

(21) were dealing only with fresh, normal cow's milk.

- 20 -
Table VI

The Effect of Dilution Upon the Hydrogen Ion
Concentration of Milk of Increasing Acidity.

 

I n d i c a t o r s

 

 

sample Undi- Brom Brom Correct-

No. luted. Phenol thymol cresol Error ing

Milk Red purpl . factor
l;20 1:100 1:100 1:20 1:100
PH pH pH pH DE DE

1 7.5 7.4 7.5 +0.1,+0.2 0.54
2 7.3 -+7.5 7.2 l.0,+0.1 0.54
5 7.2 7.2 +7.2 ........ 0.54
4 6.56 7.1 7.1 7.1 ........ 0.54
5 6.46 7.0 7.0 7.0 ........ 0.54
6 6.56 6.9 6.9 6.9 ........ 0.54
7 6.56 6.9 6.9 6.9 ........ 0.54
8 6.56 6.9 6.9 6.9 ........ 0.54
9 6.26 6.8 6.8 6.8 ........ 0.54
10 6.26 6.8 6.8 6.8 ........ 0.54
11 6.16 6.7 6.7 6.7 ........ 0.54
12 6.06 6.6 6.6 +6.6 ........ 0.54
15 5.96 6.6 +6.6 6.6 ........ 0.54
14 5.96 6.6 6.5 6.6 +0.1,0.0 0.54
15 5.86 6.5 6.4 6.5 +0.1,... 0.54
15 +5.76 6.5 +6.5 6.5 +0.2,... 0.54
17 +5.76 6.5 +6.5 6.5 -+0.2,... 0.54
18 5.66 -6.5 6.2 6.4 +0.2,... 0.54
19 5.48 6.4 6.0 6.4 .o.4,... 0.52

Samples 1, 2,
give the desired pH, and samples lOto

and 5 are the same milk but made alkaline to
19 are made acid.

- 21 -

Pasteurization.

Pasteurization of the samples was made in a water bath
heated with flowing steam and the amount of steam was regu-
lated by means of a valve. The temperature was kept constant
by means of melted paraffin on the surface of the bath to
prevent radiation.

After the pH values were adjusted, the tubes contain-
ing the samples were immersed in the bath. Pasteurization
time started as soon as the temperature of the milk reached
that of the water bath. This was determined by placing a
thermometer in a tube containing the same amount of milk.

The samples were pasteurized for 50 minutes at 62.5° C.

Plating and Counting.

After pasteurization and when the tubes were suf-
ficiently cooled, dilutions were made and 1 cc. of the de-
sired dilution was plated. Duplicate plates were used. The
plates were incubated at 57‘ C. for 48 hours and counted at
the end of the incubation period. The plates showing the
average growth of the different dilutions made, were counted.

The medium used for plating has the following compo-
sition.

Agar (market, not oven dried).......... 1.5%
Beef extract (Bacto) .................. 0.5%
Peptone (Bacto) ....................... 0.5%

Distilled Water.

,- 22 -

Table VIIe

The Effect of Different pH Values on the Bacterial
Content of Milk During Pasteurization.

 

Sample:0rigi-: Original :

N00 :nal PH: Count Count on Adjusted pH Values

 

 

:1-100 :

i7.4;_74,2 ; 7.0 ; 6.8 L 6.6 .L' 6.4
1 - 6.8 : 55,000: 500: 700: 400: 600: 400: 400
2 : 6.8 - 52,000: 1,200: 1,200: 1,500: 400: 500: 200
5 - 6.9 : 910,000: 5,400: 500: 4,000: 500: 200:5,900
4 6.8 : 170,000: 1,700:67,000:22,ooo:12,000: 500:1,000
5 : 6.8 . 120,000: 5,900: 2,200: 2,500: 2,000: 2,400; 700
6 6.8 : 550,000: 2,900: 2,900: 2,600: 5,800: 200: 200‘
7 . 6.7 ; 7,100,000: 200: 200: 200: 200: 100: 100
8 . 6.8 : 240,000:11,000:11,000: 7,900: 7,000: 2,400:l,800
9 : 6.8 :12,000,000: 1,200: 2,000: 2,000: 2,000: 1,300: 500

10 . 6.9 : 4,500,000:58,000:69,000:50,000: 7,000:59,000:1,100
ll . 6.8 : 5,400,000: 4,100:11,000: 7,200: 8,400: 5,000:2,500
12 : 6.8 : 1,700,000: 2,500: 1,500: 1,500: 1,500: 1,500: 800
13 : 6.8 - 210,000: 8,700: 7,500: 7,700: 6,800: 6,500:6,900
l4 : 6.9 : 180,000: 1,000: 1,000: 500: 600: 400: 500
15 ; 0.9 : 2,220,000: 800: 4,500: 1,700: 400: 600: 200
16 ; 6.8 : 4,900,000:21,000:20,000: 4,400: 4,800: 4,6oo:1,000
17 : 6.9 : 57,000: 600: 1,500: 600: 400: 400: 200
18 : 6.8 : 240,000: 4,400: 1,200: 900: 700: 500: 200

I‘I

. y‘-

0.

-23-

Table VII. (Con.)

The Effect of Different pd Values on the Bacterial

Content of milk During Pasteurization.

I.

Samp1890rigi-: Original Count on Adjusted_pH Values

No. nal pH Count

 

:1—100 : :7.4 7.2 7.0 6.8 6.6 6.4
19 ° 6.9 ° 14 ,000: 1,100:1fl00 ‘500. 1,000: 500; 200
20 ' 6.8 ' 470,000 2 ,200 .'1, 600.1, 900: 1,800. l,700:1,800

21 ° 6.9 270,000 1, 500 1, 500: L 600 1,100' 1,200 1,200

22 6.9 ' 67 000 1,100 :1, 200: 1, 200: 1,100' 1,500 1,200
25 ° 6.8 ° 64 000 200" 200 :1 000 1,400' 1,700' 200

24 6. 8 240,000 1, 600 1, 000 800, 1,100 800 800
Ave. count...l 575 ,472: 5 468 :8, 456 :4 ,964£ 4,252; 5,056;1,104
Per cent reduction.....99.656:99.482:99.69%:99.75%:99.812:99.95%

;24-

Bacterial count per cc.

06.000n
00.0001
00.000
H0.000
H0.0001
H6.000
Hm.000
90.000
0.000
0.000
0.000

0.000

I

 

01’th 4/ Coal? f

 

4 574:4 72

.dve.

 

 

7.4

 

2
o
7

 

 

0 8

7 6
kM¥d§ns .x\n\

6.6

6.4

 

IIIL

Bacterial count on milk at different pH values.

Fig. 1.

Per cent decrease

9973

98/w

 

-25-

 

 

/
l l I I 1 J
7,4 7.2 7.0 6.8 6.6 6.4 6.2
Adjust erl pH value 8
Fig. 2. The per cent decrease of bacteria in milk at different

pH values, pasteurized at 62.5° C. for 30 minutes.

-26-

DISCUSSION OF DATA.

Studying carefully the data presented, it can be seen
that the different counts for the corresponding pH values
used, differ somewhat and indicate differences in degree of
effectiveness of pasteurization. The pH on the adjusted
values corresponding to the original pH value of the milk
may be taken as a basis for comparison. It seems, as the
data indicates, that samples having pH values nearest to the
original have approximately the same number of colonies, ex-
_cept at pH 6.6 in which case there seems to be a noticeable
decline in the bacterial count. For instance, in the sample
having pH value 7.2, the bacterial count on the graph was
higher but beyond pH 7.2, there was a sharp decline on the
bacterial count. This then, seems to indicate that at pH
7.4 and at pH 6.6, there was anoticeable effectiveness of
the pasteurizer. This efficiency was not so noticeable in
some of the individual samples. This was due to the micro-
flora of the milk itself. In.most cases, where there was a
high count, and the efficiency of the pasteurizer was low,
the microflora of the milk was made up of organisms in which
spore formers predominated and also some heat resisting,
organisms. Brannon and Prucha (5) isolated 70 different kinds
of bacteria from milk, pasteurized them at 62.5“ C. for 55
minutes, and found that 47 organisms were non.spore formersza
and only two of these survived pasteurization. In two
instances larger counts were obtained after pasteurization.

They attributed this increase to the breaking up of the clumps

of spores, since none of the organisms growing at a
temperature of 50° 0., no thermophiles could be demonstrated.

In this work some of the pasteurized samples were
placed at 57° C. for incubation to determine the presence
of the organisms that survived pasteurization and develop at
57° C. In some instances no fermentation took place at the
end of 24 to 48 hours, and some showed the presence of
lactose-splitting organisms causing stormy fermentation with
the evolution of gas. as far as it could be determined from
the type of fermentation, the organisms causing it belong to
the sporogenes group.

The data shows that there was a noticeable decrease in
the bacterial count under pH values 7.4 and 6.6. This at
first was thought to be due to the germicidal effect of
the alkali or the acid added, but it was found that it was
not the case. Plates were made before pasteurization of
the samples adjusted to the above pd values and the count
showed a greater number of colonies deveIOping in 48 hours
of incubation than the original sample at the original pH.
This increase may be attributed to the shaking of the
sample as the alkali or the acid was added, in which the
bacterial clumps were broken thereby giving a higher
bacterial count. Inasmuch as milk is coagulated at pH 5.6
(undiluted) when heated, it is possible then that at pH
6.4 (pH 5.96 undiluted) there is not enough acid present
to destroy the organisms present in the milk. It is a

well known fact that most of the organisms in milk survive

the acidity produced by Bact. lactis acidi which is enough
to coagulate the milk without heating. This source of error
may then be neglected.

Another source of error may arise in deciding whether
the low count on pH 7.4 and pH 6.6 is due to the alkali
agglutination or acid agglutination. The alkaline agglu-
tination may be eliminated since the pH 7.4 taken after di-
luting the milk to 100 volumes, assuming that the error in
the dilution, is about 0.56 of a pH according to Sharp and
MoInerney (21), would mean that the actual pH of the undi-
luted milk is only 6.86 which lies within the pH values of
normal milks as obtained by various investigators as shown
in Table II. Bacteria also agglutinate at certain points
of acidity in a medium. Szent-Gygrgi (15) place the isoelec-
trio point of agglutinins between pd 5.69 and 5.59. These
values have a greater i—ion concentration than that of pd
6.6 (pH 6.04 undiluted) or pH 6.4 (pH 5.86 undiluted).
Therefore, while there is no agglutination there is a
probable tendency for agglutination.

By examining the curve on the percentage of reduction
it is evident that there is a steady increase in the
efficiency of the pasteurizer and a subsequent decrease of
the bacterial count as the H-ion concentration of the milk
increases except at pH 7.4. The difference in the per-
centage of reduction between the different pd values is
very small and almost insignificant, but this difference

even in so small a fraction may be enough to determine the

-29..

acceptability of the milk at dairy plants. according to
Rice and.Markley (18), milk with higher acidity contains
more of all nutrients and is particularly high in phosphates
and casein. They concluded, "High acid milks under the same
condition of storage, reach the coagulating point later

than milk of low acidity". For the same reason, milks
during the summer months may owe part of their keeping
quality to high acidity and that bacteria which otherwise
survive the pasteurizing temperature may be destroyed.
Therefore, the high acidity of the milk may serve as a

safety factor.

-50..

s mum RY

Pasteurization does not have any appreciable
effect on the H-ion concentration of milk. That there
is no evidence of appreciable increase in the H-ion con-
centration does not mean that there is no acid formed
from the sugar in the milk. There is probably some in-
crease in acidity due to the lactose being broken down
but the buffer action prevents the change, but the
colorimetric determinations using the comparator block
is not delicate enough to show the change.

Carbon dioxide is driven off during pasteurization,
but is a very small factor so that its absence does not
have any apereciable effect on the pH value of the milk.
As shown in Table III, a decrease of ten per cent in the
carbon dioxide content of the milk after heating does not
change the pH value of the milk.

Milk stored for four days at a temperature of 4° - 6°
C. does not show enough change in the H-ion concentration
to be of any practical experimental value. The fact that
cold hinders to a certain degree the growth of bacteria,
accounts for the very small changes in the pH values of
the samples of milk.

Diluting milk to 100 volumes does not show any
appreciable change in pH from that diluted 20 volumes,
provided the values lie between the pH 6.56 and 6.06
true acidity, or pH 7.2 to pH 6.6 diluted to 100 volumes.

With any further decrease or increase in the H-ion con,
centration beyond these points, the indicators used begin
to show slight deviations and as the acidity or alkalinity
increases the greater is the deviation from the normal pH
of the milk. For testing the pH of normal milk, it is
possible to dilute the milk to 100 volumes with neutral
distilled water and with the use of correction factors
like those given by Sharp and McInerney (21), one is able
to determine the approximate pH of fresh milks.

The bacterial count on the different pH values of
increasing acidity shows a very marked decrease as the
acidity increases if the normal pH is taken as a basis
for comparison. A large percentage of the samples show
this decrease while in a few samples in which Spore
forming bacteria preuominate, the count does not show a
marked decrease and the efficiency of the pasteurizer is
low. The acid or the alkali added does not show any
germicidal effect on the bacterial content even to pH 7.4
and 6.4, diluted. Instead, the count increases due to the
breaking up of the clumps of bacteria during the shaking
of the samples as the acid of the alkali is added.

The percentage of reduction of bacteria in the
samples with increasing acidity is very small when com-
pared to the per cent reduction of the milk sample at its
normal pH after pasteurization. However, this small per-
centage of reduction in the bacterial count due to acidity

may be considered as a safety factor eSpecially during the

.- 52 -

summer months when the bacterial content of milk is at

its peak.

1.

2.

5.

CONCLUSIONS.

According to the results obtained from the eXperiment,
milks having a H-ion concentration higher than the
normal, have a lower bacterial count than the normal
after pasteurization at 62.5° C. for 50 minutes.

On the average, the per cent decrease of the bacterial
count on the adjusted pH values is very small as shown
by the graph. However, this small decrease may be an
important factor during the summer months when the
bacterial counts on milks are high.

The increase in the efficiency of the pasteurizer, as
far as the adjusted pH values are concerned, is not
enough to warrant any practicable application of it in
a big scale inasmuch as the temperature used in pasteur-
ization can still be maintained higher thereby increas-
ing the efficiency of the pasteurizer with but a slight

change in the constituents of the milk.

l
()3
()3
I

ACKNOWLEDGHLNTS.

The writer wishes to eXpress his sincere gratitude
to Dr. Ward Giltner, Dean of Veterinary Division, to
Dr. E. Devereux, Research Assistant and assistant
Professor of Bacteriology, to Mr. W. L. Hallmann, Re-
search Assistant.and assistant Professor of Bacterio-
logy, and to Mr. F. W. Fabian, Research.Associate and
Associate Professor of Bacteriology, for their guidance,
valuable criticisms, and suggestions which made this
work possible.

The writer also wishes to eXpress his indebtedness
to Mr. P. H. lucas, Professor of Dairy Manufactures, for
his cOOperation, and permission to the writer to obtain

from the college Dairy,the samples used in this experi-

mental work.

e1.

2.

4.

5.

6.

8.

9.

10.

1
(>1
.9.
I

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, Law“. a
i .9