mu ROLE OF LBUCOCYTII
IN THI. SBDIMENT
POflMATION IN HOMOOINIZBD MILK

The“: fat tho Decree of M. !.
MICHIGAN STATE COLLEGE

Isaac I. Peters

I944

 

 

 

 

 

'fi‘hu NA .

 

 

This is to certify that the

thesis entitled

"The Role of Leucocytes in the Sediment
Formation in Homogenized Milk"

presented by

Mr. Isaac I.Peters

has been accepted towards fulfihnent
of the requirements for

Master'sdemwe urtnirz Husbandry

% >77. (Zen/4

Major professor

Date June 2nd, 1944.

 

 

 

THE ROLE OF LEUOOCYTES IN THE SEDIMENT FORMATION

IN EMOGENIZED MILK

THE ROLE OFULEUOOCYTES IN THE SEDIMENT FORMATION

IN HDMOGENIZED MILK

by

ISAAC I. germs

A.THESIS

Submitted to the Graduate School of Michigan State
College of Agriculture and Applied Science in
partial fulfilment of the requirements for
the degree of
MASTER OF SCIENCE

Department of Dairy Husbandry

1944

ACKNOWLEDGMENTS

The writer wishes to express his sincere appreciation to Dr. Earl
Weaver, Head of the Dairy Department, for.making this study possible;
and to Dr. G. M. Trout for his kindness in planning and directing this
investigation, as well as guiding the preparation of the manuscript.

Gratitude is also expressed to Dr. I. A. Gould for his many timely
suggestions in certain phases of this work, and to Dr. R. an Langham for
making the micrOphotographs.

The author wishes to thank Prof. P. S. Lucas for the use of the
crewmary facilities in perfonming the exPerimental work, and.Mr. D. M.
Hendrickson for his splendid soaperation in securing the various samples

required in this study.

I ,"—.1 ‘3' If‘ 0“,

~~v * .s ' ‘~ .
2. r‘ (\p; ““3? (J a

per.

TABLE OF CONTENTS

moweTIoN O’COCOOOOOOOOOOO0.0.0.0.0...0.00.00.00.00.0.00.00.00...

Rmm 0F LIW .00.00.0.0...OOOOOOOOOOOOOOOOOOOOO...0.00.00...

Reasons for the occurrence of sediment ..........................

Factors affecting sediment formation ............................

1.
2.
3.
4.
5.
6.

7.

Cellular constituents of milk ............................
Stability of‘ milk solids ................................
Foreign particles ........................................
Effect of heat on sedimentation ..........................
Effect of pressure on sediment formation .................
Effect of agitation on sediment formation ................

Effect of storage temperature on sediment formation ......

&urce8 or sediment OOOOOIOOOOOOOOOOOO..0...OOOOOOOOOOOOOOOOOOOOO

l.
2.

3.

Cell constituents entering milk during secretion .........
Denatured.milk solids ....................................

Farsi-y particles .00....00....OOOOOOOOOOOOOOO0.00.0000...

contml measures 00.00.000.000.00.000.000.00.0000000000000000000.

1.
2.
3.
4.

5.

Selection of’milk ........................................
Filtration ...............................................
Clarification ............................................
Protein stability ........................................

Storage temperature and agitation ........................

WM PmCEwRE OOOOOOOOOOOOOOOOOOOOOO0.00000000000000000000.

&ur300fm11k .0...0..00......COO...OOOOOOOO’OOOOOOOO0.0.00.0...

page

10
10
11

11

“Oth 0f handling .OOOOOOOOOOOOCOOOOOOOOOOOOOOO0.0000000000000000

&“60 or leucocytea 0.00.00.00.00...QOOOOOOOOOOOOOOOOOO0.0.90....

lethod of adding leucocytes ......................................

“ensuring the various layers .OOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOO.

Examination for intensity of sediment ............................

Method of obtaining various portions of the liquid ...............

momscopic examinations OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0...

Photographic preparations 000......OOOOOOOOOOOOOOOOOOOO00.0.0.0...

Treatment or data 0.00.00...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOO

MWAL BELTS 0.00.00.00.00.0000000000000000000.0.00.00.00.00.

1.

2.

3.

4.

5.

6.

7.

8.

9a.

9b.

Effect on the migration of leucocytes when various amounts
separator slime were added to nonhomogenized and to homo-
80111296. milk 0.00.0000...0.00.00...OOOOOOOOOOOOOOOOOOOOOOOO

Effect of adding various amounts of washed leucocytes to
fresh pasteurized milk on the creaming of the milk ........

Effect of reversing the electrical charge on the fat
globules upon the migration of washed leucocytes ..........

Effect of pasteurization and homogenization temperatures
upon the number and distribution of leucocytes in milk ....

Effect of homogenizing before or after pasteurization on
the number and distribution of leucocytee in milk .........

Effect of delivery-route agitation on the formation of
sediment in homogenized milk oeoeooooooeeeoooooeoooeooeoeco

Effect of normal homogenization pressure on the leucocyte
count or milk .000.000.000.00.IOOOOOOOOOOOOOOOOOC0.00......

Effect of repeated highspressure homogenization on the
1811000310 count or Milk OOOQOOOOOOOOQOOOOIOOOOCOOOOCOIOOOO.

Effect of continuous highkpressure homogenization on the
leucocyte Count or milk .OOOOIOOOOOOIOOOOOI0000.00.00.00...

Effect on sedimentation by adding increasing portions of
continuous high-pressure-homogenized.milk to normal
mogenizOd milk .OOOOOOOOOQOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO.

P886
11
12
12
12
13
13
14
14
15

16

16

24

56

45

54

61

73

76

P389

10. Effect of temperature of clarification on the intensity
of sediment in homogenized milk eeoeoeooeooeeeeeeoeeeoeeet 82

11. Effect of clarification at different temperatures and at
different stages of processing on the weight of dry
matter refiloved COO...OOOOOOOOOOOOOOOOOOOOOOOO00.00.0000... 86
12. llicroscopic observations of sediment in homogenized milk . 92
NM$ION 0.0.0.0...OOOOOOOOOOOOOOOQOOOOOOO..00....0.0000000000000. 99

mm.COOOCOOOOOOOOOOOOO00.000.090.000...OOOOOOOOOOOOOOOOOOOOOOOO 102

”W CITE 0.0.0....OOOOOOOOOOOOOOOO0.00000000000000COOOOOOOOC 104

 

 

 

' I III." 'r 1 [.u In
J

INTRONCTION

In many instances the practice of homogenizing milk for fluid milk
consumption has created the problsn.of sediment formation in the bottled
product. This sediment appears often as a fine ring of silt or sediment
at the bottom of the bottle, varying in intensity and color.

While the grayish and grayishryeIIOW'colors are perhaps the most COMP
non ones, other colors such as whitish,‘brounish and reddish may be ob-
eerved, depending on the main source of the sedhnent. Although this sedi-
ment nay not lower the food value of the Iilk or impair the healthfulness
of the product, from the esthetic point of view it is an important defect
in the eyes of the consumer. The distributor is, therefore, confronted
with the difficult problen.of preventing or removing the suspended particles
in the halogenized milk, which upon storage settle down and fan the sedi-
neut.

Both, the selection of clean.nilk, according to the standards of the
sediment test, and the filtration of nilfprior to homogenization are not
reliable:neasures to prevent sedhuent formation in every case. Power clari-
fication, however, before or after homogenization'lill remove most of the
suspended particles and thus render the milk sedhuent free.

Since the results of previous workers have shown that leuoocytee,
epithelial cells and cell debris make up a large part of the sedhnent in
nearly every case, a study was undertaken to investigate more closely the
behavior of leucocytes in.nilk. The probleu.vas attacked from various angles,
all Iith.the purpose in mind, that after a more complete understanding of the
behavior of the cells had been obtained, a shnple and effective remedy'night

be suggested.

REVIEW OF LITERATURE

mm; f9; the comes of sedm . The main reason for the occurrence
of sediment in homogenized milk scan to be due indirectly to the inhibited

er retarded fat arising in the processed milk. Homogenization decreases the
size of the fat globules from 2.25.11.0 microns to about 1.2 microns, using
2,000 pounds pressure (Tracy, 1936). This in turn increases the number of
fat globules and their total surface area. Each fat globule before and
after honogenization is surrounded by a casein membrane (Titus et al, 1928).
Iiegner (1914) found that the thickness of the absorbed nubrane was the
same both in the fat of nonhonogenized and homogenized milk. He showed that
about 2.27 per cent of the total casein present in milk would surround the
fat globules in nonnal milk, .while in homogenized nilk 25.2 per cent of the
total casein would surround the fat globules. The increased proportion of
casein to fat increases the specific gravity of the fatdwhich coupled with
the reduced size of the fat globule prevents thu from rising, thus allow-
ing for an even and fairly stable distribution of the fat. According to
Breed (1912) and Wilcox (1912) leucocytes will adhere to the rising fat
globules. Bechhcld (1929) attributed this preperty to the Opposite elec-
tric charges of the leucocytee and the fat globules. If no fat rising
takes place, then the leucocytes due to their higher specific gravity (Bab-
cock, 1934a, 1959, 1940) will settle to the bottom carrying some fat with
tho. Dean (1938) stated: 'chogenization tends to agglutinate particles
of fine dirt, bacteria and cellular elements of milk, and to cause then to

settle."

WW- Several factors have been found to

affect sedimentation in homogenized milk. Since they vary widely they are
being discussed separately. '

l. W Dean (1938) attributed the amount
of sediment produced in homogenized milk primarily to the number of cells
present in the milk. Tracy (1935) also considered bo:y cells as a factor
affecting sediment formation. Babcock (1940) claimed that if milk contained
fewer than 100,000 leucocytes per cc. of milk no trouble with sediment would
be encountered.

Plastridge et al (1939) found that out of 2,125 samples of milk fm
health cows, 80.2 per cent contained less than 100,000 leucocytes per cc.
with an average count of 75,000. 0n the other hand, in milk containing non-
huclytic staphylococci the average count was 220,000 leucocytes per cc. with
only 50 per cent of the samples below 100,000 leucocytes per cc.

Becker (1942) analyzed 30,331 samples of milk from 8,000 cows and found
that 68 per cent of the samples contained less than 500,000 leucocytes per
cc. and 10 per cent more than 1,000,000 per cc. Ninety-eight per cent ‘of
the cows yielding milk with counts over 500,000 per cc. showed the presence
of streptococci and 92 per cent of cows yielding milk with the same counts
gave positive reaction with Bram Thymol Blue. Baker and Breed (1920) found
that "decreasing ludrogen ion concentration of fresh milk bears a relation-
ship to increase of number of leucocytes.” Other workers, Burr et a1 (1908),
Rolling (1910), Boyberg (1911), and Varrier-J'ones and Camb (1924) all showed
that pathological conditions of the udder resulted in increased leucocyte
counts. Thus the use of milk from infected udders apparently would contain
counts above 100,000 leucocytes per cc. and according to Babcock (1940) sedi-

ment formation would take place. ‘

-4-

2. WM Dean (1929) stated that homogenization
destabilized proteins in the presence of fat. This effect increased with

fat concentrations and with efficiency of homogenization. He claimed that
part of the loss of protein stability was due to the increase in hydrogen-
ion concentration during homogenization. A lowering of pH by 0.0445 was ob-
served by homogenizing pasteurized milk at 2500 pounds pressure. Tracy (1935)
attributed unstable proteins as partial cause of sediment formation. Rowland
(1933) has shown that pasteurization will denature and coagulate some protein.

Charles and Boner (1934, 1935) rcported the white sediment in milk to
be coagulated milk solids. The addition of sodium and calcim salts, as
shown by Charles (193i) and Hahn and Tracy (1940) would affect the stability
of proteins, the addition of calcium resulting in a less stable protein.
Tracy (1941) stated that ”by destabilizing the protein by the addition of
calcium chloride it is possible to increase the degree of sedimentation,

”and by stabilizing the protein with sodium citrate the amount of sediment
can be reduced.‘

According to Bodansky (1934) proteins obtain their stability from
their electric charge and the water of hydration. They are amphoteric in
nature and thus may be either basic or acidic. At their isoelectric point
which is pH 4.6 for casein, (Rogers, 1935), pH 4.? for lactalbumin and pH
5.5 for globulin (Bechhold, 1929) they are only stabilized by the water of
indication. If water of hydration is removed by heat, coagulation of the
micelles will take place (Bodansky, 1934). Salts affect the electric charge
of the micelles and thus in turn affect the stability of the protein (Bech-
hold 1929). According to Davies (1936) the rucval of the electric charge
will result in agglutination, while dehydration will cause coagulation of

the protein. Sosmler (1938) claimed that while lactalbumin and lactoglobulin

retain their stability at the isoelectric point, casein was not sufficiently
twdrolyzed to prevent agglutination taking place.

3. 29:2ng pagicle . Trout and Halloran (1933) found that part of
the sediment was made up of very fine dirt which could not be moved by
ordinary filtering. Charles and Sommer (1934) reported that the gray color
of the sediment was due to foreign dirt particles. According to Dean (1938,
1940) agglutination would facilitate settling, thus producing a grey sedi-
ment. The foreign particles resuble leucocytes in their behavior in milk
in that they are carried up with the rising fat, and settle if no fat rising
action takes place.

4. Efegt 9; heat pp gagimentatipg. As pointed out earlier, heat has
a destabilizing effect on the milk proteins since it tends to remove the water
of hydration, thus causing coagulation. According to Bechhold (1929) heat
changes milk protein from hydrcphilic colloids to hydrOphobic colloids. 'Bo-
dansky (1934) mentioned heat as one of the factors responsible for denature-
tion of proteins. He claimed that denaturation of proteins might take place
at hydrogen ion concentrations far runovcd from their iscelcctric point. Pre-
cipitation or flocculation, however, occurred at the isoelectric point. Bow-
land (1933) found that holding milk at 63° 0. for 30 minutes resulted in the
coagulation of 10.4 per cent of the lactalbumin. He observed that, "for each
rise of 1° 0. between 63° c. and 75° 0. the rate in increase of denaturation
was constant, the tuperaturs coefficient of the reaction being 1.5." Davies
(1936) also mentioned the fact that precipitation of albumin and globulin by
heat favors molecular association of these proteins. This would tend to in-
crease sedimentation, since the larger particles would be less stable.

5. W The decree of pressure
to which milk is subjected in the process of homogenization also scans to

affect sediment fomation. Bauer (1938) stated that a pressure of 2500
pounds would increase the temperature of milk from 5 to 10° F. This would
tend to increase the coagulation of proteins if homogenization were carried
out at pasteurizing tuperature.

A lowering in pfi'of milk by 0.0445 as a result of homogenization was
observed by Doan (1929). Dahle et a1 (1930) found that as the homogenization
pressure increased the pH of the ice cream nix decreased. A lowering of the
pH increases the twdrogen ion concentration, thus reducing the electric charge
on the protein molecules and rendering thu less stable. Bonner (1938) ex-
plained the increase in hydrogen ion concentration to be due to the precipi-
tation of tricalcim phosphate thus liberating free hydrogen ions. However,
Istaz and von Soest (1907) could not observe an increase in titratable acid-
ity after homogenization.

Dean (1938) found that piston-type homogenization gave greater deposits
of sediment than did the Bump Rotary type when run at the seas pressure.
Higher pressures increased the sediment content due to the greater destabiliz-
ing effect on the proteins. The most important function of the homogenizer,
however, is the shearing. action of the pressure valve which reduces the size
of the fat globules, together with the increased proportion of the total
casein adsorbed at the fat surface, tends to stabilize the fat, as was non-
tioned previously. Tracy (1941) reported that with increasing pressure the
percentage of nitrogen increased which he believed was due to the destabiliz-
ation of proteins.

6. W The workoraahnand
Tracy (1941) showed that agitation of homogenized milk, especially at high
tuperatures tended to speed up or increase sedinentation. Tracy (1941)

claimed that agitation would cause clmping of cells, and thus facilitate

settling.

Trout et a1

 

(1935) observed that heat shocking of homogenized milk seemed to favor sedi-
mentation. Tracy (1935) reported that sediment was more likely to fans when
milk was warned. Hahn and Tracy (1940) also found that increased tempera-
tures after bottling would increase sedimentation. Tracy (1941) pointed out
that storage temperatures of 40° 1". were more desirable than higher tapere-
tures so far as freedone of sedimentation was concerned. Samar (1938)
stated that low tuperatures allowed for fat clumping and an increase in
viscosity of the milk by the swelling or hydration of proteins, which would
sea in the case of homogenized milk to tend to inhibit sedimentation in
part at least.

W. According to the results of sediment analyses made by
various workers the sources of sediment may be divided into the following
three main groups, each of which will be dealt with separately.

1- WWW run as drawn
from the udder of the cow contains various numbers of leucocytes, as shown
by Russell and Hoffman (1907, 1908), Cupbell (1909), Plastridge et a1
(1939), and Husker (1942). According to Babcock (1934a, 1934b, 1934c, 1939,
1940) large numbers of leucocytes are present in the sediment of bottled
homogenized mu. Tracy (1941) and Trout (1942) also reported the presence
of leucocytes in the sediment. Baker and Bread (1980) found that milk with
a high leucocyte content showed also an invariably high number of epithelial
cells and cell debris. Brudny (1914) mentioned the fact that large nmbers
of epithelial cells may be present in normal milk. Both Babcock (1934a, 1934b,
1939, 1940) and Tracy (1941) reported the presence of epithelial cells in the
sediment. Tracy (1941) also included cell debris and erythrocytes as occa-

sional constituents of the sediment. Brudny (1914) stated that the presence

of erythrocytes could be detected by the reddish color of the sediment,
while leucocytes and epithelial cells will give a yellowish sediment. Thus,
leucocytes, erythrocytes, epithelial cells and cell debris may be regarded

as sediment constituents entering milk during secretion.

2. W. The work of Rowland (1933) showed that
pasteurization would coagulate some of the albumin. Trent and Halloren
(1933) and Trout et a1 (1935) analyzed sediment by the Hojonnier method
and found that it contained fat and solids-not-fat. Charles (1934) raported
that the white sediment was made up of casein. Charles and Sommer (1935)
found the presence of "substances related to milk solids" in the sediment
of hasogenized milk. Tracy (1935, 1941) attributed some of the sediment to
unstable proteins or particles of milk solids. ‘

3. W Foreign particles include any dust, spores or
other insoluble particles entering the milk from the time it is drawn until
bottling, which cannot be removed by straining or filtering. Brudny (1914)
mentioned that dirty milk would give a grey sediment. Trout and Halloran
(1933) found very fine dirt to be a partial cause for sediment. Similar
results were obtained by Dean (1938, 1940). Charles and Sonar (1934,, 1935)
concluded that grey-colored sediment in homogenized milk was due to dirt
entering milk during handling on the farm.

W The following control measures have been suggested by
various workers:

1. W. The selection of milk appeared to be the first
step for controlling sediment in the processed product. This was advocated
by Jones (1929). Trout and Balloren (1933) advised clean milk production,
especially in dry weather. Babcock (1934b) suggested selection of low-cell

count milk in order to prevent sediment fonation by leucocytes. The re-

sults of'many workers, previously listed, showi.that mastitic milk contains
large numbers of blood cells, which invariably would show up in form of
sediment.

2. 1111235122., Trials conducted.by Dahlberg and.larquardt (1924)in
which they compared the removal of cells by filtration and clarification,
showed that filtration removed about 28 per cent of the total number, while
clarification lowered the count by 66 per cent. Trout (1933) showed that
the sediment test did not serve as a reliable guide in the selection of'milk
for homogenization. lilk with sediment scores of 9.3 would show dark sedi-
ment, while milk with a sediment score of 8.3 would show less sediment hav-
ing a light yellowish color. Other experiments conducted (Trout 1934) with
cream showed that filtration did not have as great an effect on removal of
sediment as did clarification.

3. W The most common measure used to prevent sediment
formation is by the use of clarifiers. lclncrney (191'?) reported that about
99 per cent of the insoluble dirt in milk is moved by clarification. Ac0
cording to»flieder (1936) clarification reduced the cell count of’milk by
67.53 per’cent. Trent and Bhlloran (1933) recommended power clarification.
Bebcock (1934b) suggested the same method. Hood and White (1934) advised
power clarification at 40° I. which will eliminate entirely or reduce to a
negligible quantity sednnent deposits in.homogenized milk. Opinions as to
the time and temperature for clarification vary; Babcock (1934b, 1939, 1940)
reconended: clarifying, homogenizing, pasteurizing, while Charles and Summer
(1934), Tracy (1935), Hahn and Tracy (1940) advise clarification after he-
mogenization.

With respect to clarifying.temperature, Hood and White (1934) found

that 40° 1'. would give satisfactory results. Charles and Sommer (1934)

-10-

recs-aided clarification after homogenization while the milk was still hot.
Marshall and Hood (1918) found that using 55° 3., 75° 3'. and 100° 1'. as clari-
fying tanperatures the dry weight of slime increased with increase in tu—
perature. Using 900 1., 110° 1"., 125° 1. and 140° 1'. the same trend was ob-
served. J’acobsen and Olson (1931) found also an increase in reduction of
cells with an increase in clarifying temperatures. Hammer (1916) in his
studies on the clarification of milk, stated that, "the percentage of cells
eliminated appeared to bear no relation to the original number of cells
present, the tunperature of the milk or the percentage of fat.” By using
the Dean-Buckley method he found on 52 samples of milk the percentage of
cells eliminated to vary from 7 to '73 per cent with an average of 39 per
cent. Ha-er and Hauser (19l8) obtained similar results. Work done at the
Idaho Station (1926) showed that the clarification temperature seemed to
have no effect on the amount of visible dirt moved.

4. W Doan and linster (1930) recommended the method
of double homogenization or the use of a two-stage valve in order to in-
crease the protein stability. They claimed that double homogenization would
increase the pH slightly as capared with the effect of single-stage ho-
mogenization. Charles (1934) found that by the addition of sodium citrate
the white sediment could be eliminated. Hahn and Tracy (1940) observed the
same results. ‘

Hahn and Tracy (1940) found

 

that holding bottled homogenized milk at tmnperatures between 40450 F.
would help to prevent sediment formation. The same authors found that agi-
tation aould cause clumping of suspended particles and thus facilitate sedi-

ment formation.

EXPERIEENTAL PROCW

The outline of the general procedure used in performing the experi-
mental work in this particular research problem was as described below.
Special mention is made in the particular emperiments of any changes in
procedure and the reasons for tho.

W All milk used in this work was obtained from the
College Creamery. The homogenized milk, both regular (3.8$ fat) and ”Vita-
min D" (4.5% fat) {33-3 from the college herd where machine milking was x
practiced, and a. high degree of cleanliness was observed. The pasteurized
milk was a mixed milk from regular College Creamery shippers.

W Hill: used for‘pasteurizaticn was filtered at the
incoming temperature which ranged from so to 60° 1'. A rotary pulp was used
to force the milk through the a von Gonton filter on its upward noveuant to
the pasteurizing vat. alall lots were gravity filtered, using a regular
milk filter cloth.

The milk to be used for homogenization purposes was clarified at the
receiving tmnperature, using a DeLaval power clarifier. For clarifying
all lots of milk a laboratory size, DeLaval power clarifier was used.

All milk was pasteurized at 142° to 144° F. for 30 minutes and cooled to
40° 1'. over a sweetwater surface cooler.

Homogenization was performed by either of two Union Steam Pup, Duo-
Visco valve viscolizers. This was done after pasteurization and prior to
cooling, at a pressure of between 2000 and 2500 pounds per square inch.
Both units were of sanitary construction, were washed regularly after each
day's use, and kept in good working condition.

The milk was bottled directly after leaving the cooler, into clean

sterile quart bottles and stored at 40° 1'. usually for 48 hours before

~12-

emaminations of any kind were made.

W The leucocytes were obtained from the separator
or clarifier slime at the College Creamery and were used either in the form
of slime, or as washed leucocytes. Care was taken to collect and use only
that portion of slime which resmnbled the leucocytes in color, omitting the
reddish deposits close to the interior wall of the separator or clarifier
bowl.

In preparing the washed leucocytes, a method similar to that suggested
by Schuppius (1907) and used by Strynadka and Thornton (1938) was uployed.
The fresh slime was suspended in a physiological salt solution (8.5 gms. of
HaCl in one liter of distilled water), centrifuged in a high-speed centri-
fuge, and the process repeated until the liquid remained fairly clear and
the sediment showed a light greyi sh or grayish-yellow color. Iicroscopic
observations of such preparations showed leucocytes only, with no other par-
ticles present. This sediment contained approximately one-hundred million
leucocytes per milliliter.

W In both cases, whether slime or washed
leucocytes were used, portions were weighed out into clean beakers and milk
was added to produce a suspension. A spatula or rubber policnan was used
to break up any clamps, and in addition, if it were felt necessary, the
mixture was passed through a hand homogenizer to insure homogeneity. The
so-prepered suspension was added to the respective portions of milk and
mixed well before any samples were taken. The bottled product was shaken
25 times before sampling as in making a bacteriological examination.

W In order to determine the volume in the
layers produced upon the storage of milk a cardboard measuring ruler was

made for each of the quart and the pint bottles. The cardboard was cut to

fit the shape of the bottle, marked in inches to i” divisions. By filling
an mapty bottle with water using a graduated cylinder, the volume in each
division was obtained. This ruled cardboard could thus be placed conven-
iently close to the bottle and readings made without disturbing the con-
tents of the bottle. The volume was then recorded in inches or in milli-
liters.

matign for igtensity of sediment, Sediment examinations were
made after holding the bottles for 48 hours from the time of bottling. The
milk having a taperature of 40° F. was carried into the laboratory room
and examinations were made immediately afterwards by one or two persons be-
fore any changes in temperature occurred. Any shaking or tilting was
guarded against, so as not to disturb any sediment present. The intensity
of sediment was recorded as follows:

0 :: no sediment

slight sediment

distinct sediment

pronounced sediment

l
2
3
4 very pronounced sediment
No higher value than 4 was given in any case, although the amount of sedi-
ment may have been even greater than the standard established for this group.
[93mg of gptainigg £331on portion; of the 1.191119: A suction-siphon
arrangement was mnployad in drawing off the various layers formed, or in re-
moving one-third portions of milk from each bottle. Clean bottles were used
to collect the siphoned liquid. Care was taken not to intermix the individual
layers, or portions, if such danger existed. This was especially necessary
with pasteurized milk in which the upper layer usually carried more leuco-

cytes per milliliter than did the lower portions.

01‘-

ngmggpic examinations. Of the three methods for counting leuco-
cytes as listed by Campbell (1909) and Salus (1912), the volumetric method

using the counting chamber seemed to be the most preferred one. Russell
and Hoffman (1907) no compared the Stokes-Stewart wear-stain method with
the Dean-Buckley volumetric method found that the latter showed almost uni-
formly higher results than the former. They found that ”so far as the tech-
nique is concerned the volumetric method (Donn-Buckley) is more accurate.

It can be made as rapidly as the shear sediment and is less trying on the
eyes.‘ They used the counting chamber method (1908) and so did Campbell
(1908) in his work on leucocytes in milk. Accordingly, the counting
chamber method was employed in this study.

The leucocyte counts were made according to directions recommended
by the Spencer Lens Coupany, using Toisson's Fluid as a stain and Bright-
Line Improved Heubauer Counting Chamber with 0.1 m. depth between the
surface of the counting chamber and the cover glass. White-cell blood :
pipettes were used. Each sample was transferred to the counting chamber,
filling the squares on both sides of the moat. The cells were counted in
the four-corner, 1 mm. squares and in the central ruled area on both sides
of the hamscytometer (10 square mu. in all) thus giving a factor of
20,000. In making counts on sediment, the sample had to be diluted and a
correspondingly larger factor had to be used. Wright's stain was used in
preparing smeared stains and a procedure followed as outlined in the ”Atlas
of Huatology" by Osgood and Ashworth (1937).

W In some instances, where photographs were
desired, suitable adjustments in the method of procedure were made. The
method used by Homer and Hauser (1914) was employed to show the cream layer.

The milk was added to a dry pint bottle, ' containing 10 cc. of a saturated

alcohol (95$) solution of Sudan III. The mixture was then shaken until all
the color was uniformly dispersed. Thus the cream was of a reddish color
and no difficulty was encountered in making the photographs. In photograph-
ing the washed cream in heat-treated whey mixture the light was adjusted
fron behind the bottles, so as to show the clearness or transparency of the
whey. The cream, being less transparent than the whey showed thus a deeper
shade on the picture.

The slides used for nicmphotographs were prepared at the time the
experheent was made, and stained with‘lright's stain, as mentioned previous-
ly.

greatment of 92th. All leucocyte counts of two or>nore trials were
treated logarithmically. The figures are based on the logarithmical aver-
age values shown in the tables. In some cases the data of one trial are
shown only, although additional trials had been run, showing the sale trend

of results.

-15-

 

n12; gm were gddgg to goghomogenizeg and to homogenized gig,
The purpose of this experiment was to show the migration of leuco-

cytes in nonhomogenized and in homogenized milk. By adding known weights
and numbers of leucocytes to each quart bottle it was felt that the limit
of the “carrying capacity” of leucocytes in the homogenized milk could be
found. The nonhomogenized milk was used as a control, showing the behavior
of leucocyte migration when the fat globules were not reduced in size.

Five trials were conducted, using fresh clarified, pasteurized, non-
homogenized and regular homogenized milk. Fresh separator slime was added
at the rate of 0.0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 grams per quart to
quart bottles of nonhomogenized and homogenized milk. The bottles were held
for 48 hours at 40° 1., after which time examination for sediment and leuco-
cyte counts of the upper, middle and lower one-third portions of each bottle
were made.

for each gram of fresh separator slime added per quart of milk, an in-
crease of approximately 800,000 leucocytes per ml. was observed in both the
nonhmogenized and in the homogenized samples. This is shown in table 1,
column 1. The distribution of leucocytes after storage, in numbers per
millimeter and in per cent of total is shown in table 1 and figure 1 re-
spectively. The data show that while in the nonhomogenized milk from '78 to
98.4 per cent of the total number of leucocytes were carried in the upper
one-third portion of the bottle, the same portion in the homogenized milk
contained only from 30.8 to 11.7 per cent of the total leucocytes per bottle.

The homegeni zed milk, however, showed large increasing numbers in the lower

-17-

one-third portions, with greater additions of slime per bottle. Thus,
upon the addition of 5 as. of slime per quart 70.4 per cent of the total
leucocytes, or over 8,000,000 per ml. were found in that portion.

The difference in the distribution of leucocytes in the bottles to
which no slime was added is attributed partially to the low initial count
and partially to the fapt that clarification had removed the larger leuco-
cytes, thus leaving the smaller, more stable leucocytes in the milk. It
can be seen that there exists a fairly uniform distribution of leucocytes
throughout the bottle in the homogenized milk to which no slime was added.
There was, however, a slight settling and it might be said that the limit
of the "carrying capacity” of leucocytes by this particular milk had been
reached.

Upon the addition of 0.5 an. of separator slime, 59.8 per cent of the
total number of leucocytes were found in the lower one-third portion and
slight sedimentation was observed, as shown in table 2. The same intensity
of sediment, namely 1.4, was not observed in the nonhomogenized milk until
3 gas. of slime were added to a quart of milk. This sediment, however, al-
though given the same intensity rating, showed only a couparative .811
number of leucocytes, namely 73,500 per ml., as compared with 1,334,000 per
ml. in the homogenized milk showing the same intensity of sediment.

The rising of the leucocytes with the rising fat, and the settling in
the absence of fat rising is vividly shown in figure 2. These cclxmns are
based on the average per cent of total leucocytes in each one-third portion
of the bottle after storage, upon the addition from 0.5 to 5.0 as. of slime
per quart of milk. The strong attraction between the fat and the leucocytes
in the nonhomogenized milk is clearly shown. The uniform distribution of fat

in the homogenized milk might be a factor in holding some of the leucocytes

-13-

in suspension, thus a relatively larger number is carried in the upper and
middle portions, as compared with the middle and lower portions of the non-
homogenized milk, resulting in a lower column in the lower third portion of
the homogenized milk. ‘

The intensity of sediment in the homogenized milk increased in direct
preportion to the number of leucocytes present in the lower portion. In
the nonhmogenized milk, however, the leucocyte count in the lower third
portion of the bottle showed no relation to the intensity of sediment pro-
duced, as shown in figure 3. The sediment in the nonhomogenized milk,
therefore, was not due primarily to leucocytes, while the same cannot be

said in the case of the sediment in the homogenized milk.

-19-

Table 1. The distribution of leucocytes in nonhomogenized and pasteurized

homogenized milk (Average of five trials)

 

Separator Leucocytes Leucocytes per ml. after storage at 40U F. for 48

 

 

 

 

 

 

 

 

slime per ml. hours in the
added before Upper one-third Iiddle one-third Lower one-third
mlgm 559mg of bottle 9f bottle 9f bottle
Nonhomoggni ggg
1 of f of i of
total total total
0.0 131,000 328,400 78.0 55,700 13.2 36,700 8.8
0.5 493,000 948,200 89.0 63,500 6.0 53,700 5.0
1.0 967,200 1,658,000 95.9 32,000 1.9 38,900 2.2
1.5 1,374,000 2,214,000 95.1 , 34,900 1.5 79,400 3.4
2.0 1,890,000 3,318,000 96.2 60,800 1.8 68,800 2.0
3.0 2,439,000 5,142,000 97.3 68,400 1.3 737,500 1.4
4.0 2,905,000 6,157,000 96.8 58,500 0.9 142,000 2.3
5.0 4,091,000 8,290,000 98.4 47,000 0.6 91,900 1.0
W '
0.0 260,000 236,400 30.8 241,000 31.4 289,300 37.8
0.5 632,000 424,500 19.0 472,000 21.2 1,334,000 59.8
1.0 980,000 475,800 15.3 691,000 22.2 1,942,000 62.5
1.5 1,402,000 729,500 17.0 885,000 20.6 2,676,000 62.4
2.0 1,813,000 820,000 15.7 1,083,000 20.8 3,308,000 63.5
3.0 2,650,000 1,234,000 16.6 1,383,000 18.6 4,823,000 64.8
4.0 3,580,000 1,191,000 11.7 2,024,000 19.8 6,980,000 68.5
5.0 4,438,000 1,601,000 13.5 1,919,000 16.1 8,380,000 I10.4

Table 2. Sediment in nonhomogenized and in homogenized.milk to which

separator slime had been added. (Average of five trials)

 

Eeparator slhme

 

 

added ntens t of s t‘w h we
W W
0.0 0.0 0.0
0.5 0.2 1.4
1.0 0.6 2.2
1.5 0.8 2.4
2.0 0.8 3.2
3.0 1.4 3.6
4.0 1.6 4.0

5.0 1.4 4.0

/OC7

%
Q

0,
Q

.20

LEUCOCYTES (PERCENT TOTAL!

 

 

K‘UPPER

HOMOGE/V/ZED "'"""'""
NONHOMOGEN/ZED

"‘

’

-,-___-vtiowae

’-- -

\\
\: ............ (M/DDL—E.

\ ‘--

 

 

/ 2 ' 64'

SL/ME ADDED IGMS/OZ'I

Mgnel.

The distribution of leucocytes in the upper,
middle and lower thirds of bottled nonho-
mOgenized and homogenized milk having a wide

variation in the number of leucocytes.

/ 00

k Os 00
Q Q Q

LEUCOCVTES (PERCENT 70m:
N
O

 

WA

HQMOGE/V/ZEC - 701/.
N 0 NHOMO GEN/ZED - -

 

 

 

 

 

 

UPPER M/DDLE LOWEP

TH/PDS 0F. OUAPT 80 TTLE

kigure 2.

The percentege distribution of leucocytes in the
upper, middle and lower thiPCS of bottled non-

homogenized and homOgenized milk.

‘x
Q

HO/UOCEN/ZED —————
NO/VHCMOGEN/ZEO

(h

MEIiT/ ’ ’ I

 

L EUCO CY 7'55 Will. A /0/v5)

 

'0 7 2'” 3 4 5
sum: ADDED (GMSIO 7.":

figure 3. The relationship between sediment and the
number of leucocytee in nonhomOganized end

in homOgenized milk.

 

 

SED/MENT INTENSITY

 

nt e ft

Since previous trials showed the migration of leucocytes with the
fat. it sealed of interest to find whether the fat would follow the leuco-
cytes if sufficiently large amounts of washed leucocytes were added to the
111k. Such a procedure would make possible a closer study of the attrac-
tien between the fat and the leucocytes.

Iresh washed leucocytes were added to pint bottles of fresh pas-
teurised milk in which creaming had not taken place. Increasing amounts
of washed leucocytes were added as shown in table 3. For photographic pur-
poses ten .1. of saturated Sudan III was added to each pint bottle. Each
bottle was shaken twenty-five times and placed in storage at 40° 1'. for
sufficient length of tine to allow for the natural undisturbed migration of
both the fat and the leucocytes. After two weeks time the bottles were
examined for cross: volume, sediment formation, distribution of leucocytes,
and the percentages of fat and total solids in the upper middle and lower
thirds of each pint bottle. The results are shown in tables 3, d, 5 and 6
andinfiguresd. 5,6. 7,8and9.

The addition of washed leucocytes had various effects on the ores-ing
ability of 1111: depending upon the numbers added. The depth of the crean
layer increased with added moments of washed leucocytes until finally both
upper and lower layers were formed. Ihile no sediment was observed upon the
addition of up to 12.5 as. of washed leucocytes per pint of milk, the cream
7011-0 was increased noticeably, as shown in table 3 and figure 9. At this
point the upper third of the bottle contained over one hundred million leu-

cocytes per Il., as shown in table 4, which was 96.8 per cent of the total

- 25'-

number present in the milk. These were associated with 92.8 per cent of
the total fat present.

Upon the addition of 2.5 ans. more of leucocytes the “breaking point"
of the ores-line was reached, the leucocyte count drOpped in the upper layer
to approximately thirty-five million per ml. and continued to decrease in
numbers upon the further addition of increasing weights of leucocytes, as
shown in column 2, table 4. The divided crean.volume was associated with
marked decrease of fat and total solids in the upper layer.

The data in figure 8 show clearly the action of the leucocytes in
general upon the cream volume. Data showing the migration of the fat with
the leucocytes towards the bottom of the bottle are presented in table 5
and figure 5. The correlation befieen the number of leucocytes and the
percentage of fat is shown in figure 6. It is of interest to note that
the fat curve follows the leucocyte curve, although it tends to lag behind
from the “breaking point“ of the leucocyte curve on toward the right. How-
ever, upon.the addition of between 30 and 40 gas. of added leucocytes, the
upper and lower fat curves cross and thus, from the 4.0 gm. addition on,
there was more fat in the lower one-third of the milk than there was in the
upper one-third. From this graph (31g. 6) it can be seen also that the fat
had a strong tendency to remain in the upper part of the bottle, but that
the stronger downward sweeping action of the leucocytes prevented the rising
of the fat. The data show that there:mnst exist a wary strong attraction
betwaen the fat and the leucocytes, otherwise the fat would obey the laws
of gravity and rise to the top.

The date in table 6 and figure '7 show the distribution of total solids
in the milk to which various amounts of washed leucocytes had been added.
The upper and lower total-solid curves follow closely the leucocyte and fat

curves respectively and cross at the addition of between 20 and 25 gas. of

washed leucocytes per pint. A steep slope was observed between 12.5 and 15
as. samples in all three curves, due to the shifting of both the fat and
the leucocytes.

The cream volumes, both on the top and bottom of the bottle showed
a grayish-red color upon the addition of increasing weights of leucocytes.
In general, the color of the 'cream' in the lower layer was of a darker

grayish-red hue than that of the upper layer.

Table 3. The influence of added leucocytes on the crooning ability of milk

 

 

 

feucocytes added Volume n e nt t
a, [pint C a er s ent 1a

0.0 53 412 0

8.5 58 417 0

5.0 56 409 0

7.5 70 405 0

10.0 'M. 401 0
12.5 '77 398 0
15.0 4.5 390 40
20.0 40 390 45
25.0' 35 385 55
30.9 30 390 60

. 45.0 23 355 8‘7

60.0 15 360 100

 

Table 4. The influence of the number of leucocytes in.milk on their

distribution

 

Leucocytes Leucogxtes per*ml, after storage in the

 

Leucocytes per 101. Upper one-third Middle one-third Lower one-third
added before of pint bottle of pint bottle of pint bottle
nt ate a NoL _j£_r NQ.;' _~j£fi, N91; .i!L_
0.0 400,000 1,440,000 71.3 200,000 9.9 880,000 18.8
2.5 9,000,000 10,000,000 94.2 50,000 0.4 920,000 5.4
5.0 20,000,000 44,800,000 93.5 80,000 0.1 5,060,000 8.4
7.5 28,400,000 84,000,000 92.8 180,000 0.2 d,820,000 7.0
10.0 59,600,000 83,200,000 93.5 120,000 0.1 5,660,000 6.4
12.5, 48,000,000 111,200,000 95.8 380,000 0.5 4,520,000 5.9
15.0 56,500,000 34,400,000 25.0 2,1so,000 1.5 101,000,000 75..
20.0 74,500,000 52,200,000 16.8 560,000 0.5 159,200,000 82.9
25.0 101,000,000 51,400,000 15.1 540,000 0.2 175,800,000 84.?
30.0 109,500,000 21,700,000 8.? 280,000 0.1 228,800,000 91.2
45.0 175,000,000 11,200,000 2.2 500,000 0.1 492,000,000 97.?

60.0 220,000,000 6,600,000 1.8 60,000 0.0 544,000,000 98.8

 

Table 5. The influence of the number of leucocytes in milk on the dis-
tribution of the fat after storage (Babcock test)
Distributign 0: fat in the

Upper one-third liddle one-third Lower oneothird
Leucocytee 2f pint bottle of pint bottle of p195 bottl;__
added Fat Gms.of of Fat Gms.of % of Fat Gna.0f i of
W 1 fat tote; fl fgt total 5; {at totgI
0.0 10.1 16.463 93.5 0.3 0.469 2.8 0.4 0.652, 3.7
2.5 10.1 16.463 94.4 0.3 0.489 2.8 0.5 0.489 2.8
5.0 10.1 16.463 92.6 0.4 0.652 3.7 0.4 0.652 3.7
7.5 10.0 16.300 95.2 0.2 0.326, 1.9 0.3 0.489 2.9
10.0 9.7 15.811 91.5 0.5 0.815 4.7 0.4 0.652 3.8
12.5 10.3 16.789 92.8 0.4 0.652 3.6 0.4 0.652 3.6
15.0 7.1 11.573 68.9 0.5 0.815 4.9 2.7 4.401 26.2
20.0 6.7 10.921 63.8 0.4 0.652 3.8 3.4 5.542 32.4
25.0 5.8 9.454 54.2 0.2 0.326 1.9 4.7 7.661 43.9
30.0 5.9 9.617 56.2 0.2 0.326 1.9 4.4 7.172 41.9
45.0 3.5 5.705 34.3 0.2 0.326 2.0 6.5 10.595 63.7
60.0 4.4 7.172 39.6 0.2 0.326 1.8 6.5 10.595 58.6

~—

 

 

 

 

 

 

-29-

 

 

 

Table 6. The influence of the number of leucocytes in milk on the dis-
tribution of the total solids after storage (lojonnier method)
mgtmbguop 2r tgtal solids in thg A A
Upper one-third Middle one-third Lower one-third
Leucocytss nt ott e f int b tt 0 0 nt bottle
added T.S. Gms.of i of T.S. Gms.of % of T.S. Gms.of i of
nt ‘5 '1' S tot T S tot
0.0 18.32 29.8616 49.4 9.26 15.0938 25.0 9.51 15.5013 25.6
2.5 17.86 29.1118 48.6 9.32 15.1916 25.4 9.56 15.5828 26.0
‘ 5.0 19.55 50.9255 49.2 9.55 15.7295 25.2 9.73 15.9414 25.5
7.5 17.69 28.8347 48.6 9.49 15.4687 26.1 9.20 14.9960 25.3
10.0 17.97 29.2911 48.4 9.60 15.6480 25.9 9.52 15.5176 25.7
12.5 17.91 29.1933 48.6 9.29 15.1427 25.2 9.67 15.7621 26.2
15.0 14.52 23.6676 41.1 9.36 15.2568 26.5 11.41 18.5983 32.4
20.0 14.30 23.3090 38.6 9.51 15.5013 25.7 13.22 21.5486 35.7
25.0 13.47 21.9561 36.7 9.18 14.9634 25.0 14.01 22.8363 38.5
30.0 13.19 21.4997 35.4 9.22 15.0286 24.8 14.81 24.1403 39.8
45.0 12.03 19.6089 30.8 9.11 14.8493 23.4 17.85 29.1118 45.8
60.0 11.11 18.1093 28.4 9.05 14. 7515 23.1 18.96 30.9048 48.5

 

 

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Figurea

The creaming of milk to which various amounts of leucocytes had been
added. From left to right respectively additions of 0, 7.5, 15, 30, and

60 gas. of washed leucocytes were made.

 

Figure 9

The influence of certain amormts of added leucocytes on the creaming
ability of milk. From left to right respectively 0, 12.5, 15.0, and 60

gas. of washed leucocytes were added.

-35-

3. t f rs th lect c c the at ob s u
t sh e 000 .

Since fat globules carry a negative charge normally, (ll-sen, 1932)
with its isoelectric point at pH 4.3 (North and Samar, 1935) below which
point they obtain a positive charge, and the leucocytes carry a positive .
charge (Bechhold 1929), it was felt that by lowering the pH below the iso-
electric point of the fat globules, the behavior of the similarly charged
leucocytes toward the fat globules could be observed. If the rising of the
leucocytes with the fat were due only to the sweeping action of the fat,
than the number of leucocytes migrating to the top of the bottle should be
the same in both cases, whether the fat and the leucocytes carried the same
or opposite charges. If, however, the rising was due to attraction of cp-
pcsite charges, then the leucocytes should rise with the negatively charged
fat globules, but settle down, when the fat carried a positive charge.

Pasteurized milk, held for 24 hours at 40° 1'. was tempered to 80° F.
and separated. The cream was diluted with physiological salt solution at
80° 1’. and separated again. This was repeated two times, or until the skill
portion was fairly clear. The ski-111k was heated to 86° 1., rennet added
to coagulate the casein, and the curd cooked, raising the tuperature to
120° 1'. in order to expel most of the whey. The whey was strained and
heated to 100° 1., 50.1.0 to 00° 1., and clarified in order to remove .11
suspended matter.

The whey and washed cream were remixed, washed leucocytes added at the
rate of 0.5 a. per quart and the mixture acidified using N/lficl, taking
samples at decreasing pH values, down to pH 1.5. Pint bottles were used and

milk ranging in pH from 6.5 to 1.5 was stored for 48 hours at 40° 1., after

which time examinations were made as to cream volume, sediment formation,
distribution of leucocytes and so forth. The results are presented in
tables 7’and 8 and in figures 10 to 14 inclusive.

The effect of acidifying the artificial preparation of washed cream
in sweet whey showed an interesting picture in the behavior of both the fat
globules and the leucocytes. At pH 4.3, the approxhnate isoelectric point
of the fat globules, fat rising was most complete, leaving a elear whey be-
low, with no sediment at the bottom (Figure 12).

lith increasing and decreasing pH values the cream volume was less
(Table 7, Figure 12) until at both extremes the volumes were of equal size,
the whey portion showed the same milky color and some white sediment was
found at the bottom.of the bottles.5

The distribution of leucocytes in the upper :middle and lower one-
third portion of each pint bottle is shout in table 8 and figure 10. Above .
the isoelectric point the leucocyte count followed the distribution of the
fat, while at lower pH values, the leucocyte count in the cream layer de-
creased, which seemed to indicate that the fat, although rising toward the
top had failed to carry the leucocytes with it. Data in figure 11 illus-
trates the behavior of the leucocytes with respect to the amount of fat ris-
ing. As more acid was added the positive electric charge on the fat globules
and the leucocytes, which carry a positive charge (Bechhold, 1929) became
more pronounced, preventing the leucocytes to be carried up by the sweeping
action of the rising fat.

The data in figure 11 show that above the isoelectric point of fat the
leucocyte curve excels in slope the cream-volume curve, indicating that the
_leucocytes are carried up proportionally more than the fat. .At pH values be-

,low the isoelectric point, however, the leucocytes are carried up at decreas-

ing proportionally less than the fat, particularly at pH 3.0 or below where
the difference is most pronounced.

While it can be seen from table 7 and figure 12 that at pH 6.0 and pH
3.0, 2.5, 2.0, 1.5 the cream volumes are all the same, namely 4.5 m1., yet
the leucocyte count in the upper one-third protions ranged from 540,000 to
30,000 per ll. Asming that fat rising with clumping had taken place at
both pH 6.0 and 3.0 then it would follow that equal numbers of leucocytes
could be expected in the two cream layers. The only reason for the differ-
ence in the behavior of the leucocytes, or the fat must then be due to the
fact that both carry the same electric charge and thus repel each. other. -
This would tend to explain the decreasing number of leucocytes carried into

the upper portion with decreasing pH values.

Table '7. The influence of pH on fat rising in a washed-cream heat-treated

 

 

 

whey mixture

pH of Green volume 2 . he, 1;... .9 . _owe a. . . L—
m lfliL avg-n of .5;- ntt»:,~ 0 an. 2

6.5 53 milky 1.0

6.0 45 " 1.0

5.0 182 fairly clear 0.0 .

4.5 212 clear ' 0.0

4.0 190 fairly clear 0.0

5.5 55 milky 0.5

3.0 45 " 1.0

2.5 ‘5 " 1.0

2.0 45 "' 1.0

1.5 45 " 1.0

i‘The sediment had a white color.

-39-

 

 

 

 

 

Table 8. The influence of pH on the migration of leucocytes in a washed-
cream, heat-treated whey mixture
on c es 1111 n the
pH of Upper one-third Middle one-third Lower one-third
we of pig}; bottlefl of pint bottle of at bottle
i of 9; of L—i-o?’
total total total
6.3 300,000 15.6 80,000 4.2 1,340,000 80.2
6.0 340,000 25.5 100,000 4.7 1,480,000 69.8
5.0 1,420,000 83.3 200,000 11.7 80,000 4.8
4.3 1,860,000 88.5 200,000 9.5 40,000 2.0
4.0 1,100,000 80.9 120,000 8.8 140,000 10.3
3.5 260,000 43.3 40,000 6.7 300,000 30.0
3.0 20,000 1.3 40,000 2.6 1,480,000 96.1
2.3 20,000 1.3 20,000 1.3 1,300,000 97.4
2.0 80,000 4.9 40,000 2.4 1,520,000 92.7
1.3 40,000 2.4 10,000 0.6 1,600,000 97.0

 

 

 

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LEUCOC‘VTES (M/LL/ONSJ

 

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CREAM VOLUME /N MLS.

 

Figure 12

Pint bottles of washed-cream, heat-treated whey mixtures, having pH values
from left to right of 6.3, 6.0, 3.0, 4.3, 3.3, 2.3 and 1.3 respectively.
Note the cream volume in each bottle and also the clearness of the whey in

the acuple 4, having a pH of 4.3, the isoelectric point of fat.

 

Figure 13

Pint bottles of washed-cream, heat-treated whey mixtures having pH values
from left to right of 6.0, 3.0, 4.3, 4.0, and 3.0.
Note the decrease of crean.volume in the samples having pH values above

and below that at the isoelectric point and also the clearness of whey

in the lower portions.

 

Figure 14

Pint bottles of washed-cream, heat-treated whey mixtures ranging in pH
from 6.5 to 2.3.

From left to right:

(1) pH 6.5 - normal range. Fat carries a negative charge.
(2) pH 4.3 - isoelectric point of fat. No charge.

(3) pH 2.5 - high-acid range. rat carries a positive charge.

Note the cream volumes and the similarity of opacity of the lower portions

in bottles 1 and 3.

4. Effect of pasteurization and homogenization temperatures upon the

aggggr and distribution of leucocytes in milk,
The work of’Russell and Hoffman (1908), and of Campbell (1909) had

shown that when portions of the same milk were heated to increasingly
higher temperatures an increase in the leucocyte count took place. Since
this work was done with nonhomogenized milk only, it was felt to be of
interest to repeat their experiments using both nonhomogenized and he-
mogenized.milk.

Three trials were run, using raw clarified milk to which fresh sepa-
rator slime was added at the rate of 1.3 to 3.0 gms. per quart of’milk.
The milk was divided into five gtwo-gallon portions. Temperatures of 140°,
150°, 160°,~170° and 190° r’. were employed, with a holding time of so
minutes. Each portion, pasteurized at a different temperature, was ho-
mogenized separately at 2500 pounds pressure. Samples were taken before
and after homogenization. All samples were held for 48 hours at 40° F.
before examination of any kind was made.

Increasing the pasteurization temperature resulted in increased
leucocyte counts both in the nonhomogenized and homogenized portions of
milk (Table 9, column 1 and figure 15). The results are similar to those
obtained by Campbell (1909) with nonhomogenized.mi1k. The homogenized milk
counts are lower than those of the nonhomogenized milk at all temperatures.
This is due, as will be shown later, to the destruction of some of the leu-
cocytes during the process of homogenization. Otherwise the slopes are
identical.

While it has been known for a long time that only nature white blood

cells enter the blood stream, which do not multiply'(8abin, 1923) and the

-45..

incubation trial by Slanetz and Naghski (1939) showed the same results,
it must be assumed that raw milk actually contains a higher. leucocyte
population than that generally obtained by counting procedures. Campbell
(1909) used both the volumetric Deane-Buckley method and the Stokes-Stewart
smear-stain method, and found that higher counts were obtained in each case,
when milk was heated to higher ranges of increasing temperatures. Thus it
would seem that some leucocytes will not stain unless heat treatment is
practiced. Sabin (1923) and Sabin et a1 (1925) who practiced vital stain-
ing on living cells obtained from the blood, found that a certain group,
called ”non-motile form" would not stain with neutral red, which was com-
monly used. They gave the following emplanation. “As seen in the living
preparations, the cell first stOps moving and rounds up, and the granules
no longer stain with neutral red. It is obvious that these granules have
either lost all power of reacting to vital dyes, or that the cell membrane
has become impermeable to the dye." Schilling, as quoted by the same
authors (1923) referred to this type of cell as ”physiological death of
leucocyte." The probability exists that some leucocytes found in milk may
possess some such pmperties which prevent their staining, unless heat
treatment is practiced.

lhile the leucocyte counts increased with increasing pasteurization
tuperatures resulting in a fairly uniformly inclined slepe, the percentages
of total leucocytes in the upper, middle and lower thirds of the bottle in
the nonhomogenized milk showed broken curves (Fig. 16). Failure of the fat
globules to climp and form a cream layer at 160° 1'. resulted in an abnomal
distribution of leucocytes in the nonhomogenized sample.

Data in figure 16 show the various curves, while those in table 10

and figure 17 show the relation of cream volume to the per cent of total

leucocytes in the upper one-third portion of the five samples of nonhomog-
anized milk. The data in this table show that the depth of the cream vol-
ume is a decisive factor in the distribution of leucocytes, and is in agree-
ment with the observations made previously that the leucocytes tend to rise
with the fat globules, while in the absence of fat rising they will settle
to the bottom.

Increasing the pasteurization temperature resulted in an increased
intensity of sediment, both in the nonhomogenized and homogenized milk
samples. ‘Eowevsr, the intensity of the sediment in the homogenized.milk
was greater in each case than that of the correspondingly heat-treated non-
homogenized milk sample. Data in table 11 and figure 18 show the relation
of the intensity of sediment of the nonhomogenized and homogenized milk to
the pasteurization temperature employed. The maximum increase in intensity
of sedhment was between the pasteurization temperatures of 130° and 160° 1.,
resulting in a large increase of sediment. Since all milk portions were held
at the respective pasteurizing temperatures for 30 minutes, it can be ex-
posted that some coagulation of albumin, such as observed by Rowland (1933)
took place at higher temperatures. This albumin, although white in color
would tend to settle and carry with it suspended matter, which otherwise
would not have been deposited. This would explain the reason for the in-
tensity of sediment in the milk pasteurized at 170° and 180° 1?. respectively,
since both of these lots showed cream rising, but still had a sediment equal
in intensity to that of the sample pasteurized at 160° I. where no fat ris-
ing had taken place.

It is of interest to note that the same relation exists between the
intensity of sedhmant and the per cent leucocytes in the lower.one-third

portion of the bottle, in both the nonhomogenized and in the homogenized

milk as was reported in section 1. While the sediment in the homogenized
milk is due primarily to the settling of leucocytes, such is not the case
in the nonhomogenized samples, especially at the pasteurizing tainperatures

of 170° and 180° 1‘.

Table 9. The influence of the pasteurization temperature on the distri-
bution of leucocytes in nonhomogenized and in homogenized milk

(Average of three trials).

 

 

 

 

 

 

 

 

Past. Leucocytes per Leucocfigs per g, gftg; 850mg 13 the
temp. m1. before Upper one-third Middle one-third Lower one-third
2!, stem: 91' bottle gf bottlg g; bottlg
Nonymog an; god
1. of $ of for
total total total
140 2,087,000 3,962,000 94.9 139,000 3.3 72,680 1.8
150 2,158,” 6,240,000 94.0 23.400 0.4 574,200 506
160 2,582,000 4,638,000 71.0 942,400 14.4 959,000 14.6
170 2,372,000 7,006,000 89.9 248,300 3.2 335,800 6.9
180 3,370,000 6,741,000 88.0 199,000 2.6 718,800 9.4
flogenizeg
140 945,800 585,400 20.9 532,700 19.0 1,684,000 60.1
130 1,433,000 223,200 6.2 421,800 11.6 2,978,000 82.2
160 1,676,000 396,600 809 453.500 10.1 3,619,000 81.0
170 2,042,000 693,800 11.0 643,100 10.2 4,977,000 78.8

180 2,696,000 748,300 9.1 805,400 9.3 6,690,000 81.1

 

-49-

Table 10. The relationship between the cream volume and the leucocyte
count of the upper one—third when the milk was pasteurized

at different temperatures (Average of three trials).

 

 

 

Past. tap. Per cent of total leucocytes Cream volmne per
J}. g mper one-third of bottle bottle (min)
140 94.9 155 '
130 94.0 113.5
160 71.0 0.0
170 89.9 127.3
180 88.0 122.3

Table 11. The influence of the temperature of pasteurization upon the
intensity of sediment in nonhomogenized and in homogenized

milk (Average of three trials).

 

 

 

Past. temp. Qtensitz 9f aging} pp bgttg g; gmrt bottle
J1: _= ' _, t - -. :s -
140 ~ 1.0 _ 3.3
130 1.3 3.3
160 2.3 3.6
170 ‘ 2.6 3.6

180 2.6 3.3

LEUC‘OCVTES fM/LL/ONS)

 

 

 

 

 

4.0 T 1 r
HO H065 M’ZED ———————
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3.0 ._ _
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PASTEUP/Z/NG 75,14,0pr Tupifqr}

LEUEOEVTES fPEPfE/V 7 TOTAL)

 

 

 

     
       

 

 

 

 

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PASTEUP/Z/NG TEMPEPA TUPE (”F)

LEUC‘OFVTES fPEPCE/V 7' 7'0 TAL)

 

 

 

 

 

/00 T 1 I 200

@LEUCOFVEES
so fix, ~ /60

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PAS TEUR/Z/NG TEMPEPA TUPE for?)

.0" ‘L. unis-28:42

CPEAM VOLUME //V MLS.

LEUCOFYTES (PERCENT TOTAL)

/00

 

 

 

 

       

 

 

T T T
HOMOGEN/ZED------
NON H O M Ob E. .xV/ZE .9 ——
_____ K
80r- / ', ------------------- 4.1;:
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PASTEUP/Z/A/G TEMPE/PA TURE (OF)
,h a he: \' 1’35 3T)
1 - l 1 A.) d L x 1 :T. 1‘ C11.
I‘LL-’lhho‘i’griqiiij. ‘d Ind ‘95.“; :3 H

5. nfggt of homogegizim bgfore or after pasteui_-i_zation on thg mm:

W

Since homogenization before or after pasteurization are both prac-
ticed commercially, and a difference in opinion exists in the preference
of one or the other method, it was felt that a comparative study of the
two methods would be of value. By varying the sequence of the procedure,
special attention was to be paid to the number of leucocytes after process-
ing, and to the intensity of sediment produced in each case.

To ten gallons of fresh, raw, clarified milk was added fresh sepa-
rator slime at the rate of 1.3 gms. per quart. One-half portion of the
milk was heated to 100° 1., homogenized and pasteurized, while the other
one-half was pasteurized and homogenized. Each one-half portion was di-
vided into five equal lots. Pasteurizing tuperatures of 140°, 150°,
160°, 170° and 180° 1. were «played, holding the milk for 30 minutes.
Bomogenizatien was performed at 2500 pounds pressure. Samples (taken at
various stages of processing were held for 48 hours at 40° if. before
examinations of any kind were made.

The data, shown in table 12 and figure 19, indicate that homogen-
ization before pasteurization reduced the leucocyte count to a greater ex-
tent than if homogenization after pasteurization were practiced. This,
however, was not the case when pasteurization temperatures of 140° to 150°
1'. were used. No definite conclusions could be reached as to why this was
so, but the fact ranained that all three trials showed the same trend. The
leucocyte count for the pasteurized-homogenized milk yielded a slope similar
to that observed when the milk was heat-treated at a high temperature as was
shown in part 4 figure 15. Thus the difference in the slaps of the leuco-

cyte-count curve of the homogenized-pasteurized milk was due to the reversed

-55-

sequence of treatment. Some explanation might be found in the following:
Ihile all the homogenized-pasteurized milk was homogenized at the same
tuperature, namely 100° F. , the pasteurized-homegenized milk was homogen-
ized at a different temperature, namely at the pasteurizing tanperature of
each respective lot of milk. The distribution of leucocytes after storage
was very much the same with both treatments. Some difference was encoun-
tered at tunperature treatments above 160° F. as shown in figure 20. This
difference in behavior was attributed to a variety of factors, which re-
quire further study.

While all saluples showed a large amount of sediment, the intensity
varied with the individual lots (Table 13). A slight increase in the in-
tensity of sediment was observed between 140° and 150° F. heat treatment
of pasteurized-homogenized milk, while the othersof this series were
given the same score. All samples of the homogenized-pasteurized milk
showed a uniform intensity of sediment, which was greater than the high-
est value given to the pasteurized-homogenized lots. Thus, according to
the results obtained from the above trials, pasteurization at 140-150° 1".
before homogenization would be preferable to homogenization prior to

pasteurization.

-53..

Table 12. The leucocyte counts in pasteurized-homogenized and homogenized-
pasteurized milk suples, before and after storage, pasteurized
at different temperatures for thirty minutes (Average of three

trials) .

 

e coc as e to s 0
Upper oneothird Iiddle one-third Lower one-third

Past. Leucocytes per
tap. :1. before

 

 

 

 

 

 

or, gtgmg 9f bottle W W
at e ofiomo ze
Par Par Per
cent cent cent
140 775,100 429,400 17.2 410,000 16.5 1,651,000 66.3
150 1,080,000 299,500 10.2 396,600 13.9 2,188,000 75.9
160 1,113,000 477,400 11.8 447,300 11.0 3,137,000 77.2
170 1,545,000 563,000 12.8 767,500 17.5 3,067,000 69.7
180 1,865,000 729,300 13.6 389,000 7.3 4,242,000 79.1
A 0 en - t e
140 1,098,000 353,400 13.8 395,400 15.5 1,806,000 70.7
150 1,140,000 317,500 11.0 338,800 11.8 2,219,000 77.2
160 820,000 245,000 9.5 455,700 17.8 1,863,000 72.7
170 856,700 179,300 8.4 168,700 7.9 1,792,000 83.7
no 1,315,000 599, 700 18.2 640,600 19.4 2,060,000 62.4

 

 

-57-

Table 13. The intensity of sediment in pasteurized-homogenized and
homogenized-pasteurized milk, pasteurized at different

. tanperatures (Average of three trials).

 

 

Past. Tap. nten t of s n bott o ttle of l
J; A s ze -homo Ho e - a e ze
140 2.3 _ 3.0
150 2.6 3.0
160 2.6 3.0
170 2.6 3.0

1% 2.5 3.0

 

 

J r- PASTfHOMO. “““““ _,
HOMO—PAS T.—- ------

LEUCOFVTES fM/LL/ONS)
N
I

 

 

 

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I40 /50 I60 I70 /80

PAS TEUR/Z/NG

TEMPERA TURE FOP)

LEUCOCY 7‘55 (PERCENT TOTAL)

 

 

 

 

T T T
./'~.-1_0W£"r?
80 " ./. . I:
_ I':4:-:-,“& ..... ‘ 5< \ If,
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I40 /50 I60 I70 /80

P45 TEUR/Z/NG TEMPERA TURE (OF)

»-1

LEUCOCYTES {PERCENT TOTAL)

867

460

4t?

‘20

0

I40

 

)SEDhMENT‘

PAS 77- HOMO. — - — -- — -—
HOMO—PAW? _. _____

i_ 1 4L

(3}: f;£"r/Alljfi/7n

a.
INTENS/ T?”

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I50 I60 I70

I80

PASTEUR/Z/NG TEA/I PER/3 TUNE (0.5")

I)

2‘ . a. I' L ,
.i'I-‘ (9;. E‘f“"‘xr‘3":"j'¢y, r.‘ '

{l~ 1‘ *p- b‘\‘ \ ~ -, '—- a. ‘. _
. e ‘fétl ., .JZICffiTLI/i LLKJI x11 t"c 1*rhccgfiqte a

1n .n- lo a unwth1ra of bo.tlea $13}

A ' .,. 1
-:’.‘ulut':.'h a? £124}

6. Iffect‘of e1 ve ~route tati n on the formation of sad ant
mammal

Since agitation of homogenized milk, such as in unavoidable during
delivery had been found to aid the settling of leucocytes, as reported by
Hahn and Tracy (1940), a close study was made over a period of time, using
clarified, filtered, and nonclarified-nonfiltered homogenized milk to ascer-
tain the specific effects of route agitation on sedimentation.

Twenty-four trials were conducted over a period of five months, using
regular clarified homogenized milk. Six trials were made with.nonc1arified,
filtered regular homogenized.milk, and nine trials with nonfiltered, non-
clarified vitamin D milk, over a shorter period of time.

Quart bottles were picked at random during the bottling process, two
at a tmse, one of which was used as control and held at 40° F. for 48 hours,
while the other one was taken on the delivery route, but returned and placed
with the control bottle for the remainder of the 48 hours. Examinations as
to formation of sediment and leucocyte count were made at the and of the
holding thme. ltperiments were started late in July, and carried on with
intervals until the end of December.

The influence of delivery route agitation was Observed. All three
sets of trials conducted showed higher leucocyte counts in the lower portion
of the'bottle in the return milk as compared with the control. This differ-
ence in counts was attributed to the agitating effect that the milk had to
undergo during delivery. Although the danger existed that heat shocking,
such as reported by Tracy (1935), would also produce similar results, tem-
perature observations showed that the milk leaving at 40° F. would return

at a temperature not higher than 50° F. Thus the effect of a change in

- '52 -
temperature, although possibly a factor, was considered to be negligible.
The data are shown in tables 14, 15 and 16, and in figures 22, 23, and 24.
While the increase of the number of leucocytes per milliliter in the lower
portion of the bottle upon delivery-route agitation was only slight, the
increase in the intensity of sediment was more pronounced. This was es-
pecially the case with the nonclarified series, as shown in table 17 and
figure 25. The beneficial effect of clarification is clearly shown in
these trials.

The higher intensity of sediment in the regular filtered, nonclari-
fied milk, as compared with the intensity of sediment observed in the vita-
min D, nonfiltered, nonclarified milk may be partially due to the higher
leucocyte count in the former>milk, as shown in table 18. The data show
that if milk is not clarified, high intensities of sediment may be ob-
tained even with leucocyte counts below 100,000 per'milliliter. Hahn and
Tracy (1940) found that if the milk were normal in every respect than it
was possible to eliminate sedimentation when the milk contained approxi-
mately 100,000 T. 25,000 cells per milliliter. The above results are in
agreanent with their observations.

no special study was made on influence of heat shocking on the
settling of leucocytes and the intensity of sediment produced. However,
it was generally observed that in examining the milk in the laboratory,
with a temperature of 70° to 85° F. in the room, the milk from the cooler
having a temperature of 40° I. would increase in temperature during the
examination period. Observations made in l5-minute intervals showed that
the intensity of sediment in the control samples of the nonclarified quart
bottles of'milk would increase within the first 15 minutes and show the

same intensity of sediment as was found in the return samples on the first

- 53 -

observations. Since little agitation had taken place, the change in in-
tensity of sediment was attributed to the effect of increasing temperature
of the milk. The sediment of all the nonclarified milk studies was usual-

ly of a yellowishegrey to grayish color.

Table 14. The influence of delivery-route agitation on the migration of
leucocytes in regular clarified homogenized milk (Average of

24 trials).

 

Leucocytee per*ml. in the upper,.middle and lower one-third por-
gions, after storage, in regular glarified, homogenized milk.

 

 

 

 

 

 

Control : Returg_
Trial Upper one- Middle one- Lower one» : Upper one- Middle one- Lower one-
up, thirg wthird third, : third thirgj third
1 10,000 20,000 120,000 120,000 100,000 100,000
2 120,000 80,000 80,000 40,000 40,000 40,000
3 20,000 10,000 80,000 60,000 10,000 40,000
4 80,000 120,000 160,000 20,000 20,000 80,000
5 40,000 10,000 60,000 20,000 20,000 60,000
6 60,000 60,000 40,000 20,000 10,000 160,000
7 40,000 20,000 60,000 20,000 60,000 280,000
8 40,000 40,000 100,000 10,000 60,000 80,000
9 40,000 80,000 100,000 60,000 100,000 100,000
10 40,000 10,000 40,000 60,000 20,000 20,000
11 80,000 20,000 80,000 60,000 20,000 120,000
12 60,000 60,000 120,000 20,000 10,000 260,000
13 100,000‘ 10,000 220,000 100,000 80,000 160,000
14 40,000 60,000 220,000 40,000 60,000 160,000
15 20,000 60,000 60,000 20,000 20,000 20,000
16 40,000 60,000 60,000 60,000 20,000 20,000
17 20,000 20,000 20,000 20,000 60,000 60,000
18 40,000 40,000 60,000 20,000 40,000 60,000
19 40,000 160,000 200,000 80,000 40,000 360,000
20 40,000 10,000 180,000 40,000 120,000 80,000
21 40,000 60,000 60,000 80,000 10,000 320,000
22 10,000 10, 000 80,000 10,000 10,000 20,000
23 100, 000 80,000 240,000 20,000 40,000 140,000
21819.999 199.000 £139,000 _910.000 910.000 100,999
W00 904% M450 2 .600 8.4M.
Per cent
f t 2 0 22.6 23 2 9 9 96,9

 

 

-55-

Table 15. The influence of delivery-route agitation on the migration of
leucocytes in filtered, nonclarified regular homogenized milk

(Average of six trials).

 

Leucooytes per ml. in the upper, middle and lower one-third por-

 

 

 

 

 

 

 

ns af sto te n ncla f ed homo enized m.lk
antrgl 3* Return ~_
Trial Upper one. Middle one- Lower one. :‘Upper one- Middle one- Lower one-
H9, tggg tgrg tug-g : tn; rd tmLcL third
1 40,000 220,000 460,000 80,000 60,000 340,000
2 60,000 80,000 340,000 80,000 160,000 480,000
3 140,000 80,000 200,000 10,000 40,000 260,000
4 180,000 160,000 260,000 40,000 140,000 400,000
5 220,000 220,000 640,000 260,000 200,000 640,000
9 90,999 120.000 994900.000, 100,000 100,000 269,000
Leg.
e 320 3 .600 318 300 63 640 10 400 376 000
Per cent
91 90991 17,9 24.9 58.0 91198 ‘19.8 69.4___

Table 16. The influence of delivery-route agitation on the migration of
leucoyctes in nonfiltered, nonclarified homogenized vitamin D

ldlk (Average of nine trials).

 

Leucocytee peerl. in the upper,:lidd1e and lower one-third por-
tions, after storage, in nonfiltered, nonclarified, homogenized

 

 

 

 

 

 

 

aw
M = Rem
Trial Upper one- liddle one- Lower one- : Upper one- Hiddle one- Lower one-
t thir t d : thir thir t

1 140,000 180,000 640,000 220,000 160,000 520,000

2 80,000 20,000 220,000 ' 60,000 40,000 200,000

3 20,000 20,000 60,000 40,000 10,000 120,000

4 40,000 100,000 300,000 20,000 140,000 180,000

5 120,000 80,000 180,000 180,000 60,000 180,000

6 80,000 60,000 160,000 40,000 80,000 80,000

7 10,000 40,000 10,000 10,000 10,000 40,000

8 120,000 80,000 ‘300,000 120,000 120,000 300,000
-2.___.in_9.___1-00 0:000 24204200 W
,Log.

330 930 3 600 55 700 6 000

IPer cent

WM 20 180 41.0 W

 

Table 17. The influence of deliverybroute agitation on the intensity of

sediment in quart bottles of homogenized:milk

 

Type and treatment of milk The intensity of sediment in the

 

 

pug; to Mpgenization Contggl Bgtgzg

Regular, clarified 0.0 .2

Regular, filtered, not clarified 0.5 3.0

Vitamin D, not filtered, not 0.35 2.33
clarified

 

Table 18. The relation between the number of leucocytes per’milliliter

in quart bottles and the intensity of sediment observed

 

Control : Re
Type and treatment of’milk Leucocytes Intensity' :Leucocytes Intensity

3110; to homogenization peg ml. of aggent, per g. 9f ggdimgt

 

 

Regular, clarified 56,260 0.0 49,580 0.2

Regular, filtered, not 183,070 0.5 180,220 3.0
clarified

Vitamin D,:not filtered, 86,420 0.55 91,100 2.33

not clarified

 

LEUCOCYTES (PERCENT TOTAL)

80

60

40

20'

 

CON T901. -

RETURN

 

 

 

'7

 

 

 

   
 

  
  
 

 

 

/
Z

/
Z

  
   
 

    
  
 

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/x.

 

 

Zl

UPPER MIDDLE LOWER

TH/RDS 0F QUART BOTTLE

The Hay cent cistriOutipa of leuco«
cytcs in the tyfiec, middle ans )0 er

one-thivd ;orticn5 of ;u£rt battles

0f Feguia: Glorified. homogenized milk.

LEUCOCVTES (PERCENT TOTAL)

60

6'0

40

20

 

CO/V r901 -

U

95 ram/v 711/. C“

\

 

 

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UPPER MIDDLE
TH/RDS OE QUART BOTTLE

 

 

 

 

LOWER

 

 

LEUCOCYTES (PERCENT TOTAL)

fie
C)

Ch
C)

in
C)

N
9

 

T-

CONTROL - T

m: TURN

 

 

 

 

 

 

 

 

UPPER MIDDLE LO IVER

TH/RDS OF QUART BO TTLE

Ihe For aenL ciFtriouLiwn of leucocytes
in the u-:03, miiale rad loxe: one—third

0

portions or_;UL;t bottles or nonfiltered,

noncirrified honogcti7cd Vitamin L milk,

'Tu

 

CONTROL -

RETURN

 

 

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‘

 

samplee of control find return Womo~

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7. finest of normal homogenization pressure on the leucocyte cggt g;
sills...

The observation was made, while conducting the various trials, that
the homogenized milk usually contained a lower leucocyte count per milli-
liter, than the homogenized samms of the same milk. Thus it was felt
of interest to find out what the average percentage reduction in the number
of leucocytes was when normal homogenization pressures were employed.

All milk used in this experiment was pasteurized at 142° - 144° F.
for 30 minutes and homogenized at pressures ranging from 2000 to 2500 pounds
per square inch. Leucocyte counts were made from samples of milk collected
before and after homogenization. A total of thirty trials were conducted.
The data secured are presented in table 19.

The data show that in all but four cases the number of leucocytes
found in the nonhomogenized milk exceeded the count in the homogenized milk.
The inconsistency of these four trials, however, might be explained by the
fact the counts were within the range of experimental error, and were made
also on low count milk to which no leucocytes had been added.

The reduction, based on the logarithmic average of the above trials,
was found to be 41.28 per cent.

The decrease in the number of leucocytes in the homogenized milk must
be attributed to the destructive action of the homogenization process. No
other explanation can be given, since in making the counts, it was much
easier to see the leucocytes in the microscopic field when homogenized milk
was used, than with nonhomogenized milk. Thus, as far as observing the
leucocytes in making the counts was concerned, no lower numbers should have

been encountered.

-72..

 

 

 

 

 

 

Table 19. The influence of normal homogenization pressure on the
destruction of leucocytes
Trial Lgucgcytes per ml, in milk when
39,, Nonhomogenizsg wfilil’mnOgenized
1 3,900,000 2,320,000
2 1,620,000 480,000
3 1,440,000 760,000
4 1,140,000 1,040,000
5 1,800,000 640,000
6 1,000,000 700,000
7 1,060,000 460,000
8 880,000 700,000
9 940,000 760,000
10 2,240,000 960,000
11 640,000 440,000
12 260,000 340,000
13 100,000 120,000
14 80,000 100,000
15 180,000 100,000
16 440,000 160,000
17 160,000 100,000
18 200,000 140,000
19 120,000 180,000
20 320,000 320,000
21 140,000 120,000
22 2,260,000 580,000
23 3,000,000 1,140,000
24 4,060,000 2,160,000
25 1,460,000 700,000
26 1,900,000 560,000
27 1,580,000 460,000
28 960,000 480,000
29 1,060,000 500,000
30 800,000 480,000
av' s 725.600 426,000
c t uct o 41 28

 

 

 

e. WWWCOO a c
m

' Since the results of previous trials had shown that normal homogeniza-
tion pressures reduced the leucocyte count per milliliter in the homogenized
milk, an experiment was conducted to show the influence of repeated high
pressure homogenization on the leucocyte count of milk. It was felt that
such.precedure would demonstrate the destructive action of homogenization
on the leucocytes in the milk, at high homogenization pressures.

Three trials were conducted, using raw, clarified milk to which was
added fresh separator slime at the rate of 1.5 gms. per quart of milk. The
milk was pasteurized at 142° - 144° F. for 30 minutes and homogenized, first
at 2500 pounds pressure and then followed by five repetitions at 5000 pounds
pressure. Quart samples were collected, which were cooled and held for 48
hours at 40° F. at the end of which period examinations were made.

The leucocyte count psrnmilliliter decreased rapidly with the first
two trials of high pressure homogenization which reduced the count over 80
per cent. After that the rate of reduction was markedly less. The data are
shown in table 20 and figure 26. The highest total reduction obtained after
the last homegenization was 92.4 per cent.

Although the leucocyte count decreased with repeated high pressure
homogenization, no reduction in the intensity of sediment was observed. All
bottles of homogenized.milk showed sediment of a grayish color and were given
an intensity rating of 3.0. Some difference was noticed in the general ap-
pearance of the sediment. The sediment deposits of the milk homogenized from
one to five times resubled sand in appearance, while the sediment of the

last bottles was smooth and even throughout. The conclusion was reached that

~74-

the broken leucocytes tended to settle down and ferm.sediment, similarly

to the nonbroken leucocytes found in milk.

Table 20. The influence of repeated high pressure homogenization on the
leucocyte count and the intensity of sediment in quart bottles

of homogenized.milk (Average of three trials)

 

lumber Homogenization . Leucocyte Intensity
of’times pressure Leucocytes reduction of

en a s. s n. No. 0 tota ed t

0 0 957,000 0 0.0

1 2500 625,400 34.6 3.0

2 5000 357,200 62.7 3.0

3 5000 159,100 83.4 3.0

4 5000 106,300 88.9 3.0

5 5000 84,300 91.2 3.0

6 5000 72,700 92.4 3.0

k >.\M\<\Q.wm, KQ kk\W>\.wk\<\

 

 

 

 

 

 

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sediments

-75-

9a. Effect of continuous, high-pressgre homogenization on the leucocyte

gynt of milk,
Since previous experiment had shown the destructive action of high-

pressure homogenization upon leucocytes, a trial was conducted with the
purpose of attempting to accomplish the complete destruction of leucocytes.

To five gallons of pasteurized milk was added fresh separator slime
at the rate of 1.5 gas. per quart. The milk was heated to 140° F. and
homogenized, first at 2500 pounds pressure, and again at 5000 pounds press-
ure for ten minutes continuously. Qnart samples of milk were collected
before and after the first homogenization as well as at two-minute inter-
vals during the continuous process. All samples were cooled and held at
40° F. for 48 hours at the end of which time examinations for sediment and
leucocytes were made.

The data, shown in table 21 and figure 27, give the results obtained
in this experiment. Virtually complete destruction of leucocytes was ac-
complished. The leucocyte count was reduced from 2,240,000 per milliliter
to 20,000 per milliliter as a result of homogenizing at 5000 pounds press-
ure for 10 minutes. MicroscOpic examination of the homogenized milk showed
the last samples to contain leucocytes of small size only. The leucocyte
fragments, although not readily stained could be seen as small particles in
the microscopic field. The intensity of sediment was found to be the same
in all samples of homogenized milk. This would indicate that the broken
leucocytes settled and fomed sediment, similarly to the nonbroken leuco-
cytes.

Since the temperature of the milk increased during the process of
homogenization from 140° F. to 190° F. it might be expected that the sedi-

ment of the latter samples contained some destabilized portions together

- 77 -

with the fragmented leucocytes. Also, the high temperature must have
liberated more leucocytes thus resulting in a higher percentage of total

reduction than the 99.1 per cent as calculated.

Table 21. The influence of continuous high pressure homogenization on

the leucocyte count and on the intensity of sediment

 

 

Time Homogenization Leucocyte Intensity
homogenized pressure Leucocytes reduction
min bs. er s in. ergml. E£_of total} sediment

0 0 2,240,000 0 2.0

0 2500 960,000 57.1 3.0

2 5000 320,000 85.8 3.0

4 5000 180,000 92.0 3.0

5 5000 100,000 95.5 3.0

8 5000 40,000 98.3 3.0

10 5000 20,000 99.1 3.0

 

LEUC‘OCVTES (MILL/0N5)

 

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95. Effect on sedimentation of adding increasigg portions of continuous,

ngh-pmssgpe-hgmogenigeg milk to normg_l homogenized milk,
The object in this experiment was to find out whether or not mixtures

 

of normal homogenized milk and milk subjected to a continuous high press-
are treatment, which was high in leucocyte fragments, would fem sediment
of greater intensity.

To a series of eleven quart bottles containing decreasing amounts
of normal homogenized milk, were added increasing amounts of the continu-
ous, high-pressure-homOgenized milk prepared earlier in this experiment.
All samples were mixed well, and allowed to remain undisturbed for 48 hours
at 40° F. Sediment studies were made at the end of the storage period.

The intensity of sediment increased with the addition of increasing
portions of continuous, high-pressure-homogenized milk. This is shown in
the data of table 22 and figure 28. The sediment was not due primarily to
leucocytes, since none of the samples contained a leucocyte count above
80,000 per milliliter. The sedinmnt curve in figure 28 follows the straight
line of the milk mixture. Thus no doubt can exist as to the settling of

the broken leucocytes in normal homogenized milk.

Table 22. The influence of adding increasing portions of continuous
highepressure treated milk to normal homogenized milk on

the intensity of sediment produced

 

Per cent of normal Per cent of continuous Intensity

 

Sample homegenized milk highrpressure treated of
No. per bottle milk:per botgle :Eediment
1 100 0 0
2 90 10 ‘ 1
3 80 20 3
4 70 30 3
5 60 40 3
6 50 50 3
7 40 60 4
8 30 70 4
9 20 80 4
10 10 90 4
11 0 100 4

 

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- 32 -

10. Effgct 9f tggpgzgtgre of clazifigation 9n thg intensity of sedimgnt

1g hgmogenized milk,
While the results of previous workers (Jacobson and Olson, 1931) had

shown that the temperature at which.milk was clarified plays a role in the
efficiency of the removal of leucocytes from.milk, a further study seemed
to be of interest in view of sedimentation in homogenized milk.

Six gallons of fresh raw milk were divided into three equal lots.

The three lots of milk were clarified at temperatures of 60°, 100° and 145°
1. respectively. Washed leucocytes were then added to the clarified milk
at the rate of 2 gms. per quart. The mixtures were pasteurized at 142° to
1440 F. for 30 minutes, followed by homogenization at 2500 pounds pressure.
Quart samples were collected, cooled and stored at 40° F. for 48 hours.
Examinations fer sediment formation and leucocytes were made at the end of
the storage period.

The use of lower clarification temperatures resulted in slightly lower
leucocyte counts in the clarified milk, as shown in the data of table 23.
This difference in count, based on the results of three trials only, seems
not to be of any significance. All the clarified milk had very low leuco-
cyte counts, and no sediment formation would have taken place, according to
the results obtained in previous trials.

The addition of similar weights of washed leucocytes to the milk which
had been clarified at different temperatures with subsequent pasteurization
and homogenization showed a similar trend in the leucocyte count to that
observed in the clarified milk. Lower leucocyte counts were obtained in

milk samples which had been clarified at lower temperatures.

-s:s-

The correlation of the leucocyte count per milliliter of milk after
homogenization to the intensity of sediment produced is shown in the data
of table 23 and in figure 29.

Whether or not the slight increase in leucocyte count at the higher
tunperature and the increase in the intensity of sediment were due only
to the clarification temperature employed, is not certain. Since, however,
the same trend, namely a higher count with higher clarification tempera-

ture, was observed in the clarified milk, both before and after the addi-
tion of washed leucocytes, and the intensity of sediment increased also
with higher clarification temperatures it would follow that low clarifica-
- tion tunperatures would be more desirable.

The data in table 24 show the distribution of leucocytes in the upper,
middle and lower one-third portions of quart bottles of milk after storage.
The distribution, calculated as percentage of total number of leucocytes
per bottle, in the upper, middle and lower portions were about the same in

all three lots of milk.

- g4 -

Table 23. The influence of the temperature of clarification on the
leucocyte count and the intensity of sediment observed

(Average of three trials)

 

Temperature of Intensity

clarification‘ Leucogytes per*milliliter 9f the milk when of
(°F.) Raw Clarified E

omogenized 'sediment

 

60 31,740 10,000 589,600 2.66
100 31,740 12,600 683,400 3.0
145 31,740 15,870 781,200 3.66

l"2 gms. washed leucocytes per quart omeilk were added prior to
homogenization.

Table 24. The influence of the temperature of clarification on the
distribution of leucocytes in quart bottles of homogenized
milk to which washed leucocytes were added after clarifica-

tion (Average of three trials)

 

Thmperatfire of w”wigucgcytss p822milliliter in the
milk at clari- 'Upper one-third iniddle one-third Lower one-third

{ication (°F.) of quart bottle of upgrt bottlgr, of guart bottle

 

fiof $of fiof

total total total

60 252,000 17.2 405,100 27.7 807,500 55.1
100 300,000 16.5 464,500 25.5 1,055,000 58.0

145 433,200 19.9 546,000 25.0 1,199,000 55.1

(I: I (:1.

 

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1.132; . .

11. Effect of clarification at different temperatures and at different
gtgggg g: pppcessing on thp weight of dz: matte; psmoveg,

Since clarification removes the unstable suspended particles it was

 

thought to be of interest to study the weight of dry matter removed from
equal weights of milk, when clarification was performed at different tem-
peratures of the milk and at different stages of processing. The weights
of dry matter removed per given quantity of milk would then serve as an in-
dication as to the efficiency of the clarifying process at different tem-
peratures and at different stages of processing.

To twelve gallons of fresh raw milk were added washed leucocytes at
the rate of 0.5 gms. per quart. The milk was divided into six equal por-
tions or lots. Lots 1, 2, 3 and 4 were clarified at temperatures of 40°,
60°, 100° and 145° 1“. respectively. Let 5 was clarified at 145° 1?. after
pasteurization, and lot 6 at the same temperature after pasteurization and
homogenization.

All six lots of milk were pasteurized and homogenized in the regular
manner, quart samples collected, cooled and stored for further studies.
The clarifier slime of each lot was collected separately by scraping and
rinsing the bowl with small amounts of distilled water, until perfectly
clean. The slime and the washings were placed in petri plates and dried
in a drying oven at 100° 0., cooled and weighed.

The reduction in leucocyte count due to clarification was very great
in all cases, ranging from 85.2 to 97.2 per cent of total number of leuco-
cytes as shown in the data in table 25. The difference in leucocyte count
between the individual lots of milk, however, were not considered signifi-
cant enough in order to pay too much importance to these values. The same

might be said about the slight differences in numbers of leucocytes per

- 87 -

ldlliliter of‘milk in the clarified, homogenized samples of the different
lots of milk. The differences in leucocyte count are all in the range of
experimental error.

The data in table 26 and in figure 30 show the grams of dry.matter
removed by clarification of each two—gallon lot of milk. The weight of the
total dry matter removed increased with increasing heat treatment, but de-
creased with homogenization. This would tend to indicate that more effi-
cient clarification can be obtained by clarifying the milk at pasteuriza-
tion temperatures, prior to homogenization.

According to totalosolids analysis made by the Mojonnier method the
washed leucocytes contained 21 per cent total solids. Since each lot of
milk contained four grams of washed leucocytes, which would mean 0.84 gms.
of dry matter, the rest of the removed matter must have been present in the
milk prior to the addition of the washed leucocytes. The high values ob-
tained in the two lots of milk clarified at 145° 1. before and after pas-
teurization would show the effect of heat treatment, both on_the leucocytes
and also on the milk proteins.

The fact that the pasteurized homogenized lot showed a lower value
must be attributed to the homogenizing action upon the suspended particles.
The possibility exists, that some particles must have been reduced to a small
enough size so that they would not be removed by the centrifugal force of
clarification.

Fat tests made by the.Mojonnier method on each lot of dry matter
showed a decrease in percentage fat with higher clarifying temperatures. A
slight increase, however, took place with homogenization. The data are
shown in table 26 and figure 31. Some difficulty was encountered in.making

the tests, since the dry matter was used and it seemed difficult to dissolve

the dry slime. Since all samples were treated in the same manner it was
felt that the data would show the trend of fat removal with clarification
at different temperatures of’milk, at different stages of homogenization.

The slight increase in the percentage of fat in the dry matter ob-
tained from the homogenized lot over that of the nonhomogenized lot, clari-
fied at the same temperature might be attributed to the reduced size of the
fat globules in the homogenized milk. The smaller size of the fat globules,
together with their increased specific gravity would aid in removing some
of the fat during the process of clarification.

According to the fat analysis made on washed leucocytes by the Mo-
jonnier method it was mund that 23.58 per cent of the total solids were
made up of fatty substances. This value is much higher than the highest
value found in the dry separator slime, which would indicate that a large
percentage of the dry separator slime was made up of fat-free substances.
As the milk was clarified at higher temperatures the percentage of fat in
the dry separator slime decreased, showing that the percentage of solids-

not-fat increased.

- 39 -

Table 25. The influence of clarifying temperature and subsequent ho-
mogenization on the leucocyte count of milk (Average of
three trials)

Stage of processing Leucocytes per milliliter in the milk

 

 

 

and temperature at Before After clarifying After clarifying
Ipich glgrizied clarifying ggi reduction &.homogenization
1. Raw at 40° F. 446,000 31,740 92.9 25,400
2. Raw at 60° F. 446,000 18,170 95.9 25,200
3. Raw at 100° F. 446,000 12,600 97.2 18,600
4. Raw at 145° F. 446,000 25,200 94.3 20,000
5. Pasteurized at 145°F.446,000 66,000 85.2 31,300

6. Homagenized at 145°F.446,000* 28,810 93.5 12,600

I"Leucocyte count before homogenization.

Table 26. The influence of the temperature of the milk and the stage of
processing on the weight of dry matter removed by clarifica-

tion (Average of three trials)

 

Stage of processing and

 

 

temperature at which Dry matter removed from Per cent fat in
W 2 gallons of milk fags.) dry matter

1. Raw at 40° F. 1.5277 13.7

2. Raw at 60° F. 1.8720 11.6

3. Raw at 100° F. 2.2325 9.0

4. Raw at 145° F. 3.1616 5.1

5. Pasteurized at 145° F. 3.1671 4.2

6. Hemogenized at 145° F. 2.2459 4.6

 

70 7341. 501. IDS IGMSJ

 

 

 

 

   

  

 

 

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- 92 -

12. Microscopic observations of sediment in homogenized milkL

Many microscopic fields showing the sediment in homogenized milk had
been studied while performing the previously described experimental work.
Some of the more common and illustrative views were photographed and are
presented in figures 32 to 36 inclusive.

The influence of the temperature of clarification upon the removal
of cellular elements in raw milk is shown clearly in figures 32, 33 and
34. Higher clarification temperature allowed for the more complete re-
moval of the large cellular elements, which usually tended to settle in
the homogenized milk, and thus contributed to sediment formation. A.tem-
perature of 145° F., therefore, seemed to be more suitable for clarifica-
tion, as far as the removal of large cellular elements was concerned.

This could be attributed to the fact that the milk would be less viscous
at a high temperature, and thus allow for greater ease with which the
suspended cellular elements could be thrown out by the centrifugal force.

The clarifier slime obtained by clarifying the decanted milk, with-
out disturbing the sediment in the bottom of the bottle was very similar
in all three cases. Few leucocytes and epithelial cells were found in the
microscopic fields of milk clarified at 60°, 100° and 145° F. respectively.

The sediment of nonclarified homogenized milk was commonly observed
to be of a nature as shown in figure 35. Leucocytes and large epithelial
cells, with few small fragments were predominant. The clarifier slime
obtained by clarifying the same milk, which had been decanted without dis-
turbing the sediment showed few large epithelial cells but many leucocytes

and some smaller fragments.

- 93 -

While it had been shown previously that continuous high pressure
homogenization reduced the leucocyte count by 99 per cent, the stained
sediment showed very many small fragments, with few leucocytes. The
small fragments were believed to be parts of the broken leucocytes. Fig-

ure 36 shows the typical view of the sediment.

 

Figure 32.
A. Sediment of homogenized milk which had been clarified at
60° 2. and to which washed leucocytes were added at the
rate of 2 gms. per’quart of milk, followed by pasteurize-

tion and homogenization.

B. Clarifier slime obtained by clarifying the above milk,
which had been decanted without disturbing the sediment.

(lagnification 550:).

 

A.

B.

-95-

 

Figure 33.
Sediment of homogenized milk which had been clarified at
100° 1". and to which washed leucocytes were added at the
rate of 2 gas. per quart of milk, followed by pasteuriza-

tion and homogenization.

Clarifier slime obtained by clarifying the above milk,
which had been decanted without disturbing the sediment.

(lagnifi cation 5501:) .

 

A.

B.

-95-

..

 

 

 

Figure 34.

Sediment of homogenized milk which had been clarified at
145° F. and to which washed leucocytes were added at the
rate of 2 gms. per quart of milk, followed by pasteuriza-

tion and homogenization.

Clarifier slime obtained by clarifying the above milk,
which had been decanted without disturbing the sediment.

(Hagni fi cation 550:) .

- 97 -

 

Figure 35.

A. Sediment of nonclarified homogenized milk. A.predominance

of leucocytes and large epithelial cells was observed.

B. Clarifier slime obtained by clarifying the above milk, which

had been decanted without disturbing the sediment.
(lagnification 550:).

 

Figure 36.

Sediment found in milk which had been homogenized
continuously for ten minutes at 5000 pounds prou-
ure. Fragments of broken leucocytes conned to make
up the largest part of the sediment.

(Magnification 5501) .

-99-

DISCUSSION

While performing the experhmental work in the studies reported, the
main emphasis was placed on the behavior of leucocytes in nonhomogenized
and in homogenized.milk under various conditions of temperature, pH, and
homogenizer pressures. A.careful study of the various results obtained
allows for a better understanding of the behavior of leucocytes in.milk
and their influence upon sediment formation in homogenized milk.

There exists no doubt that leucocytes play an important part in sedi-
ment fermation in homogenized milk. Thus, selection of milk with low leu-
cocyte counts would eliminate this source of sediment, and may, therefore,
be regarded as the first step in the prevention of sediment formation in
homogenized milk. However, other sources of sediment exist and it is possi-
ble to have sediment formed in homogenized milk with a very low leucocyte
count. It follows then that the selection of milk wholly on the basis of
leucocyte count is a rather unreliable method to assure absence of sediment
in the homogenized milk. In addition, other more dependable methods shotdd
be employed to eliminate sediment.

Ordinary filtration is not a satisfactory method to eliminate leuco-
cytes and other suspended particles in milk in large enough quantities so
as to render the average homogenized milk sediment free. Therefore, filtra-
tion should not be considered as a protective measure against sediment forma-
tion in the homogenized milk.

The most reliable method to be employed to prevent sediment formation
to take place in homogenized milk is power clarification. This should be

done at a temperature and at a stage of processing which would allow for

the most complete removal of the suSpended particles found in milk. Clari-

- 100 -

fication at the pasteurizing temperature before or after the holding
period and previous to homegenization is recommended. At this temperature
the fat is in the liquid state, and the milk is less viscous which allows
for the easy removal of the suspended particles. The high temperature also
helps to free the leucocytes by lowering the attraction between them and
the fat, and thus allows for greater deposition in the clarifier slime.
Since pasteurization tends to denature some proteins, especially albumin,
and makes them unstable, clarification after pasteurization would remove
also the possibility of this source of sediment. Clarification should be
performed before homogenization since many leucocytes and other suspended
particles are broken up during the process of homogenization, and thus may
be too finely divided to respond to clarification.

Deepite the fact that higher than usual temperatures of holder pas-
teurization liberate more leucocytes to be thrown out in subsequent clari-
fication, it is believed regular pasteurization temperatures of 142° to
144° F. for 30 minutes are more desirable than higher temperatures for the
same length of time for the reason that less protein will be destabilized
and removed by clarification. Pasteurization should be done before clari-
fication and homogenization in order to render the milk sediment-free.

While heat treatment, such as employed in pasteurization, tends to
destabilize the proteins to some extent, it is felt that homogenization
should follow pasteurization, rather than precede it. The chances are that
if’milk is pasteurized after homogenization more proteins will be denatured
during pasteurization, since homogenization tends to destabilize the pro-
tein to some extent. If on the other hand, the milk is pasteurized before

homogenization, the milk proteins enter the pasteurization process in a

- 101 -

more stable condition than if it were homogenized prior to pasteurization
and the effect of homogenization upon the milk proteins, followed by im-
mediate oooling of the milk, is less pronounced.

Since delivery-route agitation and heat shocking from.the time the
milk is bottled until it is consumed are commonly unavoidable, these con-
ditions should be taken into account when processing the milk. To be
assured of a satisfied customer a satisfactory product should be offered.
This can be done only if all precautions are taken to remove the largest
amount of suspended particles of the milk. There should be no traces of
any kind of sediment whatsoever.

In summarizing, the procedure recommended according to the present
knowledge, all milk should be pasteurized at regular pasteurizing tem-
peratures, clarified at the pasteurizing temperature after pasteurization
and homogenized at a high enough pressure to assure homogeneity of the
milk, followed by immediate cooling to 40° F. or lower. Such milk should
be able to reach the consumer sediment free and thus uphold the consumer's

confidence in the product.

- 102 -

SUMMARY

The behavior of leucocytes in nonhomogenized and in homogenized
milk has been studied from various angles.

The addition of 0.5 gm. of separator slime per quart of clarified,
pasteurized, homogenized milk was sufficient to produce a noticeable
sediment upon storage of the milk.

Adding increasing increments of washed leucocytes to freshly
pasteurized, nonhomOgenized milk resulted in increasing the depth of the
cream layer to its ”breaking point" beyond which a "cream layer" composed
of fat and leucocytes formed in the bottom of the bottle.

When the electric charge of the fat globules was reversed from nega-
tive to positive, the leucocytes failed to be carried up by the ”sweeping
action” of the fat.

Higher leucocyte counts and a somewhat higher intensity of sediment
were noted when higher pasteurization temperatures were employed.

Homogenization prior to pasteurization resulted in a slightly lower
leucocyte count and in a higher intensity of sediment as compared with
similar milk homogenized after pasteurization.

Delivery-route agitation had a pronounced influence on the settling
of leucocytes and other suSpended matter in homogenized milk, and thus
aided in the formation of sediment. Heat shocking had a similar effect to
that of delivery-route agitation.

Normal homogenization pressures destroyed large percentages of leuo
cocytes in milk. Results of thirty trials showed a reduction of 41.28 per
cent. Rehomogenizing the milk five times at 5000 pounds pressure reduced
the leucocyte count 92.4 per cent, with little effect on the intensity of

sediment.

- 103 -

Milk homogenized at 5000 pounds pressure for 10 minutes showed a
reduction of leucocyte count of 99.1 per cent. The intensity of the sedi-
ment was the same in all homogenized samples. The additions of increasing
portions of this milk to normal clarified homogenized milk resulted in a
correSponding increase in the intensity of the sediment in the milk mix-
ture.

Milk clarified at 60°, 100° and 145° F. respectively, showed slight
differences in the leucocyte count of the clarified milk. This difference,
however, was felt to be in the range of experimental error and thus was
considered to be of little significance.

The weight of dry total solids removed from equal portions of
similar milk when various clarification temperatures were used at differ-
ent stages of processing, showed that milk clarified at 145° F. before or
after pasteurization, resulted in the largest weights of total solids re-
moved. The percentage of fat in the dry total solids decreased with in-
creasing clarification temperatures.

‘Hicroscopic examinations of clarifier slime showed that more large
cellular constituents were removed at clarification temperatures of 100°
and 145° F. than at 60° F. In general, the sediment of nonclarified ho-
mogenized milk showed more large epithelial cells and other large frag-

‘ments, than did the clarifier slime of the same decanted clarified milk.

-104-

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