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SOME FACTORS AFFECTING THE ASCORBIC
ACID CONTENT OF MILK AND ITS POSSIBLE

ASSOCIATION WITH THE OXIDIZED FLAVCB

SOME FACTORS AFFECTING THE ASCORBIC ACID CONTENT
OF MILK AND ITS POSSIBLE ASSOCIATION WITH THE

OXI DI ZE D FLAVQ

Thesis

Respectfully submitted to the Graduate School of
Michigan State College of Agriculture and Applied
Science in partial fulfillment of the requirement

for the Dagree of Master of Science.

By
Erlend C. Gaessing

1938

THESIS

 

ACKNOWLEDGMENTS

The author of this thesis wishes to express his gratitude
to Professor 1:. L. Anthony, Dean of the Division of Agriculture,
through thom this study was made possible.

The writer is greatly indebted to Doctor G. M. Trout,
Ibparhment of Dairy Husbandry, for his suggestions and careful

criticism in the writing of this thesis.

1-15986

TABLE OF CONTENTS

page
INTRODUCTION...... ................. . ......... ....... ...... .... 1
REVIEW OF LITERATURE.......................................... 3
General Discussion......................................... 3
Methods of determining ascorbic acid in milk by titration.. 4
Amount of vitamin C in raw milk. 4
Effect of breed......................................... 6
Effect of stage of lactation and season................. 7
Effect of mastitis....................................... 7
Effect of bacteria.. .......... .......................... 8
Presence of dehydroascorbic acid in milk................- 8

The effect of heat treatment upon the stability of
ascorbic acid........................................... 8

Effect of metal contamination on the stability of
ascorbic acid........................................... 11
Influence of aeration upon the ascorbic acid content of milk 12
Influence of light on the ascorbic acid content of milk..... 14
Effect of freezing on ascorbic acid......................... 15
Different relationships found in respect to ascorbic acid... 15
Relationship between ascorbic acid and oxidized flavor...... 15
Relationship between vitamin C and flavor in milk........... 16
Relationship between ascorbic acid and enzymes.............. 16
Relationship between ascorbic acid and glutathione. ........ . 18
Other aspects of ascorbic acid.............................. 20
PROCEDURE............. ........... ... ..... ...................... 21

page

mmImTAL RESULTSOOOOOOOOOOOO....OOOOOQOOOOOO;OOOOOO000...... 23

Part I. Processing, ascorbic acid, and the oxidized

flavor of milk............................................ 23
The effect of copper upon the stability of ascorbic acid

and.upon the develoPment of the oxidized flavor when the

milk was pasteurized.at high and low temperatures......... 23
The effect of flash heating of milk at various temperatures

upon the stability of the ascorbic acid................... 85
The effect of various heat treatments upon the ascorbic acid

content of milk and upon the development of oxidized

flavor when copper was added.before and after heating...... 48
The effect of capper and irradiation upon the ascorbic

acid content of raw milk when the cold milk was passed

over a surface cooler..................................... 56
Relationship between the oxidized flavor and the ascorbic

acid of milk.............................................. 65
Part II. Ascorbic acid occurring naturally in milk under

various conditions........................................ 80
The effect of season upon the ascorbic acid content of

streptococcic and non-streptococcic milk.................. 80
The ascorbic acid content of commercial pasteurized milk,

irradiated milk and raw milk throughout the year.......... 92
Influence of leucocyte count of the milk upon the

ascorbic acid content............. ...... .................. 100
Correlation between the bacteria count, the leucocytes,

the reduction time and the stability of ascorbic acid

in milk-g00.......0.0............OOOOOOOOOCOI0.000........ 107

A study of the relationship between the per cent
lactic acid, the flavor score and the ascorbic
acid of milk..........................................
Influence of sodium titrate and citric acid upon the
ascorbic acid content of milk and upon the develOpment
of the oxidized flavor................................
The effect of the addition of c0pper and glutathione
upon the ascorbic acid content of milk and.upon the
deve10pment of the oxidized f1avor....................
IHSCUSSION..................................................

smgiiARYoeoeeo000000000000000000.0000000000000000.0000.000000

LITWME CITEDOOO000......O......OOOOOOOOOOOOOOO0.0.0.0...

page

114

116

123
127
133

135

INTRODUCTION

Recent deve10pments in the chemical aspects of vitamin C. in
its isolation and.in the exact chemical methods fer its determines
tion have made it possible to estimate rapidly and with a high degree
of accuracy the amount of vitamin C. ascorbic acid. in a great
variety of food products.

The importance of vitamin C in human nutrition is a well
established fact. That of average milk is considerably below the
human requirements and especially those of the child. However. the
amount of vitamin C as such is not necessarily the chief object
sought in its estimation. Recent years' studies on the structure
and chemistry of vitamin C have shown characteristics regarding its
oxidation-reduction potentials. its activation and inactivation by
specific enzymes. as well as its response to metals. The role of
vitamin C in the regulation of colloidal conditions and. recently.
the suggestion that the vitamin itself might act as a catalyst are
also of considerable interest to ascertain phases of market milk
investigation.

These observations indicate that the determination of vitamin
C under various conditions might throw some light on some of the
physical and chemical problems in milk to which.vitamin C undoubtedly
is closely related and which are of major importance in the dairy
industry.

Lately. several investigations have dealt with the vitamin C
in milk as affected by breed. lactation. season and flavors. However,

very little work has been done extensively or in detail in regard to

- 2 -

the relationship between flavor and vitamin C in milk from individual
cows. or to the effect of various heat treatments of the milk upon the
stability of vitamin C. The object of this experiment has been to study
the vitamin C of various milks and to correlate, if possible. the amount

of this vitamin with several factors incident to production.

- 3 -
nsvxsw or LITERATURE
GENERAL DISCUSSION

Ssent-Gyorgi (1932) undoubtedly was the first investigator
who isolated vitamin C from the adrenal cortex. He named it hexuronic
acid and did not try to identify it with vitamin C. The first identi-
fication appears to have been done by Vaugh and.Ray (1931). Shortly
thereafter. Sverbely and Szent-Gyorgi (1932) proved that hexuronic acid
mnd.vitamin C were identical. Later work by these and other investigan
tors has substantiated the identity of vitamin C and hexuronic acid. or
ascorbic acid. the name which will be used frequently in this presenta»
tion. There seems to be no reasonable basis for questioning the identi-
ty of the vitamin now.

Two separate methods have been generally used in determining
the amounts of vitamin C in food products. These are, by biological
assay and by chemical titration.

Tillman and Hirsch (1932) were the first to show the close rela-
tionships which existed between the data secured when the amount of
vitamin C was determined by titration with 2-6 diehlorophenolindOphenol
method.and.by biological assays. These close correlations were later
confirmed by Bessey and King (1934) who made improvements over Tillman's
method. Harris and.Bay (1933) also confirmed these findings.

Several other methods. Tauber and Kleiner (1935). Tauber (1935),
Tamber and Kleiner (1935), Gothlin (1933), Martini and Bonsignore (1934)
amd.Joseph (1936) have been develOped in the determinations of vitamin

C. However. the titration method is by far the most extensively used

-4-

within recent years. Some factors obtain in the titration method which
may give rise to errors. King (1936) classified them as follows:

'(a) Other substances may be present which reduce the reagent: (b) a
portion of the vitamin C may be present in the reversibly oxidized form
(which is still biologically active): (c) substances may be present
which interfere with the reaction of either the oxidising or reducing
agent. In only a few cases has there been evidence of serious error
when the titrations were carried out rapidly in acid solution.‘ He
believed. therefore. that due caution should be used in the interpreta-

tion of results obtained by the titration method.
Methods of determining ascorbic acid in milk by titration.

Sharp (1938) preposed a method for determining ascorbic acid in
milk in which the milk was titrated directly after being acidified.
This method was rapid and checked fairly well when ascorbic acid was
added to milk and then redetermined.

Ihitnah and associates (1936) and Russell and associates (1936)
precipitated the proteins with trichloroacetic acid and then titrated
the filtrate with 2-6 dichlorophenolindophenol. Both of these procedures

were proposed originally by Bessey and King (1934).
Amount of vitamin C in raw milk.

1. Effect of ration. The laboratory syntheses of vitamin C is
accomplished by the use of carbohydrates. Micheal and associates (1934),
in one of the technical syntheses of vitamin C. used d-glucose as the
initial product. According to these investigators. it was un-

reasonable to suspect that the living organism might be able to

-5“

synthesize vitamin 0 under certain conditions pertaining to the diet.
Guha and Ghash (1936) showed that subcutaneous and intravenous inJec-
tions of mannose in rats was followed by an increase in the vitamin C
content of the various organs. However, the ability or non-ability

of the living organism to synthesize vitamin C was not fully clarified.

No very extensive work has been done on the effect of the
ration of the dairy cow on the amount of vitamin C in milk. Riddell
and associates (1935) have made studies of the influence of the ration
on the, ascorbic acid content of the milk. They fed a dry ration plus
silage and pasture to one group of cows and a dry ration plus no silage
or pasture to a second group, using the double reversal system. They
concluded from the results obtained that the rations studied had no
‘significant influence on the ascorbic acid content of the milk.

Russell and associates (1936). in a study of the effects of
breed characteristics and stages of lactation on the ascorbic acid
content of the milk, stated that after the first two months of lactation
the ascorbic acid content of the milk was apparently dependent solely
upon the ascorbic acid content of the ration fed the cow.

lhitnah and associates (1936) regarded an increased intake of
ascorbic acid in the feed to be accompanied by an increased excretion
of ascorbic acid in the urine. but with no increased secretion in the
milk. According to Riddell and associates (1936) no significant in-
crease in the ascorbic acid content of the milk occurred when the cow's
rations were changed from a good.winter ration to pasture. They ex-
plained this on the basis that the cow was able to synthesise ascorbic

acid from the feeds of the winter ration.

-5-

In studies on the relationship between off flavors and.the
ascorbic acid content of milk, Brown and associates (1937) showed
that feeding tomato Juice or lemon Juice‘removed the milk's suscep-
tibility to become oxidised. They explained this as due to the in-
creased ascorbic acid content of the milk. Anderson (1937), in the
feeding of carrots to cows. secured similar data. explaining them as
did the above investigators.

Stanislaw and associates (1937) stated that their work indi-
cated there was no evidence of any effect of pasture feeding on the

amount of vitamin C in the milk.

2. Effect of breed. Russell and associates (1936) found on the

average the following amounts of vitamin C in milk from cows of differ-

ent breeds:
Holstein, Av. 10.0 mg. ascorbic acid per quart
Ayrshire. ' 12.9 ' z ' ' '
mm.eyg I 13.0 . . ' .
Jersey. ' 13.3 ' ' ' ' '
Brown Swiss, ' 14.8 ' ' ' ' '

They claim that this difference in the ascorbic acid content could not
be explained on the basis of the different amounts of milk produced by
the cows as two of the different breeds (Brown Swiss and Holstein) dur-
ing the experimental period.produced the same amount of milk.

lhitnah and Ridden (1936) found that the average values of
ascorbic acid for the different breeds were as follows:

Holstein, Av. 23.5 mg. ascorbic acid.per liter
e

Ayrshire, " 24.1 " " "
Guernsey. ' 26.0 ' ' ' ' '
Jersey. ' 29.2 ' ' ' ' '

These investigators stated that the high values for this particular

- 7 -

Jersey herd which was investigated in this work was significant. The
average ascorbic acid content of milk for all cows and for all months
tested.was 25.5 mg. ascorbic acid per liter.

Stanislaw and associates (1937) concluded that the vitamin C

content of milk was not affected by'breed.

3. Effect of stggg_9f lactation and season. Russell etal. (1936) con-
cluded from their work that the stage of lactation appeared to have a
more definite effect upon the ascorbic acid content of milk than the
breed differences.' The ascorbic acid content was found to be relatively
higher during the early stages of lactation. but decreased to a minimum
after about two months of lactation. and then increased to a maximum
with later stages of lactation. In the same work a few samples of colos-
trum were also titrated and in each case the vitamin C content was found
to be unusually high. This high ascorbic acid content was believed to
indicate a certain degree of storage of this substance by the cow during
the preparturition period.

shitneh and Biddell (1937) stated that the early stages of lac-
tation coincided with the low ascorbic acid content of the milk;

Stanislaw and associates (1937) stated that in three out of four
cases studied the ascorbic acid content of colostrum was higher than that
of average milk.

Ferdinand (1936) has shown that the vitamin C content of cows
and humans milk. estimated by the methylen blue titration, was lower in

the early spring than in winter.

4. Effect of mastitis. Stanislaw and associates (1937) found that the

quarter infected.with mastitis yielded milk definitely poorer in ascorbic

- 3 -
acid than that secreted by the normal udder.

5. Effect of bacteria. Little work has been done to show the effect
of bacteria upon ascorbic acid. Thatschenko (1936) showed that lactic
acid bacteria of the type Bulgarians acidcphilus and.B. acidolphilus
leichmani reduced the reversible oxidizable form of ascorbic acid to
dehydroascorbic acid. Lominski (1936) showed that ascorbic acid in
high dilution inactivates bacteriOphage. This was probably due to the
reducing acition of ascorbic acid since other reducing substances pro-
duce similar effects. Gagyi and UJsagly (1936) found that certain
bacteria possessed.a capacity for destroying vitamin C. In experiments
carried out by incubating suspensions of virulent organisms with vitamin
0 solutions, they found that the destruction was a partly reversible
process and that a certain quantity of vitamin 6 would be restored on

reduction with hydrogen or hydrogen sulphide.

6. Presence of dehydrascorbic acid in milk. Stanislaw and associates
(1937) showed. in determining the two forms of vitamin C. ascorbic acid
and dehydroascorbic acid. by treating milk with hydrogen sulphide. that
as milk was drawn from the normal udder it contained only the reduced
form of ascorbic acid. Their results. in part. are as follows:
Reduced ascorbic acid. 2:17 mg. ascorbic :cid.per 100 m1.
Total ascorbic acid. 2.21 ' ' ' '

The effect of heat treatment upon the stability of ascorbic acid.

Considerable work has been done on the general effect of heat on
ascorbic acid. However. little of this work has been done in respect to

specific time-temperature relationships.

~9-

La Her and associates (1922) were probably the first ones to
study the effect of different temperatures on the vitamin C content
of vegetable Juices. They studied.the effect of heating upon the
destruction of the vitamin C in tomato Juices heated for periods of
one to four hours at 60°. 30° and 100° c. The results showed that
the velocity of destruction. under the conditions of the experiement.
decreased with the time and to a greater degree than would be expected
if the reaction followed the unimolecular law of the square-root rule
of Schultz. The percentage destroyed varied emperically as the square
root of the time.

Schwartse and associates (1930) found by biological assays that,
by boiling lightly three quarts of milk for five minutes in a glass
beaker or in an aluminum pan. the vitamin C content was reduced
approximately 20 per cent. In another experiment Schwartzs (1931) and
associates studied pasteurization of milk aerobically in aluminum.
copper and tinned cepper tubular pasteurizers. The destruction of
vitamin C in aluminum pasteurizers was from 20 per cent to 40 per cent.
This reduction was larger than that found in their previous experiments
on boiling milk for five minutes. These results were rather to be ex-
pected as the duration of the exposure of pasteurization was much longer
and was strictly aerobic. Tinned copper gave slightly greater losses.
Pasteurising in exposed copper cans resulted in losses of the vitamin 0
up to 80 to 90 per cent.

King and Waugh (1984) proved that there was no significant
destruction of vitamin c in milk by the Electropure and Han Vik (flash

contact) methods of pasteurizatbn when all aluminum equipment was used.

-10-

This, according to their theory, was due to: first, a very short heating
time: second, to the methods of heating: third. to a protection from the
atmospheric oxygen during heating: and fourth. to a minimum exposure to
metals having catalytic effects. The findings are based.upon two series
of animal assays of the raw and pasteurized milk and also upon the ti-
tration of the vitamin by means of 2-6 dichlorophenolindOphenol method.
In this experiment they also pastuerized some milk in the same types of
tank for 30 minutes at 143 to 145° r., which resulted in a significant
destruction of vitamin C.

Except where specified. as in the work of Vaugh and King, the
vitamin C determinations in the three researches reported above were
done by biological assays. In the research reported below the determina-
tions were made by the titration methods.

Whitnah and associates (1935) concluded that the stability of
ascorbic acid in fresh raw milk was greater than that either in the raw
or in the pasteurized samples secured at commercial pasteurizers. No
immediate loss was found to result from pasteurization by the short-time
high-temperature method. The 30 minutes holding process of pasteurizas
tion was not well adapted fer the conservation of ascorbic acid in milk.
Among all the commercial types using 30 minute holding periods only the
glass lined vat produced.pasteurized milk where less of ascorbic acid on
storage was as small as in the raw milk;

Sharp (1936) demonstrated that an insignificant amount of ascor-
bic acid was destroyed by heating for 30 minutes at 63° 3. providing the
milk was not metal contained.. After three days the pasteurized milk con-

tained more ascorbic acid than did similar milk raw.

- 11 -

nut and setterfield (1937) regarded the 2-6 dichlorophenolindrophenol
titration method used by Sharp for the estimation of ascorbic acid in milk
reliable. but only when applied to fresh milk. Difficulties with ascer-
taining the correct endpoint would seem to render Sharp's method less
reliable for milk samples stored for three or more days.

Ion (1937) stated that pasteurization by the holder method destroyed
the reversibly oxidized ascorbic acid but did not affect the reduced form
of ascorbic acid in milk. The amount of destruction of ascorbic acid
caused by pasteurization in the absence of catalytic metals was thought to
depend upon the previous exposure of the milk to light.

neednan (1937) stated that milk pasteurized for 30 minutes by the
holding method retained over 70 per cent of its original ascorbic acid
content. Schlimmer and associates (1932) stated that short-time cooking
did not reduce the ascorbic acid very much. In this experiment the

holding method gave lowered ascorbic acid content.
Effect of metal contamination on the stability of ascorbic acid.

nearly all investigators are agreed that small amounts of capper
catalyze oxygen uptake of the vitamin. several other metals will
naturally have the same effect although.probably not so pronounced as the
capper.

Kellie and Zilva (1985) showed that ordinary laboratory distilled
water contains enough quantities of copper and iron. especially copper.
to catalyse the irreversible oxidation of dissolved ascorbic acid. Sharp

(1936) showed the accelerating effect of soluble capper on the oxidation

- 13 -

of ascorbic acid. The following figures show their average results
expressed as mg. ascorbic acid.per liter.
Fresh.pasteurizcd milk........6....................20.l
After holding for 3 days at 85 F.

h'OOOOOO00.000.000.000...000......0....0.00.00.00011. 3

Past. (1450’. “wmin.)OOCOOOOOOOOOOIOOOOO00.00.00.11.0
- 0 13 mg. Cu per liter..... 1. 7

lhitnah and associates (1936) demonstrated that the addition
of copper produced increased losses of ascorbic acid both in raw and
in pasteurized milk. However. the presence of copper did not result
in a serious loss in the raw milk. ‘At least 20 times as much iron or
20 times as much chromium or nickel as capper were required to pro-
duce comparable destruction of ascorbic acid. copper (or brass) was
probably 100 fold more soluble in milk than stainless steel. 0f the
four metals studied. copper. iron, nickel. and chromium. capper was
the only one which brought about a decreased ascorbic acid content in
both.pasteurized and in stored milk. This fact substantiated the
earlier inference that excessive losses of ascorbic acid after
pasteurization and storage indicated contamination with copper. Copper -
supplements did not at any time produce a detectable difference in the
losses of ascorbic acid content of fresh raw milk or freshly pasteurized
milk.

Schlimmer and associates (1932) have shown that the metals.
copper and silver. are harmful to the ascorbic acid content of milk.
nickel. chromium. and aluminum did not show any effect upon the ascorbic

acid content of milk.

Influence of aeration upon the ascorbic acid content of milk.

A report of research on the effect of aeration upon the ascorbic

-13-

acid content of milk or other food products could not be found in the
literature. It is obvious. though that our present knowledge of vita-
min 0 and its chemical characteristics permits us to conclude definitely
that aeration will destroy ascorbic acid.

Already Event-Gyorgi (1931) showed that hexuronic acid (ascorbic
acid) showed a fairly high reduction potential. It lost two hydrogen
atoms upon mild oxidation and. if once slightly oxidized. could be re-
duced again by treatment with hydrogen sulphide.

That ascorbic acid can be oxidized several ways. both reversibly
and irreversibly. is a well known fact.

Roe and Barnum (1936) demonstrated that reversibly oxidized
ascorbic acid has approximately one-fourth the antiscorbutic potency of
ascorbic acid in its reduced form when administered to guinea pigs.

Tauber and Kleiner (1935) have advised a method for determining reversibly
oxidized ascorbic acid. This is accomplished by treating the substrate
which contains the reversibly oxidized ascorbic acid.with hydrogen sulphide
which reduces the oxidized form. The reduced form then can be titrated
as‘usual.

Sharp (1938) recently recommended a method for determining
reversibly oxidized ascorbic acid in milk in which method the same principle
is used.

Smith and King (1931) found that the purified preparations of
ascorbic acid can be held for a period of two or three weeks without
serious loss of activity. if preperly protected from oxidation during
laboratory manipulations and subsequent storage by the use of an atmos-
phere of nitrogen or carvon dioxide. Day ice proved to be particularly

suitable.

_ 14 -

Tillman and associates (1932) demonstrated that ascorbic acid
could be oxidized reversibly and irreversibly. The first kind of oxi-
dation could be brought about by iodine. by hydrogen peroxide. or by
2-6 dichlorOphenolindophenol and.then reversed with hydrogen sulphide.

whereas oxidation by atmospheric oxygen produced irreversible oxidation.
Influence of light on the ascorbic acid content of milk.

Kon and Watson (1936) have shown that milk giving a positive
chemical test for vitamin C failed to reduce endOphenol reagent after
exposure to daylight through glass. The reducing power of the ascorbic
acid could be restored to a varying degree by treatment with hydrogen
sulphide. but irreversible losses always took place. Visible light.
composed of rays of short wave length. chiefly blue and.violet. was
mainly responsible for the reaction. Ultraviolet rays were probably
active. Yellow and red rays were almost always without effect. The
action of light did not take place in the absence of oxygen. The
change which took place when milk was exposed to sunlight was oxidation
which was an actinic activation of oxygen. The ascorbic acid was
oxidized to dehydroascorbic acid in the reversible form. Synthetic
ascorbic acid added to milk behaved towards light in the same way. Pint
bottles of milk exposed for one-half hour in the sun and then stored for
one hour in the dark lost fully one-half of the original antiscorbutic
properties. It is interesting to note the statement in this work that
the amount of destruction of ascerbic acid caused by pasteurization in
‘the absence of catalytic metals depended upon the previous exposure of

the milk to light.

- 15 -

Ion (1987) confirmed the above findings. Riddell and lhitnah
(1936) observed that exposure to sunlight might be destructive to

ascorbic acid.

 

Effect of freezipg on ascorbic acid.

Lillegren (1986), by histolOgical comparison of the degree of
healing induced in scorbutic guinea pigs by daily administration of
milk and orange Juice both fresh and stored at --30° c. for 24 to 36
days. demonstrated that the animals receiving the fresh material
reached a given stage of healing in about two-thirds of the time needed
by those receiving the frozen material.

Granat reported (1936) that lemons. frozen in their natural
state and stored from October to May at temperatures below -40° 0.. re-
tained their antiscorbutic activity as shown by the prevention and cure

of scurvy in guinea pigs by daily dosages of three grams.
Infferent relationships feund in regpect to ascorbic acid.

Relationships between ascorbic acid and oxidized flavor. According to
Sharp and associates (1936) there was a positive correlation between the
rate of oxidation of ascorbic acid and the rate of deve10pment of the
oxidized flavor.

In this work the addition of 100 mg. of ascorbic acid per liter
greatly retarded the deve10pment of the oxidized flavor in milk.

lhitnah and associates (1937) in a study of the naturally
occurring vitamin C in milk of different breeds found that the frequency
with which oxidized flavor occurred.was in the following order: Holstein.

Ayrshire. Guernsey. Jersey. Within the breed there was no relation

- 15 -

between the amount of ascorbic acid in the milk and the development of
the oxidized flavor.

Anderson (1937) found that carrots fed at the rate of five pounds
daily greatly reduced susceptibility of the milk to become oxidized.
Carrots were fed to one cow in this experiment and apparently did not
give oxidized flavor in milk. This is explained according to Anderson as
being due to the fact that carrots were a fairly good source of vitamin C.

Brown and associates (1937) have shown that feeding tomato Juice
or lemon Juice reduced or eliminated the susceptibility of milk to become
oxidized and have explained this effect on the vitamin 0 contained therein
since they fbund.that the feeding of 0.6 grains of ascorbic acid daily

greatly reduced the milk's susceptibility to become oxidized.

Relationship between vitamin C and.flavor in milk. It might be expected
that a high vitamin C content in foods such as milk would.give good flavor.
Garret (1937) and associates found a significant correlation coefficient
of over 0.6 based on data from all cows,‘28, indicating that a definite
relationship existed between vitamin C and flavor of milk from individual
cows. Ihen the vitamin C content was high the flavor of the milk was

usually good.

Relationship between ascorbic acid.and enzymes. Szent-Gyorgi (1932) gave
evidence that there was in the cabbage leaf a highly active enzyme which
in the presence of oxygen rapidly oxidized hexuronic acid (ascorbic acid)
to its reversible oxidation.product. The name hexoxidase was used for
this enzyme.

Ianber and Ileiner (1935) succeeded in preparing from hubbard

squash an enzyme which caused the ascorbic acid to react directly with

free oxygen.

-17—

Bessey and King (1934) in the chemical determination of vitamin
C used trichloracetic acid in macertaing and extracting the tissues. as
the acid protected the vitamin C from rapid oxidative reaction which
occurred upon liberation of the active enzymes from the macerated tissue.
cells.

Shrinivasan.(1935) proved the presence of an enzyme in drumsticks
Juice which was inactive when treated with trichloracetic acid. Roe
and Barnum (1936) attributed the antiscorbutic effect of reversibly oxi-
dized ascorbic acid to an enzyme in the blood which had the power to re—
duce the reversibly oxidized form of the ascorbic acid.

Stotz and associates (1937) attributed the catalytic activity of
squash and cauliflower Juice on the oxidation of ascorbic acid to a
specific oxidase. which was probably a compound of the capper present
and some protein materials. The catalytic prOperties of capper were
changed greatly by the presence of proteins. A mixture of cOpper and
albumin assumed the characteristic preperties of the enzyme. It dis-
played.an optimum pH similar to that of the enzyme: was inactivated.by
heat and acid: and showed similar velocity relations to the quantity of
substrate. ,

Sharp (1986) believed the oxidation of ascorbic acid in milk
could be explained by assuming the presence of an ascorbic acid oxidase
the action of which was markedly accelerated by traces of dissolved
copper. He believed that slight destruction of the enzyme ascorbic
acid oxidase occurred by 30 minutes' treatment at 62° to 63° C. Heating

for 30 seconds or longer at 77° 0. destroyed the enzyme completely.

..18-

Hopkins and Iorgan (1936) have reported the isolation of an
enzyme which oxidized.ascorbic acid from the cauliflower. which they
believed was identical with the hexoxidase of cabbage described by
Szent-Gyorgi (1932) and probably also with the similar enzyme isolated
from squash by Tauber and xieiner (1935).

Sharp and associates (1986) explained the rate of oxidation
of ascorbic acid in milk by assuming that the milk contained an enzyme
which catalyzed the oxidation of ascorbic acid. This was probably not
the same which catalyzed the oxidation of the fat to produce oxidized
flavor. However. the two enzymes were quite similar in their preperties.

Dam and Satterfield (1937) stated that the postulation by Sharp
(1986) of the presence of an ascorbic acid oxidase in milk'was difficult
to reconcile with the observation that raw milk lost only 50 per cent
of its reduced ascorbic acid on standing for three days at 3° C.

However. in view of the fact that several investigators have
found enzymes that did oxidase the ascorbic acid. it could be Justly
expected that milk also contained an enzyme that acted similarly. That
this enzyme was the same as that which Reads (1932) ascribed as causing

the oxidation of the fat in milk remains to be demonstrated.

Relationship between ascorbic acid and glutathione. Gara and Giani (1934)
found in the outer layers of the renal gland a substance which inhibited
oxidation of ascorbic acid. Glycokoll alanin.showed some inhibiting
action: aldenyl acid showed a little stronger inhibiting action: while
cystine. cysteine. and.aspecially glutathione. exhibited a very strong

inhibiting action. Glutathione. even in a concentration of 1/100,ooo

-19-

molar. showed a very strong inhibiting action. Bessey and King (1934)
showed that there appeared to be some relationship between the ascorbic
acid and glutathione contents of the suprarenals. Probably the auto—
oxidation of ascorbic acid.under the catalytic influence of heavy
metals is impeded by glutathione. Similarly the auto-oxidation of
adrenalin is inhibited by ascorbic acid and also by glutathione and
cysteine. Bessey and.King also demonstrated the protective action of
the glutathione in retarding the auto-oxidation.of ascorbic acid.
Either substance alone readily took up oxygen. They stated that the
stability of the heavy metal complex linkage decreased in the following
order: thio. ascorbic acid. adrenalin.

Borsook and Jeffrey (1936) in experiments on blood. urine and
tissue slices of animals showed that the protective action of ascorbic
acid consisted of a reduction of the reversible oxidized ascorbic acid.
This reduction can be affected rapidly by glutathione. Guzman and
associates (1936) showed that fluids of animal and of some vegetable
origin. which contain appreciable quantities of ascorbic acid. belong
to a group of biological fluids which possess inhibiting mechanism. thus
protecting the ascorbic acid from-oxidation. This inhibiting action.
which is caused by a variety of catalysts. mostly cOpper and hemochromogen.
was probably due to glutathione.

Iodometric titrations for determination of glutathione in tissue
extracts has been described by Gladys and associates (1932) Gara and
Giani (1934), and Queneei and Wacholder (19:35).

Buruinana (1937) reported that he cauld.not find glutathione in

humans'. cows'. or buffaloes' milk. but was able to find it in maret'

milk.

-20-

.cher aspects of ascorbic acid. Recent studies on ascorbic acid have
shown that under certain conditions ascorbic acid could function as a
catalyst or co—enzyme. Hopkins and Morgan (1936) demonstrated that the
enzyme which oxidized ascorbic acid rapidly to dehydroascorbic acid
brought about the oxidation of reduced glutathione only in the presence
of ascorbic acid. Under these conditions the oxidation of the ascorbic
acid itself began only when that of the glutathione was almost complete.
This was due to the continuous reduction of the dehydroascorbic acid.
Stotz and Harper (1937) made similar conclusions which might
indicate that the ascorbic acid was an important catalyst in biological

oxidations.

- 21 -

PROCEIURE

The procedure followed in the direct determination of ascorbic
acid in milk was the same as recommended by Sharp (1938). This method
was checked occasionally by adding commercial ascorbic acid to milk and
then redetermining the amount of ascorbic acid present by titration.
The redetermination always checked within five per cent. On a few
occasions the proteins were precipitated with trichloracetic acid. How»
ever. this method did not give results any better than those obtained by
direct titration.

The method employed by Gladys (1936) in determining blood glu-
tathione was used in studying the glutathione of milk. The method was
checked by adding glutathione to water. In the redetermination. the
glutathione checked within five per cent.

Leucocytes and bacteria were counted according to the Breed
method as outlined in the Standard Methods of Milk Analysis. The
methylene blue tests were made also as outlined by the Standard Methods
of Milk Analysis.

The per cent lactic acid was determined according to the Mann‘s
acid test.

Aeration of milk was accomplished by passing the milk over an
experimental surface cooler.

Holder pasteurization of small lots of milk was accomplished
in glass bottles which were partially submerged in a hot water bath.

The commercial milk was pasteurized in glass or allegheny metal lined

- 22 -

vats. Flash pasteurization was brought about in a specially constructed
pasteurizer in which the milk was heated to any desired temperature be-
low 100° C. and cooled to below 5° C. in the course of 5 to 15 seconds.
The apparatus was so constructed that all milk was heated and could be
controlled. when desired. to a temperature within one-tenth of a degree.
Controlled auction was used to draw the milk through the apparatus at
any speed and. hence at any temperature desired.

The experimental irradiation of milk was accomplished by pass-
ing the milk over one side of a surface cooler toward which ultra violet
rays from the Alpine Sun Lamp of the Hanovia Chemical Manufacturing
Company was directed. The irradiation was performed at the highest in-
tensity.

The copper solution was made up as follows: one gm. of CuSo45H20
was made up to 100 ml. using distilled water. thus forming the stock
solution. One ml. of the stock solution was then diluted to another 100
m1.. using distilled water as before. One ml. of this second solution
was finally added to 200 m1. of the milk. which resulted in the addition
of 13 mg. per cent of copper to the milk.

The milk which was used in studying the seasonal effect upon the
ascorbic acid was obtained both from the dairy barn and from the college
creamery. The milk from the dairy barn was always titrated within two to
four hours after it was drawn from the cow. The milk was stored in a
refrigerator at 49 C. and titrated daily for four consecutive days. The
milk from the college creamery was titrated within six to eight hours
after being processed.

The oxidized flavor of milk was classified as follows: e + a

strong to very strong flavor: + + distinct to pronounced oxidized flavor;

+ slightly oxidized flavor; ? doubtful oxidized flavor: - no oxidized flavor

O

EIPERIMENTAL RESULTS
Part I

PROCESSING, ASCOEBIC ACID,‘AND THE OXIDIZED FLAVOR OF MILK

The effect of copper upon the stability of ascorbic acid and
upon the development of the oxidized flavor when the milk was
pasteurized at high and low temperatures.

Individual samples of milk were obtained from 20 cows of the
college herd and were cooled and treated at once. Each sample was di-
vided into five lots: one lot served as a control: two lots were
pasteurized at 63° C. for thirty minutes. after which 0.13 mg. per liter
of capper was added to one lot: while the two remaining lots were
pasteurized at 75° C. for thirty minutes. after which copper was added
to one lot as above. Titration. for the ascorbic acid were made daiky.

The milk was studied organoleptically for oxidized flavor on the fourth
day. The data obtained are presented in Tables 1. 2, 3. 4. 5 and 6, and
summarized in Table 7, and are shown graphically in Figures 1 and.2.

An examination of the data of Table 3 shows the catalyzing effect
of copper upon the oxidation of ascorbic acid when the milk was pasteurized
at 63° C. for thirty minutes. This effect was very marked after the second
and third days. On the fourth day no appreciable amount of ascorbic acid
remained. Coincident with the marked decrease in ascorbic acid, the
strong develOpment of oxidized flavor was usually noted. However. this
observation was not consistent in all samples as later it will be shown
that with some milk one may observe very pronounced decreases in the

ascorbic acid content. also in the presence of copper, without any notice-

- 24 -
able develOpment whatsoever of the oxidized flavor.

Some destruction of ascorbic acid. resulting from 63° C. holder or
pasteurization. without c0pper contamination. was apparent especially on
the first and second days. However. on the third day the pasteurized
milk. although relatively low in ascorbic acid. had a larger amount of
ascorbic acid than did the control samples. In other words. the stability
of ascorbic acid was slightly less in raw milk over a three day storage
period than similar milk pasteurized by the holder method.

Pasteurization of milk at 75° C. for thirty minutes resulted in
a slightly greater initial destruction of the ascorbic acid than
pasteurization at 68° 0.. but. on the other hand. the stability of the
ascorbic acid present. after the high heat treatment. was greatly in-
creased over that when the low temperature was employed. However. there
was a slight decrease in the amount of ascorbic acid present during
storage as shown in Table 4 and Figure l. The increased stability of
ascorbic acid at the 75° C. exposure was shown more strikingly in the
cases where copper was added to the milk. There was a slight decrease
in the ascorbic acid present compared with the milk that had been heated
to the same temperature. but without any addition of cOpper. Oxidized
flavor did not develop at all in the samples thus high heat treated even
in the presence of copper.

An examination of Tables 1. 2. 3. 4 and 5 shows that milk from
certain cows exhibited a continuous and remarkable stability in respect
to ascorbic acid. regardless of the different treatments. This stability
was especially noticeable in the case of cows number 80. 111 and 116. a

Guernsey and two Jerseys. respectively. This outstanding stability of

the ascorbic acid of some cows' milk is demonstrated in Table 6. In
view of these observations. and mindful of the relationship between
the destruction of ascorbic acid and the development of oxidized flavor.
it might be expected that these samples would not show any tendency to
develop the oxidized flavor. However. an examination of the oxidized
flavor data on milk of the different treatments showed that milk from
these three cows had Just as great a susceptibility for oxidized flavor
as did that from any other cows. In fact. milk from cow number 116 had
already shown. while raw. a tendency to develop the oxidized flavor.
These effects of capper and heat treatments upon the stability
of ascorbic acid in milk from individual cows are summerized in Table 7

and are presented graphically in Figures 1 and 2.

Oxidized
flavor after

-25-
mg./L)
*Third

Dal

Second

my

Ascgrbic acid (

8
8

 

First

 

oxidized flavor in raw milk from individual cows after

The ascorbic acid content and the deve10pment of the
various storaggpperiods.

 

 

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Table 1.
Cu

(Ho.

9.. 9.. 9.. ?. ?.
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1 12211 111..

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565551151500505505

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22108803497628.7737
121111222111111111

16.5

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6&8e80fl144n51enue6nve1n6e09e21flw
1211122222 112221221

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11111

111
116
117
197
287
238
239

no
mo.

 

 

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Table 2. The ascorbic acid content and the deve10pment of the
oxidized flavor in holder pasteurized milk from
individual cows after various stor_a§e_p_eriods.

8 8 —

Cow x_ Ascorbic acid (mg. IL) : Oxidized

(No. ) 8 Second 8 Third 8 Fourth 8 flavor

: Day 8 nay 8 my 8 after
8 8 8 8 four days
8 8 8 8
l3 8 16.5 8 9.3 8 5.9 8 «-
30 8 22.0 8 19.7 8 12.7 8 -
36 8 9.0 8 2.9 8 1.9 8 -
37 8 9.0 8 3.9 8 1.9 8 'f
41 8 17.5 8 14.7 8 10.8 8 -
42 8 14.0 8 8.8 8 5.9 8 -
100 8 20.0 8 14.7 8 12.8 8 -
111 8 18.5 8 18.7 8 18.7 8 7
116 8 23.0 8 20.6 8 18.1 8 -
117 8 19.5 8 16.7 8 8.8 8 1- 7
134 8 14.0 8 10.8 8 6.8 8 -
142 8 16.0 8 10.3 8 5.9 8 -
174 8 11.0 8 5.4 8 2.4 8 -
180 8 18.0 8 12.3 8 9.8 8 -
190 8 17.5 8 13.3 8 11.3 8 «-
197 8 18.5 8 13.7 8 10.8 8 -
237 8 10.5 8 2.9 8 2.9 8 -?
238 8 17.0 8 12.9 8 10.8 8 -
239 8 16.0 8 7.8 8 3.9 8 -
300 8 161; 8 14.7 8 11.8 8 -
8 8 8 8
Ave. 8 15.9 8 11.3 8 8.7 8

 

- 28 -

 

The ascorbic acid content and the development of the

oxidized flavor in copper contaminated.holder

 

pasteurized milk from individual cows after various

storagepperiods.

 

 

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- 29 —

 

 

 

 

 

 

 

 

Table 4. The ascorbic acid content and the development of the
oxidized flavor in higpptemperature (75° C. - 30 min.)
pasteurized milk from individual cows after various
storgge periods.

8 8

Cow 8 Ascorbic acid (Mg/L) 8 Oxidized

(No.) 8 Second 8 InfiThird 8 ’Fburth 8 flavor after
8 thy 8 Ihy 8 Day 8 four days
8 8 8 8

13 8 13.5 8 11.3 8 10.3 8 -

3O 8 19.0 8 18.2 8 16.2 8 -

36 8 11.0 8 9.3 8 8.8 8 -

37 8 12.5 8 10.8 8 9.3 8 -

41 8 15.5 8 12.8 8 12.8 8 -

42 8 14.5 8 13.7 8 11.8 8 -
100 8 17.0 8 16.2 8 14.7 8 -
111 8 18.5 8 18.7 8 16.7 8 -
116 8 18.0 8 17.2 8 14.7 8 -
117 8 17.0 8 13.7 8 11.8 8 -
134 8 15.0 8 12.8 8 10.3 8 -
142 8 11.0 8 10.3 8 10.3 8 -
174 8 12.5 8 10.8 8 10.3 8 -
180 8 14n5 8 14.3 8 12.2 8 -
190 8 17.0 8 14.3 8 13.7 8 -
197 8 14.5 8 14.3 8 12.8 8 -
237 8 13.5 8 11.8 8 9.3 8 -
238 8 15.5 8 14.3 8 12.8 8 -
239 8 16.0 8 14.3 8 12.2 8 -
300 8 12.5 8 12.8 8 10.8 8 -

8 8 8 8

Ave. 8 14L9 8 13.6 8 12.1 8

 

- 30 -

The ascorbic acid content and the development of the

"’ 80 mine)

 

(750 c.

oxidized flavor in high temperature

 

Table 5.

 

pasteurized copper contaminated milk from individual

cows after various storage periods.

 

 

 

flavor after

three days

Oxidized

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variation in the stabilitx_of ascorbic acid of milk from

Table 6.

 

individual cows as shown bx_high heat treatment and storage.

Ihfference as a result
of heat treatment
Increase : Decrease

 

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117
134
174
180
190
197
237
288
239
300

142

 

Ave.

 

- 32 -

Table 7. She effect of various treatments of milk from individual

cows on the stabilitzpof the ascorbic acid. {Average of

gilk from 20 cows).

 

Treatment : Ascorbic acid (Mg./L) after
3 First ray : Second Day : Third lay : Fourth Eaz_
: x : :
Control : 20.7 x 17.1 : 11.0 : 6.2
: : : :
Past.°62.8° c. - 30 min. : - : 15.9 : 11.7 : 3.7
: : x :
Past. 62.8° c. - so min. : : : :
plus copper : - : 7.7 : 2.8 : 0.0
8 8 8 8
Past. 75° c. - 30 min. : - : 14.9 : 13.5 : 12.1
Past. 75° C. - 30 min. : : : :
plus c0pper : - : 14.6 : 12.9 3 10.9

 

-33...

 

 

 

 

 

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Figure 1. Showing the relative stability of ascorbic acid in milk
processed at different temperatures with and without the
addition of copper.

- 34 -

25"

 

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Figure 2. The stability of ascorbic acid in milk from individual
cows when treated for one-half hour at 63° C. and 75° C.

The effect of flash heatinggof milk at various temperatures

upgn the stability of the ascorbic acid.

Samples of milk from individual cows were flash pasteurized at
various temperatures from 600 to 97° C. for, and cooled within,a
period ranging from five to fifteen seconds. Following pasteurization,
the samples were stored and titrated daily for ascorbic acid. The data
secured are presented in Tables 8 and 9 and.are shown graphically in
Figures 8 and 4.

An examination of these data shows that at least six effects
were noted as a result of flash pasteurizing. at high temperatures, milk
from individual cows. These are: first, the milk from all cows did not
respond identically as to the stability of the ascorbic acid at similar
heat treatments: second, flash pasteurization temperatures ranging from
85° to 95° C. had the most pronounced stabilizing effect upon the ascor-
bic acid; third, the ascorbic acid stability passed through a mixima as
the flash exposure extended from 60° to 97° 0.: fourth, flash exposures
below 750 C. had a less stabilizing effect upon the ascorbic acid than
that existing naturally in the raw milk; fifth, the critical flash ex—
posure minima for ascorbic acid stability were between 75° and 85° c;
and sixth, milk having a naturally low ascorbic acid content usually
showed the largest increased stability of ascorbic acid upon flash heat-
ing than did those samples having a naturally high ascorbic acid value.

These relationships can best be observed in Figures 5 and 6.

Increase or

- 36 -
November.

L—

Effect of flash heating at different temperatures upon the
ascorbic acid content of milk from individual cows during_

 

 

 

Table 8.

decrease from
control (%)

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t 0000000 0257990 2488754 0002770 0000480
M5 1111111

1 00 00 00 00 00 00 00 .0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 .0 .0 00 '0 00 00 00 00 .0 00 00 .0 00
. 0031510 3.532235 5994.940 33.59285 0959499
gm eeeeeee eeeeeee eeeeeee eeeeeee eeeeeee
M N 0027580 3534425 5789897 2380425 0080000
(4 1111 1111111 111 111
d 0. 00 00 0. 0. 00 00 00 00 .0 .0 00 .0 00 00 .0 00 .0 0. 00 00 .0 00 00 00 0. 00 00 00 00 00 00 0. 00 00 00 0. 00 00
.1 9451159 5927234 1594900 5517739 9549438
Cd eeeeeee eeeeeee eeeeeee eeeeeee eeeeeee
arm 1178881 9055541 5990998 9955541 1521123
03 111111 1112111 11111 1111
1 De .0 to ee 00 00 00 00 O. I. 00 00 ee 06 00 ee 00 O. 00 90 00 00 90 00 00 O. 00 00 00 ee 00 O. .0 00 00 00 0. 00 .0
.0 9887224. 2511527 5050555 5155555 8757773
rdV1 eeeeeee eeeeeee eeeeeee eeeeeee ee eeee
o.m.u «0390.1.1.nv9. «unusuac5.4.9. ounc1.nvmw 5.4.7.9.svsu4. 3.1.4qququoun.
” 111 1111111 1222 1111111 111111
A 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0. 0. 00 .0 00 00 0. 0. 00 00 00 0. 0. 00 00 00 00 00 00 00
88883810 788.585588 7777777 3385587 3383333
.5 e eeeee eeeeeee. eeeeeee eeeeeee eeeeeee
S 1 34535 8784758 3232223 1999 01 2544455
1 1111111 1111111 2222222 2111 22 1111111
00 .0 00 00 00 00 .0 00 00 00 .0 00 00 00 00 00 00 00 0. 00 00 00 00 00 00 .0 00 00 00 00 .0 00 00 00 00 00 00 00 00 0.

5 1 555

0

U) % 7 w .59 5 7 M“ 4. .1
2855157 3775157 75885 2555059 1358047
0 5789991 5789991 57 9 1 57899 1 57 9991
r0 ' 0' 0' on 0' .0' n 0
0....5-910- ..n. .rO- - - r0 . a - u. Ion. ...r
O C tC .....C 55 t0 5.....C 5 t
I\ 5 39 .50. 544935n 54 e e473 55594. on 24. e935n
54.58390 5788990 5739990 5788950 5738990
7 9 C C 55 c 90 8 0

 

Table 8 continued from previous page.

 

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64- 67
74-75
83- 85
90- 91
94- 95
96- 97
Control

 

- 33 _

 

Effect of flash heating at different temperatures upon the
ascorbic acid content of milk from individual cows durin

Table 9.

g

-:——_

Julz.

 

 

Ascorbic acid (MglLfi

( )

Increase or
: decrease
: from control
0
O

2

day

Ave.

to 00 00 o.

 

Cow 246

60 - 63
7O - 72

 

Te?gega§ure

3549
1.1.1.
216
660.0.
1
. . . o
r
nvooau+u
.8.8.9 n
o
no

 

Cow 226

60 - 62
69 - 71
79 - 81

nonvnvnunu

O 0 O 0 0

9.1. ”(.6

. 2 3 3

00 00 00 00 .0 00

.o.o...6.4_o

0 0 0 0 0 O

ouavnunuanU

1.1.1.1.1.1.

00 00 00 00 00 .0

7.0.0.1uqu92

0 0 0 0 0 0

”(no.o.o.o.8
1.1.1.1.

00 00 00 00 00 00

nonunca2n2.o

0 0 0 O 0 0

88 - 90
94 - 96
Control

Cow 46

 

199991.
0 o o o o 0
1.33231
1.1.1.1.1.1.
”099.4.1
5789

1
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F
00084....
67889m

C

 

Cow 184

61 - 62

71 - 74
79 - 81

88 - 90

2
0
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6
9
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C

 

Cow 14
60 - 62

4.9u1.
2

976
2

761..

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79990
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8O - 83

160

o o o o
6900
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169

one.
7809
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88 - 90

96 - 97
Control

 

Cow 146

60 - 62

00 00 00 00

832777

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6899

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70 - 73
79 - 81

88 - 91

94 - 96
Control

 

0

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- 39 -

 

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4.] ‘¢‘}/*7

Figure 4. Showing the effects of various flash pasteurizing temperatures
upon the ascorbic acid content of milk from cow No. 116 when
stored for several days.

 

 

7

 

Figure 3. Showing the effects of various flash pasteurizing temperatures
upon the ascorbic acid content of milk from cow No. 144 when
stored for several days.

- 41 -

 

 

 

 

 

Jo £5" 7w 7.5“ ’0' If“ 70’ “’7
f . W /"/’/

Figure 5. Showing percentage va iations from the control samples of the
ascorbic acid content of individual cows milk when treated at
different temperatures (November).

-42..

 

 

 

 

 

 

*40
i
E
3
+40 * u *
+30 ~ ~ ~ ~ ,
+20 a ._1 a - a-
\v/a a g ..- a-
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1 I . I 1'
! 9 s ; i ‘
’40 ' ‘ ‘ '
(0' 55" 7a ' 7r” J’a " if d 90 ’ 95"

Figure 6. Showing percenteg: variations from the control sample of

the ascorbic acid content of individual cows' milk when
treated at different temperatures (July).

743'

The effect of various heat treatments upon the ascorbic

acid content of milk and upon the development of oxidized

flavor when copper was added before and after heating.

Stability of ascorbic acid. This experiment was run in June. 1937. The
milk was obtained from the College Dairy Barn and was mixed from three
different breeds. namely. Jersey, Guernsey and Brown Swiss. The milk
was treated and the ascorbic acid value determined the same day as milk-
ing.

The milk was divided into two groups, I and II. of six lots each,
A. B, C. D. E and F. The lots were again divided into six portions,

1, 2, 3, 4. 5 and 6 of 200 ml. each. To the six 200 ml. portions 0. 13,
26, 39. 52 and 65 mg/% of copper were added respectively. The copper
was added to the milk comprising Group I before heating and to that of
Group II after heating. Lots A. B, C, D, E and F of both the Groups I
and II. were heated for exactly ten minutes at 65°. 70°. 75°, 80°. 85°
and 90° C.. respectively.

The milk was heated in glass bottles in steam-heated water baths,
and following heat treatment, was stored at 4-0 C. When the capper was
added after heating, it was added to the milk while still warm immediately
after heating, and then the milk was put into the refrigerator.

The determinations for ascorbic acid were run on four consecutive
days. The samples were scored for oxidized.flavor after three and after
four days.

The experimental data are tabulated in Table 10 and plotted on
Figures 7. 8. 9 and 10.

Figure 7 represents the picture obtained by plotting the ascorbic

-fi”

acid content of milk against temperatures for the different amounts

of copper added before heating. From this figure may be observed that
80° C. was found to be the optimum temperature for the stability of
ascorbic acid regardless of the different amounts of cOpper added.

When increasing amounts of cepper were added. the ascorbic acid content
decreased, but, in all cases, 800 C. was the Optimum. temperature of
ascorbic acid stability.

From Figure 8. representing similar heat treatments except that
the cepper was added after heating. may be seen also that at 80° C. the
maximum stability of ascorbic acid.was secured. However. as it may be
seen. the ascorbic acid did not decrease much after the temperature of
80° C. had been exceeded.

In studying these figures some characteristic differences
worthy of note should be mentioned. The figure representing the samples
to which the copper was added after heating shows very little difference
in the ascorbic acid content for the different additions of copper at
temperatures at 80° C; and over, while the figure representing the
samples to which the copper was added before heating shows a very wide
variation in the ascorbic acid content for the different additions of
copper at temperatures of 80° C. or over. These differences between the
two series show up under all conditions studied with no exception.

In the figures representing copper additions before and after
heating. similar wide variations in the ascorbic acid content of milk
heated to temperatures below 80° C.. for instance. 65° C., were noted.

However. when the temperature of 80° C. had been exceeded, those samples

O

- 45 -

which had had copper added after heating showed very little variation
in the ascorbic acid content for the different additions of c0pper.
while those that had had copper added before heating showed the same
wide variation as those heated to temperatures below 800 C.

From the above discussion it follows that a temperature of
80° C. for 10 minutes seems to be an exposure during which marked
changes take place in milk as far as factors affecting the stability
of ascorbic acid are concerned.

The ascorbic acid content of milk to which different amounts
of c0pper had been added after heating tended to remain at a relatively
high level at the 800 C. exposure. This tendency may be noted from the
data presented in Tables 11 and 12.

All the data indicated that copper, when added below temperatures
of 80° 0., whether before or after heating. had the same destructive
effect upon the ascorbic acid. When the milk was heated at temperatures
beyond 80° C., capper. regardless of the time added, had little effect
upon the destruction of ascorbic acid.

COpper added before heating had quite a different effect at the
800 C. exposure on the ascorbic acid stability than when added before
heating. The decrease in ascorbic acid was smaller at temperatures above
800 C. than at temperatures below 80° C.. but was considerably larger
than those that had had the copper added after heating.

The differences in the ascorbic acid values between the controls
and the samples to which copper was added increased after the second day

in the same relative amount both in those samples which had had the copper

-46-

added before or after heating at temperatures above 800 C. This
peculiarity can Justify the conclusion that the difference between
the samples which had had the c0pper added before or after heating at
temperatures above 80° C. was due to causes during heating while the
copper was present. and was due to a very small extent. if at all.
to any other cause.

Data showing the stability of ascorbic acid, when c0pper was
added after heating, are illustrated in Figure 9. It appears from
these graphs of this figure that a temperature of 80° C. for ten minutes
is consistently the most favorable to the stability of ascorbic acid.
Temperatures of 85° c. and 90° c. are also good for stabilizing the
ascorbic acid ranking in respective order as to their importance. All
three of these temperatures. with 80° C. ranking first. apparently have
a stabilizing effect upon the ascorbic acid, regardless of the amount of
c0pper present.

A temperature of 75° 0., however, shows some peculiarities.
This temperature is apparently able to stabilize the ascorbic acid just
as well in general as the three above mentioned temperatures when cOpper
is present in amounts of 26 mg/% or less, but if the copper content is
higher. the temperature exhibits a sudden drop in its stabilizing effect.
In other words, 750 C. with heating time of 10 minutes. is one of the
temperatures at which important changes take place regarding ascorbic
acid. Probably these changes occur between 75° C. and 80° C.

The lines indicating the ascorbic acid values for 55° c. and
70° C. are equally interesting, as noted in Figure 9. These temperatures

showed eXtremely small ascorbic acid stabilizing power. Their lines

lying considerably lower show entirely different characteristics than
do the lines for the other temperatures. Also they follow each other
very closely.

The temperatures show the same characteristics in general in
regard to the stability of ascorbic acid when capper was added before
heating (Figure 10). Those of 65° C. and.70° C. follow each other
closely and show different characteristics that do the other tempera-
tures. That of 80° C. also was the temperature at which the most
stabilizing effect regarding ascorbic acid occurred. The other tem-
peratures, 85° 0., 90° C. and 75° C. rank in the same order, as above

mentioned, in importance.

Oxidized flavor. The theory has been generally accepted that heating

 

to sufficiently high temperatures prevents development of oxidized
flavor even when c0pper was present. Ibta secured on the deveIOpment
of oxidized flavor are presented in Table 10.

The data show that the deve10pment of oxidized flavor tended
to follow the destruction of ascorbic acid, but did not reach a minimum
point of greatest development at any of the temperatures studied. As
the temperatures increased. the milk became less and less susceptible
to oxidized flavor. even in the presence of large amounts of copper.
At temperatures over 85° C. the milk did not develop any oxidized flavor,
when capper was added before heating. When capper was added after heat-
ing. some of the milk became oxidized even at 90° C. as the data of
Table 10 indicates. but to a smaller degree than any milk heated to

lower temperatures.

An important difference between the stability of ascorbic
acid and the deve10pment of oxidized flavor was noted. The ascorbic
acid reached its maximum stability at a temperature below 85° C., at
80° 0. in this experiment. while the oxidized flavor reached a
minimum or failed completely to develop at a temperature above 850 0.,
at 900 C. in this experiment. This observation is of especial interest
in view of the theory that has been prOposed that the enzyme which
causes oxidized flavor is similar in many respects to the one which
oxidizes ascorbic acid. This theory is not here being retired or a new
one prOposed. but it does seem that, upon heating. the stability of
ascorbic acid and the milk's susceptibility to acquire oxidised flavor

have two entirely different critical temperatures.

:flavor

:acid
:(Mg/L)

 

z:Ascorbic:Ascorbic:Oxidized
(Ms/L)

Oxidized :

=(Mg/L)

:C0pper added before heating :zggpper added before heating
:acid

gppn the stability of the ascorbic acid and upon the develop:
:(Mg/L)

ment of the oxidized flavor of milk.

 

 

The effect of adding copper before and after heat treatment

 

Copper:Ascorbic:Ascorbic

:added" :acid

10 min.):

 

 

Table 10.
Treatment
Temp.

(° C.

e. +.+
h +++4+ .70.? +§F 6.0.9. 4.4...-
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r 7?. 7.7.
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00 00 00 00 00 00 00 00 00 .0 00 00 00 00 00 00 00 00 00 00 0O 00 00 00 00 00 00 00 00 00 00 00 00 00
00
w 99949944949944.9499 9499994999999
0. 000 0. 00 00;. 00.. 0 00 00 0. 0 .000 0 O
4.nemuou1.1.ncoe1uoe1.1.7.4.4.1.ac1. 5.4.4.4.92susuqu161.nvcuss
“m 1. 1. 1.1.1.1. 1.1.1.1.1.1.1.1.1.1.1.1.1.
.0 00 00 00 00 00 00 00 00 00 3 00 00 00 00 .0 00 .0 00 00 00 00 00 00 00 00 00 .0 .0 00 00 00 00 00 00
w O5.5050105505055000.30505000000000
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84197694209698864908797789887677
.M 1.1.1. 1.1.1.1. 1.1.1.1.1. 1.1.1.1.1.1.1.1.1.1.1.1.1.1.
1
0000000000000000000000000000300000.000000000000.000000000000000:00000000
nu nu uu nu nu nu uu nu nu nu u. uu nu uu nu uu uu uu uu uu uu nu uu nu nu uu nu nu uu u. nu uu uu nu uu uu un
h +4. ——~+.+.T ,+f——
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M00000...0000000000000000000000000000000000000000800830.3000.0000
r.wl . e . . . . . . . . . . . . . . . . . . . . . . . . . +3» . .
no
00 00 .0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 0O 00 00 00 00 00 00 00 00
.w 994.95 999994944999999990.00004599
0 0 000 O 00 0 0 000000900. .0 Q0 0 O 0 00 0 0
58311 58411275274175431743120643
.M 1 1 111 1.1.111 11111 11..
2
00 00 00 00 00 00 00 00 0. 00 00 00 00 00 00 00 00 00 00 00 00 00 00.00 .0 00 00 00 0. 00 00 00 .0 00 00
WW 00505....05050500005050000050055000
d eeeeeSeoeeeoeeoeeseeeeoeeeeeosee
95165094174299631798755277422077
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.l
00 00 0.0 00 00 00 00 00 00 00 00 00 .0 00 00 00 00 00 00 00 00 00 00 00 00 00 .0 00 00 00 00 0O 00 00 00
12345612345612345612345612345612
00 00 00 00 00 00 '0 00 00 00 00 00 0O .0 00 00 .0 00 00 .0 00 00 00 00 00 00 .0 00 00 00 00 00 00 00 00
5 O 5 O 5 O
6 7 7 8 8 9

 

 

 

 

 

 

6.9
4. 39 Mg. 9
N

8
5. 52
6. 65

10.5

Control
2. 13 Mg %
26 ll

: l.
3.

 

.Key

...

It

- 50 -

Table 11. The difference in ascorbic acid content between control
sample and samples to which 52 mg. % copper was added
at different temperaturesj for the first and second day:
of storage.

 

 

 

 

 

 

 

 

 

 

Sample : Ascorbic acid lst day (Mg/L)

: Temperature (6 C. - lO miniTi

8 65 : 7O 8 75 2 80 : 85 8

: Cu added before heating

: : x a : :
Control : 19.0 x 19.0 : 19.0 : 19.5 : 17.4 : 1
52 mg.% : 5.4 : 4.0 : 11.4 : 14.9 : 10.5 :
Diff. : 15.6 : 15.0 : 7.6 : 4.6 : 6.9 : 1

Cu added after heating

Control : 18.0 : 19.0 : 19.0 : 18.5 : 18.0 : 1
52 mg.% : 7.5 : 9.0 : 14.0 : 17.4 : 17.0 : 1

z : : : : -
Diff. : 10.5, : 10.0 : 5.0 x 1.1 : 1.0 :

a

: Ascorbic acid 2nd day (Mg/L)

: Temperature (0 C. - lO min.y

3 Cu added before heating
Control : 15.9 : 15.9 : 17.9 : 17.9 : 17.0 :
52 mg.% : 1.5 : 1.8 : 4.9 x 11.9 : 10.4 :
Diff. : 14.4 : 14,1 : 13.0 3 6.0 : 6.6 :

: Cu added after heating
Control 3 14.9 : 16.6 : 17.5 : 17.9 : 15.9 :
52 mg.% : 1.9 : 1.9 : 6.9 : 14.8 : 13.8 :

x : : : : :
Diff. : 13.0 : 14.7 : 10.6 : 2.2 : 2.1 :

 

Table 12.

- 51 -

The difference in ascorbic acid content between control
sample and sample to which 65 ggf% copper was added at
different temperatures for the first and second day of

storage.

 

 

 

 

 

 

 

:
Sample : Ascorbic acid lst day (Mg/L)
: Temperaturefi(‘U_C. - 10 min.)
: 65 z '70 z 75 : 80 z 85 : 90
: Cu added before heating
Control 3 19.0 : 19.0 : 19.0 : 19.5 : 17.4 : 17.0
65 mg.% : z 2.5 : 7.0 : 12.0 : 10.0 : 9.5
: : : : : :
Diff. : : 16.5 : 12.0 : 7.5 ' : 7.4 : 7.5
Cu added after heating
Control : 18.0 : 19.0 : 19.0 : 18.5 : 18.0 : 17.0
65 mg.% : 6.0 : 6.5 : 9.0 : 17.0 : 16.0 : 15.4
x : : : : :
Diff. : 12.0 : 12.5 : 10.0 8 1.5 : 2.0 : 1.6
: Ascorbic acid 2nd day Tug.7L)
8 Cu added before heating
Control : 15.9 8 ,15.9 8 17.9 8 17.9 : 17.0 : 15.0
65 mg.% : : 2.5 : 1.9 x 7.9 : 6.5 : 4.9
x : ‘ : : : :
Diff. : 3 15.4 : 16.0 3 10.0 : 10.5 : 10.1
: Cu added after heating
Control : 14.9 : 16.6 : 17.5 : 17.0 : 15.9 : 15.8
65 mg.% : 1.9 : 1.9 : 1.9 : 12.9 : 10.9 : 11.4
Diff. : 13.0 : 14.7 : 15.6 : 4.1 : 5.0 : 4.4

 

- 53 -

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- 55 -

The effect of c0pper and irradiation upon the ascorbic acid
content of raw milk when the cold milk was passed over a
surface cooler.

 

Milk used in this experiment was secured from individual cows
at the College Dairy Barn during the month of August. Immediately after
sampling. the milk was put into a refrigerator at 4° C. where it was
held until treated the next day. The ascorbic acid content was deter-
mined daily thereafter for four days. Likewise, the milk was tested
organoleptically to note the deve10pment of the oxidized flavor. The
data secured are presented in Tables 13, 14, 15, 16 and 17. inclusive.
and are summarized in Table 18 and are shown graphically in Figure 11.

A comparison of the data in Table 14 indicates that aeration of
the cold milk alone was sufficient to cause a considerable reduction in
the ascorbic acid content of the milk. Not only was this effect true as
an average, but also of each lot of milk from the 17 different cows. The
addition of cepper prior to aeration further reduced the ascorbic acid
content of the milk. as shown in Table 15. However, as shown in Table 16
irradiation of the milk had little effect upon the ascorbic acid content
of the milk beyond that resulting from aeration. Likewise, as shown in
Table 17. irradiation of cOpper contaminated milk had little effect beyond
that resulting from the copper alone. These comparisons can be made more
readily from the summaries presented in Table 18. In these eXperiments
irradiation was carried out similarly to that employed in some commercial
plants. namely, irradiation of the milk raw, cold and pasteurizing after-
wards. These data indicate that irradiation, in itself, as it is per-

formed under practical conditions, has no deleterious effect upon the

57-

ascorbic acid present. However, the addition of c0pper to the milk
followed by aeration resulted in a drop in the ascorbic acid consider-
ably below that of the control samples and that of the samples irradiated.
In the case where the milk had capper added to it and then irradiated,
about the same drop in ascorbic acid was noted as in the case where
cepper alone was added. This is another indication that irradiation it-
self has little effect, if any. upon the ascorbic acid of milk.

In these experiments the samples were scored for oxidized flavor
also. However, it was rarely possible to detect any oxidized flavor,
although the milk was irradiated, copper contaminated, and was held as
long as six days.

Despite the drastic reduction of ascorbic acid values resulting
from aeration and from the addition of c0pper, oxidized flavors in milk
were noted rarely, another indication that the develoPment of oxidized
flavor and instability of ascorbic acid are not so closely related as
might be expected.

In this experiment the same interesting peculiarity regarding
different milk's stability in respect to ascorbic acid was noted. Cows
number 25, 80, 111 and 116 seemed to produce a milk which was markedly

stable in respect to ascorbic acid despite the different treatments.

-58—

 

 

 

 

 

 

Table 13. The ascorbic acid content of raw milk from individual cows
when stored at 46—0. for several day_;'
Cow 8 Ascorbic acid (Mgll) -
(NO.) 8 First 8 Second 8 Third 8 Fourth
: daL 8 day : day : day_
14 8 18.0 8 15.0 8 10.6 8 3.2
23 8 16.0 8 12.5 8 7.4 8 2.1
25 8 26.0 8 24.0 8 21.0 8 12.7
30 8 25.0 8 24.5 8 22.3 8 19.1
37 8 21.0 8 19.0 8 15.9 8 9.0
97 8 20.9 8 15.0 8 9.0 8 2.1
111 8 24.0 8 21.5 8 18.1 8 12.2
116 8 23.5 8 22.0 8 19.6 8 14.9
117 8 22.0 8 19.0 8 15.4 8 19.1
134 8 20.5 8 17.0 : 12.2 8 6.4
144 8 18.5 8 16.0 8 11.1 8 6.4
190 8 20.0 8 16.0 8 13.3 8 6.9
226 8 23.0 8 20.0 8 16.9 8 10.6
246 8 23.0 8 19.0 8 14.9 8 6.4
252 8 23.0 8 19.0 8 19.1 8 12.7
261 8 20.5 8 20.5 8 16.0 8 10.1
300 8 23.0 8 19.0 8 15,4 8 5.3
Ave, 8 21.5 8 16.8 8 14.4 8 9.4

 

 

Table 14. The ascorbic acid content of raw milk from individual cows
when cooled over a surface cooler and stored.

 

 

 

 

Cow 8 Ascorbic acid EMg/L)
(No.) 8 First 8 Second 8 Third
8 day 8 day 8 day
3 3 3
14 3 13.5 3 7.9 3 2.7
23 3 13.0 3 7.9 3 1.1
25 3 22.5 3 18.1 3 9.6
30 3 22.0 3 19.5 3 15.4
37 8 14.5 8 7.9 8 3.7
97 3 16.0 3 9.6 3 2.1
111 3 18.0 3 13.3 8 4.2
115 3 15.5 3 11.7 3 2.1
117 3 17.5 3 12.7 3 5.3
134 3 15.5 3 12.7 3 5.8
144 3 15.5 3 9.6 3 4.2
190 3 14.5 3 9.6 3 3.7
226 8 14.0 8 6. 4 8 1.6
246 3 16.5 3 9.5 3 3.2
252 3 19.5 3 1459 3 12.2
251 3 17.5 3 13.3 3 6.4
__500 : 19.0 8 12.7 : 4.2
3 3 3
Ave. 3 16.9 3 11.5 3 5.1

 

The ascorbic acid content of raw milk from individual cows

then cooled over a surface cooler. copper contaminated and

stored.

 

 

 

Table 15.

 

 

 

d 00070007000000000 4.
r 00000000000000... 0
WM 00030003000000000 0

) 00 00 00 00 00 .0 00 .0 00 .0 00 00 00 00 00 00 0. 00 I. 00 00 00

W

(

d

1d

On 21867673116661777 2

a0 00 0000.0000.0000 o

cmw 32%m2135221112322 3

.13

w

0

C

8

A
0000.0 '0 0000.... 00 .0 O0 00 00 00 .0 0. .0 00 00 00....
+0
8 0.5050550550505505 0
r7. 0000000300....o.. 0
.1.@ 8.5468513956487090 9
F 11.. 1...... 1 1

0. 0. DO 00 00 O. I. 00 00 O0 00 0. 00 00 00 00 O. .0 00 00 0. .0 00
) 43507 4 o
0 7167 06 210 e

'0 1223391113M“92%%60 V

WW 11111122 28 A

 

_4-

when cooled over a surface coolerj irradiated and stored.

- 51 -
The ascorbic acid content of raw milk from individual cows

 

 

 

Table 16.

1140361677771769—é
0... 000000000

.0 0. .0 .0 .0 .0 00 0. 00 00 00 00 00 00 00 00 00 00 00 00

38451214154010933
.0000...0000.003.

m 55.590445087919444.
111 111!— 111

50.0003009000000000000.00000000000000

Ascorbic acid (Mg/L)

.... 55550550055555555
8 000.000.000.00...
r 10805119853258.6765
Jmu 1.111192111111111111111. 1.1.1.1.

 

 

\II
. 4350771674M06%210
w o 1192921uquoa111l112u 0.92 Rucumw
“wmw 1.1.1.1.1111n252n2n2

5.5

10.5

15.1

Are.

 

 

- 53 -

The ascorbic acid content of raw milk from individual

—_

Table 17.

 

 

cows when cooled over a surface cooler, cggper con-

tamined, irradiated, and stored.

 

 

'Third
da

econd

dag;

Ascorbic acid (Mg/L)

First

day

 

Cow
(N0.)

00010000000000000
00000000000000000
00020000000000000

0. 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00

71.747122171171700
00.00000000000000
32372233222222223

0000000000000000000.0000000000000000

05005555555055050

00000000000000000
54.465502966599081
11 11 1 1

00 00 00 00 00 00 00 00 00 00 .0 00 00 00 00 00 00 00
4350771674M05%210
122339111no 92 550
11111122223

 

 

0.1

3.0

00

8.9

Ave.

 

- 53 -

Tabl0 18. The effect of irradiation upon ascorbic acid in raw milk
with end without the addition of copper.

 

 

Treatment __ __Ascorbic acid (Mg/L)

8 1st 8 2nd 8 3rd 8 4th
__‘ ° day 8 day 8 day 8 day;k

3 3 3 3
Control 8 21.5 8 18.8 8 14.4 8 9.4

3 3 3 3
Cooled over surface cooler 8 - 8 16.9 8 11.6 8 5.1
Cooled and cOpper added 8 - 8 9.0 8 3.2 8 0.0
Cooled and irradiated 8 - 8 15.1 8 10.5 8 5.5
Cooled, copper added and 8 8 8 8

irradiated 8 ~ 8 8.9 8 3.0 8 0.0

 

‘“\J

- 54 -

1?6'

43.84% )

/

”W C 4145 (”77/

f
L,

 

Figure 11. Showing the effect of aeration. irradiation
and addition of copper upon the ascorbic acid
contait of milk after several days of storage.

~65-

Relatignship_between the oxidized flav9r_and_the ascorbic
acid ofwmilk.

 

The data reported herein are from three different experiments
carried out respectively in spring, summer and fall. Milk was se—
cured from individual cows from the College Ihiry Barn. An attempt
was made to secure milk from ten cows whose milk showed no suscepti-
bility to the deveIOpment of the oxidized flavor under various treat-
ments and from ten cows whose milk did show this characteristic.

In each experiment the milk samples were subjected to different
treatments, such as pasteurization. the addition of copper, aeration,
and so forth.

The data are classified into two groups. those cows whose milk.
as a result of the various treatments, showed little. if any, suscepti-
bility to develop oxidized flavor, and those whose milk had a pronounced
susceptibility. These are shown in Tables 19 and 20.

The average values show that milk with a tendency to deve10p the
oxidized flavor contained a higher initial ascorbic acid content than
did that of the other group.

Tables 21 and 22 include data obtained from the respective milks
of Tables 19 and 20 when both groups were pasteurized at 63° C. for one-
half hour. with the addition of cOpper. Again, the non-susceptible group
of milk showed a lower ascorbic acid content than did that of the other
group. The data were treated statistically with the results summarized
in Table 23. Very high standard errors, indicating very high significance
of the different average values, were found. These results indicate a

relationship between the susceptibility to the deveIOpment of the oxidized

- 55 -

flavor and the initial ascorbic acid content in milk. Data of Tables
19 and 20 show that milk testing high in ascorbic acid most easily
became oxidized. Data of Tables 21 and.22 indicate also that as a
result of heat treatment, even with such an oxidized flavor promoter
as cepper. the oxidized flavor was on the average most apt to develop
in those samples having the highest initial ascorbic acid content.

Data of Tables 24 and 25 show the treatments to which the milk
was subjected to determine its susceptibility to the deve10pment of the
oxidized flavor. The data furnish mufficient proof as to the different
susceptibilities of the milk toward oxidation.

Data in Tables 26 and 27 indicate that not only was the per-
centage drop largest in the group which showed no susceptibility to
oxidized flavor, but also the actual drop. It must be noted that this
drop was calculated also on the samples which were pasteurized and to
which copper was added. Furthermore. the ascorbic acid values were de-
termined before the samples were scored for oxidized flavor which was
usually on the third day. Throughout the experiment it has been noticed,
with a few exceptions, that milk which has any oxidized flavor of note
usually has no ascorbic acid.

In the first series of these three experiments temperatures of
heating were in one case as high as 75° C. and this temperature was em—
ployed as long as one-half hour. The data in Tables 28 show the compare!
tivs amounts of ascorbic acid in non-susceptible and susceptible milk
under various heat treatments. These average values are illustrated in

Figure 12. Heating at 75° C. for one-half hour increased the stability

- 57 -

of the ascorbic acid content tremendously for the samples that showed
no susceptibility to the deveIOpment of the oxidized flavor, but had

little influence upon the ascorbic acid of the susceptible samples.

- 68 _

 

 

 

 

 

 

 

Table 19. The ascorbic acid content of different cows' milk showing
pronounced susceptibility to the deve10pment of the
oxidized flavor.

Cow 8 Ascorbic acid (Mg[L) _:

(No.) 8 First 8 Second 8 Third 8 Fourth
8 day 8 day 8 day 8 day
8 8 8 8

50 8 20.2 8 16.3 8 14.4 8
79 8 17.3 8 6.3 8 1.5 8
146 8 23.5 8 24.0 8 16.3 8
156 8 18.3 8 13.0 8 10.6 8
197 8 20.2 8 15.4 8 12.5 8
226 8 20.6 8 15.4 8 13.0 8
240 8 26.4 8 22.1 8 20.2 8
248 8 29.7 8 21.1 8 20.1 8
254 8 17.8 8 16.8 8 13.0 8
259 8 20.6 8 14.4 8 12.5 8
261 8 21.1 8 16.3 8 16.0 8
23 8 15.8 8 10.4 8 6.9 8
25 8 27.2 8 19.8 8 14.8 8
30 8 21.7 8 18.3 8 16.8 8 13.8
111 8 20.7 8 16.8 8 14.3 8 9.8
116 8 19.8 8 17.3 8 12.4 8 8.4
117 8 17.8 8 14.8 8 11.4 8 7.9
144 8 16.8 8 12.8 8 8.4 8 5.9
252 8 21.7 8 16.8 8 14.8 8 9.8
261 8 19.3 8 16.3 8 10.4 8 7.9
300 8 19.3 8 16.3 : 10.9 : 7.4
30 8 23.6 8 22.6 8 19.6 8
41 8 19.3 8 18.5 8 13.7 8
42 8 21.7 8 18.1 8 6.8 8
111 8 24.5 8 23.5 8 23.1 8
117 8 21.2 8 19.5 8 12.8 8
134 8 20.2 8 17.0 8 10.8 8
142 8 18.8 8 16.0 8 8.8 8
180 8 20.7 8 16.0 8 10.8 8
190 8 21.2 8 17.5 8 12.7 8
197 8 22.2 8 17.5 8 11.8 8
238 8 22.2 8 17.5 8 13.7 8
300 8 18.8 8 16.5 8 13.7 8
8 8 8 8
Ave. 8 21.0 8 17.0 8 13.0 8 7.1

 

- 5g _

The ascorbic acid content of different cows' milk showing

 

Table 20.

no susceptibility_to the development of the oxidized flavor.

 

 

Fourth

d
r
1
mm
)
L
loo
m5
(
ma
a m
C
08
.18
b
r
O
C
MOO

First

 

Cow
(N0.)

0.

..

 

4.
O
3

0. 0. .0 0. C. .0 .0 .0

39950734.

00......

63011774.
1. 1.

O. 0. O. 0. 0. O. .0 '0

58308024
2270085.].
1.11?”

99949947
0 s o e 0

see
56m75441

9934.94.99
0.00....

mononueoo.nov.au
.11111.

38383388
0000000.
44535329
1.1.1.1111111.
00 00 00 00 O. 00 00 00
3378783
00......
88070768
11212111
7774.06
349392wm
11222

12.5

16.

13

10.5
20.1
12.5
13.0

5

e
1
1...

O. O. 0. .0 .0 .0

34.331477

0 o e e o s
884691
112112

35
37
100
174
237
239

 

5.7

C.

7.4

11.5

17.4

Ave.

 

.-

- 7o -
Ascorbic acid content of individual cow's milk showing

susceptibility to oxidized flavor development when

holder

 

 

Tab]. 6 21 0

per contaminated and stored.

c0p

pasteurized,

Ascorbic acid (Mg/L)

 

 

Cow

TFSurth

Third

8 Second

First

—_

 

(No.)

50

 

00:00

-5385
1

79
146
156

197
226
240
248

1

1

254
259

O. O. O. I. .0 O. .0 O. .0 O. CO 00 00 00 Os O. O. .0 00

4.4.4.0,0.4.nwooqscoaouons5959.5.9.9

wiqwauoaqu1inw1l1l9~5v921155921.1.1.

1.1. 1‘1.

0. O. O. O. .0 I. O. 00 00 O. 00 .0 00 00 00 O. O. 00 00

4.7.Ronquauooquosno
O O O O O O 0 O O O
manvnvaoauq3n04.9~4.
1.9211111111111.1.1l

1lwuaunv11557. 9911 11 1.7.4.0 nu
55929213111.1iuuavavmwmw4.ww1i1lzcawnv
52 11111111929213 11111.1.1_

9
o
0

240788399709349944494055005550
0 000000 use...
554.3.7.wm:wnhmmnwnwsun4”0,558.0.1"(sundae.5.4

o
8

190
'197

0.9

4.5

1.9
0.9

O
o
7

238
300

9.0

 

.0 0.

2.7

8.6

14.9

Ave.

 

_ 71 -

Ascorbic acid content in samples of individual cows' milk
showing susceptibility to oxidized flavor development

Table 22.

when holder pasteurized, cepper contaminated and stored.

 

Ascorbic acid (MglL)

Cow

Third

 

(No.)

 

4.94.9900nuo09549999

2.111100001231100

84592469944949980850500

551094144472Q¢o2212330235

25
28

73
100
108
180
252

44.34.334.994

0. O.
81.140.586.96
1111

14
37
47
97
134
190
226
240
246
13
36
37
100
174
237
23

 

1.9

4.6

9.8

Ave.

 

- 73 -

Table 23. Statistical treatmagt of data from Tables 19 and 20 and
from Tables 21 and 22.

 

Ascorbic acid (Mg/L)

 

 

 

 

 

: z
8 8
Treatment 8 Calculations : Nonesusceptible : Susceptible
None : a First day
Control :Calculated ave. : 17.46 I 4.55 : 19.92 I 3.04
j_tandard errors : 3.23
3 :
: 2 Second day
:Calculated ave. : 11.45 1 4.72 : 17.11 I 3.5
:Standard errors 2 _ 4.80
: : Third day
:Calculated ave. : 7.41 3: 4.16 : 14.05 i 4.3
:Standard errors : __5.77
x : First day
Past : :
63° c. :Calculated ave. : 4.46 1 2.71 : 8.84 1 3.77
plus :Standard errors : 5.10

capper :

- 73 -
Samples of individual cows' milk showing susceptibilitz
to oxidized flavor development when subiected to several

treatments.

Table 24.

 

 

 

 

Oxidized flavor development when the milk was

-—

(636 C - 30 min.7"and

Pasteurized

 

flapper
and

 

aeration

 

e 4 +

+ + +
4'1“!-

raw-

4 w r

+ + +
+ h +

«Inns

8

.7 .

... _

*

+ r v

e + r

4:...

.0

.p

..\ .0

117

134
142
180
190
197
238

y.
8 4 + 4

+ +
+.+

 

- 74 -

 

 

 

 

 

 

 

Table 25. gal-ppm of individual cows' milk showing no susceptibilitx
to oxidized flavor deveIOpment when subJected to several
treatments.

Cow 3__9;idized flavor development when the milk was

(No.) 3 Raw plus 3 Pasteurized (88° C. - 80 min.) and
3 capper 3 Control 3 Copper 3 Aeration 3 Copper
3 3 3 added 3 3 added
3 3 3 3 3 aeration
3 3 3 3 3

25 3 - 3 + 3 3 :
28 3 - 3 + 3 3 3
73 3 - 3 - 3 3 3
100 3 - 3 - 3 3 3
108 3 - 3 - 3 3 3
180 3 - 3 - 3 3 3
252 3 - 3 - 3 3 3
14 3 3 - 3 - 3 - : _
37 3 3 - 3 - 3 - 3 -
47 3 3 - 3 - 3 - 3 -
97 3 3 - 3 - 3 - 3 -
184 3 3 - 3 - 3 - 3 -
190 3 3 - 3 - 3 .. g _
226 3 3 - 3 - 3 - 3 -
240 3 3 + 3 - 3 - 3 -
246 3 3 - 3 - 3 - : ..
13 3 3 3 - 3 3
36 3 3 3 + 3 3
37 3 3 3 - 3 3
100 3 3 3 - 3 3
174 3 3 3 + 3 3
237 3 3 8 + 3 3

239

 

Q

Table 26. The decrease in ascorbic acid of non-susceptible and
susceptible milk from individual case during the

month of ME;-

 

 

 

 

 

 

 

 

 

Cow 3 Decrease in ascorbic acid from raw milk
(No.) 3___ Per Cent 3 %§.Aper liter
3 Past. (63” C. 3 4?ast. plus 3 Past. 63V’C. 3 Past. plus
3 30 min.) ' copper 3 (30 min. 3 c0pper
3 Stored 2 da. 3 Stored 3 da. 3 Stored 2 da. 3 Stored 3 da.
3 3 3 3
3 Nonsusceptible
13 3 64 3 88 3 10.5 3 14.4
36 3 90 3 81 3 16.4 3 14.8
37 3 90 3 81 3 16.4 3 14.8
100 3 47 3 59 3 11.3 3 14.1
171 3 85 3 85 3 14.0 3 13.9
237 3 85 3 85 3 16.8 3 16.7
239 3 82 3 75 3 17.8 3 16.2
3 3 3 3
Ave. 3 77.6 3 79.1 3 14.7 3 15.0
3 Susceptible
30 3 46 3 32 3 10.9 3 7.6
41 3 44 3 40 3 8.5 3 7.8
42 3 72 3 65 3 15.8 3 14.2
111 3 24 3 35 3 5.8 3 8.5
117 3 58 3 43 3 12.4 3 9.2
134 3 66 3 73 3 13.2 3 14.5
142 3 69 3 71 3 12.9 3 13.3
180 3 53 3 79 3 10.9 3 16.3
190 3 47 3 62 3 9.9 3 13.2
197 3 51 3 79 3 11.4 3 17.7
238 3 51 3 70 3 11.4 3 14.5
300 3 37 3 52 3 7.0 3 9.8___
3 3 3 3
Ave. 3 51.5 3 58.4 3 10.9 3 12.2

 

Mnggr lite-1"-“H ’

. Past. plus
copper
Stored 2 da.

 

Past. plus
copper
Stored 3 da.

Decrease in ascorbic acid from raw milk
Per Cent

Past. (63° C.

30 min.)
Stored 2 da.

 

 

susceptible milk from individual cows duringgthe month of

 

The decrease in the ascorbic acid of non-susceptible and

 

 

 

Table 27.

(No.)

Cow

4.4.0.1uo.¢wo.4.4. qu 0.0.auncoc4.4.zu4.q.
00.0 0 00.0.0000
1m1u1u2unanu44no.o .4 ocauo.1anwwmw:uqunu
1.1.1.1.1.1.1.1.1. 1. 1. 1.1.1.
.0 .0 O. .0 O. O. 00 .0 .0 O. O. .0 O. .0 O. O. O. O. .0 .0 .0 O. .0
14.5wo.o.owqunwaw4. a. A.4.4.4.:.4.0.4.a34.
O 0 000000....
muooo.nw:unwoonwawmw 7. Aucaauaucuauauo.auo.

t O

D. 1.

e .D

C .1
oo 8 00 oo oo oo oo oo 0o oo oo 00 oo 00 .00 oo oo oo oo oo oo oo oo oo oo

u D.

s e

... w

m m
nu99041u03cunrna.o .O 7.1.o.nco.7.nvfivd.4.
000...... L 000.000...
035433329 8 32 1M93096
7.7.7.259596nunuoo a. no.6 .o non/"1.6.0
'0 .0 O. O. .0 O. O. .0 .0 O. .0 .0 .0 .0 .0 O. O. O. .0 O. I. O. .0
7.075.799.88 3 0691834173
000 o o 0000......
7.mm7.9wqcuu 0.8. MW 934.4.Avou Rod. R.
nonano no.0 noaoncnéno "one no

0
4.77 406 e 35 1.67 210
1.q34.mwzuoeoumwmw v ncqcmw1.1.11H_b.omw
11222 A 1.1.1.122

 

 

12.1

6.6

61.4

32.8

Ave.

 

 

table 28. The ascorbic acid values of non-susceptible and susceptible
milk on the third day'of storage.

 

 

 

 

 

 

Cow 3 Ascorbic acid (fig/L)
(No.) 3 3 Pasteurized 3 Pasteurized
3 Control 3 (63° c. - 30 min.) 3 (750 c. - 30 min.) _
3 3 3
3 Non-susceptible
13 3 6.8 3 9.3 3 11.3
36 3 2.5 3 2.9 3 9.3
37 3 3.4 3 3.9 3 10.8
100 3 12.8 3 14.7 3 16.2
174 3 7.8 3 5.4 3 10.8
237 3 2.5 3 2.9 3 11.8
239 3 6.8 3 7.8 3 14.3
3 3 3
Ave. 3 6.1 3 6.7 3 12.1
3 3 3
_ 3 Susceptible
30 3 19.6 3 19.7 3 18.2
41 3 13.7 3 14.7 3 12.8
42 3 6.8 3 8.8 3 13.7
111 3 23.1 3 18.7 3 18.7
117 3 12.8 3 16.7 3 13.7
134 3 10.8 3 10.8 3 12.8
142 3 8.8 3 10.3 3 10.3
180 3 10.8 3 12.3 3 14.3
190 3 12.7 3 13.3 3 14.3
197 3 11.8 3 13.7 3 14.3
238 3 13.7 3 12.9 3 14.3
300 3 13.7 3 14.7 3 12.8
Ave. 3 13.2 3 13.9 3 14.2

 

u

- 79 _

 

 

 

 

 

 

 

 

 

.25”
2 0
~\\
\\\\
§{\
\i:

1: /f"
p
3
k
2 3‘

Tg /b
§§

f
3h2fi42}¢¢§5¢f?
_._.- 75671411244273422Q5
67
XMW a 3 ' 7 .
7"
L/4779144;Z;{7uet/
Figure 12. Showing the ascorbic acid values of milk susceptible and

non-susceptible to the development of the oxidized flavor
following different heat treatments.

- 30 -
Part II
ASCQBIC ACID OCCURRING NATURALLY IN MILK UNDER VARIOUS CONDITIONS

The effect of season upon the ascorbic acid content of
streptococcic and non-streptococcic milk;

The time of the year in which the milk from streptococcic and
from non-streptococcic cows was examined for ascorbic acid.is given in
Table 29. The ascorbic acid data obtained are presented in Table 30.
and are shown graphically in Figure 13.

The data in Table 30 are treated statistically according to the
law of variance and the values obtained are tabulated in Table 31.

Data in this table indicate, by the large value of F, for monthly means.
that there are significant differences between the monthly averages of
ascorbic acid; and also, the size of I, for replications of cows, indi-
cate significance between replications, or cows.

0n examining the monthly means of ascorbic acid for the first
day. it is seen that the means for December, January, February and March
were significantly smaller than.that for the rest of the months. This
was done for the fourth day also with the same conclusions.

.Ascorbic acid values of milk from normal and from mastitic cows
respectively are presented in Tables 32 and 33. The average ascorbic
acid value of the milk from eaCh cow was calculated and are assembled in
Tables 34 and 35. The data demonstrate clearly that the mastitic cows

have a decidedly lower ascorbic acid content than that of the normal cows.

The ascorbic acid values of the milk from non-mastitic and
from mastitic cows per month are given in Tables 36 and 37 and.are
shown graphically in rigure 14. These data indicate clearly that
the mastitic milk show the greatest variation in ascorbic acid

throughout the year.

-82-

 

 

 

 

 

 

 

 

Table 29. The different periods in which the __cows' milk were
used for ascorbic acid determinations.
Cow 3 Normal r
139. ) 3 Breed 3 Started 3 Ended
3 3 3
230 3 Brown Swiss 3 1st December 3 2nd July
_ 150 3 Ayrshire 3 2nd April 3 24th November
146 3 Ayrshire 3 let December 3 2nd April
116 3 Jersey 3 7th July 3 15313 October
37 3 Guernsey 3 20th October 3 24th November
3 : . ;
Cow 3 Mastitic J
(No. ) 3 Breed 3 Started 3 Ended
3 3 3
239 3 Brown Swiss 3 4th March 3 24th November
207 3 Holstein 3 9th December 3 26th April
174 3 Holstein 3 29th April 3 24th November
63 3 J erseL 3 9th December 3 19th March

 

- 33 _

Table 30. The average daily values of ascorbic acid (Mg/L) in
milk for both normal and mastitis cows throughout
the year when stored for several days.

 

 

Month 3 First 3 Second 3 Third 3 Fourth
3 day; 3 day 3 day 3 day
January 3 16.1 3 11.6 3 8.9 3 7.5
February 3 12.9 3 10.3 3 9.2 3 9.2
March 3 15.4 3 11.1 3 7.6 3 5.4
Apri1 3 17.3 3 12.5 3 8.9 3 8.3
may 3 18.5 3 15.1 3 11.2 3 7.9
June 3 19.8 3 16.4 3 13.2 3 10.7
July 3 18.3 3 15.4 3 12.5 3 11.2
August 3 20. 5 3 17. 9 3 15. 4 3 13. 7
September 3 18.3 3 16.6 3 13.4 3 12.0
October 3 21.0 3 17.9 3 15.5 3 12.0
November 3 19.1 3 16.2 3 13.0 3 11.3
Ipcember 3 15.6 3 11.7 3 8.9 3 7.3

 

- 34 -

 

 

 

jfmaifljj
M

Figure 13. Showing average values of ascorbic acid in milk for both
normal and mastitis cows throughout the year.

 

- 85 -

Table 31. Agalyses of variance pertaining to seasonal variations
of ascorbic acid in raw milk from both normal and
mastitis cows (lst day titrations7.

 

 

 

 

Source 3 Degrees 3 Sums 3 3
of 3 of 3 of 3 3
variation 3 freedom 8 squares 3 Variance 3 F
3 3 3 3
Total 3 47 3 394.5 3 3
3 3 3 3
Between 3 3 3 3
month 3 11 3 233.5 3 21.2 3 6.23
means 3 3 3 ‘ 3
Between 3 3 3 3
replication: 3 3 47.4 3 15.8 3 4.64
means 3 3 3 3
Error 3 33 3 113. 6 3 3. 4 3

 

- 86 -

Table 32. The average ascorbic acid content (Mg/L) of normal covs'

milk throughout the year (874 determination—).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3
Month 3 .1 No. 37 -_Ouernsey _
3 First 3 Second 3 Third 3 ourth
3 day; 3 day 3 day 3 day
October 3 20.5 3 17.4 3 13.8 3 9.6
November 3 19.9 3 16.4 3 12.2 3 11.6
3 3 3 3
Average 3 20.1 3 16.7 3 12.6 3 11.3
3 No. 16, Jersey
July 3 19.1 3 16.3 3 13.4 3 11.8
August 3 20.8 3 18.3 3 15.9 3 13.9
September 3 19.2 3 17.6 3 16.7 3 12.9
October 3 22.5 3 19.2 3 15.2 3 13.4
3 3 3 3
Average 3 20.0 3 17.5 3 15.0 3 12.6
3 3 3 3
3 No. 146 - Ayrshire
December 3 16.8 3 12.9 3 12.1 3 11.4
January 3 17.2 3 12.9 3 12.9 3 13.2
February 3 16.0 3 14.7 3 14.9 3 15.6
March 3 17.8 3 15.6 3 10.3 3 10.3
3 3 3 3
Avergge 3 17.2 3 14.2 3 12.4 3 12.5
3 3 3 3
3 No. 150 - Ayshirc
April 3 17.3 3 13.2 3 10.8 3 8.7
May 3 19.6 3 17.4 3 14.9 3 13.6
June 3 18.5 3 16.5 3 13.8 3 10.9
July 3 17.6 3 15.4 3 13.3 3 12.4
August 3 19.8 3 17.4 3 15.4 3 15.0
September 3 15.0 3 13.4 3 11.4 3 9.1
October 3 .18. 6 3 15. 4 3 14. 2 3 9. 8
November 3 19.7 3 16.2 3 13.1 3 10.2
3 3 3 3
Average 3 18.4 3 15.7 3 13.7 3 11.1
3 3 3 3
3 No. 230 - Brown Swiss
December 3 18.0 3 14.0 3 10.7 3 9.4
January 3 16.7 3 13.1 3 9.8 3 8.0
February 3 14.2 3 11.3 3 9.9 3 9.1
March 3 15.9 3 12.9 3 10.0 3 6.6
April 3 17.4 3 13.5 3 11.1 3 11.6
May 3 16.9 3 13.6 3 11.6 3 8.5
June 3 l9. 8 3 14. 4 3 12. 0 3 10. 5
3 3 3 3
Average 3 17.0 3 13.3 3 10.7 3 9.1

 

C

O

V.

The average ascorbic acid content (Mg[L) of mastitis cows'

milk throughout the year (922 determinations).

 

 

Table 33.

 

 

 

 

 

 

 

 

 

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Aveggge

.0

The averagg_ascorbic acid (MglL) content of raw milk
from normal cows.

 

Tab10 34s

 

 

3 Brown Swiss

3 Guernsey
3 Jersey

3 Ayrshire
3 Ayrshire

15.5 12.9 11.3

18.6

 

 

 

37
116
146
150
230

Aver

from mastitis cows.

 

The average ascorbic acid (IglL) content of raw milk

Tab1o 35.

 

Breed

 

Cow
$19.)

 

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a

-89-

 

 

 

 

Table 36. he averagg values of ascorbic Ici d (lglL) in milk from
eggmal cows throgggut the year .
3 3 3 3 3
Month 3 Tire t 3 Second 3 Third 3 l'ourth 3 Average
3 day 3 day 3 any 3 day 3 of all
3 3 3 3 3 (lg!
3 3 3 3 3
January 3 16. 9 3 13. O 3 11. 3 3 10. 6 3 13. 0
February 3 15. 1 3 13. O 3 12. 4 3 12. 3 3 13. 2
Harsh 3 16. 8 3 14. 2 3 10. 1 3 8. 4 3 12. 4
April 3 17. 3 3 14. 2 3 10. 9 3 10.1 3 13. 1
may 3 18. 2 3 13. 3 3 13. 2 3 11. 1 3 14. 0
June 3 l9. 1 3 15. 5 3 12. 9 3 10. 7 3 l4. 6
July 3 18.3 3 15.4 3 13.3 3 12.1 3 14.8
Aug]. 0‘ 3 20.3 3 15.9 3 1507 : 1&4 8 1606
September 3 17.1 3 17.8 3 14.1 3 11.0 3 15.0
October 3 20.5 3 l7. 5 3 14.4 3 10.7 3 15. 8
lovember 3 19.8 3 16.3 3 12.7 3 10.9 3 15.0
December 3 17.4 3 13.4 3 11.4 3 10.4 3 13.2
3 3 3 3 3

 

 

inmmfmn

mastitis cows througholit the leer.

 

 

The ewergggfyalue of ascorbic acid (Mglél

mdble 37.

 

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q

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mm.

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977

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13.8
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Figure 14.

 

- 91 -

 

15sel

"1 1 H‘

W; ‘ § i1
------- W‘ I

g/VI/ alt / 0”

Showing monthly average values of ascorbic acid in milk
throughout the year for normal and for mastitic cove.
Each month's value represents the average found each day
for four days average.

-92..

The ascorbic acid content of commercial pasteurized milk,
. irradiated milk. and raw milk throughout the year.

Samples of Grade A raw, of regular pasteurized and of irradiated
milk were titrated for ascorbic acid daily throughout the year. he
Grade A raw milk was produced at the College hiry Barn. The irradiated
milk was. high grade. big: testing milk produced at the College hiry Barn
and was irradiated in the 01’ unit at the College Creamery. The regular
pasteurized milk was mixed milk testing approximtely 3.7 per cent fat
from approximately thirty patrons delivering milk to the College Creamery.
The milk was pasteurized by the holder process in Pfaudler glass-lined
and in Allegheny metal pasteurizers. The percentages distribution of the
ascorbic acid contents of these milks are given in Tables 38. 39 and 40.
The average seasonal values are given in Table 41 and from this Table
the graphs of l'igures 15. 16 and 17 are constructed.

m the basis of these findings it can be stated that as far as
the irradiated and Grade A milk were concerned there was a very definite
correlation between the season and the ascorbic acid content of the milk.
This was very pronounced in the irradiated milk in connection with which
it must not be overlooked that the oxidized flavor was not noticed from
the first of April to the first of November.

The seasonal variation did not show up very well in the pasteurized
milk. However, this milk was mixed milk from thirty different patrons, of

various quality, and was at least 24 hours old before titration.

r

L

or dquEhen milk was held four days at 6° 0. Ci,

‘2

 

 

 

Percentage distribution of ascorbic acid in irradiated.milk throughout thggyear. by seasons.
Distribution of ascorbic acid

 

 

- Tabl. 380
Ascorbic 3

-93-

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3.6:
7.23

3323.5 3
3311.7 3
3317.6 3
33 6.9 3

317.6 3 2.9

33 8.7 3 5.0 3
3316.7 3 5.0 3
3321.7 3 2.5 3
3319.4 3 2.6 3

3315.2 3
33 4.3 3

33

3

3316.3 3 4.8 3

3318.4 3
3328.6 3
33 2.0 3
33
33

10 - 11 3 6.8 3
ll - 12 3

12 - 13 3

13 - 14 3

14»- 16 3

15 - 16 3

16 - 17 3

17 - 18 3

19 - 20

 

Q.

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day; at 5° 0. 5g)

 

held four

Summer

3 lot 3 20d 3 3rd 3 4th 33 lot 3 2nd 3 3rd 3 4th 33 1st 3 2nd 3 3rd 3 4th 33 1st 3 2nd 3 3rd 3 4th

33 day»: d§y_3_g&z;3 day 33

9.43 15.033

 

dal;ghen milk was
3

Sprung

33
33
33

a distribution of ascorbic acid in regulargpasteurized milk throughout the year by seasons.

 

Winter

Distribution of ascorbic acid per

Percent

 

 

3 day 3

 

 

Table 39.
Ascorbic 3

acid
(ls/L)

,3

C>FJCQ8039IO

3 29.1
5.33 11.8

3.23 15.83 11.8

3 2.6 3 14.23 63.633

3 10.53
9.4: 10.5:

5.733
6.63 31.433
3.23 22.933
3 19.43 25.733
2.63 16.13
2.63 12.93
7.73 19.43
3 15.43 16.13
3 28.23
3 20.53

33 6.8 3 12.83

33 9.1 3

3.23

2.63

14.33 22.733
25.03 4.533
17.93 4.533 2.1
7.23 4.533

1 3
3 3
7 3
3 3

5
10
7
O

11.8

5.33
3.23 21.13 11.8

9.43
3315.8 3 16.13

33 2.6 3

3.23
9.43

33 5.3 3
33

8.633
2.933 2.6 3

3.033 2.3 3
3.033

15.6
9.43

3
3
3
3

3 4.5 3 18.83

3 9.1
33 8.3 325.0 3
3312.5 322.7 3
33 4.2 311.4 3

3310.4 3 6.8 3

33
33

7.23
3.63

9.1315.4 3 10.73

4.5310.3 3
9.1317.9 3

7
8

5 -
7 -

3 11.8

5.33

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e
e
3
3

9 3

10 - 11 3 15.93
11 - 12 3 13.63

8 -

3.23 21.13

9.43 5.33
33 7.9 3 19.43
3318.4 3 12.93

33 5.3 3
3310.5 3

3.23

5.13

3.13 33
3

3325.0 3 2.3 3

3314.6 3 4.5 3
3312.5 3

2.33 2.6 3
3
3
12 - 13 3 20.53
13 - 14 3 4.53
14 - 15 3

33 6.3 3

3313.6 3

3 4.2 3

15 - 16 3
16 - 17 3
17 - 18 3

33 7.9 3 3

33 5.3 3

3

33 4.5 3

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{table 41. Average seasonal values of sscorbic acid ( L) in the
three types of milk used in this experiment 1594

 

 

 

 

 

 

 

 

 

de terminati one).
8
Seasons 8.: First 8 Second 8 Tnird 8 Fourth
8 d9; 8 (lg 8 day 8 day
8 8 8 8
8 Irrsdisted milk
linter 8 7.2 8 2. 5 8 0.4 8 0.0
Spring 8 11.1 8 6.1 8 2.0 8 0.7
Summer 8 14.2 8 9. 1 8 4. 7 8 2.2
1.1L 8 1029 8 6. 1 8 2.8 8 1.2
8 8 8 8
Averggg 8 10. 9 8 6. O 8 2. 5 8 1. 0
8 8 8 8
8 Grade A milk
Winter 8 6.0 8 4.8 8 1.3 8 0. 6
Spring 8 10. 5 8 6.0 8 3.4 8 l. 8
sum- 8 8. 8 8 6.4 8 4. 7 8 3. 3
Il'sll 8 7. 7 8 4. 2 8 2. 5 8 l. 2
8 8 8 8
Avsfle 8 8. 3 8 5. 3 8 3.9 8 1.7
8 8 8 8
8 Pas tenrized milk
[inter 8 9.9 8 5. 8 8 3. l 8 0. 8
Spring 8 12. 9 8 7. 6 8 4.0 8 2.2
Sumner 8 18. 0 8 9. 5 8 5. 8 8 3.3
1311 8 13.0 8 9.4 8 6.8 8 3.4
8 8 8 8
Average 8 12. 2 8 8. l 8 4. 9 8 2. 4

 

0‘

- 97 -

 

 

 

 

 

 

9 ’ 4

figure 15. Showing the seasonal values of‘ascorhic acid in irradiated
milk after different periods of storage.

- 93 -

[3*

} -—-.§z¢¥

' "-4u¢7 ¢4'

 

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629¢zn~¢%¢24Zooaf/hh72291/

 

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Figure 16. Showing the seasonal values of ascorbic acid in
pasteurized milk after different periods of storage.

-99-

 

A)”

a

 

 

 

 

 

 

 

Figure 17. Showing the seasonal values of ascorbic acid in
Grade A raw milk after different periods of storage.

-100-

Influence of the leucoczge count of the milk upon the
ascorbic acid content.

Different experiments were carried out during the spring of
1937 to study the effects of leucocytes on the ascorbic acid content
of milk.

llixed nilk. both from mastitis and from non-mastitis in-
fected animals. from the different breeds of cattle were secured.
The milk studied was obtained from 91 cows of which 28 were Guernsey:
17. Jersey: 8. Ayrshire: 18. Holstein: and 20. Brown Swiss.

The data secured are tabulated in Tables 42 and 43. rearranged
in another grouping in Table 44, treated statistically in Table 45.
and presented graphically in' figure 18. hta secured on the Iilk
having a leucocyte count below 500,000 per 8881. are included in Table
43 while those from milk having a count of above 1,000,000 per ml. are
included in Table 42. from these tables, as well as Figure 18. it can
be seen that the higher the leucocyte count the lower the ascorbic
acid value. The rate of oxidation of ascorbic acid in the two groups
of nilk upon storage appeared to be parallel. as noted in Tigers 18.

The standard errors between the means of ascorbic acid values
for the first day was found to be extremely high, indicating great
significance, as shown in Table 41. As the milk was held in storage, 1
the difference in the means showed a lower standard of error. but the
value for the fourth day was yet significant.

In hble 44 the data are grouped differently from those in
Tables 42 and 43. In this case. five different classes were established

as follows: leucocyte count below 0.5 million per ml. (151 determinations):

- 101 -

between 0.5 and 2 million (88 determinations): from 2 to 4 million
(36 deterninations): from 4 to 6 million (17 determinations) and over
6 millions (30 determinations).

The results indicate that a relationship exists between the
leucocyte count and the ascorbic acid values of the milk. The higher
the leucocyte count the lower the ascorbic acid value and vice versa.
However. the differences in the ascorbic acid values for milk of
different groups, as indicated by the leucocyte count. diminished
from the first to the fourth day. .As pointed out in Table 45, the
standard error decreased from the first to the fourth day, indicating
the decrease in the significance of the difference. The graph illus-
trates this difference in another way. on the first day the difference
in the ascorbic acid values between the two extreme groups, below 0.5
million and above 6 million leucocytes per ml. was large. being
approximately 6 mg., while on the fourth day the difference was small.

being apnroximately 2.5 mg.

- 102 -

The ascorbic acid content (g5;_of individual cows' milk

 

 

having;leucocyte count over one million per ml. after

different periods of storage.

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- 103 -
The ascorbic acid content (Mg/L) of individual cows' milk

having;a leucocyte count below 0.5 million per m1. after

different periods of storage;

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-104-

Table 44. The ascorbic acid content (Mg/L) of milk having varioue
leucocyte counts after different periods of storage
T7322 determinations).

 

Lecorbic acid value (lg/L) when leucocyte count was in

 

 

 

8
8
Star a 8 millions per m1.
((1833 8 Below 8 Fitween 8 Between 8 ,Fetween 8 Uver
8 (L5 8 0.5 - 2 8 3 - 4 8 4 - 6 8 6
8 8 8 8 8
l 8 22.1 8 21.2 8 18.6 8 16.8 8 16.2
2 8 16.8 8 16.0 8 15.1 8 13.2 8 11.2
3 8 12.5 8 10.0 8 8.2 8 10.8 8 7.9
4 8 9.0 8 7.5 8 6.9 8 6.5 8 6.8

 

- 105 -

mable 45. Statistical treatment of data from Tables 38 and 89

 

.Lecorbic acid valuee in low and high leucocyte
count milk after varioue periods of storage (ESZL)

 

 

 

 

 

 

 

8

8

8

8 Below 0.5 million 8 Over 1.0 million
Calculations 8 leucocytea.per ml. 8 leucocyte: per m1.

8 Iiret flay *
0.16. mean 8 22.26 t 2.86 8 18.41 - 6.16
Standard error: 8 _;__r 5.21

8 Second Day
Cale. mean 8 17.20 3: 3.70 8 16.64 1‘ 3.95
fitgndard errore 8 .:._ 3.96

8 Third Hwy
Calc. mean 8 12.79 t 4.89 8 9.48 t 8.69
standard errore 8 3.10

8 Fourth
0.16. mean 8 9.06 I 4.5 8 6.27 t 3.71

Standard errors

2,87

 

- 106 -

 

 

 

 

 

 

 

Figure 18. Showing the average values of ascorbic acid (lg/L) in milk
having various leucocyte: counts after different periods
of storage (322 determinations).

-107-

Correlation between the bacteria count, the leucocytes. the

reduction time and the stabilitL of ascorbic acid in milk.

Studies were made on individual seaples of milk from 19 cows
of the college herd and upon 38 lots of mixed milk delivered at the
College Creamery. The methylene blue reduction periods. the bacterial
counts, the leucocyte counts and the dain ascorbic acid values are
presented in Tables 46. 47 and 48.

the data in Table 46 were obtained from studies on individual
cows' milk from the College Herd. hose milks were obtained in glass
and cooled immediately. Consequently. the bacterial counts were ex-
tremely low. Likewise, the methylene blue reduction periods were high,
averaging 6.5 hours for the 19 samples. the ascorbic acid values ranged
from an average of 20.3 mg.per liter on the first day to 9.8 lg. per
liter on the fourth day, indicating an inverse relationship between the
reduction time and the oxidation of the ascorbic acid.

he data in Tables 47 and 48 pertain to studies on mixed milk
delivered at the College Creamery. Here the relationship between the
amount of ascorbic acid present. its drop, the bacterial counts, and the
leucocyte counts of mixed milk of average quality will be noted.

The large number of bacteria seemed tohave a protective effect
upon the stability of ascorbic acid while a high leucocyte count had a
harmful effect. High bacteria and leucocyte counts in the same sample
seemed to have a counteracting effect upon the ascorbic acid, therefore,
resulting in its greater stability. In several cases in which the

bacteria count was high. the milk not only had a high initial ascorbic

- 108 -

acid content, but in some cases the ascorbic acid value, instead of
decreasing from day to day, remained constant, or actually increased.
Conversely, if the milk had.a high leucocyte count, the ascorbic acid
value was likely to decrease to a greater extent than in those samples
showing a low leucocyte count.

The data of Tables 4? and 48 are reclassified according to the
decrease in ascorbic acid from first to third day and are presented in
Tables 49 and 50. Two groups of milk, those which showed.a decrease in
ascorbic acid between 0 and 5 mg.per liter and those which exhibited a
decrease between 6 and 13 mg. per liter, are listed.

The average results from these Tables show clearly that the
group of samples exhibiting the largest decrease. 6 to 13 mg. per liter
of ascorbic acid, had the longest reduction time and the lowest bacterial
count, while those samples showing the smallest decrease, 0 to 5 mg. per
liter, had the shortest reduction time and the highest bacterial count.
The leucocyte count was about the same in each class, therefore, their
effects upon the ascorbic acid might be considered to be similar.

The above data seem to indicate that the main factor affecting
the stability of the ascorbic acid of the milk in this experiment was
chiefly the bacteria. The data reported in Table 46 obtained from studies
of milk in which the bacterial count was very low, showed that apparently
no relationship existed between the long methylene blue reduction periods
and the stability of the ascorbic acid. while data of the two other ex-
perimeats, summarised in Tables 47 and 48, showed that a very high bacterial
count, therefore, a short reduction period, resulted in the smallest de-
crease of ascorbic acid. The bacterial papulations of milk were thus able

to influence the ascorbic acid stability in milk to a marked degree.

Reduction

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The ascorbic acid content of individual cows' having_

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- 110 -

the ascorbic acid content of mixed milk from individual

Table 47.

patrons compared with the methyleneblue test, with the

bacteria count and with the leucocyte count;

 

 

 

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~111-

the ascorbic acid content of mixed milk from individual
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- 112 -

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table 49.

I

 

than 5.0 wr liter after three days‘ storage.

 

 

8 Bacteria count 8 Leucocyto cOunt

Decrease in ascorbic 8 Reduction

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- 113-

Table 50. me reduction timeL the bacteria count. and the leucocyte
count of nilk showigg a decrease in ascorbic acid of ncre
than 5.0 mg. per liter after three day? storaggL

 

 

 

8 8 8
Ascorbic acid 8 Reduction 8 Bacteria count 8 Leucocyte count
(Mg/1.) after 8 time 8 8
three days 8 hours 8 (Mill. per m1.) 8 (Mill. per m1.)
8 8
6.0 8 2.55 8 0.02 8 4.20
5.1 8 1.55 8 0.50 8 0.80
5.8 8 2.20 8 0.70 8 1.20
5.9 8 7.05 8 0.30 8 2.10
5.5 8 8.15 8 0.04 8 0.60
7.1 8 6.45 8 0.20 8 0.50
7.1 8 6.45 8 0.03 8 1.20
7.0 8 8.45 8 0.07 8 1.40
10.5 8 4.15 8 0.40 8 1.30
13.1 8 5.05 8 0.60 8 0.50
11.0 8 5.05 8 0.08 8 2.90
7.4 8 3.00 8 0.50 8 2.90
8.9 8 7.30 8 0.02 8 0.70
10.5 8 7.05 8 0.02 8 1.50
6.9 8 6.00 8 0.03 8 0.90
9.9 8 4.50 8 0.13 8 0.70
6.5 8 4.45 8 O. 8 8 0.40
9.9 8 6.15 8 0.04 8 2.70
8 8 8
7.57 8 5.30 8 0.21 8 1.5

 

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- 114.-

.A stggz_pf the relationship between the per cent lactic

acid. the flavor score and the ascorbic acid of milk.

Individual samples of milk from approximately 30 cows of the
College Herd were obtained.and cooled immediately over a surface cooler.
Samples for study were secured before and after cooling. The titratable
acidity. expressed as per cent lactic acid, the ascorbic acid content
and the flavor score were determined. The data are assembled in Tables
51 and.52.

The data indicated that a high flavor score was associated with
a large amount of ascorbic acid: likewise. that a high per cent of

lactic acid in milk was associated with a low ascorbic acid content.

- 115 -

 

 

 

 

 

 

 

Table 51. ghe relationship between the per cent of lactic acid
and the ascorbic acid content of raw milk;
8 8
Lactic 8.8811 8 Number of 8 Ascorbic 8cm wig.)
( f ) 8 samples 8 Before going 8 fter going
8 8 over cooler 8 over cooler
8 8 First 8 Second 8 first 8 Second
8 8 déy 8 day 8 jg5;_ 8 day
8 8 8 8 8
Above 0.180 8 48 8 15.3 8 8.6 8 14.1 8 7.2
gelow 0.180 8 67 8 17.6 8 10.0 8 15.9 8 7.6
Thble 52. 159 relationship between the flavor score4apd ascorbic

 

acid content of raw milk.

 

 

 

 

8 8
Flavor score 8 number of 8 :‘_ Ascorbic acid (gglh)
8 samples 8 Before going 8 After going
8 8 over cooler 8 over cooler
8 8 First 8 Second 8 First 8 5econd
8 8 2!! 8 95!: 8 9!! 8 9!!
8 8 8 8 8
Above 22.5 8 60 8 17.4 8 10.0 8 15.8 8 7.8
Below 22.5 8 65 8_:1g.9 8 9,0 8 1419 8 7.4

 

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- 116 -

Influence of sodium citrate and citric acid upon the ascorbic

acid content of milk and.upon the development of the oxidized

flavor.

This experiment was conducted to determine what relationships,
if any. existed between the citric acid or its salts and the ascorbic
acid content of milk. The study was made also to note what effect the
presence of citric acid or its citrates had upon the oxidized flavor
of milk. However. in the latter study it was not possible to trace any
relationship inasmuch as the oxidized flavor did not develop in the
original milk, or to a very small extent. even by additions of large
amounts of copper.

The data secured are presented in Tables 53 to 57 inclusive. The
first three tables represent data secured on the influence of the addi-
tion of sodium citrate. The milk was obtained from the College Herd and
was treated the same day. The data in Table 58 show the effect of the
addition of sodium citrate upon the stability of ascorbic acid in milk.
with and without the addition of capper. The milk was not heat treated
in this experiment. The data indicated that the addition of sodium
citrate did not have any appreciable effect upon the stability of ascorbic
acid when copper was present. 0n the contrary, the data seemed to indicate,
when sodium citrate was added to the milk without the addition of copper,
that the sodium citrate contributed slightly to the destruction or loss
of ascorbic acid. Ihen copper was present also the same trend.was noted.
In the presence of copper the destruction of ascorbic acid.was rapid and
on the second day had disappeared.

The data in Tables 64 and.55 indicated that the same general re-

- 117 -

results were obtained on holder pasteurized milk as when obtained on
raw milk. Here it will be noted that the addition of sodium citrate
in increasing amounts, both with and without the presence of capper.
increased the loss of ascorbic acid.

The data in Tables 56 and.57 were secured on similar mdlk, raw
and pasteurized, to which citric acid rather than sodium citrate was
added. Under these conditions a slightly different result than that
noted above was obtained. There was a slight indication that citric
acid might retard the destruction or loss of ascorbic acid. This can
be explained as due partly to the change in the pH value or as due in
part to the possibility that the citric acid actually did function as
a hydrOgen donator.

The data in both Tables 56 and 57 show that the addition of
citric acid to milk, with or without copper present. was apt to give a
slight increase in the ascorbic acid content. As the addition of citric
acid increased, however. this increase was very small and.undoubted1y
without any significant value.

The conclusions from this experiment must be negative. The
amounts of citric acid and sodium.citrate added.were far beyond the
amounts present in milk even under abnormal conditions. Even with the
addition of these excessive amounts. the stability of ascorbic acid was

not maintained.

- 118 -
opper were added.

 

The ascorbic acid content of raw milk to which sodium

 

citrate and c

 

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Additions
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- 119 -

The ascorbic acid content of raw and of pasteurized

Table 54.

 

 

milk to which sodium citrate and copper were added.

 

 

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0 O
t t
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t t
.1 - .1
OIIIIIIIIICIIIIIIIII
a a
NIIIIOIIIINIIIIIIIII
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£133.0019uen04“
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63° c.
for 30 ,
min.

 

’0

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(3:15le
3&1.

*4”

 

First 8 Second : Third

Ml

opper were added.
: Ascorbic acid

 

- 120 -

accorbic acid content of raw and of pasteurized
Addition

 

 

milk to which sodium citrate and c

Q»

 

 

Table 55.
Heat
flaunt

9551400000
0 e e e o e e e e J
mm76400000

0000000000000:
1184559788
Mil 1n033llL.

9943475249

8886520983
1‘1.1.1.1.1.1.

“0.".

dazllll

my”...

3
lulll

+++++

citrate

a

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dolll ll ll ll l
01.234.01.234.
0000......
0000000000

0.000000000000000...

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. 0 . 0 . 0 0 0
9776£OOOO

00.000.000.000:

saw—Daoobezaoe

lOenUeBQIeZZIO
1.1.1.

0.000000000000000.

4.R.ncAv9.o.¢u¢unv
.0000...

:w1.1.au¢uo.au=u1.

1.1.1.1.1.

000.000.000.000...

a...
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3
ml

3
1...

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Ha citrate

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nvalncnoienu.4.o.§
0.0.0.000
nvnvnunvncncncnvn.

000.000.000.000...

 

6367C.

for 80
minutee

 

121 -

The ascorbic acid content of_pasteurized milk to which

Thblo 56.

copper and citric acid were added before pasteurization.

 

 

 

Ascorbic acid (MglL)

 

Addition:

Third
a:

Second

“L0

8

 

9900131903 000;
00.000.00.000. 00....
00550050 050
e o o o o o o 0
11223555 212
1111

00.00.000.000... 00....

050055000050

.uixguzmawon1.nw.uawq.nw
1111 111

000.000.000.000-..

mmllanoIII
4010004001000

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6
1000 2000
++++++++

itric acid

000.. 0.0000
000000000000

 

0‘

‘I A

O.

Q.

on

- 122 -

The ascorbic acid content of raw milk to which citric

acid.and cgpper were added.

Table 57.

 

 

 

:TMM
day

 

M

m

( .0

dd

am
4cm
c&

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0

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m

55555000000000000000
2323. 300000000000000

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££565m22122333300001
11111

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00505505050000055550

88999878094£56722247
111111

WIIIWIIIIWIUII

JFIIIJPIUIIJPIIII
& . &uuun
mil. llfllm

9 2 5
Sullslullnvlflli

++¢++++¢++r+++

itric acid

CIIIIIIIIIIIIIIIIIII
do/IIIIIIII IIIIIIIIII
50

zsomoomomoamoo $500

0001 001200012

 

O . O O O C C C . .
OOOOOOOOOOOOOOOOOOOQ

 

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C

.

§C

- 123 -

Effect of the addition of copper and glutathione upon
the ascorbic acid content of milk and upon the
development of the oxidized flavor.

 

Recently much work has been done in respect to the presence
of reduced sulphur containing compounds in living tissues and.their
possibility in biOIOgical oxidations. 0f chief importance, because
widely distributed throughout different tissues, is glutathione.
Ghutathione is a tripeptide, glutamyl-cysteinyl-glycine, having in
its reduced form the following formula:

I I

2
KHZ CHQSH

Glutathione has been attributed great importance as to its

protective action against oxidation of ascorbic acid, not only in

respect to its reducing capacity because of sulphur present in the

molecules, but also due to its ability, as most disulphide systems, to

form complexes with metal ions.

From the above discussion, the conclusion would seem logical

that if glutathione were present in milk, it might affect not only the

ascorbic acid present in milk, but also, the oxidation of the fate.

Accordingly, pure glutathione was added to milk in an effort to deter-

mine its effect upon these constituents. To check the value of the

method used, glutathione was added to distilled water in known amounts

and then redetermined by titration as outlined in theprocedure. Ten
trials were run and, in each case, the amount of glutathione checked

within five per cent of the actual amount added.

- 124 -

Glutathione was then added to milk in known amounts and then
redetermined. As much as 49.8 mg. glutathione per liter were added to
milk. However, before glutathione was added to milk, titrations were
run in order to determine the blank value. Fairly high titration
values were obtained on these blanks, despite the fact that the milk
was four days old, was pasteurized, irradiated and ascorbic acid was
not present.

Immediatelywafter the glutathione was added, the milk was
titrated, and remarkably low values were obtained over the blank, con-
sidering the large amount of glutathione added. Still more surprising
was the fact that, as time pregressed, this value became smaller and
smaller, and after 2 hours the titration value was the same as the blank.
Ihe phenomena was observed in several trials.

In another experiment the glutathione was added to fresh raw
milk in which an appreciable amount of ascorbic acid was present. Two
trials were run the results of which are tabulated in Tables 58 and 59.
Data in Table 58 gave indication that the glutathione had little, if any,
protective action on the ascorbic acid, both with and without copper
present. Although the ascorbic acid value drapped rapidly, the milk
did not develoP an oxidised flavor, However, this milk was raw and.was
not susceptible to oxidation, From the data presented in Table 59 the
same conclusions as above can be drawn. In addition, the data showed
that glutathione had no effect upon the develoPment of oxidized flavor
in raw milk when capper was present.

It is fully'apgreciated that the conclusions which may be drawn
from the above two sets of data are far from being final but are given to

indicate the relationship found between added glutathione and the stability

of ascorbic acid in milk.

- 125 -

The effect of the addition of copper and glutathione

upon the ascorbic acid content of milk and.upon the

development of the oxidized flavor.

Table 58,

 

 

 

 

 

d W
m:
umm
lam
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00.0.3:
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am
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rt
03
crm
.1
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600033000004
0 . O C O . O O O O .
7000£4000001

00:00:..:::::‘:..

4.4.nwnw1u1unwawnwnwawnu

RIIOeQNQIOelllz

00003000000000.000030000

239783495278

7%0655210763
111 11111

..8..8..::O.::..:‘

Hl Ell.ll
SI Sllll
Gl Gllll

++ ¢++++

« a

d d

d d

all alllll

O

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fill do:
1.. lllll
00 0
In: My
Mmll lllll
0362 369258
0125 1.23567

 

V

- 126 -

The effect of the addition of copper and glutathione

upon the ascorbic acid content of milk and.u

Table 59.

 

pon the

_‘_

 

development of the oxidized flavor.

 

 

hird,day

iflud
1””

ft

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M

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mm

co

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+++ +
.....+4rt.??++

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:8:::::::.0:::00

99584490099444

......OIOOOOOO
00041111131111
111

3:::::::..::::..

84455011740666
0 O O O O C O O O D C C O 0
43200098620778
111111 11

:8::::..::::..:..

Mllllllllll
dPllllllllll

g

mlllll l lll l
35925836925
12356712356

+++++s

Hlllllll
”Slllllll
CGullllll

trol no addition

 

- 127 ~
DISCUSSION

Milk from individual cows studied in this experiment showed a
seasonal variation in the ascorbic acid content of the milk regardless
of the presence of streptococci infection or otherwise. However, milk
from the mastitic cows showed a greater variation in this respect than
did that from the normal cows. The mastitic milk had on the average
a lower ascorbic acid value. The ascorbic acid in milk seemed to reach
a minimum in the months of Ibcember, January, February and March.

The ascorbic acid content of the raw milk decreased.uniforma1y
upon storage and was, on the average, constant for each of the four days
studied.

Grade A raw milk and irradiated milk from the College Creamery
showed the same seasonal variation in ascorbic acid as did that studied
from the individual cows, However, the pasteurized milk of the College
Creamery failed to show such a pronounced variation. These variations
were not shown, as in the case of the raw milk, by the different months,
but by the four seasons. However, it must be remembered that this
pasteurized milk was farmeruproduced mixed milk, produced under different
conditions as to sanitation and equipment and invariably was older milk
at the time of processing. 0f the Grade A.raw milk, pasteurized milk,
and irradiated milk, the irradiated milk showed the widest variations for
the four seasons. The irradiated milk also had the highest ascorbic acid
content during the summer months, but showed a considerably small amount
during the spring and fall months. This milk was high quality milk pro-

duced at the College Dairy Barn and was irradiated in a GP unit at the

- 123 -

College Creamery. The irradiated milk from the fifteenth of October to
the first of may consistently yielded upon storage the oxidized flavor,
but such a flavor was not noted so extensively in the Grade A.raw milk
or in the regular pasteurized milk.

A.high leucocyte count in milk was apparently correlated.with
the lower ascorbic acid content of the milk. If this were due to the
leucocytes themselves or to certain abnormal conditions in the udder,
resulting in abnormal milk, is still uncertain. The leucocytes do, howb
ever, contain enzymes, such as catalyse and peroxidase, which might have
oxidized the ascorbic acid.

An abundance of bacteria seemed to have a preserving effect upon
the ascorbic acid or to increase the stability of the ascorbic acid
present in milk. Kende (1922) indicated that a large number of bacteria
in milk prevented the deve10pment of oxidized flavor which was considered
to be due to an oxidation of the fat. The above mentioned result of the
bacteria might, therefore, be expected. However, this does not mean that
the develOpment of the oxidized flavor and the decrease in the ascorbic
acid are due to the same cause, enzymes produced by bacteria, for instance.
High leucocyte counts and.high bacteria counts together seemed to have a
counteracting effect upon each other, the ascorbic acid remaining fairly
constant.

As shown by the methylene blue test, there was an indirect corre-
lation between the time of reduction and the stability of the ascorbic
acid. However, this was undoubtedly due to the enzymic action of the
bacteria present since the reduction time is only an indication of the

numbers of bacteria present.

~ 129 n

A slight correlation was noted between the ascorbic acid con-
tent or its stability and.the per cent acid, calculated as lactic acid,
originally present. Likewise, a slight relationship was noted between
the flavor score of the fresh raw milk and the original ascorbic acid
content. On the other hand, neither the citrates nor the citric acid
had little effect, if any, upon the stability of the ascorbic acid in
milkor upon the deveIOpment of the oxidized flavor. It is a well known
fact that sodium citrate as well as formaldehyde can act as hydrogen
donator in biological oxidations. Formaldehyde is not present in milk
but citrates are to a varying extent. From the above conclusions it
could rightly be assumed that the citric acid or its salts present in
milk would.affect the ascorbic acid. The citric acid would function by
transforming its hydrogen to the ascorbic acid thereby reducing the
oxidized form which is not determined in the titration method. As has
already been demonstrated the ascorbic acid is subjected to considerable
variations throughout the year and it could.be assumed that the variations
in the citric acid and its salts could possibly be the explanation of these
variations.

Surface cooler aeration of milk always decreased its ascorbic acid
content, although the eXtent varied with milk from the individual cows.
Irradiation of the raw milk, as it was performed in the commercial plant,
did not affect the stability of the ascorbic acid in milk to any appreciable
extent.

Heating milk for one—half hour at 63° C. in glass containers caused
a slight initial destruction in the ascorbic acid as compared with that

present in similar milk raw, but the ascorbic acid was more stable upon

storage. This effect showed up after the third or fourth day, as then the

heat treated milk had a larger ascorbic acid content than did the raw.
Heating for one-half hour at 75° C. gave a still larger initial destruc-
tion than heating at 63° C., but the stability was increased still more,
so that after three or four days' storage the ascorbic acid was present
in considerably larger amounts than that present in the raw milk, or in
the milk pasteurized by the holder method.

Heating milk, with and without additions of copper, for ten
minutes from 650 0.. with five degree intervals, up to 90° C. seemed to
have different effects upon the ascorbic acid value, depending upon
whether the copper was added before or after heating. In all cases the
greatest stability of ascorbic acid.was noted at the 80° C. exposure.

If the copper were added before heating, a decreased stability of ascorbic
acid at temperatures above 800 C. was noted. However, if copper were
added after heating, an increase or decrease in the stability of the
ascorbic acid beyond 800 C. did not occur. There was a very definite
indication that a critical temperature in the stability of ascorbic acid
existed between 70° C. and 80° 0. Especially was this pronounced in the
samples to which copper had been added after heating. The temperature

of 75° C. seemed to be close to the border line of this critical tempera—
ture.

In the case of flash pasteurization similar results were noted.
However, the critical temperature was found to increase slightly and lay
between 75° C. and 85° C. The optimum temperature for ascorbic acid
stability was apparently the highest temperature employed, 97° 0. Flash
pasteurization at temperatures below 75° 0. decreased the stability of

the ascorbic acid as compared to that of the raw milk.

- 131 -

Milk fran different cows showed a wide variation in the effect
of temperature treatment. Some showed a very pronounced increased
stability of the ascorbic acid while others did not. Those that did
show an increased stability were of lower initial ascorbic acid content.
This phenomena might support the theory regarding the formation of re-
ducing substances when milk is heated to certain temperatures.

The addition of copper under many conditi one, particularly of
low heat treatment, decreased the ascorbic acid content of milkin a
relatively short time. However, this effect was not noted in the milk
samples when they were heated as high as 75° C. When a ten minute ex-
posure was employed, the temperature of 80° C. was found to have the most
stabilizing effect upon the ascorbic acid. Irradiation had no further
effect upon the ascorbic acid beyond that resulting from the presence of
capper in the milk.

Addition of glutathione to milk did not have any effect either
on the stability of ascorbic acid or on the deve10pment of the oxidized
flavor.

Throughout these studies some cows' milk were observed to have
responded differently to the various treatments to which they are sub-
Jected than that of others. Some showed a very pronounced stability in
regard to ascorbic acid while others did not exhibit this stability. This
variation was noted in the raw milk as well as in the milk to which
copper had been added. Likewise, those samples showing much stability
did not show any increased stability by heating to high temperatures, for
example 75° C. for one-half hour. However, these samples under other
conditions were readily susceptible to the development of the oxidized

flavor .

- 132 -

The oxidised flavor was found to develop more readily in milk
with a higher initial ascorbic acid content, but never developed until
the ascorbic acid.was gone. The samples which were most susceptible
to oxidized flavor deveIOpment upon treatment, not only had the largest
amount of ascorbic acid when raw, but also showed, on the average, the
smallest percentage decrease and the smallest actual decrease during
storage. This was also the case when the milk samples were pasteurized,
with and without copper present, for one-half hour at 63° C. “hen copper
was present in these samples a very intense oxidized flavor was usually
noted after three days.

In one case in which a set of samples was treated at 75° C. for
one-half hour, the samples having the most susceptibility to oxidized
flavor develOpment showed by far the greatest increased stability upon
heating to such temperatures.

The observation: was made on numerous occasions that the ascorbic
acid, upon additions of supper to the milk, would decrease rapidly and
.would have disappeared entirely in the milk in the course of two or three
days without any development of oxidized flavor. This was practically
always the case in the summer months. It follows, therefore, that the
rapid decrease of the ascorbic acid is no indication of the milk's sus-
ceptibility to oxidized flavor, even in the presence of copper. When the
oxidised flavor did deve10p in the individual cow's milk, as was the case
in the late fall, winter, and early spring, it could always be predicted
that the samples with the highest ascorbic acid content would be the most
susceptible to the oxidized flavor deve10pment. Neither must it be for-

gotten the suggestions already mentioned in the review of literature that
the ascorbic acid (vitamin C) might function as a catalyst in blOIOgical

oxidations.

- 183 -

SUMMARY

.A seasonal variation in the average ascorbic acid content of
the raw milk from individual cows was found. Likewise, a seasonal
variation for the irradiated milk from the College Creamery was noted.
This variation was less pronounced in the Grade A raw milk and was not
observed in the mixed farmer—produced milk when pasteurized.

Streptococcic milk contained less ascorbic acid on the average,
with wider variations, than nonstreptococcic milk.

Milk containing a high leucocyte count had less ascorbic acid
than milk having a low leucocyte count. The ascorbic acid in milk of
high bacterial count was more stable than that of low count milk.

Slight correlations between the titratable acidity and the flavor
score of raw milk and the ascorbic acid were noted.‘ However, no relation—
ship was found between citric acid.and its salts and the ascorbic acid.

Processing had diverse effects upon the ascorbic acid values of
milk. .Aeration decreased the ascorbic acid in milk. Irradiation of raw
milk with and without capper had no effect upon the amount of ascorbic
acid. Pasteurizing at 63° C. for one-half hour caused a slight destruction
of ascorbic acid, but increased its stability upon storage. Pasteurizing
at 750 C. for one-half hour caused a still greater initial destruction
of ascorbic acid, but further increased its stability.

Heating for ten minutes at different temperatures showed a
temperature of 80° C. as giving greatest stability to ascorbic acid. When
the milk was heated for ten minutes, the critical temperature for the

stability of ascorbic acid was found to be close to 75° C.

c.134—

Flash.pasteurization at different temperatures gave the critical
temperature for ascorbic acid stability as being between 75° C. and 85° C.
Different cows' milk showed varying effects upon the ascorbic acid as a
result of flash pasteurization.

usually milk with an initial low ascorbic acid content showed the
greatest increased stability upon heat treatments. This might support
the theory regarding the formation of reducing substances when milk is
treated at certain temperatures.

Addition of glutathione to milk was found to have had no effect
upon the ascorbic acid value or upon the deve10pment of the oxidized
flavor.

.L few cows' milk showed a very great stability in respect to the
ascorbic acid. By different treatments such samples always maintained a
higher ascorbic acid value.

Milk with relatively large amounts of ascorbic acid originally
was found to be more susceptible to the development of the oxidized
flavor under various processes. Copper added before and after heating
had different effects upon the ascorbic acid. That added before had the
most destructive effect. Samples showing the smallest decrease in ascorbic
acid.after various treatments were most susceptible to the development of

the oxidized flavor upon the addition of copper.

- 135 -

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1936. Off Flavored Milk - a Problem of Animal Nutrition. Milk
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(2) Bersin, T. and Kosen, H. 2.
1935. Biochemical Relationships between Ascorbic Acid and
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(:5) Bessey, c. A. and King, 0. o.
1934. Chemical Determination of Vitamin C. Jour. Biol. Chem.
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(4) Borsook, H. and Jeffreys, C. E. P.
1936. Glutathione and Ascorbic Acid. Science 83 z 397.

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- 136 -

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1933. Strength of Skin Capillaries for Determining Vitamin C.
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Guha, B. C. and Ghash.
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Harris, L. J. and Ray, 8. N.
1933. Vitamin C and the Suprarenal Cortex. Biochem.1Jour.
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1936. Inactivation of BacteriOphage by Ascorbic Acid. C. R.
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