The autoxidative behavior of milk lipid fractions in systems of controlled composition
by Shun Ku
A thesis submittted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Agricultural Products Utilization
Montana State University
© Copyright by Shun Ku (1966)
Abstract:
Fractions of milk lipids representing the phospholipid protein complex (fat globule membrane material
from buttermilk (FGM I), and from butter serum (FGM II), the true fat (butteroil), and the intact milk
fat globules (dialyzed cream) were allowed to autoxidize in systems composed of distillated water or
milk serum or occasionally pH 6.60 phosptiate buffer. The effect of the following factors was
investigated: (1) copper ion alone, ascorbic acid alone and copper ion plus ascorbic acid (2) sul-phydryl
groups (L-cysteine), whole casein and K-casein (3) synthetic and naturally occurring antioxidants
(NDGA and α-tocopherol).
The autoxidative behavior of these milk lipid fractions was dependent on the particular fraction used
and the nature and composition of the aqueous system into which it was dispersed. Copper ion alone
and ascorbic acid alone, catalyzed oxidized flavor development by the fat globule membrane and
dialyzed cream. Copper ion plus ascorbic acid gave the highest oxidized flavor intensity with fat
globule membrane material. Copper ion from various salts gave variable catalytic power indicating that
the catalytic power of copper ion in milk serum is dependent on the dominant kinds of cuprous salt
complexes. The lowest oxidation intensities were given by the butteroil.
The fat globule membrane from buttermilk (FGM I)? differed very significantly in its oxidative
behavior in comparison to that from butter serum (FGM II). Generally FGM II gave much higher
oxidation rates and intensities than FGM I.
L-cysteine, whole casein, K-casein, NDGA and α-tocopherol behaved either as antioxidatants or
pro-oxidants depending on the milk lipid fractions in question and the aqueous medium used. The
reason for such behavior is discussed. THE AUTOXIDATIVE BEHAVIOR OF MILK LIPID FRACTIONS
IN SYSTEMS OF CONTROLLED COMPOSITION
by
SHUN KU
A thesis submittted to the Graduate Faculty in partial
fulfillment of the requirements for the degree
of
MASTER OF SCIENCE
in
Agricultural Products Utilization
Approved:
Head, Major Department
MONTANA STATE UNIVERSITY
Bozeman, Montana
December, 1966
-iiiACKNOWLEDGEMENTS
The author wishes to express his appreciation to Dr. A. M. El-Negoumy
for his guidance and help through the investigation and the preparation of
this thesis, and also his generous offer of prepared casein and Kappa casein.
Thanks are also due to Dr. J. C, Boyd for the assistance enabling
the author to pursue graduate work at Montana State University.
Sincere appreciation is expressed to Dr. Graeme Baker for his useful
suggestion in the author's experimental work.
He also wishes to thank Dr.
K. Goering, Dr. C. A. Watson and Dr. K. Emerson for helpful suggestions in
setting his study program.
The author wishes to express his sincere gratitude to his parents
for their encouragement and understanding.
I
-ivTABLE OF CONTENTS
Page
VITA
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11
ACKNOWLEDGEMENTS .
iii
TABLE OF CONTENTS,
iv
v
INDEX TO TABLES'
ABSTRACT.......
o * e « . e < e * e
INTRODUCTION
II.
•
ix
e
.......... ......................
3
. . . . . . . . . . .
3
A.
The Origin of Oxidized Flavor . . . . . .
3
B.
The Role of Milk Fat and Phospholipids
5
..................
Nature of Autoxidation
..........
Factors Influencing Development of Oxidized Flavor
A.
B.
............
. . . . . . .
8
Effect of Composition of the Aqueous Phase
on Oxidized Flavor Development
D„
6
Effect of Copper, Sulfhydryl Group and Metal
Protein Complexes . . . . . . . . . . . . . .
C.
6
The Role of Copper in Presence and in Absence
of Ascorbic Acid
Effect of Antioxidants
. . . . . . .
10
10
.............
I.
Nordihydroguaiaretic Acid (NDGA)
2»
Alpha-tocopherol............ . . . . .
....
10
11
. . . . .
12
Experimental Techniques . . . . . . . . . . . . .
13
A.
13
Ill. The Nature of the Fat Globule Membrane
IV.
I
...
LITERATURE REVIEW.
I.
*
The Use of Model S y s t e m ................ .. .
-VB„
The 2-thiobarbituric Acid (TEA) Test ..............
MATERIALS AND METHODS , , » , , e e * » » * e * * » o » * » * * * o @ *
I.
II.
III.
Preparation of Milk Lipid Fractions
. . . . . . . . . .
A.
Fat Globule Membrane Materials (FQl) ..............
B.
B u t t e r o i l .................... ........... ..
C.
Dialyzed Cream (D.Cr.) . . . . . . . . . . . . . .
.
The Basic Medium For the Systems . . . . . . . . . . . .
A.
Milk Serum ........
. . . . . . . . . . . . . . . .
B.
Phosphate Buffer ..................
C.
Distilled Water
. . . . . . . .
. . . . . . . . . . . . . . . . . .
Preparation of the Model Systems . . . . . . . . . . . .
i
IV.
Measurements of Oxidation Rates and Intensity
RESULTS AND DISCUSSION
SUMMARY
o
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APPENDIX
LITERATURE CITED
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. . . . .
. . . . . . . . .
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•
-vi-
INDEX TO TABLES
Page
Table
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
X.
The effect of Cu*-*" plus ascorbic acid on the
autoxidation of various milk lipid fractions
in different media (absorbance)...........................
25
The effect of increasing concentration of ascorbic
acid in the absence of Cu++ on the autoxidation of
milk lipid fractions (absorbance).........................
62
The effect of increasing concentration of ascorbic
acid in the presence of Cu++ on the autoxidation of
milk lipid fractions (absorbance).........................
63
The effect of Cu*""*" from various sources in the
presence of ascorbic acid in water and milk
serum systems on the autoxidation of milk lipid
fractions (absorbance)....................................
64
The effect of varying fat content (from dialyzed
cream) in presence of 4 ppm Cu++ and an increased
concentration of ascorbic acid in milk serum on
the autoxidation of milk lipid fractions (absorbance).....
65
The effect of increasing concentration of L-cysteine
in the presence of Cu++ and 2% of lipid on the
autoxidation of milk lipid fractions (absorbance).........
66
The effect of increasing concentration of casein
in the presence of Cu++ on the autokidation of
milk lipid,fractions (absorbance).........................
67
The effect of increasing concentration of Kappa
casein in the presence of Cu++ on the autoxidation
of milk lipid fractions (absorbance)......................
67
The effect of NDGA in the presence of Cu"*-*" on the
autoxidation of milk lipid fractions (absorbance).........
68
The effect of the antioxidant property of
tocopherol on the autoxidation of milk
lipid fractions (absorbance)..................j............
69
-Vi,!Figure
1.
Page
The effect of increasing concentration of ascorbic
acid in water system and in the absence of copper
ions on the autoxidation of milk lipid fractions.........
25
2 . The effect of increasing concentration of ascorbic
3.
4.
5.
acid in milk serum in absence of copper ions on the
aytoxidation of milk lipid fractions.....................
27
The effect of increasing concentration of ascorbic
acid in presence of 4 ppm Cu"*-*" in water system on
the autoxidation of milk lipid fractions.................
28
The effect of increasing concentration of ascorbic
acid in the presence of 4 ppm Cu^+ in milk serum
system on the autoxidation of milk lipid fractions.......
29
The effect of Cu+* from different sources in water
system on the autoxidation of milk lipid fractions.......
36
6. The effect of Cu*""*" from different sources in milk
serum system on the autoxidation of milk lipid
fractions.........................................
7»
8.
..
The effect of varying fat content (from dialyzed
cream) in the presence of 4 ppm Cu"*"*" and an
increasing concentration of ascorbic acid in milk
serum system on autoxidation of milk lipid fractions.....
38
The effect of increasing the concentration of
L-cysteine in the presence of 4 ppm Cu"*""*" in
water system on the autoxidation of milk lipid
fractions................................................ ..
9.
10.
11.
37
The effect of increasing the concentration of
L-cysteine in the presence of 4 ppm Cu*""*" in
milk serum on the autoxidation of milk lipid
fractions.........................................
The effect of increasing concentration of casein
in the presence of 4 ppm Cu+ *" in water system on
the autoxidation milk lipid fractions.............
The effect of increasing concentration of casein
in the presence of 4 ppm Cu"*-*" in milk serum on the
autoxidation of milk lipid fractions..............
' 41
42
..
46
47
-viiiFigures
12.
13.
14.
15.
16.
17.
Page
The effect of increasing concentration of K-casein
in the presence of 4 ppm Cu++ in water system on
the autoxidation of milk lipid fractions..................
48
The effect of increasing concentration of K-casein
in the presence of 4 ppm Cu++ in milk serum oh the
autoxidation of milk lipid fractions.......... ...........
49
The effect of NDGA. in the presence of 4 ppm Cu++
in water system on the autoxidation of milk lipid
f
r
a
c
t
i
o
n
s
.
.
.
3
The effect of NDGA in the presence of 4 ppm Cu++
in milk serum system on,the autoxidation of milk
Ixpid fractions...........................................
4
^^
The effect of the antioxdant property of
c(-tocopherol in wafer system on the auto­
xidation of milk lipid fractions........................
55
The effect of the antioxidant property of
of-tocopherol in milk serum system on the
autoxidation of milk lipid fractions.......... .
57
-ix-
ABSTRACT
Fractions of milk lipids representing the phospholipid protein
complex (fat globule membrane material from buttermilk (FGM I), and from
butter serum (FGM II), the true fat (butteroil), and the intact milk fat
globules (dialyzed cream) were allowed to autoxidize in systems composed
of distillated water or milk serum or occasionally pH 6.60 phosptiate
buffer.
The effect of the following factors was investigated: (I) copper
ion alone, ascorbic acid alone and copper ion plus ascorbic acid (2) sul=
.
phydryl groups (L-cySteine), whole casein and K-casein (3) synthetic and
naturally occurring antioxidants (NDGA and o(-tocopherol).
The autoxidative behavior of these milk lipid fractions was depen­
dent on the particular fraction used and the nature and composition of the
aqueous system into which it was dispersed.
Copper ion alone and ascorbic
acid alone, catalyzed oxidized flavor development by the fat globule mem­
brane and dialyzed cream.
Copper ion plus ascorbic acid gave the high­
est oxidized flavor intensity with fat globule membrane material.
Copper
ion from various salts gave variable catalytic power indicating that the
catalytic power of copper ion in milk serum is dependent on the dominant
kinds of cuprous salt complexes. The lowest oxidation intensities were
given by the butteroil.
The fat globule membrane from buttermilk (FGM I)? differed very
significantly in its oxidative behavior in comparison to that from butter
serum (FGM II).
Generally FGM II gave much higher oxidation rates and
intensities than FGM I .
-X -
L-cysteine, whole casein, K-casein, NDGA ando(-tocopherol behaved
either as antioxidatants or pro-oxidants depending on the milk lipid
fractions in question and the aqueous medium used.
behavior is discussed.
The reason for such
INTRODUCTION
Off-flavors resulting from lipid oxidation are among the most common
flavor defects occurring in milk and its products.
often described as:
These off-flavors are
cardboard, oxidized, metallic, oily, tallowy, or fishy
depending on the intensity of the defect.
This flavor defect causes
millions of dollars of losses every year both to the milk producers and
processors.
The bulk, of the fat in milk is in the foi^n of small fat globules
averaging 2-5 u in diameter.
The surface of each fat globule is covered
with a layer of material commonly known as the fat globule membrane (FQM).
This, membrane is a complex made up mainly of phospholipids and proteins.
From the standpoint of autoxidation, it is of importance to realize that
these globules exhibit a large surface area which is rich in oxidizable
phospholipids. The most important factors that tend to accelerate oxidized
flavor development- are metal catalysis and the presence of ascorbic acid
which provide a properly poised hydrogen accepting and donating system in
the reduced-oxidized ascorbic, acid relationship.
Research on oxidized flavor development in milk is complicated by
the fact that different milks show.extreme variation in resistance and
susceptibility.
Some milks are objectionably oxidized within a few hours
from milking, while in others, this effect cannot be induced even with
large additions of copper.
There is little doubt that the complexity and
variability of milk composition are contributing factors to the confusing
results sometimes obtained in oxidized flavor research.
In the present
investigation, model systems of known composition were used in an effort
to minimize the variables.
Various fractions of milk lipid were
-2“
incorporated in these systems with the objective of determining the source
of "off-flavors" and the compositional conditions that modify their in­
tensities.
Among the lipid fractions used were:
fat globule membrane
.
materials both from buttermilk, and butter serum; whole fat globules as
dialyzed cream; butteroil prepared by skimming (devoid of phospholipids),
and butteroil prepared by complete dehydration (rich in phospholipids)„
Comparative analyses were made to investigate the following:
(1)
The development and intensity of the flavor in various media
such as milk serum, phosphate buffer and salt-free medium
(water).
(2)
The effect of variation in the concentration of some of the
major and minor milk constituents on the course and intensity
of flavor development.
(3)
The role of copper ions and ascorbic acid in promoting or
inhibiting oxidized flavor development in the various milk
lipid fractions.
(4)
The effect of some antioxidants and the conditions leading to
their relative effectiveness.
REVIEW OF LITERATURE
I.
Ac
THE NATURE OF AUTOXIDATION
The Origin of Oxidized Flavor
Oxidized flavor was first recognized as due to autoxidation of milk
lipids by Rogers and Gray (1909).
Greenbank (1940) believed this flavor
was due to a group of unstable intermediate compounds which could be
further oxidized to flavorless substances.
The compounds resulting from
lipid autoxidation represent a complex system, and the general routes of
the reactions taking place were reported by Lea (1962) as follows:
Dimer, Higher polymer
t
polymerization
Autoxidation through
free radical
Fat
Fat hydroperoxides
further oxidation
✓
peroxides
/
,/
polymers
fission
/
hydration
\
oxidation of CH=GH
\
aldehydes
ketoglycerides
epoxides
semi-aldehydes
OH-glycerides
aldehydo-glycerides
Di-OH-glycerides
OH-compounds
acids
El-Negoumy ej: a L (1962a) indicated that the polyunsaturated fatty
acids, especially linolec and linolenic acid are the precursors for flavor
compounds. According to Farmer and Sutton (1943b) and Farmer and
Sundralingam (1943a), lipid autoxidation is initiated through a free
radical mechanism as follows:
-4-
The initial step probably concerns the removal of hydrogen from a
methylene group adjacent to a double bond. The resulting free radical may
attach an oxygen to form a peroxide free radical which then abstracts
hydrogen from another methylene group adjacent to a double bond to start
over a formation of another free radical and continue the chain reaction.
This mechanism was confirmed by chemical kinetic studies.
(Holland and
Gee 1946).
The compounds responsible for oxidized flavor were reported to be
carbonyls in nature and to include n-alkanals; 2-alkenals; 2, 4-dienals;
ketones and vinyl ketones (Keeney and Doan 1951, a,b,c,; El-Negoumy et a l .
1961 and Tharp and Patton 1960).
Malonaldehyde which is an intermediate
compound in autoxidation and which is responsible for the 2-thiobarbituric
acid color reaction, has been reported by Day (1960) to correlate well
with organoleptic scores.
Later research indicated that oct-l-en-3-one displays metallic
flavor in oxidized dairy products with a flavor threshold of one part in
q
10
in butter fat (Stark and Forss 1962) and that 2, 6 ,-nonadienal displays
the grassy flavor (Hammond et: j*L 1964) .
proposed as their respective
Linoleate and linolenate were
precursors.
-5-
Wilkinson (1964) suggested the following mechanism for the pro­
duction of oct-l-en-3-one through cleavage of the polyunsaturated fatty
acid and secondary decomposition of the autoxidation products:
CH3-(C H 2) 4-CH=CH-CH2- ^ CH=CH-CH2) 6 - C<>°
9 ,12-linoleic acid
CH3(CH2) 4-CH=CH-CH2Oh
oct-2-enol
CHi(CH2)^-C H
CH___ CH2 + +OH "
intermediate
CH3(CH2) 4-CH-CH=CH2
OH
oct-l-en-3-ol
through chain propagation or
^ hydroperoxides decomposition
CH3(CH2) 4 -C - CH=CH2
+-H-
&
oct-l-en-3-one
B,
The Role of Milk Fat and Phospholipids
From the standpoint of oxidized flavor development in aqueous media,
only the 0.2-1.0% phospholipids in milk lipid (Jenness and Patton 1959)
are of concern.
The true fat (triglycerides) part of milk fat does not
seem to be attacked in aqueous dairy products.
The phospholipids which
are present in the fat globule membrane are the site for oxidized flavor
development (Palmer and Samuelsson 1924; Thurston ^t aL 1935).
This is
confirmed by the fact that the iodine number of the polyunsaturated
”6-*
fatty acids in phospholipids was lowered substantially during oxidised
flavor development while that of the triglycerides was practically unchang­
ed (Swanson and Sommer 1940)„
The vulnerability of phospholipids to attack by oxygen was proposed
to be due. to their higher content of polyunsaturated fatty acids (Hilditch
1956; Smith and Lowry 1962) and also to their existence as a part of the
phospholipid-protein complex of the fat globule membrane which is an ad­
sorption site for copper ions and ascorbic acid (Tarassuk and Koops i960)„
The carbonyl compounds ensuing from milk lipids during oxidized
flavor development were isolated and characterized by several investigators
Day and Lillard (I960), and El-Negoumy et al„ (1961), isolated them from
autoxidized but ter oil, Forss et, aL» (1955, 1960 a,b,c,) isolated them from
skim milk and washed cream, Bassetfe and Keeney (1960) isolated them from
non-fat dry milk, while Parks and Patton (1961) isolated them from stored
dry whole milk, autoxidized under various conditions„
Fhospholipids are preferentially oxidized in an aqueous system,
while they serve as an antioxidant in non-aqueous dairy products (Patton
1962)o
II. FACTORS INFLUENCING THE DEVELOPMENT OF OXIDIZED FLAVOR
A.
The Role, of Copper in Presence and in Absence of Ascorbic Acid
The level of naturally occurring copper ions in milk is sufficient
to catalyze the development of oxidized flavor (King and Dunkley 1959;
Hunziker 1924).
The addition of ascorbic acid tq metal-free milk retarded, but
did not stop oxidized flavor development (Gorbelt and Tracy 1941).
-I -
On the other hand, in copper-contaminated milk, ascorbic acid is oxidized
during storage and at the same time cupric ions are reduced to the cuprous
state (Holmes 1953).
Cuprous ions are the active catalyst in milk lipid
autoxidation (Smith and Dunkley 1962 a,b).
This mechanism was demonstrated
by using the copper chelator, neocuproine and ascorbic acid oxidase.
The
following mechanism involving the catalytic role of the cuprous ions in
peroxide radical formation was suggested by Wilkinson (1964):
Hx
/"X
X/
CH-CH(OH) CH2OH + Cu++
X
OH 2
Cu
Z \
VX
Q-
/
C = C
O=C^
O=C
CH-CH(OH)CH2OH + Cu+
\V
0
0
C
/
CH-CH(OH)CH0OH +HO2 -
X
0
+ H+
v
WC
^\]H-CH(OH)CH-OH
-8
Tarassuk and Koops (I960) suggested that a special structural con­
figuration of the copper-protein-lipid complex may be capable of attaching
ascorbic acid to it permitting cupric ions to be converted to the cuprous
state by ascorbic acid oxidation.
The level of ascorbic acid has a direct influence on the autoxidation
rate of milk fat globule membrane (King, 1962, 1963) , El-Negoumy (1965)
suggested that both the oxidation rate and intensity in an aqueous phase
are dependent pn the kind and predominance of the cuprous salt complexes
formed„
B.
,I
Effect of Copper. Sulfhydryl Groups and Metal Protein Complexes
The sulfhy^ryl groups (-SH) in thioglycolic acid, cysteine and
glutathione catalyze the autoxidation of phospholipids and unsaturated
fatty acids (Meyerhof 1918, 1923; Harrison 1924; Hopkins 1925).
This
catalytic effect ceases when the (-SH) groups are oxidized to disulfide
(-S-S-) or sulphate (Tait and King 1936).
Catalysis of lipid oxidation
involving (-SH) groups require the presence of traces of metallic ions,
especially iron or copper, which also cafalyze the oxidation of (-SH)
Hopkins
(1925) suggested that (-SH) groups catalyze peroxide formation
■
,
through their oxidation to the disulfide form (-S-S-)„ On the other hand,
Forster and Sommer (1951) reasoned from their observation that trypsin re­
tarded or prevented oxidized flavor and that this was due to the release
of reactive (-SH) groups from whey proteins„ This is confirmed by the
fact that fishy flavor in washed cream was inhibited by addition of heated
skim milk or whey and that the inhibiting effect was removed by blocking
(-SH) with the SH inhibitor N-ethylmaleiamide (Tarassuk, Koops and Pette
-9-
1959).
Sulfhydryl compounds and proteins containing (-SH) groups are
believed to be similar in their behavior to ascorbic acid during lipid
autoxidation.
Wilkinson (1964) proposed the following mechanism for explaining
the role of cupric ions and glutathione during the course of peroxide
formation by unsaturated fatty acids:
R1
,R"
+2Cu++
S
+
SH+
Vs xi""
°,
GSSG
GSO2H
x CU+ ''
R'
H
^ Xz H
X / H
/ \
HgC
C r " + 2 H-
2 RH
\
/
C
/ \
2 H2C
C-Rm (GS)
I i!
+ 2 Cu
\ H'''
RO •, RO2, ROOH etc
RH = Unsaturated fatty acid
R = Glutamyl Radical
R = Glycinyl Radical
-10C.
Effect of the Composition of the Aqueous Phase on Oxidized Flavor
Development
The composition of the aqueous medium in which the lipid is dispers­
ed has a very significant effect on both the rate and intensity of oxidized
flavor development by milk fat. The various salts present in milk serum
may form complexes with ionic copper, which makes them vary significantly
in their catalytic power (El-Negoumy 1965).
El-Negoumy (1965) also found
the concentration of sodium caseinate, ©(-lactose and ascorbic acid to
significantly influence autoxidation of globular milk fat.
This effect
was dependent on the kind and concentration of other solubles in the system.
D.
Effect of Antioxidants
I . Nordihydroguaiaretic Acid (NDGA)
Nordihydroguaiaretic acid (NDGA), a phenolic antioxidant has the
following structure:
Its antioxidant properties result from the two hydroxyl groups in
the ortho and para configuration (Matill 1931; Stull ej: a L 1948 a, b) .
A kinetic study (Lundberg at al.1947a) indicated that in general, the loss
of antioxidant property of phenolic antioxidant in autoxidized fats did
not occur as a single reaction but was completely dependent on reaction
with the products of fat oxidation.
NDGA was found to be an effective antioxidant for various dairy
products such as frozen sweet cream (Stull at aL 1947, 1949), whole
-11milk and unsweetened frozen creep (Stull et al. 1948a), spray-dried milk
and ice crgam (Stull £t al. 1951 a, b) , butter (Krukovsky j2t al. 1949 a,b) ,
butferoil (Wyatt an4 Day 1965), and salted sweet cream butter (El-Negoumy
and Hammond 1962).
NDGA functions as an antioxidant by retarding oxygen absorption by
phospholipids and methionine.
Ascorbic and citric acids act as syner­
gists which enhance the effectiveness of NDGA.
This antioxidant shows its
greatest action near pH 6.5 (£>tull ej: al. 1951b) . NDGA retards the induction
period of metal catalyzed lipid oxidation (McDowell, 1955).
Non-oxidized and stale flavors were reported in cold storage dairy
products containing NDGA (El-Negoumy and Hammond 1962; Wyatt and Day 1965).
Thfe optimum concentration of NDGA for its most effective antioxygenic
aption lies within 0.001 to 0.01% (Stull jit al.1951b).
2.
Alpha-tocopherol
Tocopherols (vitamin E) arfe well known as antioxidants in animal
tissues (Zalkin et al.1960; Edwin et al.1961).
Alpha-tocopherol appears
to be a naturally occurring antioxidant in milk fat which contains about
0.022 mg/g. (Webb and Johnson 1965),
A significant correlation exists
between the tocopherol content of milk and its ability to resist oxidized
flavor development (Krukovsky et al.1949'a.b). Natural alpha-tocopherol
occurs in the unsaponificable material of milk fat and can be added to the
extracted fat to enhancfe its stability (Smith et al,1948).
•
'I
Addition of alpha-tocopherol to milk during cold storage at con­
centration from 0 .2-2 .4 mg per liter decreases the loss of reduced ascorbic
acid and increases somewhat the milk's resistance to oxidized flavor
"12development (Holmes 1952).
III.
THE NATURE OF THE FAT GLOBULE MEMBRANE
The bulk of the lipid material in milk exists in the form of an
emulsion of tiny spherical particles (3-5 u in diameter), or globules
dispersed in milk serum (Jenness and Patton, 1959).
A membrane surround­
ing each fat globule was demonstrated microscopically by Storch in 1897,
The phospholipids occur in the membrane as a monomolecular layer which is
associated with the membrane protein iri the form of a complex (Palmer and
Samuelsson, 1924; Palmer and Wiese, 1933; Sommer, 1951; and Brunner et al.
1953 a,b).
King (1954) visualized the fat globule membrane to be made of a
surface layer of polar phospholipids, cholesterol, and vitamin A which is
associated with the high melting triglycerides in the lipid phase of the
fat droplets and are attached to the protein in the aqueous phase of milk
serum.
Morton (1954) reported the fat globule membraine to be an accumu­
lation of lipoptotein particles, "milk microsomes", which passed into the
m|.lk from the mammary gland during milk secretion.
Brtinner (1962) considered Van der Walls force, hydrogen bonding,
and electrostatic attraction to be the major forces responsible for proteinlipid interaction in the membrane.
Harwalker and Brunner (1965) suggested
that the hydrophobic and covalent disulfide bonds may be significant forces
in the microstructure of the membrane complex.
. The milk fat globule membrane was suggested as a possible site of
lipid oxidation due to; (a) its high oxidizable phospholipids content
(Jenness and Patton, 1959); (b) its high catalytic copper content (Herald
-13-
et aL 1957b and Ramachandran et al. 1960)
Herald and Brunner (1957a) isolated and separated ,the fat globule
membrane proteins into two fractions based on their solubility in 0 .02M
NaCl„
The insoluble fraction had a brown reddish color;
properties simi­
lar to those of pseudokeratin (Brunner 1962) and a higher density than the
soluble fraction.
These workers also found the insoluble fraction to be
more tightly associated with the fat globules (Jackson and Brunner 1960)
I
than the soluble fraction.
On the other hand, the soluble fraction had
a low-density, whitish color, properties similar to glycoprotein (Brunner
1962), and higher phosphorus content than the insoluble fraction (Herald
et a I. 1957b) .
IV.
A.
EXPERIMENTAL TECHNIQUES
The Use of Model System
Model systems are used for the purpose of investigating the effect
'
of various milk constituents on the development of oxidized flavor. King
(1962) used model systems composed of pH 6.5 phosphate buffer to investi­
gate the autoxidation of fat membrane material in presence of ascorbic
acid.
El-Negoumy (1065) investigated dialyzed globular milk fat in the
presence of various salts and in both water and phosphate buffer systems.
The 2-thiobarbituric acid test was used in King's (1962) and
El-Negoumy1s (1965) work to measure the oxidation rates.
Reaction con­
ditions were standardized by King (1962) using model systems.
B.
The 2-thiobarbituric Acid (TBA) Test
2-thiobarbituric acid (TBA) has the following structure:
-14-
TBA reagent was first used by Liversedge and Kohn (1944).
They
observed that various animal tissue slices, after incubation under aerobic
conditions, developed a characteristic color which was measured spectrophotometrically at 535 mu.
The color was produced as a result of the
interaction of products of oxidation of unsaturated fatty acids with TEA.
Dunkley and Jennings (1951) found that the absorbance of the TBA
color reaction correlated closely with the organoleptic flavor score of
oxidized milk.
Several reports (Patton £t aJ.. 1951 and Biggs and Bryout
1953) discussed the usefulness of this reaction and factors influencing
the results, such as the pH, heating time, method of measuring color,
and copper contamination.
Sidwell et al.(1954) compared the peroxide value, total carbonyl
value and TBA test in the same samples and found them to be correlated
with each other.
Jennings £t al. (1955) found the spectra of pigments derived from
the TBA reaction with oxidized milk to be identical with those derived
from TBA-malonaldehyde reaction.
Sinnhuber And Yu (1958) used elemental analysis to determine the
empirical formula of TBA pigment as C ^ H g N ^ O ^ ^ .
Furthermore, the
of
one pigment derived from TBA-oxidized milk and the pigment derived from
TBA-malonaldehyde on paper chromatography were identical.
They believed
-15-
the pigment to be a condensation product produced according to the
following reaction:
HS
S^/N^jOH
1K z O H
x: -CHo-C^
/
\
H
+ 2 H2O
HCl
H2O
tl
^iT
I= CH-CH=CH
(pigment)
O
H
The exact mechanism of maIonaldehyde formation during autoxidation
is not known. Wilkinson (1964) suggested that the precursors of malondaldehyde may be derived from linoleates-2-enals and 2, 4, dienal as follows:
R - CH 2 -CH=CH-CHO
°2 + Cu++
Z
R - GH -CH=CH-CH=O
t
acid + Cu++
0
i
R - CH=O + CH(OH)=CH-CH=O
malondaldehyde (enol form)
According to King (1962), the use of trichloroacetic acid in the
TBA test has several advantages. Trichoroacetic acid is used to flocculate
the proteins and provide the acidity necessary to carry out the subsequent
reaction with TBA.
This high acidity effectively stops lipid reactions
which are particularly important when applying such a rapid raction to
milk.
They also found that lactose interferred with the TBA reaction.
This was shown by chromatographic separation and spectrophotometrie
analyses of the TBA pigments. A satisfactory color formation was
-16—
indicated at 60°C at which lactose degradation is minimized.
Results were
also presented, showing good correlation of the oxidized flavor with the
TBA test.
King (1962) also found the TBA test to correlate well with
organoleptic judgment.
MATERIALS AND METHODS
I.
Preparation of Milk Lipid Fractions
A 0 Fat Globule Membrand Materials (FQl)
The fat globule membrane material was prepared according to King
(1962)„
Fresh raw cream obtained from the University's dairy plant was
warmed to 40°C and washed four times, using four folds of its voluthe of
tap water with a De Laval Model 518 cream separator.
to remove all traces of skim milk constituents.
The objective was
The washed cream was then
cooled to S-IO0C s and churned in a laboratory churn to obtain butter arid
buttermilk.
The pH of the buttermilk was adjusted to pH 4.9 with 0.1 N
HCl followed by exhaustive dialysis against pH 4.9 water for 48 hours at
2-4 C .
The dialysis water was changed every 12 hours.
The volume of the
dialyzed buttermilk was reduced considerably by perevaporation, and the
balance of the water was then removed by freeze drying.
has been designated ,FGM I .
i
This preparation
The butter was melted at 70°C and then sep-
arated into butteroil and butter serum using a separatory funnel. The
butter serum was treated in the same manner as the buttermilk to obtain
a preparation designated as FQl II.
of these preparations;
The following is a schematic diagram
-18Raw Cream
Warm to 40°C add tap water and separate
Repeat 4 times
\l/
Washed Cream
Cool overnight at 5-10°C
v
Churning
/
Buttermilk
V
Butter
Melt at 70°C
Butteroil
Butter serum
4
pH adjusted to 4.9
(Treat as Buttermilk)
s/
Dialyzed gainst pH 4.9
water for 48 hours
Perevaporate
4
Lyophilize
4
FGM I
(from buttermilk)
B.
FGM I I
(from butter serum)
Butteroil
The methods used in preparing the various butteroils were those
used by El-Rafey £t jal. (1944).
I.
Phospholipid rich butteroil (B.O.I):
Melted butter was saturated
with NaCl and the moisture was removed by boiling.
The proteins
and membrane material were precipitated by NaCl, leaving a
golden yellow butteroil which was separated by a separatory
funnel to remove all contaminants.
did not exceed IlO0C.
The processing temperature
-19-
2.
Skimmed, 70°C, butteroil (B.O.II):
Butter was melted at 70°C,
which resulted in separation of the butteroil layer from the
butter serum.
The butteroil was skimmed off then filtered
through a layer of glass wool.
This butteroil is almost devoid
of phospholipids.
3.
Butteroil III, rich in phospholipids heated to 130°C (B.O.III):
The butter was melted and heated with continuous stirring at
IOS-IlO0G until the water had been evaporated and the non-fat
solids settled.
The temperature was then raised to 130°C.
As
the temperature approached 120°C, the butteroil formed a rela­
tively stable foam which dissolved back into the fat on cooling.
The fat had a dark reddish color and the characteristic aroma
of the butteroil prepared by the boiling-off process.
addition, it had a better flavour and odor.
In
The fat was then
clarified by centrifuging and the dark brown sediment was
removed.
C . Dialyzed Cream (D.Cr.)
Fresh raw cream was washed with tap water and then dialyzed for
48 hours.
water,.
The fat content was then adjusted to 30% by adding distilled
It was then divided into 150 ml portions which were placed
in plastic bags and frozen until needed.
II.
A.
THE BASIC MEDIUM FOR THE MODEL SYSTEMS
Milk Serum
Milk serum was prepared by dialyzing 2 liters of 5% lactose solution
against 24 gallons of skim,milk for 48 hours at Z-S0C .
The milk serum was
-
20
-
then divided into small portions, frozen and used as needed„
B.
Phosphate Buffer
■
!
Phosphate buffer (pH 6 .6) was prepared by dissolving 4.4 g NaH2P04
'
plus 2.6 g NagHPO^ per liter of distilled water.
C.
1
Distilled Water
Fresh prepared distilled water was used.
Ill.
PREPARATION OF THE MODEL SYSTEMS
All the model systems used in this investigation contained 2% lipid
material and 4 ppm copper ions per 100 ml.
These systems were basically
made of either distilled water or milk serum.
Distilled water and phos­
phate buffer were used for comparative experiments.
The other components
in these systems were added at the concentrations indicated in the individI
ual experiments.
All systems were adjusted to pH 6 .6-6.8 using either
0.10 N HCl or NaOH as needed.
Oxidation was allowed to proceed for 48
hours at 3-5°C before analysis.
Whole casein and K-casein were prepared by Dr. El-Negoumy. Whole
casein was iso-electricalIy precipitated from fresh skim milk at pH 4.60
and separated by centrifugation at 3000 x g.
This wet casein was washed
5 times with distilled, water, with the aid of an electric blender. The
washed casein was then freeze dried.
K-casein, prepared from whole casein was purified by the method
,
of Zittle and Custer (1963).
'
'I
I
Ascorbic acid," L-cysteine, NDGA, and o(-tocopherol were commercial
preparations purchased frop Nutritional Biochemicals Corporation, Cleveland,
Ohio.
-21IV.
MEASUREMENT OF OXIDATION RATES AND INTENSITY
Progress of oxidized flavor development was followed by the 2-thiobarbituric acid test, using King's method (1962) as follows:
The oxidized material (17.6 ml) were pipetted into a glass stopper­
ed flask.
After warming to 30°C, 1.0 ml of 100% trichloroacetic apid
solution was added, followed by the addition of 2.0 ml of 95% ethanol.
The flask was stoppered and shaken vigorously for 10 seconds. After
J
5 minutes, the contents were filtered through Whatman No. 42 filter paper.
;
To 4.0 ml of the clear filtrate 1.0 ml of TBA solution was added.
The
TBA solution was made by dissolving 1.4 g 2-thiobarbituric acid in 100 ml
of 95% ethanol.
The flask Was stoppered, mixed and placed in a 60oC water
bath for one hour.
I
The reaction solution was cooled to room temperature
;■
.
- -
and its absorbance measured at 532 mu using a Beckman Model B spectrophoto­
meter.
Blank determinations were made with each set of experiments using
fresh identical systems which were not oxidized.
The absorbance of the
blanks were subtracted from those pf the samples tp give the absorbance
due to lipid oxidation.
RESULTS AND DISCUSSION
In the present investigation, various fractions of milk lipid were
subjected to autoxidation in model systems composed basically of distilled
water, or milk serum, and occasionally pH 6.60 phosphate buffer.
Among
the milk lipid fractions, the phospholipid-protein complex of milk fat was
represented by the fat globule membrane material and whole globular milk
fat was represented by dialyzed cream.
Pure butteroil represents the true
fat (triglycerides) of the milk fat globule.
Six milk fat preparations
were used in this investigation:
(1)
Fat globule membrane prepared from buttermilk (FGM I):
This was a brown reddish color, a high density and retained a hygro­
scopic consistency upon freeze drying.
(2)
Fat globule membrane prepared from butter serum (FGM II):
This has a whitish appearance and a lower density than FGM I .
(3)
Dialyzed cream (D.Cr.):
This was prepared from washed cream
by exhaustive dialysis.
(4)
Butteroil I (B.O.I):
Rich in phospholipids, prepared by the
boiling-off method at IlO0C .
(5)
Butteroil II (B.O.II):
Devoid of phospholipids, prepared by
the floatation method at 70°C.
(6)
Butteroil III (B.O.III):
Rich in phospholipids, prepared by
the boiling-off method at 130°C.
The difference in physical appearance and properties of FGM I and
FGM II is probably due to differences in the amounts, and possibly the
kinds of proteins, which make up the phospholipid-protein complex.
Herald ej: al. (1957) separated 2 protein fractions from the fat
-23-
globule membrane based on their solubility in 0.02 M NaCl. One of them
was insoluble and generally had an appearance similar to FGM I prepared
here, while the soluble protein was similar to FGM II.
These authors
indicated that the soluble fraction had a higher copper and phosphorus
contend: than the insoluble fraction.
An examination of the results
obtained here with these 2 fractions indicated that they generally
differed substantially in their oxidative behavior.
Generally FGM II
exhibited much higher oxidation intensities than FGM I.
The butteroil preparations showed the lowest oxidation intensities
and rates, probably because they lacked the fat globule membrane. Dialyzed
cream, which is composed of the triglycerides and surrounded by the intact
fat globule membrane, showed oxidation rates and intensities between those
given by the globule membrane and butteroil.
The rates and intensities of oxidized flavor development were measur
ed in all model systems in terms of the absorbance of the 2-thiobarbituric
acid (TEA) color reaction.
The negative absorbance value indicate that it
was lower than that of the blank test.
(I)
The fat globule membrane:
An examination of the data presented
in Table I and Figures I, 2, 3, and 4 reveal that the fat globule membrane
materials (FGM I and FGM II) gave the highest oxidation rates and intensity
in the presence of ionized copper alone, ascorbic acid alone or ionized
copper plus ascorbic acid in comparison to other milk lipid fractions.
This is undoubtedly due to their higher contents of phospholipids and
polyunsaturated fatty acids which make them the probable site for oxidized
-24flavor development in aqueous systems„ This is supported by the data
obtained by Swanson and Sommer (1940) who found the iodine number of the
fatty acids located in milk phospholipids to be lowered substantially
through autoxidatioh, while that of the triglycerides remained practically
unchanged.
Milk phospholipids have been reported ^ich in polyunsaturated
fatty acids by Hilditch (1956) and Smith and Lowery (1962).
The polyun­
saturated fatty acids, especially linoleic and linolenic acids were shown
by El-Negoumy et: al^. (1962a) to be the precursors for oxidized flavor
compounds in milk fat.
The FGM material is also richer in its natural
copper content than whole milk (Herald et al. 1957b).
In the presence of copper alone, the oxidation intensity given by
FQl II in pH 6.60 phosphate buffer was almost double that given by FQl I
(Table I).
This behavior is indicative of the compositional differences
between the 2 preparations.
The difference may be the result of a higher
concentration of polyunsaturated fatty acids in FQl II.
Verification of
this point awaits future investigations.
Ascorbic acid alone, in the absence of copper, exhibited powerful
pro-oxidant properties in the presence of the fat globule membrane material
(Table I and Figures I, 2).
This behavior is, in all probabilities, due to
the higher natural copper content of the metallo-phosphilipid-protein comp­
lex of the fat globule membrane.
Herald et al. (1957k) reported that the
fat globule membrane material contained a higher concentration of mineral
elements than milk serum.
They suggested that these elements are possibly
constituents of the various enzymes in the membrane.
Ramachandran et: al.
(I960) showed the fat globule membrane to be rich in copper and iron.
TABLE NO. I
The Effect of Cu1-*" Plus Ascorbic Acid On The Autoxidation
Of Various Milk Lipid Fractions In Different Media (absorbance)
Milk Lipid Fractions
Reaction Medium
Distilled Water
Cu+1-
Ascorb­
ic Acid
Cu^+
As.A.
Phosphate Buffer
(pH 6.60)
Cu h "
Ascorb­ Cu"""+
ic Acid A.A.
Milk Serum
Cu h "
Ascorb­
ic Acid
Cu h +
As.A.
I
tsd
Vi
FQM I (from
buttermilk)
0.228
0.125
0.565
0.270
0.290
0.720
0.-242
0.157
0.317
FGM II (from
butter serum)'
0.217
0.477
0.477
0.430
0.135
0.835
0.298
0.181
0.478
Dialyzed Cream
0.042
0,045
0.177
0.092
0.128
0.195
0.105
0.155
0.310
B.O. I
0.014
0.024
0.053
0.001
0.014
0.031
0ioi7
0.017
0.026
B.O. II
0.006
0.008
0.040
0.006
0.018
0.106
0.020
0.013
0.042
B.O. Ill
0.015
0.008
0.017
0.004
0.004
0.035'
0.010
0.011
0.032
i
-26-
,------ FGM I
#-—
■
—
— < FGM TI
TBA (Absorbance)
^iimiwiiu i D i a l y z e d Cream
Ascorbic Acid (mg/100ml)
Figure No. I:
The effect of increasing concentration of ascorbic
acid in water System in the absence of copper ions
on the autoxidation of milk lipid fractions.
-27-
FQi I
FGM II
.« Dialyzed Cream
B.O. I
B .0. II
(Absorbance)
B.O. Ill
Ascorbic Acid (mg/lOOml)
Figure No. 2:
The effect of increasing concentration of ascorbic
acid in milk serum in absence of copper ions on the
autoxidation of milk lipid fractions.
-28-
.-------- .
FGM I
♦— — — — &
FGM XX
Dialyzed Cream
B.O. I
B .0. II
(Absorbance)
B.O. Ill
Ascorbic Acid (mg/lOOml)
Figure No. 3:
The effect of increasing concentration of ascorbic
acid in presence of 4 ppm Cu++ in water system
on the autoxidation of milk lipid fractions.
-29-
4 FGM
minimi .<./)/ „ Dialyzed Cream
B .0. I
B.O. II
TBA (Absorbance)
B.O. Ill
8
12
16
Ascorbic Acid (mg/100ml)
Figure No. 4:
The effect of increasing concentration of ascorbic
acid in the presence of 4 ppm Cu++ in milk serum
system on the autoxldation of milk lipid fractions.
-30=
Richardson and Guss (1965) found the high density membrane material to
contain one atom of copper or iron per 21 to 24 molecules of polyunsaturated
fatty acidso
The development of oxidized flavor by the fat globule membrane in
the absence of copper ions and in the presence of ascorbic acid alone
offer an explanation for development of this flavor in milk, free from
metallic contamination.
It is a well known fact that the elimination of
copper ions completely from dairy processing equipment did not give milk
supplies much protection against this defect.
As a matter of fact, bulk
milk supplies seem to be more susceptible to development of oxidized flavor
in recent years due to natural copper.
FQl I and FGM II differed very significantly in their oxidative
behavior in the presence of ascorbic acid alone.
The high oxidation
intensity given by FQl II in a salt free system (water) is again a proof
that these 2 membrane preparations a.re quite different from each other.
This is probably the result of the higher content of polyunsaturated fatty
acids and natural copper in FQl II.
It is suprisipg, however, that FQl I
reversed its oxidative trend in water and gave a higher oxidative intensity
than FQl II in pH 6.6 phosphate buffer.
'
:
This behavior may bd due to differences in the configuration of the
.
.
.
copper-protein-lipids complex in FQl I and FQl II.
It is possible that
FQl I is more able to promote the formation of the catalytic cuprous phos­
phate complex.
The copper-protein-lipid complex of the fat globule membrane
have been reported by Tarassuk and Koops (I960) to attach ascorbic acid.
-31-
In the presence of ionic copper plus ascorbic acid, the oxidation
intensities given by both FGM I and FfiM II are close in all systems„
The highest oxidation intensity of FGM preparations was in phos­
phate buffer, followed by water, and then milk serum.
Cuprous phosphate
complex has been shown by El-Negoumy (1965) to be a more powerful catalyst
for oxidized flavor development than other cuprous complexes.
An increas­
ed concentration of ascorbic acid in the absence of copper ions (Figures I
and 2) in water systems induced an oxidation intensity with FQl II which
was double that given with FQl I .
Both of the membrane preparations,
however, increased their oxidation intensity significantly with an increase
in ascorbic acid concentration.
Thg same trend was followed in milk serum
systems, except that the intensity was generally lower than that in water
systems.
The salts present in milk serum probably interact with copper,
occurring naturally in the membrane material, forming cupric and cuprous
I
complexes.
;
The catalytic effect of copper will be dependent on the kind
and amount of anions in these complexes.
According to Smith and Dunkley
(1962a), cuprous ions are the active form initiating the oxidation reaction.
Thus, the predominance of the cupric salt complexes in milk serum could be
responsible for the lowered oxidation intensity observed in this medium.
Another possible explanation for this behavior in milk serum could be the
configurational changes occurring in the natural copper-protein-phospholipid
complex of the membrane Aaterial as a result of interaction with the salts
in the milk serum.
Tarassuk and Koops (I960) suggested that naturally
occurring copper exists as a copper-protein complex which is capable of
accepting or attaching ascorbic acid upon reduction of cupric to cuprous
j
-32-
ions O
The highest oxidation rates and intensities were given by both
preparations of the membrane material in the presence of copper plus
ascorbic acid (Table I and Figures 3 and 4).
phosphate buffer and lowest in milk serum.
The intensity was highest in
The work of Freiden (1958a),
Freiden and Alles (1958b), Kelley and Watts (1957), Filtman and Freiden
(1957) and King and Dunkley (1959) showed that ascorbic acid causes rapid
reduction of cupric to cuprous copper.
The abundance of the cuprous ions
is especially manifested in phosphate buffer where the cuprous complex
formed is highly pro-oxidant. This is in agreement with the findings of
El-Negoumy (1965) who reported the cuprous salt complex formed with phos­
phate to be twice as effective in its catalytic effect as those formed
in the presence of sodium citrate.
An increased concentration of ascorbic acid in the presence of
copper ions gave an oxidation intensity which was much higher in water
> .
than in milk serum systems (Figures 3 and 4). This is in agreement with
the findings of King (1963).
Here again the copper salt complexes formed
in the presence of milk serum had an overall suppressive effect on
adtoxidation in comparison to salt free systems (water).
The presence of
copper chelators such as sodium citrate in milk serum and the predominance
of cupric salt complexes is most likely responsible for this behavior.
(2)
The intact globular milk fat in dialyzed cream;
The oxidation
intensity developed by dialyzed cream in the presence of copper alone was
dependent on the composition of the aqueous system in which it was dis­
persed (Table I ) .
Water systems gave the lowest intensity, followed by
-33-
Phosphate buffer, and then milk serum.
This is in agreement with the
results of El-Negoumy (1965) who suggested that cuprous ions initiate
the autoxidation reactions, only when they exist in the form of complexes
with anions in the system.
The higher oxidative trend of dialyzed cream
in phosphate buffer and milk serum points to the validity of this sugges­
tion.
Ascorbic acid alone initiated oxidation of globular fat, especially
in phosphate buffer and milk serum.
This behavior is probably due to the
presence of the intact membrane material surrounding each fat globule.
The meribrane material, because of its high content of phospholipids and
natural copper is probably the site of oxidized flavor development in the
intact fat globules.
Nelson and Pink (1954) and French and Monks (1961)
presented evidence indicating that copper carboxylate complexes in organic
compounds exhibit considerable ionic character.
This, if true, means that
natural copper in the metal-lipo-protein complex is capable of ionization
which would increase its catalytic power considerably.
Ascorbic acid is capable of reducing natural copper to the cuprous
state according to Smith and Dunkley (1962 a,b). The results obtained with
increasing concentration of ascorbic acid in water systems where a prooxidant effect is evident supports this conclusion.
Smith and Dunkley
(1962) have also shown a strong positive correlation between the incidence
of a spontaneous oxidized flavor development and the high levels of natural
copper in milk.
The low oxidation intensity of an increased concentration
of ascorbic acid alone in milk serum is probably due to the reduction in
the catalytic power of copper salt complexes in comparison to the cuprous
*34—
ascorbate complex.
Free ascorbic acid exhibits antioxidant properties.
The intact globular milk fat gave its highest oxidation intensity
in the presence of copper plus ascorbic acid (Table I and Figures 3 and 4).
Disregarding differences in the levels of oxidation intensities, this trend
is similar to that given by the fat globule membrane in presence of copper
ions plus ascorbic acid.
This similarity is probably a reflection of the
presence of the fat globule membrane.
An increased concentration of ascorbic acid in the presence of
copper ions gave an increased oxidation intensity in water systems, with
I
the reverse trend being true in milk serum systems (Figures 3 and 4).
Results similar to these were obtained by El-Negoumy (1965) in salt free
systems and in systems containing individual salts or their mixtures. He
suggested that, in the absence of salts, reduced ascorbic acid converts
cupric to cuprous ions in amounts proportional to its concentration.
cuprous ascorbate acts as a strong peroxidation catalyst.
The
In milk serum,
the cuprous ions complex with the salt anions, thus sparing the ascorbic
acid which then behaves as an antioxidant, especially at higher concentra­
tions »
Copper ions (4 ppm) from various salts gave variable rates and in­
tensities in similar systems containing dialyzed cream and an increasing
concentration of ascorbic acid (Figures 5 and 6).
All copper salts gave
a pronounced pro-oxidant effect in water systems with an increased concenx ,
tration of ascorbic acid.
On the other hand, no significant changes in
oxidation rates or intensity occurred in milk serum systems.
These results
are in support of El-Negoumy1s suggestion (1965) that the catalytic power
-35-
of copper induced oxidized flavor development in milk is dependent on
the kind and relative predominance of the various anions which complex
with cuprous ions.
Copper in the presence of acetate, carbonate and nitrate
anions had less catalytic power than the sulfate in water systems„
Smith
and Bunkley (1962 a,b) suggested that cuprous copper in the form of corns?
plexes is the limiting peroxidation factor which promotes spontaneous oxi­
dized flavor development„
According to Wilkinson (1964), the ionic species
depicted by many investigators to be free in autoxidation, is more likely
to exist as a complex or chelate in an aqueous medium.
The results ob­
tained here, with the various copper salts, support Wilkinson's suggestion.
Both the rate and intensity of oxidized flavor development by
■
-•.
intact globular fat are significantly influenced by the fat content of the
system.
This is clearly demonstrated by the data in Figure 7.
The reason
for the observation that concentrations higher than 0.50 mg ascorbic acid
are antioxidants in systems containing 2-4% fat, and pro-oxidant in systems
containing 6-12% fat is probably du.e to the abundance of fat globule
membrane material in the latter.
The antioxidant protection given at
higher concentrations of ascorbic acid is much less apparent in systems
containing higher fat content because of the presence of more substrate
for oxidation.
The variation in oxidation intensity observed at higher
fat concentrations is probably due to the accumulation of larger amounts
of oxidation products which usually suppress autoxidation after ,reaching
‘
|
’• i
'
a certain concentration.
,
'
I’
The oxidation products presumably do this by
intercepting and interacting with the free radicals necessary for initiat­
ing the reaction.
King (1963) suggested that the milk lipfd oxidative
-36-
Cu (CH3COO)2
CuCO
CuSO,
TBA
(Absorbance)
0.4 -
8
12
16
Ascorbic Acid (mg/100ml)
Figure No. 5:
The effect of Cu++ from different sources in water
system on the autoxidation of milk lipid fractions.
-37-
(Absorbance)
Cu(CH3COO)2
Figure No. 6:
The effect of Cu4+ from different sources in milk
serum system on the autoxidation of milk fractions,
-38—
x__. TL fat
, 47, fat
67, fat
1.0
►—
Z
— —
—— * 8% fat
»--------- »10% fat
Hist.....,.*.**"*#12%
fat
0.8
«
•l
(Absorbance)
V-1*"
0.6
....'''X
S
°*4
•V<
0.2
8
12
16
Ascorbic Acid (mg/lOOml)
Figure No. 7:
The effect of varying fat content (from dialyzed cream)
in the presence of 4 ppm Cu++ and an increasing concen­
tration of ascorbic acid in milk serum system on autoxudation of milk lipid fractions.
-39-
system is activate^ by association with ascorbic aci.d and inhibited by the
products of lipid and ascorbic acid oxidation.
El-Negoumy (1965) suggested
that at higher ascorbic acid concentrations, the lipids are spared by inter­
actions between the degraduation products of both the ascorbic acid and
lipid oxidation and cuprous, ions. This reduces the cuprous ions available
for initiating the reaction.
(3)
The true fat or triglycerides (Butteroil): Virtually no oxi­
dation took pl,ace in the 3 butteroil preparations in the presence of copper
alone or ascorbic acid alone (Table I and Figures I and 2).
B.O. I and
B.O. Ill, which, according to El-Rafey est al. (1944), include phospho­
lipids showed very little oxidation intensity in all systems.
B.O. II
which is devoid of phospholipids showed higher oxidation intensity in
phosphate buffer than in either water or milk serum systems.
El-Rafey
jit ail. (1944) showed that the improved keeping quality of B.O. I and
B.O„ III is due to the denaturing effect of heat on the phospholipidprotein complex which passes from the non-oil phase to the oil phase as a
result of the specific processing treatments.
These workers also found
the concentration of reducing substance (mostly -SH bearing compounds) to
be much higher in oils made by the boiling off process than by the low heat
treatment.
The low oxidation intensity of the butter oil preparations in the
presence of copper ions alone or copper ions plus ascorbic acid in com­
parison to the much higher rates obtained with the fat globule membrane
and dialyzed cream is in support of the conclusion that the fat globule
membrane is the site of oxidized flavor development in aqueous media.
-40-
The Role of gulfhydryl Groups (L-cysteine), Whole Casein and Kappa Casein
Compounds containing S-H links are important in oxygen transport in
lipid oxidation in biological systems, but the way in which they catalyze
lipid oxidation is not fully understood.
The role of casein and K-casein
may be dependent on their cysteine content.
Beeby (1964) has shown that
freshly prepared Krcasein contains cysteine and not cystine as was former­
ly believed.
Hill (1964) concluded from his results that casein micelles
contain cysteine and that it is likely that cystine is not present.
(I)
fractions:
The effect of L-cysteine on the autoxidation of milk lipid
The behavior of an increasing concentration of L-cysteine on
the autoxidation of the various milk lipid fractions was dependent on both
the particular lipid fraction used and the composition of the aqueous phase
in which the reaction took place (Figures 8 and 9).
Higher concentrations of L-cysteine were significantly pro-oxidant
in the presence of FGM I and FGM II in water systems.
However, the oxida­
tion intensity given by FQi II was much higher than that given by FGM I.
A completely different trend was given in milk serum, where concentrations
of L-cysteine from I x IO-lS l to 12 x IO- S l had a pro-oxidant effect in both
the FQi preparations. A concentration of 12 x IO-^M gave an antioxidant
effect, while higher concentration gave lower oxidation intensities.
This
behavior is probably explicable in terms of the changes taking place in
L-cysteine between the reduced (GSH) and the oxidized (GSSG) forms„ The
pro-oxidant properties of (-SH) groups cease when they are oxidized to
disulfide (-S-S-) or sulfate (Tait and King, 1936).
Like ascorbic acid,
cysteine is a reducing agent which converts cupric to cuprous state.
-41-
_______ .
FGM I
--------- ,
FGM I I
D i a l y z e d Cream
(A b s o rb a n c e )
in n 11iiininmiiiiiiii.
HjitltUU4
(ii1iiiJ-
L - c y s t i e n e (10"^ M /1)
F i g u r e No. 8 :
The effect of increasing the concentration of
L-cysteine in the presence of 4 ppm Cu+"** in
water system on the autoxidation of milk lipid
fractions.
-42-
•---------- •
0.4
FGM I
FCM II
Dialyzed Cream
B .0. I
B.O. II
0.3 h
(Absorbance)
B.O. Ill
a
0.2
0.1
0
—0.05
t
I
Figure No. 9:
i
_____I_____ I_____ I----- 1—
9
12
15
18
21
L-cysteine (IO-^M/I)
l
3
t
6
The effect of increasing concentration of L-cysteine
in the presence of 4 ppm Cu+"*" in milk serum on the
autoxidation of milk lipid fractions.
-43-
The following reactions were proposed by Wilkinson (1964) to explain the
action of the reduced and oxidized forms of L-cysteine:
GSH-.
% =
CS"
+ H+
Cu++
+ GR- ■;■■■-.
— A GS-
+ Cu+
GS-
+ RH
+ R-
^ GSH
These reactions indicate that the reduced form of cysteine initiates
the production of free, radicals (R) from the unsaturated fatty acids„
Oxidation with irreversible formation of (QSSG) at the pH of the milk
system (near pH 7.0) may be represented by the following overall reaction:
2 CS"
+ 2 C u + + ....
—
^r GSSG
X
I
+ 2 Cu+
Conditions favoring thg predominance of the reduced .form of
L-cysteine would make L-cysteine pro-oxidant; in nature, while conditions
favoring the oxidized form would make it antioxidant in nature. The re­
duced pro-oxidant form is mpst likely to be predominant in w^ter systems,
probably due to the dearth of milk serum salts.
In milk serum systems, as
the concentration of L-cysteine increases, the concentration of the oxidized
L-cysteine form (GSSG) increases gradually due to the predominance of cupric
salts.
%his makes L-cysteine progressively antioxidant at its higher con­
centrations , These assumptions ere supported by the findings of several
investigators.
Hopkins (1925) studied the oxidation of emulsified un­
saturated fatty acids and found that at pH 3-4 glutethione (GSH) catalyzed
lipid oxidation, but at pH 7.4 to 7.6, the gluathione was rapidly autoxidized and lipid oxidation ceased.
results using lecithin.
Tait and King (1935) obtained similar
They observe^ that when the oxidized form of
-
-.44-
glutathione (GSSG) was adde4, no catalysis of lecithin oxidation occurred.
The reduced form of glutathione, while strongly catalytic, was recovered
by them ffom the reaction mixture apparently unaltered ^t the conclusion
of oxidation.
These results were latef subtantiated by Busch and Kline
(1941) who used both cysteine and glutathione fpr the oxidation of phos­
pholipids.
The behavior of L-cysteinp in the presence of FGM I and FQl II ■
seems to follow the atiove described pattern, although compositional
differences between these two membrane preparations were apparent.
All concentrations of L-cystefpe in both the weter and milk serum
systems acted as a powerful antioxident with the tjhree butteroil prepara­
tions. These results agree witU those of ElTRafey £t al. (1944) who
found that increased concentrations of the reduced substances in butteroils made by the "boiling-off" method gave thent very high stability against
oxidation in comparison to oils prepared by the low heat treatment.
Dialyzed cream, which contains both the fat glpbule membrane and
the true fat in their natural condition, gave an oxidative behavior simirIar to that of FGM II in water systems and FGM I in milk serum.
The
oxidation intensity wag much lower than that given by the FQl material.
In all probability, phis ig a reflection of the lower concentration of
the FQl material present, which according to the results obtained in the
present investigation, are the site for oxidized flavor development.
(2)
The effect of casein and Kappa casein:
The behavior of whole
casein and K-casein during the autqxidation of milk lipid fractions was
dependent on the substrate arid the nature of fhe aqueous phase into which
-45-
it was dispersed (Figures 10, 11, 12, and 13).
The increased concentration of whole casein exerted a significant
antioxidant effect with FCM I and dialyzed cream in water systems, but
behaved as a pro-oxidant with FGM II. The behavior with FCM I and dialized
cream was dependent on the concentration of casein.
Concentrations of
0.50-1.0% were pro-oxidant but higher concentrations were antioxidant.
All three milk lipid fractions showed practically no significant oxidation
in milk serum systems at all casein concentrations.
.
Increased concentrations of K-caseiii in water systems gave the
reverse of the trend given with FGM II in water.systems.
Concentrations
up to 1.5% K-caseih were pro-oxid@nt, while higher concentrations were
antioxidant.
The autoxidation of FGM I and dialyzed cream was suppressed
substantially in the presence of an increased concentration of K-casein.
This was the same trend obtained with whole casein, which gave much lower
oxidation intensity with these lipid fractions.
The autoxidation of these three milk lipid fractions was suppressed
drastically in milk serum systems and in the presence of K-casein at all
concentrations„
This behavior is similar to that given by whole casein.
Whole casein and K-casein, when present in a salt free system,
probably act as pro-oxidant.
This was suggested by El-Negoumy (1965).
In the case of FGM I and dialyzed cream, this was true at lower concentra­
tions of these proteins where the ratio of protein to copper was enough
to form the cuprous-protein-complex. Higher concentrations of proteins
favor protein in a free form which acts as an antioxidant.
In the case
of FGM II, probably differences in its make up and composition counteracted
-46-
* FGM I
(Absorbance)
Dialyzed Cream
Casein (7.)
Figure No. 10:
The effect of increasing concentration of casein
in the presence of 4 ppm Cu++ in water system on
the autoxidation milk lipid fractions.
-47-
TBA
(Absorbance)
Dialyzed Cream
Casein (%)
Figure No. 11:
The effect of increasing concentration of
casein in the presence of 4 ppm Cu+"*" in
milk serum on the autoxidation of milk lipid
fractions.
—48—
"
niiuiti,'f Dialyzed Cream
(Absorbance)
Y
K-casein (%)
Figure No. 12;
The effect of increasing concentration of K-casein
in the presence of 4 ppm Cu++ in water system on
the autoxidation of milk lipid fractions.
49
-
TBA (Absorbance)
-
Dialyzed Cream
K-Casein (%)
Figure No. 13:
The effect of increasing concentration of K-casein
in the presence of 4 ppm Cu+"** in milk serum on
the autoxidation of milk lipid fractions.
-50-
the antioxidant effect of higher casein concentration favoring the pro­
duction of the catalytic form of copper.
Freiden and Alles (1958) showed
that proteins act as agents for the reduction of copper ions when the
latter is present at concentrations higher than I ppm.
Smith and Dunkley
(1962 a,b) substantiated these, results by detecting cuprous copper with
I
neocuproine.
Lea (1936) obtained results indicating that the copper pro­
tein ratio may influence the catalytic power of copper in the presence of
proteins. Using copper concentrations of 0.4 to 1.5 ppm with 4% albumin,
he found that copper catalysis ceases at low ratios of copper to protein.
The reason that higher concentrations of K-casein were antioxidant in
water systems with FGM II, while higher concentrations of whole casein
were pro-oxidant is difficult to explain,
The explanation probably lies
within the biochemical nature of both FQd II and K-casein.
It may be
that K-casein, which has been reported by Beeby (1964) to be the cysteine
bearing casein component, releases its -SH groups by interactions with
FQd II.
These released sulfhydryl groups may then increase the anti'
I
oxidarit properties of K-casein.
'
'
'
'
Tarassuk et al. (1959) and Koops et al.
■
(1959) found that fishy flavor (an intensified oxidized flavor) in butter
can be inhibited by adding heated skim milk or whey to washed cream from
which the buttier was made.
They attributed this effect to the released
-SH groups, since this inhibiting effect was removed by blocking the -SH
groups with N-methylmaleiamide.
Forester and Sommer (1961) also attributed
the protective action of milk serum proteins against oxidized flavor devel­
opment to their releasable -SH groups.
According to El-Negoumy (1965), the strong antioxidant behavior
-51-
of both whole casein and K-casein in milk serum systems is due to the
complexing of the copper by the anions of milk salts in the serum.
He
suggested that this spares the protein, which then exerts its antioxidant
properties, especially at its higher concentrations.
The Effect of Antioxidants
Although the present United States standards do not permit anti­
oxidants in dairy products and hence the question of their effectiveness
L
is one of theoretical interest, they are of practical interest in other
countries where their use is permitted.
(I)
The effect of Nordihydroguaiaretic acid (NDGA); the anti­
oxidant properties of NDGA varied according to the milk lipid fraction
used and the composition of the equeous phase ip which they were dispersed.
Comparison of the data in Table I and Figures 14 and 15 indicate that NDGA
showed strong antioxidant properties in both water and milk serum system
with FQeI I, F(34 II and dialyzed cream.
NDGA is a more effective anti­
oxidant with FGM I and FGM II than with dialyzed cream.
Shelton (1959)
described four mechanisms by which antioxidants function as a chain stopper
for the free radical chain mechanism of lipid oxidation.
apply to phenolic antioxidants like NDGA:
Two of these
(I) addition of the lipid to
the aromatic ring of NDGA or (2) formation of a complex between the lipid
and the aromatic ring of the antioxidant.
The reason that NDGA did not
exert its antioxidant properties with the same degree of effectiveness in
FGM I and FGM II is probably due to configurational differences between
the two.
In other words, one may be able to form complexes or add to the
NDGA more effectively than the other.
->52r*
The strong antioxidant properties of WDGA in a milk serum system
probably arise from the synergistic effects of some of the salts present
in milk serum.
According to Stuckey (1962), the effect of these syner­
gists is probably due to hydrogen radical or electron donation„ The polybasic salts, especially the citrates and phosphates are the most pre­
dominant syngergists in milk serum.
According to Badings (1961) and. Jenness and Patton (1959), the
synergistic effects of these polyhasic salts may be due to their seques­
tering effect on metallic ions, According to Privett and Quakenbush
(1954), these synergists exert their greatest influence at relatively
low levels.
(2)
The effect of o(-tocopherol;
Alpha-tocopherol (vitamin E) is
a well known antioxidant in nature. Horwitt (1961) presented lines of
evidence that the main function of o(-tocopherol is that of a lipid anti­
oxidant and pointed to the important relationship of this antioxidant and
polyunsaturated fatty acids in the diet.
Alpha-tocoperol behaved as an antioxidant with FGM I, FGM II and
dialyzed pream in milk serum systems (Figures 16 and 17).
According to
Tappel (1962), the chemical basis for vitamin E functioning as an anti­
oxidant lies in its reaction with the free radical intermediates of lipid
peroxidation and lipid peroxides through which lipid peroxidation is in­
hibited.
However, higher concentration of ©(-tocopherol were less effective
with FQl II in milk serum systems.
It is suggested that FQl II may destroy
the antioxidant properties of o(-tocopherol in the presence of milk serum
salts by oxidizing of-tocopherol to of-tocopherylquinone. According to Tappel
-53-
(1962), o(-tocopherylquinone is the major product of
oxidation during autoxidation of lipids.
tocopherol
-54-
0.3
,__________ , FGM I
FQl II
TBA (Absorbance)
,__________«
0.1
Figure No. 14:
0.2
0.3
0.4
0.5
NDGA (7o)
0.6
0.7
0.8
The effect of NDGA in the presence of 4 ppm Cu h "
in water system on the autoxidation of milk lipid
fractions.
-
55
-
0.2 r
(Absorbance)
0. 1
___________ .
FGM I
___________ _
FGM II
^ ^.11D11i in n 11/i 11ji/.
D ia ly z e d Cream
0
m
H
-
0.1
■
0.1
Figure No. 15:
0.2
0.3
0.4
0.5
NDGA (%)
0.6
0.7
0.8
The effect of NGDA in the presence of 4 ppm Cu h "
in milk serum system on the autoxidation of milk
lipid fractions.
-56-
TBA (Absorbance)
Dialyzed Cream
.^L L L L L h 111
i I I I I Iiin ^
o^-tocopherol (in acetate, ug/lOOml)
Figure No. 16:
The effect of the antioxdant property of
c < -tocopherol in water system on the autoxidation
of milk lipid fractions.
-57-
(Absorbance)
0.6
Dialyzed Cream
tocopherol (in acetate; ug/lOOml)
Figure No. 17:
The effect of the antioxidant property of
^-tocopherol in milk serum system on the
autoxidation of milk lipid fractions.
SUMMARY
Six different fractions of milk lipids were subjected to autoxidation in model systems composed of either distilled water.or milk serum,
and occasionally pH 6.60 phosphate buffer. All the model systems contained
2% lipid material, Autoxidation was allowed to proceed for 48 hours at
3-5°C. The rates and intensities of oxidation were measured in terms of
the absorbance of the 2-thiobarbituric acid test (TEA).
The effects of
the following factors were investigated: (I) Presence of copper ions alone,
ascorbic acid alorte, or copper plus ascorbic acid. (2) Presence of sulfhydryl compounds (L-cysteine), whole casein, and K-casein.
(3) Presence of
synthetic antioxidant (NDGA) and naturally occurring antioxidant
( o(-tocopherol).
The following is a summary of the most important findings:
(1)
The fat globule membrane material is the most likely site for
oxidized flavor development in aqueous dairy products.
This is demonstrat­
ed by the fact that fat globule membrane material showed a higher oxidai
'
tion intensity and rate in comparison with butteroil preparations.
(2)
Copper alone and ascorbic acid alone catalyzed oxidized flavor
development in the fat globule membrane and the intact fat globules at
rates and intensity dependent on the nature of the aqueous, medium used.
(3)
The highest oxidation intensifies took place in the presence of
copper plus ascorbic acid.
Theoretically, during the storage period of
dairy products, ascorbic acid is oxidized and in the course of doing so,
it reduces cupric ions to the catalytic-active cuprous state.
(4)
The catalytic effect of cuprous ions in initiating autoxida­
tion is dependent on the kind and amount of anions which complex with it.
-59-
Cuprous phosphate is a much more potent catalyst than cuprous citrate
formed in milk serum.
(5)
Copper ions (4 ppm) from various salts gave variable oxidation
rates and intensities in similar systems, indicating varied catalytic
power of the different anions complexing with cuprous ions.
(6)
True fat, in the form of butteroils, gave the lowest oxida­
tion intensities, indicating that it is not the primary site of oxidized
flavor development in aqueous dairy products.
The boiling-off method is
suggested as the best way to prepare pure milk fat of the best keeping
qualities.
(7)
The fat globule membrane from butter serum (FGM II) generally
gave a much higher oxidation intensity in the presence of copper ions and
ascorbic acid in.comparison to that from buttermilk (FGM I).
This is
indicative of significant compositional differences between the two mem­
brane fractions.
This difference could either be due to ,different proteins
in the membrane preparations or to differences in the concentration of
polyunsaturated fatty acids and amounts of natural copper ions.
(8)
L-cysteine behaved either as a pro-oxidant, or as an anti­
oxidant , depending on the milk lipid fraction used and on the nature of
the aqueous medium in which the lipid was dispersed.
In water systems,
it promoted the autoxidation of the fat globule membrane material, while
it inhibited the oxidation of the butteroil prepartions.
In milk serum,
L-cysteine acted as an antioxidant at its lower concentrations with both
FGfcl I and FGM II and as a pro-oxidant at its higher concentrations.
In
ttie aqueous phase of natural milk serum, salt's are probably the factor to
-60-
influence the oxidation of (-SE) groups to disulfide form and change
its pro-oxidant behavior.
(9)
Increasing the concentration of whole casein inhibited the
oxidation of FGM I and dialyzed cream in water systems but promoted the
oxidation of FQl II.
The autoxidation of these lipid fractions was com-
J
pletely suppressed by casein in milk serum systems.
Concentrations of
0.5 to 1.5% of K-casein acted as pro-oxidant with FQl II and dialyzed
cream in water systems but in higher concentrations acted as an anti­
oxidant.
K-casein was antioxidant at all concentrations with FQl I .
In milk serum systems, all concentrations of K-casein were strongly anti­
oxidant.
Milk proteins exert their antioxidant properties at higher pro­
tein concentrations and lower copper concentrations.
Their antioxidant
properties are enhanced by the synergistic effect of milk serum salts.
(10) Norhydroguaiaretic acid (NDGA) showed a strong antioxidant
effect on the milk fat globule membrane.
The effectiveness was depressed
at concentrations higher than 0.5% and was enhanced by the synergistic
effect of milk serum salts.
The naturally occurring antioxidant
o(-tocopherol behaved as a more effective antioxidant at its higher
concentrations by intercepting and interacting with the free radicals
required for initiating autoxidation of lipids.
APPENDIX
- 62TABLE NO. II
The Effect of Increasing Concentration of Ascorbic Acid
In The Absence of Cu++ On The Autoxidation Of
Milk Lipid Fractions (Absorbance)
Reaction Medium
Milk Lipid
Fractions
Water System
.
Absorbic Acid (mg/lOOml)
0.5
I
4
8
12
16
20
24
FGM I
0.014
0.096
0.365
0.555
1.155
1.005
0.995
0.105
FGM II
0.347
0.482
1:457
1.907 > 2 .00* > > 2 .00* > > 2 .00* > 2 .00*
D. Cr.
0.003
0.037
0.109
0.110
0.089
0.099
0.084
0.119
B.O. I
0.015
0.017
0.017
0.024
0.028
0.043
0.053
0.060
B.O. II
0.006
0.012
0.016
0.016
0.056
0.022
0.022
0.040
B.O. Ill
0.034
0.034
0.046
0.047
0.038
.0.045
0.044
0.043
Milk Serum System
FQl I
0.055
0.052
0.019
0.032
0.237
0.092
0.089
0.137
FGM II
0.428
0.518
1.078
0.328
1.218
1.218
1.168
1.118
D. Cr.
0.055
0.050
0.040
0.048
0.042
0.048
0.055
0.050
B.O. I
0.010
0.014
0.007
0.022
0.018
0.021
0.022
0.015
B.O. II
0.009
0.013
0.014
0.035
0.017
0.014
0.032
0.028
B.O. Ill
0.009
0.009
0.008
0.002
0.009
0.004
0.005
0.006
value larger than 2
-63TABLE NO. Ill
The Effect of Increasing Concentration of Ascorbic Acid
In The Presence Of Cu*"*" On The Autoxidation Of
Milk Lipid Fractions (Absorbance)
Reaction Medium
Milk Lipid
Water System
Fractions _____________________________ _
Absorbic Acid (mg/100ml)
0.5
I
4
8
12
16
20
24
Fm i
0.255
0.285
0.360
0.395
0.465
0.565
0.565
0.605
FGM II
0.367
0.427
0.857
1.007
0.907
0.687
0.827
0.797
D. Cr.
0.032
0.043
0.143
0.195
0.252
0.229
0.292
0.402
B.O. I
0.017
0.023
0.035
0.059
0.069
0.105
0.097
0.080
B .0. II
0.000
0.000
0.030
0.030
0.033
0.042
0.057
0.060
B.O. Ill
0.004
0.007
0.038
0.032
0.057
0.054
0.060
0.060
Milk Serum System
Fm i
0.117
0.117
0.207
0.337
0.272
0.337
0.307
0.278
F m ii
0.138
0.158
0.238
0.568
0.348
0.388
0.323
0.218
D. Cr.
0.860
0.780
0.122
0.116
0.146
0.139
0.121
0.122
B.O. I
0.008
0.014
0.040
0.098
0.117
0.108
0.127
0.149
B.O. II
0.009
0.010
0.044
0.033 . 0.067
0.075
0.067
0.050
B.O. Ill
0.000
0.003
0.032
0.055
0.084
0.095
0.075
. 0.079
-64TABLE NO. IV
The Effect of Copper Ion From Various Sources In The Presence
Of Ascorbic Acid in Water and Milk Serum
Systems On The Autoxidation Of Milk Lipid
Fractions (Absorbance)
Reaction Medium
Copper
Salts
ppm
Distilled Water
0.5
I
4
8
12
16
20
24
Cu(CH3COO)2
0.059
0.074
0.124
0.159
0.154
0.174
0.154
0.124
CuCO3
0.094
0.104
0.139
0.134
0.204
0.194
0.184
0.149
0.124
0.094
0.199
0.249
0.249
0.254
0.204
0.139
Cu(NO3)2
0.084
0.074
0.134
0.114
0.214
0.204
0.194
0.134
CuSO^
0.032
0.043
0.134
0.195
0.252
0.229
0.292
0.402
Milk Serum System
Cu(CH3COO)2
0.120
0.140
0.100
0.100
0.070
0.075
0.080
0.075
CuCO3
0.135
0.125
0.105
0.085
0.085
0.080
0.075
0.070
CuCl2
0.120
0.090
0.080
0.100
0.090
0.075
0.100
0.075
Cu(NO3)2
0.075
0.080
0.075
0.085
0.085
0.080
0.090
0.100
CUSO4
0.086
0.078
0.122
0.116
0.046
0.139
0.121
,0.122
-65TABLE NO. V
The Effect Of Varying Fat Content (from Dialyzed Cream)
In The Presence of 4 ppm Cu*-1" And An Increased
Concentration of Ascorbic Acid In Milk
(Serum System On the Autoxidation Of
Milk Lipid Fractions (Absorbance)
Ascorbic Acid
mg/iOOml
0.5
1.0
4
8
12
16
20
24
27. fat
0.055
0.050
0.040
0.048
0.042
0.048
0.055
0.050
4% fat
0.150
0.120
0.090
0.090
0.075
0.070
0.070
0.060
6% fat;
0.150
0.130:
0.135
0.170
0.170
0 .155
0.121
Q-ISOl
87. fat
0.285
0.320
0.370
0.430
0.440
0,450
0.410
0.350
107. fat
0.450
0.460
0.520
0.430
0.220
0.210
0.120
0.100
127. fat
0.350
0.330
0.460
0.545
0.760
0.950
0.650
0.700
-6 6 -
TABLE NO, VI
The Effect of Increasing Concentration of L-cysteine In The
Presence Of Copper Ion And 2% Of Lipid On The
Autoxidation Of Milk Lipid Fractions (Absorbance)
Reaction Medium
Milk Lipid
Fractions
Water System
L-cysteine 10"^ M/1
I
3
6
9
12
15
18
21
FGM I
0.145
0.146
0.146
0.152
0.345
0.350
0.351
0.350
FQl II
0.267
0.287
0.377
0.597
0.537
0.667
0.877
0.907
D, Cr,
0.065
0.054
0.048
0.045
0.045
0.044
0.054
0.058
B,0, I
-0.003
-0.003
0.007
0.013
0.008
0.005
0.007
0.004
B,0. II
+0.004
-0,005
-0.073
0.006
+0.003
-0.004
0.001
-0.602
B,0, III
-0.002
-0.002
0.010
0.008
0.007
0.005
0,007
0.008
Milk Serum System
FQl I
0.010
0.280
0.280
0.310
6.400
0.270
0.140
0.200
FQl II
0.030
0.068
0.098
0.183
0.093
-0.012
0.048
0.138
D. Cr.
0.075
0.064
0.063
0.070
, 0.065
0.050
0.055
0:052
B.O. I
0,002
-0.003
0.000
0.003
0.003
-0.003
0.007
0.007
B.O. II
0.011
0.008
0.011
0.009
0.011
0.505
0.015
0.005
B.O. Ill
0.003
-0.001
-0.005
0.007
-0.003
-0.007
-0.001
-0.001 .
TABLE NO. VII
The Effect Of Increasing Concentration Of Casein In Presence Of Copper Ion
On The Autoxidation Of Milk Lipid Fractions (Absorbance)
Reaction Medium
Milk Lipid
Fractions
Milk Serum System
Water Systein
2.0
Casein %
2.5
0.5
1.0
FGM I
0.045
0.065
-0.030
-0.040
-0.050
-0.098
-0.198
-0.092
-0.118
-0.198
FGM II
0.147
0.117
0.232
0.227
0.282
-0.102
-0.107
-0.082
-0.018
-0.032
D. Cr.
0.124
0.124
0.094
0,084
0.084
-0.117
-0.126
-0.119
-0.127
-0.197
1.5
0.5
1.0
1.5
2.0
2.5
67-
;!
TABLE NO. VIII
The Effect Of Increasing Concentration of Kappa Casein In The Presence Of
Copper Ion On The Autoxidation Of Milk Lipid Fractions (Absorbance)
____ _____
Milk Lipid
Fractions
Water System
O 5
- i Q
.
Reaction Medium_________________________
i.5
____________________________Milk Serum System
Kappa Casein %______ ___________ _______
2,0
2.5
0,5
1.0
1.5
2.0
2.5
F(M I
-0.001
0.005
=0.059
-0.030
-0.025
-0.078
-0.023
-0.063
-0.113
=0.133
FGM II
0.137
0.127
0.177
0.042
0.027
0.008
-0.087
-0.072
-0.132
-0.152
D. Cr.
0.022
0.052
-0.060
-0.100
-0.110
0.038
-0.022
-0.162
-0.187
-0.147
“68-
TABI4E NO. IX
The Effect of NDGA In The Presence Of Copper Ion On
The Autoxidation of Milk Lipid Fractions (Absorbance)
Reaction Medium
Milk Lipid
Fractions
Water System
0.1
0.2
0.3
FOl I
0.110
0.080
0.090
FCM II
0.207
0.127
D. Cr.
0.004
0.009
NDGA %
0.4
0.5
0.6
0.7
0.8
0.095
0.065
0.045
0.000
0.005
0.092
0.117
0.072
0.137
0.147
0.147
0.014
0.019
I
0.014
0.029
0.029
0.029
Milk Serum System
FOl I
-0.013
-0.930
-0.113
-0.123
-0.043
0.027
0.049
0.077
FOl II
-0.022
0.012
-0.052
-0.042
-0.022
0.012
-0.042
0.012
D. Cr.
0.050
0.040
0.040
0.030
0.040
0.055
0.045
0.045
69-
TABLE NO. X
The Effect Of The Antioxident Property o f t o c o p h e r o l
On The Autoxidation Of Milk Lipid Fractions (Absorbance)
Rea1Ctiori Medium
Milk Lipid
Fractions
_
Water System
I
• ef-tocopherol mg/IOOml
80
100
40
60
FGM I '
0.565
0.405
0.505
0.445
Fm
ii
0.237
0.177
0.167
D„ Cr.
0.093
0.123
0.088
120
140
160
0.235
0.105
0.145
0.065
0.177
0.167
0.167
0.167
0.117
0.133
0.028
0.023
0.021
0.021
Milk Serum System
FGM I
0.337
0.357
0.227
0.293
0.262
0.157
0.117
0.077
Fm
ii
0.040
0.060
0.148
0.138
0.138
0.168
0.158
0.168
D . Cr.
0.080
0.080
0.080
0.095
0.080
0.045
0.038
0.038
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