FRESH AND FROZEN EGG YOLK
PROTEIN FRACTIONS:
EMULSION. STABILIZING POWER.
VISCOSITY, AND ELECTROPHORETIC
PATTERNS

Thesis for the Degree of M.. S.
MICHIGAN STATE UNIVERSITY
ELIZABETH MILLER DAVEY
1968

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ABSTRACT

FRESH AND FROZEN EGG YOLK PROTEIN FRACTIONS:
EMULSION STABILIZING POWER, VISCOSITY,
AND ELECTROPHORETIC PATTERNS

by Elizabeth Miller Davey

The primary objective of this investigation was
to identify the emulsifying properties of three crude
egg yolk protein fractions: lipovitellin, lipovitellenin,
and livetin. Of secondary importance, was a study of the
effects of freezing and thawing upon the emulsifying prop—
erties of egg yolk and all combinations of the three
fractions.

Test emulsions were prepared by emulsifying oil
in water using combinations of the three fractions in
amounts proportional to those normally found in the egg
yolk, as well as using native yolk. Emulsion drainage
read at thirty-minute intervals for two hours provided
the test of emulsion stability. Similar emulsions,
prepared with frozen egg yolk and frozen fractions were
evaluated in the same manner. The viscosity and electro-

phoretic mobility of both fresh and frozen egg yolk and

Elizabeth Miller Davey

fractions were measured by standard procedures, and all
data reported are the average of six replications.

A comparison of the emulsion stabilizing powers of
native yolk and recombined fractions showed that, although
total drainage was small in both cases, emulsions prepared
with native yolk were more stable than those prepared with
the recombined fractions after thirty minutes. Emulsions
prepared with fresh yolk and recombined fresh fractions
drained less than those prepared with frozen yolk and
recombined frozen fractions, but this difference was
significant only for the sixty minute reading.

Lipovitellin, lipovitellenin, and livetin each
functioned to reduce emulsion drainage significantly, with
lipovitellenin providing the best overall emulsion stabi-
lization. Highly significant interactions, which occurred
when any two of the three fractions were present, indicated
that while combinations of two fractions did promote
greater stability than that of either fraction alone, the
stability of the emulsions was less than would be expected
from independent action of the fractions peg s3. Examina-
tion of the rates of drainage suggested that lipovitellenin
effectively reduced initial drainage, but increased subse-
quent drainage. Livetin and lipovitellin decreased initial
drainage somewhat, and interacted with lipovitellenin to

reduce subsequent drainage. The best emulsion stability

Elizabeth Miller Davey

was observed when all three fractions were present
producing an effect which equaled independent action of
the fractions peg s3.

Determinations of relative viscosity, before and
after lipovitellin, lipovitellenin, livetin, and native
yolk were frozen and thawed, showed that freezing and
thawing greatly increased the mean relative viscosity of
lipovitellin, lipovitellenin, and native yolk, while the
relatiVe viscosity of livetin was essentially unchanged.
The change in the mean relative viscosity of frozen lipo-
vitellin indicated that protein denaturation had occurred,
but paper electrophoresis was unable to detect any change
in the mobility of the frozen lipovitellin protein. Lipo-
vitellenin, however, produced electrophoretic patterns
which indicated that a majority of its proteins were
changed by freezing and thawing in such a manner that

they became non-mobile after freezing.

FRESH AND FROZEN EGG YOLK PROTEIN FRACTIONS:
EMULSION STABILIZING POWER, VISCOSITY,

AND ELECTROPHORETIC PATTERNS

BY

Elizabeth Miller Davey

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Foods and Nutrition

1968

CHRZOQ

ACKNOWLEDGMENTS

The author wishes to extend acknowledgment to
Mrs. Mary Ellen Zabik for her guidance and counsel in
this study and in the preparation of the manuscript.
Sincere appreciation is expressed to Mrs. Carol Weaver
for her assistance with the chemical analyses, to
Dr. Francis Magrabi and Dr. Charles Cress for aid in
preparing and interpreting the statistical analyses, and
to Dr. L. E. Dawson for procurement of the eggs used in
this study and for information concerning their produc-
tion. The writer is also grateful to Dr. Kaye Funk for
her helpful suggestions and to the many graduate and
undergraduate laboratory workers who assisted in this
research.

Finally personal thanks are extended to my
husband, Jim, for cooperation and inspiration throughout
this study, and to my parents for the work that made their

educational hopes for their daughter a reality.

ii

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . ii
LIST OF TABLES . . . . . . . . . . . . . . . . . Vii
LIST OF FIGURES . . . . . . . . . . . . . . . . . Xi
INTRODUCTION . . . . . . . . . . . . . . . . . . 1
REVIEW OF LITERATURE . . . . . . . . . . . . . . 3
Composition of Egg Yolk . . . . . . . . . . . . 3
Water . . . . . . . . . . . . . . . . . . . . 5
Carbohydrate . . . . . . . . . . . . . . . . 5
Lipoprotein and protein . . . . . . . . . . . 6
Early studies . . . . . . . . . . . . . . . 6
Recent studies . . . . . . . . . . . . . . 7

1. Lipovitellin . . . . . . . . . . . . 7

2. Phosvitin . . . . . . . . . . . . . . 9

3. Lipovitellenin . . . . . . . . . . . 10

4. d-, B— and y-livetins . . . . . . . . 10

Lipid . . . . . . . . . . . . . . . . . . . . 11
Phospholipids . . . . . . . . . . . . . . . 12
Sterol . . . . . . . . . . . . . . . . . . 15
Effect of hen's diet on egg yolk lipid . . 15
Embryological studies . . . . . . . . . . . . 15

iii

Freezing of Egg Yolk . . . . .
Gelation . . . . . . . . .
Factors influencing gelation . . . .
Theories of gelation . . . . . . . .
Prevention of gelation .
Salt . . . . . . . . . . . . . .
Sugars . . . . . .-. . . . . . .
Water . . . . . . . . . . . . .
Homogenization . . . . . . . .
Other methods of reducing gelation
Emulsifying Properties of Egg Yolk . .
The emulsion system . . . . . . . .
Theories of emulsification . . . . .

Egg yolk as an emulsifier . . . .

The effects of freezing on egg yolk's emulsify-

ing properties . . . . . . . . . .
Tests of emulsion stability . . .
Objective Tests . . . . . . . . . . .

Paper electrophoresis . . . . . .

General theory of paper electrOphoresis

Electrophoretic mobility of egg yolk

proteins 0 O O O O O O O O O O O

Electrophoretic patterns of frozen egg

yolk . . . . . . . . . . . . . .

Measurement of viscosity of egg yolk

iv

Page
l7
17
18
18
20
21
21
22
22
22
23
23
24

25

26
27
27
28

28

31

33
34

Page

EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . 38
Design of Experiment . . . . . . . . . . . . . . 38
Procurement of Eggs . . . . . . . . . . . . . . 39
Preparation of Egg Yolk . . . . . . . . . . . . 39
Separation of Egg Yolk Fractions . . . . . . . . 40

Lipovitellin . . . . . . . . . . . . . . . . . 41
Lipovitellenin . . . . . . . . . . . . . . . . 42
Livetin . . . . . . . . . . .~. . . . . . . . 44
Freezing of Egg Yolk and Egg Yolk Fractions . . 44
Chemical Evaluation . . . . . . . . . . . . . . 45
Moisture . . . . . . . . . . . . . . . . . . . 45
Protein . . . . . . . . . . . . . . . . . . . 46
Objective Measurements . . . . . . . . . . . . . 46
Emulsion stability . . . . . . . . . . . . . . 47
Emulsion formula . . . . . . . . . . . . . . 47
Preparation of emulsions . . . . . . . . . . 49
Measurement of emulsion stability . . . . . 52

pH of emulsion mixtures, native yolk, and

yolk fractions . . .-. . . . . . . . . . . . 52
Viscosity of native yolk and yolk fractions . 52
Paper electrophoresis . . . . . . . . . . . . 54

Analyses of Data . . . . . . . . . . . . . . . . 55
RESULTS AND DISCUSSION . . . . . . . . . . . . . . 56
Chemical Analyses . . . . . . . . . . . . . . . 57
Moisture . . . . . . . . . . . . . . . . . . . 57

Page

Protein . . . . . . . . . . . . . . . . . . . 60
pH . . . . . . . . . . . . . . . . . . . . . 61
Emulsion Stability . . . . . . . . . . . . . . 62
Recombined fractions vs. native yolk . . . . 62

Combinations of lipovitellin, lipovitellenin,
and livetin O O O O O O O O O Q C O O O O O 67

Effects of lipovitellin, lipovitellenin,

and livetin on emulsion drainage . . . . 67
Drainage between storage treatments . . . . 74
Drainage variance among time intervals . . 75

Interaction of drainage time and fraction

combinations . . . . . . . . . . . . . . 76
Relative Viscosity . . . . . . . . . . . . . . 85
Native yolk . . . . . . . . . . . . . . . . . 87
Lipovitellin . . . . . . . . . . . . . . . . 87
Lipovitellenin . . . . . . . . . . . . . . . 88
Livetin . . . . . . . . . . . . . . . . . . . 89
Paper Electrophoresis . . . . . . . . . . . . . 89
Native yolk . . . . . . . . . . . . . . . . . 91
Lipovitellin . . . . . . . . . . . . . . . . 93
Lipovitellenin . . . . . . . . . . . . . . . 93
Livetin . . . . . . . . . . . . . . . . . . . 96
SUMMARY AND CONCLUSIONS . . . '. . . . . . . . . . 98
LITERATURE CITED . . . . . . . . . . . . . . . . 103
APPENDIX . . . . . . . . . . . . . . . . . . . . 114

vi

LIST OF TABLES
Table Page
1. Approximate composition of egg yolk . . . . . 4

2. Composition and properties of white and
yellow yolk (Romanoff and Romanoff, 1949) . 4

3. Composition of egg yolk phospholipids
(Rhodes and Lea, 1957) . . . . . . . . . . 14

4. Protein composition of egg yolk determined
by paper electrophoresis (Evans and
Bandemer, 1957) . . . . . . . . . . . . . . 32

5. Formulae used in preparation of native yolk
emUlSionSa I I Q 0 O O O O O O O O O O O O . 48

6. Formulae used in preparation of yolk
fraction emulsionsa . . . . . . . . . . . . 50

7. Analyses of variance for differences in
protein and moisture content among native
yolk, lipovitellin, lipovitellenin, and
livetin with two storage treatments--
fresh and frozen . . . . . . . . . . . . . 58

8. Treatment means, standard deviations, and
significant-differences for moisture and
protein content of lipovitellin (A),
lipovitellenin (B), livetin (C), and
native yolk (Y) with two storage
treatments» . . . . . . . . . . . . . . . . 59

9. Means and standard deviations for pH deter-
minations of fresh and frozen samples of
native yolk, lipovitellin, lipovitellenin,
and livetin . . . . . . . . . . . . . . . . 61

10. Analyses of variance for differences in
emulsion stabilizing power between fresh
and frozen native yolk and fresh and frozen
recombined lipovitellin, lipovitellenin,
and livetin . . . . . . . . . . . . . . . . 63

vii

Table Page

11. Treatment means and standard deviations for
drainage at four time intervals from
emulsions prepared with fresh and frozen
native yolk (Y) and fresh and frozen
recombined lipovitellin, lipovitellenin,
and livetin (ABC) . . . . . . . . . . . . . 66

12. Analysis of variance for determination of the
effects of fractions, storage treatment,
replications, and drainage time on the
stability of emulsions prepared with all
possible combinations of lipovitellin,
lipovitellenin, and livetin . . . . . . . . 68

13. Interaction responses of lipovitellin and
lipovitellenin to one another . . . . . . . 70

14. Interaction responses of lipovitellenin and
livetin to one another . . . . . . . . . . 71

15. Interaction responses of lipovitellin and
livetin to one another . . . . . . . . . . 71

16. Mean drainage and standard deviations for
drainage from emulsions prepared from fresh
and frozen lipovitellin (A), lipo-
vitellenin (B), and livetin (C) and read
at four time intervals . . . . . . . . . . 73

17. Drainage from zero to thirty minutes and the
increase in drainage from thirty to one
hundred and twenty minutes for emulsions
prepared with combinations of lipovitellin,
lipovitellenin, and livetin . . . . . . . . 84

18. Means and standard deviations for the rela-
tive viscosity of both fresh and frozen
lipovitellin, lipovitellenin, livetin,
and native yolk . . . . . . . . . . . . . . 86

19. Composition of ration identified as LB-63
(Dawson, 1968) . . . . . . . . . . . . . . 115

20. Chemical analyses of ration identified as
LB-63 (Dawson, 1968) . . . . . . . . . . . 116

21. Percentage of total protein contributed by
each of the three fractions . . . . . . . . 116

viii

Table

22.

23.

24.

25.

26.

27.

28.

29.

Page

Spindle size and viscometer speeds for
measurement of the relative viscosity of
lipovitellin, lipovitellenin, livetin,
and native yolk before and after frozen
storage over.six replications . . . . . . 117

Replicate averages for fractions and
storage treatment for chemical determina-
tion of moisture content . . . . . . . . 118

Replicate averages for fractions and
storage treatment for chemical determina-
tion of protein content . . . . . . . . . 119

Replicate averages for fractions and
storage treatment for the determination
Of pH 0 O O O O I O O O I O O O O O O O O 120

Replicate averages for drainage from
emulsions prepared with fresh and frozen
native yolk (Y) and recombined lipo-
vitellin, lipovitellenin, and livetin
(ABC) at four time intervals .0. . . . . 121

Replicate averages for pH, means and
standard deviations for pH of emulsion
formulae prepared with fresh and frozen
native yolk and fresh and frozen recom-
bined lipovitellin, lipovitellenin, and
livetin . . . . . . . . .~. . . . . . . . 122

Replicate averages for emulsion drainage
at four time intervals from emulsions
prepared with no stabilizer (O) and with
fresh lipovitellin (A), lipovitellenin
(B), and livetin (C) combinations as
stabilizers . . . . . . . . . . . . . . . 123

Replicate averages for emulsion drainage
at four time intervals from emulsions
prepared with no stabilizer (O) and with
frozen lipovitellin (A), lipovitellenin
(B), and livetin (C) combinations as
stabilizers . . . . . . . . . . . . . . . 124

ix

Table

30.

31.

32.

33.

34.

35.

Main effect and significant interaction
means from the analysis of variance for
determination of the effects of fractions,
replication, storage treatment, and
drainage time on the stability of oil-
in-water emulsions . . . . . . . . . . . .

Replicate averages for relative Viscosity
of lipovitellin, lipovitellenin, livetin,
and native yolk, before and after frozen
storage . . . . . . . . . . . . . . . . .

Calculated average migration distances,
mobile and non-mobile protein fractions of
fresh and frozen native yolk as separated
by paper electrophoresis . . . . . . . . .

Calculated average migration distances,
mobile and non-mobile protein fractions of
fresh and frozen lipovitellin as separated
by paper electrophoresis . . . . . . . . .

Calculated average migration distances,
mobile and non-mobile protein fractions of
fresh and frozen lipovitellenin as
separated by paper electrophoresis . . . .

Calculated average migration distances,
mobile and non-mobile protein fractions of
fresh and frozen livetin as separated by
paper electrophoresis . .-. . . . . . . .

Page

125

127

128

129

130

131

Figure

10.

LIST OF FIGURES

Page

Typical electrophoretic mobility patterns

of egg yolk's high density fraction

(Bernardi and Cook, 1960a) . . . . . . . 33
Amount of drainage plotted against drain-

age time for emulsions stabilized by

fresh and frozen native yolk and fresh

and frozen recombined fractions . . . . 65
Bar-graph of emulsion drainage depicting

the main effects of lipovitellin, lipo-

vitellenin, and livetin in emulsion

stabilization . . . . . . . . . . . . . 69
Mean drainage from all emulsions plotted

against the times at which the readings

were taken . . . . . . . . . . . . . . . 76
Interaction of drainage time with

lipovitellin and lipovitellenin . . . . 78
Interaction of drainage time with

lipovitellenin and livetin . . . . . . . 8O
Interaction of drainage time with

lipovitellin and livetin . . . . . . . . 81
Interaction of drainage time with

lipovitellin (A), lipovitellenin (B),

and livetin (C) o o o o o o o o o o o o 83
Typical dyed paper strips for samples of

both fresh and frozen livetin, lipo-

vitellenin, lipovitellin, and native

YOlk I O O O O O O O I O O O O O O O O O 90
Typical paper electrophoretic patterns of

fresh and frozen native yolk . . . . . . 92

xi

Figure

11. Typical
fresh

12. Typical
fresh

13. Typical
fresh

Page

paper electrOphoretic patterns of
and frozen lipovitellin . . . . . . 94

paper electrophoretic patterns of
and frozen lipovitellenin . . . . . 95

paper electrophoretic patterns of
and frozen livetin . . . . . . . . 97

xii

INTRODUCTION

The protein and lipoprotein complexes which are
present in the egg yolk are responsible for its action
as an excellent emulsifier in many food emulsions (Snell
gt 31., 1935). Vincent et 31. (1966) found that the
livetin and lipoprotein fractions of the egg yolk con—
tribute to its low surface energy which in turn is neces-
sary for emulsion formation.but may not influence the
emulsion stability.

The use of frozen egg yolk increases efficiency
in commercial emulsion production. However, the utility
of freezing pure egg yolk is limited by the irreversible
reaction which egg yolk undergoes as'a result of freezing
and thawing, resulting in a product of greatly increased
viscosity which is difficult to combine with other.
ingredients. Although-methods and additives reduce this
reaction, its mechanism is unknown, and use of treated
yolks is limited to products in which the treatment
itself does not have deleterious effect.

There is no agreement on the influence of
freezing and thawing egg yolk to be used in an emulsify-

ing capacity. Miller and Winter (1951) found that frozen

yolk was a more efficient emulsifier in mayonnaise than
fresh yolk, while Kilgore (1935) suggested that more
frozen yolk than fresh yolk was required to produce a
mayonnaise of acceptable viscosity.- No recent studies of
the emulsifying properties of frozen egg yolk have been
conducted.

Although the pure proteins and lipoproteins of
the egg yolk have not been characterized, the fractions
which are easily separated by physical means deserve
further study. Studies of egg yolk protein fractions
have neither dealt with the functional properties of the
fractions nor the effect of freezing upon these fractions
and their properties. Knowledge of the functions of
specific egg yolk fractions could provide a means of
improving the quality of egg yolk products.

The present investigation was primarilyconcerned
with determining the emulsifying properties of egg yolk
and three crude egg yolk protein fractions: lipovitellin,
lipovitellenin, and livetin. A secondary objective was
to study the effects of freezing and thawing upon the
emulsifying properties of the egg yolk and the three yolk
fractions. Measurements of viscosity and electrophoretic
mobility of the native yolk and fractions before and after
freezing were conducted to determine the effect of the

gelation reaction upon these variables.

REVIEW OF LITERATURE

The egg yolk was the subject of extensive research
before study of the egg white was attempted. Much of this
early work, however, was invalidated or supplemented by
later studies employing modern equipment and techniques.
As-research continued the egg yolk was found to be more
complex than formerly indicated.

This review summarizes literature on egg yolk
proteins--their composition and emulsifying propérties,--
and the effects of freezing egg yolk. Objective tests-
which measure viscosity and electrophoretic protein

mobility are also discussed.

Composition of Egg Yolk

Egg yolk is composed primarily of fat, protein
and moisture (Table 1). Minor components include
carbohydrate and ash (Ziemba, 1955; Sweetman-and McKellar,
1959; Watt and Merrill, 1963). The ash of the egg yolk
is composed oprhosphorus, calcium, magnesium, chloride,
potassium, sodium, sulfur, and iron (Romanoff and Romanoff,

1949).

TABLE 1. Approximate composition of egg yolk

 

 

Component Percentage
Water 49.4 - 51.1
Lipid 30.6 - 31.9
Protein 16.0 - 16.7
Carbohydrate 0.2
Ash 1.0 - 1.7

 

Examination of the egg yolk reveals a unique
physical structure. It is composed of alternating layers
of white and yellow yolk globules (Table 2), white yolk
constituting only three to four per cent of the total yolk
material. Since separation of yellow and white yolk is
extremely difficult, it is seldom attempted in egg yolk
studies.

TABLE 2. Composition and properties of white and yellow
yolk (Romanoff and Romanoff, 1949)

 

 

 

Component Granule-
Water Lipid Protein Diameter
% % % mm
Yellow Yolk 45.4 36.4 15.0 0.015 - 0.025

White Yolk 86.0 3.5 4.6 0.004 - 0.075

 

Water

The amount of water in the egg yolk is influenced
after laying by two factors. Water is lost through the
shell of the egg. Passage of water from the white to the
yolk occurs as the white becomes thinner-due to loss of
carbon dioxide (Triebold and Aurand, 1963).

Water exists in food products in three forms:
free, bound, and mechanically occluded. All three forms
occur in egg yolk. Romanoff and Romanoff (1949) stated
that one-quarter of the water in the egg yolk exists in
the bound form and does not freeze at -35°C. Lea and
Hawke (1952) indicated that water is a structural.component

of the egg yolk.

Carbohydrate

Levene and Mori (1929) studied the carbohydrate of
the egg yolk. They found 34.8 per cent of the sugar pres-
ent as glucose. Levene and Rothen (1929), in a study of
egg yolk polysaccharides, found four trisaccharides each
with an approximate molecular weight of 500 on the basis
of diffusion coefficients. They hypothesized a structure
consisting of one molecule of glucosamine and two molecules
of mannose, which could form four.different combinations.
These trisatcharides are associated with yolk proteins in

an undisclosed manner.

LipOprotein and protein

 

The protein, lipid and phosphorus of the egg yolk
occur in complexes which presently defy elucidation,
although theories of molecular orientation are prevalent.
Much of the protein of the egg yolk occurs as lipoprotein.
LipOproteins are water soluble proteins conjugated with
lecithin, cholesterol, cephalin, neutral lipid, and other
similar compounds. The proteolipids are distinguished
from the lipoproteins by the solubility of the former in
organic solvents instead of water (White 25 31., 1959).
Omission of the lipo- prefix from the name of a lipoprotein

indicates that the lipid has been removed.'

Early studies.--In 1842, Dumas and Cahours»

 

discovered a protein in egg yolk which they named vitellin,
and for which they proposed a simple formula. Osborne and
Campbell (1900)precipitated egg yolk protein with brine
and extracted it with ether. Their nucleovitellin (now
known as lipovitellin) consisted of protein, lecithin,

and phosphoric acid. Osborne and Jones (1909) determined
the prOportion of amino acids in vitellin.

Plimmer (1908) presented the name livetin for a
new egg yolk protein discovered in the solution after
vitellin was removed. The slow precipitation of livetin
in a solution of ammonium sulfate, magnesium chloride,

and sodium chloride indicated to Kay and Marshall (1928)

that more than one protein might be present. Jukes and
Kay (1932) published the quantities of basic amino acids
present in livetin and vitellin. Jukes (1933) determined
the quantities of additional amino acids and found carbo-
hydrate associated with the proteins, with livetin being
composed of four per cent and vitellin two per cent carbo-
hydrate.~

Fevold and Lausten (1946) isolated lipovitellenin
by extracting the supernatant from the lipovitellin pre-
cipitation with ether. Centrifugation yielded three
layers: ether—lipid, aqueous livetin, and a suspension
(of the new protein.

Phosvitin was discovered to be a high phosphorus
(ten per cent) protein contaminating previous lipovitellin
lyreparatiOns (Mecham and Olcott, 1948). Further studies
showed a ratio of one phosphate to two amino acid residues
annd indicated that nearly all the hydroxyamino residues of

Eflnosvitin are phosphorlyated (Mecham and Olcott, 1949).

Recent studies.-—In recent studies using modern

 

analytical techniques, each of the previously determined
'PrOteins has been found to contain more than one protein
I“Diety. The molecular orientation of these moieties has

also been studied.

 

1. Lipovitellin.—-When egg yolk is centrifuged

at high speed, approximately forty per cent of the yolk

protein sediments (Schmidt et 31., 1956). This
precipitate includes all of the protein phosphorus and
eighty-seven per cent of the calcium and iron of the yolk,
but less than thirty per cent of the yolk's total phospho-
lipid. Lipovitellin and phosvitin are held in this high
density fraction by ionic or secondary forces (Burley and
Cook, 1961). Chargaff (1942a) recognized vitellin (or
lecitho—vitellin) as the lipoprotein, lipovitellin. In
his study of the nature of the lipid combination in this
complex, he found that approximately nineteen per cent of
the phosphatides were firmly bound to the protein, but
could be removed by alcohol extraction and were essentially
the same as the "free" lecithin and cephalin removed by
ether extraction.

The hydrolysis of lecitho-vitellin (lipovitellin)
try pepsin and trypsin kinase indicated that the protein
Exortion of the molecule contained two widely separated or
(iissimilar phosphoric complexes, one of which was resistant
txa enzymic attack (Blackwood and Wishart, 1934). Electro-
IAhoretic and chromatographic studies show that lipovitellin
<flDnsists of two lipoprotein moieties--a- and B-lipo-
vitellin (Joubert and Cook, 1958a; Bernardi and Cook, 1960a,
1960b, 1960c). Both lipovitellins have the same protein
lipid ratio, which is approximately four to one, but protein

Phosphorus is more abundant in a-lipovitellin.

The presence of two N-terminal amino acids in
B-vitellin indicated the presence of more than one
polypeptide chain. In aqueous buffer and urea solutions,
the lipovitellins each dissociate into two subunits in-
volving only a negative entrophy change which could be
due either to changes in structure or to changes in
hydration. The bonds between protein and phosphate groups
are not broken during this dissociation (Sugano, 1959;
Burley, 1962; Burley and Cook, 1962a, 1962b). A flexi-
bility of structure allows the lipovitellins to absorb
chloroform and B-lipovitellin in this medium gels at pH 11,
perhaps because of an alteration in the position of its
phospholipids. Radomski and Cook (1964) stated that the
heterogeneity of a-lipovitellin arises from the hybridiza-
tion of several monomers. A monomer-dimer system has been
found operative in B-lipovitellin dissociation with sub-

5

lJnitS of MW = 2.27 x 10 (Cook and Wallace, 1965).

2. Phosvitin.--Although the actual structure of

 

:phosvitin is unknown, Wallace EE.El' (1966) indicated that
.it is complexed with the lipovitellins in the granules and
that they may be sub-fractions of a similar protein with
differing levels of phosphorylation. Phosvitin is pre-
Cipitated by proper concentrations of magnesium chloride.
It forms soluble complexes before precipitation and thus,
may act as a bridge in the granule structure (Joubert and

Cook, 1958b).

10

3. Lipovitellenin.--When the low density fraction

 

obtained from the centrifugation of egg yolk is extracted
with ether to remove "free" lipid, a lipoprotein with
approximately forty per cent bound lipid-is precipitated.
The lipid removed by extraction is not really a free form,
but acts to stabilize the lipOprotein (Turner and Cook,
1958; McIndoe, 1959b). The presence of more than two N-
terminal amino acids in the protein moiety (Smith and
Turner, 1958) and the variable lipid content of the low
density fraction indicate the presence of several chains
or separate protein molecules held by lipid (Martin gt gt.,
1959). Saari 2E.3l' (1964a) showed that two low-density
lipoproteins were present in their ultracentrifugal flota-
tion patterns of the fraction previously known as lipo-
‘vitellenin. Both fractions contained carbohydrate, but

1:heir resolution by paper electrophoresis was not possible.

4. d-, B- and y-livetins.--Shepard and Hottle

 

(1949) obtained three electrophoretic peaks when they
<axamined livetin and designated them as a, B, and y-
livetins. Although the molecular weight of B-livetin is
Sindlar to ovalbumin, the two proteins differ in respect
to composition and mobility. The immunological identities
of the livetins are related to those of serum globulin of
the hen (Mok and Common, 1964) but, the solubility of a-

livetin is not identical to that of serum albumin. Martin

11

et a1. (1957) found that much of the y-livetin is
precipitated with lipovitellins. Low lipid and phosphorus
content differentiate y-livetin from the lipovitellins,
although its separation is difficult (Martin and Cook,
1958).

Research concerning the livetins is incomplete at
the present time for starch gel electrophoresis resolves
sixteen zones, four of which can be identified with B-
livetin from paper electrophoresis (Hui and Common, 1966).

The remaining twelve zones have not been identified.

Early studies of the egg yolk lipid often refer to
free lipid and its removal. Weinman (1956) centrifuged
egg yolk under conditions in which chylomicron lipid would
float to the surface. Since little lipid came to the top,
he concluded that nearly all lipid of the egg yolk is
bound to protein. Numerous studies support his conclusions
(Vandegaer gt gt., 1956; Evans and Bandemer, 1957; Sugano
and Watanabe, 1961).

The lipid of the egg yolk occurs as neutral lipid
associated with protein and in the phosphatide portions of
the lipoproteins. Fisher and Hill (1964) found that lipid
held by electrostatic and hydrOphobic-bonds is easily
removed. They postulated that the remaining fatty acids

are bound to protein in association with organic phosphate.

12

(200k and Martin (1962) found two types of complexes in

‘the egg yolk--the high protein lipoprotein of a- and B-
lipovitellin consisting of a protein matrix with lipid
interaction and the low protein lipoprotein of lipo-
'vitellenin, which follows a micellar model consisting of

a lipid sphere surrounded by a film of protein. Martin
gt_gt. (1959) discovered that ether extraction of lipo-
vitellenin removes lipid essential to the maintenance of
original stability and solubility, and they concluded that
the amount of protein-free micellar lipid in lipovitellenin
must be small. The lipid of lipovitellenin is held in two
manners such that approximately forty per cent of the lipid
remains after ether extraction (Turner and Cook, 1958;
Evans and Bandemer, 1957).

Because of their abundance in egg yolk, phospha—
tides and lipids must be taken into account in the con-
sideration of the egg yolk's functional properties. It
is interesting to note that even without complete struc-
tural elucidation, the egg yolk was the most common source
of both lipoprotein and phosphatides until soybean

technology made them uneconomical.

Phospholipids.--Phospholipids or phosphatides
which occur in nature are glycerol derivities frequently
containing a nitrogenous base in the a-position, with

fatty acids in the a'- and B-positions (White gt gt., 1959).

13

Interrelationships of the phospholipids and lipoproteins
:may account for the structural complexities of the egg
yolk constituents. Witcoff (1951) described a variety of
possible linkages and the resulting compounds.

Studies have been conducted to determine the
nature of the phospholipids which equal approximately
twenty per cent of the weight of egg yolk and are associ-
ated with the lipOproteins. Schmidt gt gt. (1956) found
that more than seventy per cent of the phospholipids
remained in the high density fraction when egg yolk was
centrifuged. The research of Smith and Turner (1958)
indicated that the differences in solubility of the
components of the high density fraction could be due to
the amount and nature of lipid association.

Chargaff (1942a) found that the phospholipids of
lipovitellin had a lower Iodine Value than those of the
entire yolk. A differing rate of radioactive phosphorus
incorporation in the egg yolk led Chargaff (1942b) to an
explanation of the metabolic link between phospholipids
and phosphoproteins. He hypothesized that protein
phosphorylation at serine hydroxyls is followed by
esterification with diglyceride. Peptide linkages are
then broken, freeing serine phosphatides which can undergo
decarboxylation to cephalin, and other compounds.

In 1954, Hanahan stated that in egg yolk lecithin,

the d'-ester is unsaturated (mainly linoleic acid) and

14

‘that the B-ester is always stearic acid. Rhodes and Lea
(1956) found egg lecithin more complicated. They found
that not more than one-third of the lecithin could have a
simple di-C18 structure, the remaining saturated acid being
palmitic. They also concluded that phosphatidyl ethanola-
mine has the same type of fatty acid distribution as
lecithin. Hanahan's positions of unsaturation were con-
firmed by Rhodes and Lea (1958) but challenged by Privett
gt gt. (1962) who found, using thin-layer chromatography,
that most but not all lecithin of the egg yolk is in the
a'-saturated B-unsaturated form. Chromatographic separa-
tion of the phospholipids of egg yolk on alumina and
separation on silicic acid gave the composition listed

in Table 3.

TABLE 3. Composition of egg yolk phospholipids (Rhodes
and Lea, 1957)

 

 

Component Percentage
Phosphatidyl choline 73.0
LySOphosphatidyl choline 5.8
Sphingomyelin 2.5
Phosphatidylethanolamine 15.0
Lysophosphatidylethanolamine 2.l

Inositol phospholipid 0.6

 

15

Sterol.--Sterols are crystalline, neutral,

‘unsaponifiable alcohols of high melting points (Mattil

‘gt gt., 1964). They are associated with lipid either in
the free form or as esters of fatty acids. Saari gt gt.
(1964a) found that the lipid of the low density lipoprotein
(lipovitellenin) contains approximately three per cent
sterol. They precipitated the yolk sterol by digitonin,
and the infrared spectra of this steroid material were
found identical to the infrared spectrum of pure choles-

terol.

Effect of hen's diet on egg yolk 1ipid.--Diet
affects the fats and phospholipids of egg yolk. Ostrander
gt gt. (1960) found that feeding hens corn oil increased
the unsaturated fatty acid content of the yolk, while the
feeding of beef tallow had the opposite effect. The
influence of dietary fat is perhaps the cause of conflict-

ing data on yolk lipid composition.

Embryological studies

By tracing the development of the egg yolk, its
components may be related to the components of hen serum.
Since the egg yolk provides the nourishment for the grow-
ing chick embryo, study of yolk protein utilization may
give information about the protein composition and

structure.

l6

Calvary (1929) found that in the developing egg amino
nitrogen decreases while non-amino nitrogen increases and
that the amino acid content of the egg undergoes change
(Calvary, 1930; Calvary and White, 1931-32). McCully
gt gt. (1959) found by zone electrophoresis two lipo-
proteins which were similar in hen sera and egg yolk.
McIndoe (1959a) precipitated a lipophosphoprotein from
laying hen plasma which he felt was responsible for the
transport of plasma lipid and almost half of the plasma
phosphOprotein to the yolk, as related compounds were
isolated from the egg yolk (McIndoe, 1959b).

Schjeide and Uhrist (1960) indicated that the
yolk proteins are synthesized in the liver of the hen,
transferred by blood plasma to the ovary and deposited
as granules. Lipovitellin (MW 2 200,000) appears to be
one-half the size of its serum precursor.

To determine if some yolk components were used by
chick embryos in preference to others during incubation,
egg yolks were removed and replaced with yolk components.
The percentage survival for each component was published
(Walker, 1967). The floating fraction was found to be a
potent nutrient as were the granules. Supernatant plasma
was not nutritionally useful. When the granules
and floating fraction were combined, life support was

equal to that of whole yolk.

l7

Freezing of Egg Yolk

When the temperature of egg yolk is reduced below
-6°C, an irreversible change takes place which results in
a stiff paste-like putty when the egg yolk is thawed
(Moran, 1925). The causes of this reaction are unknown in
spite of extensive study. Various explanatory theories
have been proposed and methods have been devised to

partially prevent the reaction from occurring.

Gelation

The irreversible change which egg yolk undergoes
upon freezing is termed gelation. It is often classified
as a form of denaturation. Scheraga (1961) defined de-
naturation as the "process in which a protein or poly-
peptide is transformed from an ordered to a disordered
state without the rupture of covalent bonds." Gortner
(1949) defined denaturation as "any non-proteolytic
modification of the unique structure of native proteins
giving rise to definite changes in chemical, physical or
biological properties" and gave the following prOperties
indicative of denaturation:

l. Decreased solubility.

2. Increased digestibility by proteolytic enzymes.

3. Exposure of oxidizing and reducing groups, notably
the sulfhydryl (-SH) groups.

4. Loss of enzymatic prOperties if the protein is an
enzyme.

18

5. Modification of the specific immunological
prOperties.

6. Decreased diffusion coefficient and increased
intrinsic viscosity of the protein.

Factors influencing gelation

The freezing point of egg yolk is 0.65°C but no
gelation occurs until the temperature falls below -6°C
and_super-cooling results in no change in the yolk's
fluidity (Moran, 1925). LOpez gt gt. (1954) found no
change in pH between fresh and frozen egg yolks; however,
adjusting the pH to 3.2 before freezing resulted in a
highly viscous thawed mass. The damage is greatly reduced
by rapid freezing and thawing (Lea and Hawke, 1952), but
not by rapid freezing or thawing alone (LOpez gt gt.,
1954).

Thomas and Bailey (1933) stated that the maximum
gelation of egg yolk is achieved in sixty to one hundred
and twenty days of storage at -21 to -l8°C. Freezing has
no effect on protein, amino nitrogen, or reducing sugar
content of the egg yolk (Pearce and Lavers, 1949; Evans

and Davidson, 1953).

Theories of gelation

The formation of ice crystals when egg yolks are
frozen seems to be a requisite for gelation (Fennema and
Powrie, 1964). Moran (1925) ascribed gelation to the

solution of lecithovitellin by the concentrated salt

19

solution present in frozen yolks and its precipitation on
thawing. Urbain and Miller (1930) also indicated that
gelation is due to the dehydration and coagulation of a
lecithoprotein. They found however that sucrose which
inhibits gelation of egg yolk had no effect on lecithin
alone. Thomas and Bailey (1933) found that lecitho-
vitellin is insoluble in sugar solution and that the
addition of commercial lecithin to egg yolk prior to
freezing has no effect on gelation.

A protein or protein complex is thought to be
responsible for gelation (Vandegaer gt gt., 1956; Lopez
gt gt., 1955). Some damage may be due to the crushing or
spearing action of ice crystal growth, but lipid-protein
complexes rearrange and aggregate during freezing
(Lovelock, 1957). In protein separations, dialysis and
dilution cause protein precipitation by reducing salt
concentration. A reduction in temperature causes pre-
cipitation at higher salt concentrations. A change in pH
occurs if buffering salts crystallize and this pH change
may account for protein denaturation, however lipovitellin
is damaged more slowly at -20°C than at -3°C suggesting
that it is not sensative to suspension in a solution of
high ionic strength (McNally, 1959).

Lea and Hawke (1952) stated that a lowering of pH
alters lipovitellin and Burley and Cook (1962a) showed that

d- and B-lipovitellins dissociate with changes in hydration

20

or structure at specific pH values. Possibly sulfhydryl-
disulfide interactions during dissociation and association
of B-lipovitellin affect its structure (Burley and Cook,
1962b). Fevold and Lausten (1946) indicated that lipo-
vitellenin was involved in gelation, and electrophoretic
changes are found in the lipovitellenin from frozen egg
yolks (Powrie gt gt., 1963). Saari gt gt. (1964b) isolated
the low-density lipoprotein (lipovitellenin) from frozen
yolk plasma. They found that freezing and thawing
decreased the solubility of some plasma lipoproteins in
sodium chloride solution. The insoluble fraction had a
high free fatty acid content. At pH 4.0 their lipoprotein
solution formed a gel after freezing and thawing and it

failed to migrate in paper electrophoresis.

Prevention of gelation

 

It is desirable to inhibit gelation because thawed
egg yolk is difficult to combine with other ingredients
(Jordan gt gt., 1952). Gelation may be partially inhibited
by mechanical and chemical treatments which are used
commercially. The use of additives usually results in an
egg product of limited utility. Most research involving
the functional properties of frozen egg yolk has dealt with
treated yolks. Methods to inhibit gelation, including
explanations of the mechanisms thought to make the methods

successful, follow.

21

§gtt.—-The addition of sodium chloride (1-5%) to
egg yolks increases their viscosity before freezing, but
causes a depression of the freezing point and reduces the
viscosity after thawing (Jordan and Whitlock, 1955; Marion,
1958; Powrie gt gt., 1963). This treatment limits the
yolks to use in products where the flavor of the added
salt is desirable (Marion, 1958). Sodium chloride also
inhibits gelation in low-density lipoprotein solutions
(Saari gt gt., 1964b). The mechanism of its action may
be solely the reduction in freezing point, or it may
inhibit aggregation by stabilizing charged groups on the

egg yolk proteins (Powrie gt_gt., 1963).

Sugars.--A variety of sugars prevent gelation.
The mechanism of this inhibition is unknown, but may be
related to the chelation of iron or copper in the egg
yolk which subsequently could reduce the cross-bonding
of protein structures (Meyer and Woodburn, 1965). Sugars
which are effective include: arabinose, galactose,
.glucose, fructose, sucrose, maltose, and raffinose
(Urbain and Miller, 1930; Thomas and Bailey, 1933; Marion,
1958; Powrie gt gt., 1963). Marion (1958) observed that
9.1 per cent sucrose was necessary to produce a gelation
inhibiting effect equal to that observed when only 4.76

per cent sodium chloride was used, although on a molar

22

basis, sucrose was more effective than sodium chloride

(Powrie gt gt., 1963).

Wgtgt.--L0pez gt gt. (1954) stated that dilution
or concentration of egg yolk does not affect gelation.
However, Marion (1958) repdrted that addition of more
than four per cent water did inhibit gelation somewhat.

Meyer and Woodburn (1965) substantiated this report.

Homogenization.--Homogenization of egg yolk in a
milk homogenizer minimizes gelation (Tongur and Ragosin,
1944). Using homogenization pressures of 0, 1000, 2000,
and 3000 pounds per square inch, Marion (1958) found that
the viscosity of frozen-defrosted egg yolk decreased to
the same degree using the three higher pressures. When
no pressure was applied, gelation was not inhibited. He
also found that repeated homogenization was only slightly

effective in further reducing gelation.

Other methods of reducing gelation.--When the
yolks of fresh shell eggs are inoculated with Bacillus
ggtgg and incubated at 37°C, a hardening of the yolk takes
place (Colmer, 1948). The explanation given is that a
lecithinase produced by the bacteria breaks down a
lecithoprotein, and with the loss of lecithin's binding
action the fat and protein change from their dispersed

state. However, Crotoxin (rattlesnake venum lecithinase A)

23

prevents or reverses gelation (Feeney and Hill, 1960).
Lopez gt El° (1954) found the following enzymes effective
in reducing gelation: papain, pepsin, trypsin, and
rhozyme. Cystiene addition to egg yolks before freezing
results in a slightly greater inhibition of gelation than

does water (Meyer and Woodburn, 1965).

Emulsifying Properties of Egg Yolk

Ziemba (1955) stated that "the primary use of egg
yolks is for emulsification." Inconsistencies exist in
the literature in regard to the component responsible for
egg yolk's emulsifying properties; indeed "there exists
no single coherent theory of emulsion formation and

stability" (Becher, 1965).

The emulsion system (Sutheim, 1947)

 

"Emulsions are intimate mixtures of two immiscible
liquids, one of them being dispersed in the other in the
form of droplets" (Sutheim, 1947). Each of the liquids is
a one-phase system but a two-phase system exists after
emulsification. The discontinuous or internal phase is
the one which exists as separate globules in the emulsion.
The continuous or external phase is the liquid enclosing
the droplets. The two liquids are referred to as oil and
water, dispite the fact that one or both liquids may

contain other material. There are two types of emulsions,

24

oil-in—water and water-in-oil, with the former type being
most prevalent in food emulsions. The external phase of
an emulsion determines its properties. The emulsion can
be diluted only by the external phase. Coloring can be
achieved by using a dye soluble in the external phase.
The emulsion type can seldom be determined by visual
examination, but it may be determined by the behavior of
emulsions toward different thinners and dyes as well as

tests of conductivity.

Theories of emulsification

 

Many theories have been advanced to explain
emulsification (Clayton, 1928). Bancroft (1913, 1915)
recognized the importance of the interfacial film between
the two liquids to be emulsified. Recognizing different
surface tensions on the two sides of the film, Bancroft
and Tucker (1927) theorized that by reducing tension on
the water side of the interface, the film would curve to
enclose oil droplets and prevent coalescence. Mechanical
energy affects the size of the encased droplets, and
coalescence is minimized as droplet size is decreased
(Clayton and Morse, 1939).

Emulsifying agents have two functions; they aid
in forming the emulsion and in stabilizing it. The
emulsifier lowers the interfacial tension with greatest

reduction on the continuous side of the film (Lowe, 1955).

25

(Sharged molecules covering the film act to keep interfacial
tzension to a minimum. An emulsifier acts as the separating
[phase and its absorption at the interface is vital (Becher,
3.965). Harkins and Zollman (1926) showed that a decrease
:in interfacial tension eases emulsion formation, but sur-
:Eace active agents may not stabilize an emulsion (King,

21941; Yeadon gt gt., 1958).

1399 yolk as an emulsifier

Egg yolk promotes oil-in-water emulsions, however,
the minimum amount of egg yolk to stabilize a given inter-
facial area between water and oil is not known (Clayton,
1932). Many of egg yolk's components are emulsifiers,
namely, proteins, phospholipids, and cholesterol. Becher
(1965) stated that in 50:50 oil-in-water emulsions
inversion to the water-in-oil type will occur when the
lecithin/cholesterol ratio falls below 8:1. Cholesterol
promotes water-in-oil emulsions and mayonnaise is weakened
‘when forty-four times as much cholesterol as is normally
present is added (Snell gt gt., 1935). Lecithin was long
thought to be the constituent of egg yolk responsible for
its ability to emulsify oil in water. Snell gt gt. (1935)
added lecithin to mayonnaise and found that a product of
poor stability resulted. Yeadon gt gt. (1958) found that
1.2 per cent purified egg lecithin did not promote emulsion

stability unless fatty acids and cephalins were added. It

26

vmas concluded that a lecitho-protein of the egg yolk is
the emulsifying agent (Snell g E” 1935) .

Vincent gt gt. (1966) studied the surface energy
c>f yolk, plasma, and dispersions of yolk fractions. They
found that the plasma surface—active agents effectively
Ireduced surface energy. They indicated that the livetin
ifractions contribute to the low surface energy and that
fiurther reduction was due to the phospholipids released
lay the low-density lipoprotein for interfacial absorption.
.JOrdan gt gt. (1960) found that the stability of oil-in-
Vvater emulsions was not affected by using egg yolks from
liens fed diets containing different fats, even though the

Iodine Values of the yolk lipids differed significantly.

The effects of freezing on egg
yolkTs emulsifying properties

In 1935, Kilgore stated that more frozen (salted
cor sugared) egg yolks were necessary to produce mayonnaise
similar in consistency to that produced from fresh yolk.
ILeClerc gt gt. (1940) indicated that the functional prop-
«erties of egg yolks were altered by freezing. Miller and
‘Winter (1951) found that the viscosity of mayonnaise was
increased when frozen yolks were used, and they concluded
that frozen yolk could be reduced from 13.5 per cent to
8.6 per cent of the formula with no effect on the viscosity
or stability of the mayonnaise. A comparison of oil sepa-

ration from mayonnaises made from fresh, frozen,

27

:Ereeze-dried, and spray—dried egg yolks revealed decreased
Inayonnaise stability when any of the processed eggs were
Ilsed (Rolfes gt gt., 1955). The contradictory reports in
'the literature may be due to differences in formulae,

Inethods and materials (Lowe, 1955).

frests of emulsion stability

 

Simple systems are used to test the stability
imparted by an emulsifying agent. Mayonnaise is fre-
<quently used to test the stabilizing power of egg yolk
(Snell gt gt., 1935; Miller and Winter, 1951; Rolfes
gt gt., 1955). Stability is determined by recording
'whether or not the mayonnaise emulsion breaks during
storage or mechanical shaking.

Drainage from simple oil and water emulsions with
a high proportion of water may be measured to indicate
the stability imparted by an emulsifier. The greater the
drainage, the less stable is the emulsion system. These
emulsions have been used to quantify differences between
emulsifying agents (Harkins and Zollman, 1926; Yeadon

gt_gt., 1958; Jordan gt gt., 1962).

Objective Tests

Objective tests are those tests which depend on
some measure other than the human senses and include chemi-

cal, histological, and physical tests (Griswold, 1962).

28

tPhey give quantitative measurements and are reproducible,
vvith their accuracy being a function of the tools and
rnethods employed. In the study of egg yolk proteins,
selectrophoretic mobility and viscosity determinations are

(zonducted to measure changes in the proteins characteristic

c>f denaturation.

I?aper electrophoresis

Paper electrophoresis has been used extensively
.in the study of egg yolk proteins, both to explore their
<:omposition and to monitor separation techniques. Changes
in the electrophoretic patterns of egg yolk proteins which
have been frozen are apparent. Differing conditions which
influence ionic migration have led to variation in the
electrophoretic patterns of egg yolk proteins, dependent

‘upon the laboratories from which the patterns were pro-

duced .

General theory of paper electrophoresis (Block
.EE;E£'I l955).--Paper electrophoresis or ionophoresis is
a.useful tool in the study of protein mixtures. The basic
3principle of electrophoresis is that charged particles in
solution migrate in an electrical field. When particles
possess either quantitatively or electrically different
charges, they migrate different distances on paper strips.

Convection is prevented by draining the paper strips to

29

produce optimum resolution. Factors which govern ionic
migration include:

1. Those characteristics related to the ion itself,
namely, its charge (sign and magnitude), size,
shape, tendency to dissociate, and amphoteric
behavior, if any.

2. Those factors related to the environment in
which the ion is being studied, such as the
electrolyte concentration, ionic strength,
dielectric properties, chemical properties, pH,
temperature, viscosity, and the presence of non-
polar molecules which may influence viscosity or
dielectric properties of the electrolyte or which
may interact to form charged complexes.

3. The character of the applied field, its intensity,
purity (presence of alternating current compo-
nents), and the distribution along the migration
path (Block gt gt., 1955).

Electrolytic dissociation and reactions of acids
or bases are the processes commonly encountered to charge
particles for electrophoresis. Uncharged molecules or
amphoteric materials can show apparent mobility as a
result of endosmosis, a phenomenon based upon the electro-
kinetics of liquid-solid interfaces. A viscous medium
provides a retarding force opposite to the movement of the
particle in a constant field.

Many types of apparatus are used for paper
electrophoresis, ranging from simple to highly complex.
All embody the same basic principles; the sample is applied
to a buffer impregnated filter paper strip and a current is
passed through the strip, allowing the charged ions of the

sample to migrate in the electrical field. The protein is

then denatured, usually by drying. Control of drying

30

temperature, current, and evaporation from the strips are
important factors in the selection of apparatus. Common
buffer solutions are used as the electrolyte and the choice
of solution depends upon the sample to be tested. A
solution with a pH value higher than the isoelectric point
of the protein in the sample prevents the adsorption of
positively charged ions on the filter paper.

Irreversible adsorption of sample components on
the paper, especially prevalent when mobilities are low,
causes "tails" which remain superimposed on the other
components and make quantification impossible. On the
other hand, reversible adsorption yields comet-shaped
migration bands which do not interfere with resolution.

Since paper electrophoresis is an analytical
technique, methods of staining have been devised for the
quantitative determination of proteins, lipids, and glyco-
proteins. The dyestuff must be bound stoichiometrically
and permit easy elution unless the strips are to be
analyzed by direct scanning. Protein dyes which give
good results include bromphenol blue, Azocarmine B, and
Amidoschwartz 10B.

Stained strips are analyzed by direct densitometry
or by cutting the strips into the visible zones, eluting
the dye and analyzing the solution colormetrically. The
amount of dye bound is largely a measure of the basic

groups present and cannot be correlated with other

31

properties. Different affinities for dye may also
influence results. Unless complete resolution is obtained,
cutting the strips into bands is a highly subjective pro-
cedure.

Direct scanning techniques are rapid and conven-
ient, but direct scanners should be calibrated so that
numerical values will correspond with values obtained by
elution techniques, so that comparison between labora-
tories is possible. Scanning may involve errors. Lack
of homogeneity of the paper may result in large errors
where contrasts between background and dye are small.

Due to the great number of variables involved in
paper electrophoresis, comparison of quantitative results
can only be made if experimental techniques are precisely

the same.

Electrophoretic mobility of egg yolk proteins.--

 

In 1957, Evans and Bandemer studied egg yolk and egg yolk
protein preparations by paper electrophoresis, using
diethyl barbiturate buffer (pH 8.6, u = 0.05). The
migration distances of seven bands were determined. Using
elution techniques, they reported the protein composition
of egg yolk (Table 4). Evans gt gt. (1958) noted that

the yolk proteins of eggs from individual hens varied

greatly in speed of migration.

32

TABLE 4. Protein composition of egg yolk determined by
paper electrophoresis (Evans and Bandemer,

 

 

1957)
Protein Fraction Total Protein %
A Ovalbumin 1.7
B Livetin 5.5
C Livetin 3.1
D Lipovitellenin 41.7
E Lipovitellin (Non-Mobile) 34.5
F Lipoprotein 11.9
G 1.6

 

Using carbonate buffer (pH 9.8, u = 0.10) to
increase the resolution of low-mobility components, Saito
gt gt. (1965) were able to separate ten proteins and lipo-
proteins from the egg yolk by electrophoresis. Electro-
phoretic study of the egg yolk's high density fraction
using veronal buffer (pH 9.0, u = 0.3) yields mobility
patterns shown in Figure 1 (Bernardi and Cook, 1960a).
Differences in mobility in the ascending and descending
limbs result from the multiplicity of components widely
different in charge density.

Sugano (1959) found that the mobility of lipo-
vitellin depends upon the strength of the buffer solution.
Meyer and Woodburn (1965) indicated that ions are important

in lipovitellin migration.

33

 

4,5

 

 

L].
F————-$ desc. acs. <—————4

 

 

cmz/sec/V x 105
1 a -10 to -12
b -8.5 phosvitin
c -7.5
2 -5.4 a - livetin
-3.7 B - livetin
4 -3.1 a - lipovitellin
B - lipovitellin
5 -2.4 y - livetin

 

Figure 1. Typical electrophoretic mobility patterns of
egg yolk's high density fraction (Bernardi
and Cook, 1960a)

Livetin shows three electrophoretic peaks named
a-, B-, and y-livetin in terms of descending mobility
(Shepard and Hottle, 1949; Martin and Cook, 1958). The
three peaks overlap a- and B-lipovitellin in many studies
(Sugano, 1958). Martin gt gt. (1957) found y-livetin
similar in mobility to the lipoproteins. Phosvitin pre-

cedes the livetins in migration (Sugano, 1957, 1958).

Electrgphoretic patterns of frozen egg yolk.--
Solubility studies indicated that lipovitellenin is altered
during freezing (Fevold and Lausten, 1946). Powrie gt gt.

(1963) electrophoretically isolated two major lipoprotein

34

complexes from egg yolk. Lipovitellin did not migrate
from the point of application before or after freezing.
Unfrozen lipovitellenin migrated 28 mm. After freezing
and thawing, only a slow-migrating fraction (14 mm) was
found. Optical density values showed that the slow-
migrating lipoprotein was not lipovitellenin which was
presumably altered by freezing and thawing and became non-
mobile. In the fresh sample, the slow-migrating protein
was evidently carried along to 28 mm with the lipo-
vitellenin. Under the same conditions, Meyer and Woodburn
(1965) found that the mobile protein fraction decreased
with an increase in the non-mobile protein fraction when
the egg yolk was frozen and thawed. The addition of sodium
chloride or cysteine hydrochloride to the yolks before

freezing reduced this difference.

Measurement of viscosity of egg yolk

 

Viscosity is defined as a liquid's resistance to
flow and is caused by intermolecular attraction. Absolute
viscosity is expressed by the work necessary to maintain a
flow rate and is measured by the poise unit which equals
the force of one dyne per second per square centimeter.
Relative viscosity compares flow rate with that of a
reference liquid, usually water.

Quantitative determinations of viscosity provide

an index of denaturation (Gortner, 1949). Measurement of

35

viscosity before and after freezing and thawing can
determine the amount of egg yolk gelation, as the phenome-
non is accompanied by an obvious increase in viscosity.

Various tools have been employed to measure egg
yolk viscosity. Viscosity is dependent upon the tempera-
ture and moisture content of the sample (Payawal gt gt.,
1946).

Some of the simplest methods employed in the
determination of viscosity are based upon the time neces-
sary for the egg product to flow through a certain distance
in an Ostwald or a Mohr pipet with the tip removed (Barmore,
1934; Jordan and Whitlock, 1955). This method is highly
dependent upon an accurate timing devise and the dexterity
of the investigator. The small sample requirement is an
advantage and the method may be quite successful if the
sample is not too viscous.

The capillary viscometer (Payawal gt gt., 1946) or
Bingham plastometer (Bateman and Sharp, 1928) measures the
pressure required to pass a sample through a length of
uniform bore capillary tubing. By this method, Payawal
gt gt. (1946) found the viscosity of egg yolk (49-49.5
per cent water) to be 800 centipoises at 25°C.

After gelation has taken place, the increased
viscosity of the sample necessitates the use of some other
instrument. The Gardner-Parks mobilometer measures the

time necessary for a weighted piston to fall a pre-determined

36

distance into the sample (Pearce and Lavers, 1949).
Forsythe gt gt. (1953) found this instrument satisfactory
for measuring the viscosity of frozen whole egg fortified
with yolk. In a study of the viscosity of frozen whole
egg magma, Thomas and Bailey (1933) found each successive
plunge of the mobilometer piston to be more rapid than
the‘previouS-plunge.

Jordan and Whitlock (1955) used the MacMichael
viscometer to measure the viscosity of egg yolk. This
instrument measures the angel of torque of a disk sus-
pended by a wire into the sample which is rotated at a
constant speed. The Stormer viscometer measures the
rotation rate of a cylinder moving through the sample
impelled by a uniform force. Miller and Winter (1950)
used this instrument to study the viscosity of frozen
pasteurized whole eggs.

The Brookfield viscometer successfully measures
the relative viscosity of egg yolk which has undergone
gelation (Jordan and Whitlock, 1955; LOpez gt_gt., 1954;
Marion, 1958). A spindle of chosen size is rotated through
the sample at a predetermined number of revolutions per
minute and the force exerted against rotation by the sample
is read from the instrument and converted to centipoise
units by means of a table, dependent upon rotation rate and
spindle size. Meyer and Woodburn-(1965) reported a mean

relative viscosity of 27.4 poises for fresh yolk at 24°C

37

and a mean relative viscosity of 907.9 poises for thawed
yolk frozen at -25°C for 20 to 24 hours. Powrie gt gt.
(1963) reported a mean relative viscosity of 24 poises
for fresh yolk at 25°C and of 628 poises for thawed yolk
which had been frozen at -l4°C for 150 minutes. Fresh
yolk viscosity is influenced by the temperature and
moisture content of the sample, whereas thawed yolk
viscosity is dependent upon the additional factors of
rate, time, and temperature of freezing and the rate and
temperature of thawing (Marion, 1958; Powrie gt gt.,

1963).

EXPERIMENTAL PROCEDURE

Design of Experiment

This experiment was designed to study the emulsion
stabilizing ability of native egg yolk and three crude egg
yolk fractions: lipovitellin, lipovitellenin, and livetin.
Oil-in-water emulsions, stabilized by the yolk fractions,
were prepared according to a 24 factorial design. The
four factors were the three fractions and the storage
treatment. The two levels of the three yolk fractions
were their absence or presence in the emulsion mixture.
The two levels of storage were designated as fresh and
frozen. Thus, there were sixteen treatment combinations
for each replication of the design. Emulsions prepared
with fresh and frozen native yolk served as a control for
comparison with emulsions containing recombined lipo-
vitellin, lipovitellenin, and livetin.

To determine the effects of freezing on the physi-
cal properties of egg yolk and the three yolk fractions,
samples of both fresh and frozen native yolk and fractions
were analyzed for moisture, protein, pH, and viscosity.

Studies of electrophoretic behavior were conducted on

38

39

native yolk and the three yolk fractions both before and
after they were frozen. The entire design was replicated

six times.

Procurement of Eggs

The requirement for fresh egg yolk and the amount
of time required to complete this study obviated the method
of mixing and storing egg yolk sufficient for all replica-
tions. Therefore, eggs were obtained from a controlled
group of sixty-five S.C. White Leghorn hens (Foreman
Strain) for each replication.

Hens were approximately eighteen months of age,
housed in experimental floor pens and fed an all-mash
laying ration designated as LB~63, the formula of which
is included in the Appendix.

Eggs were gathered two times each day and held at
room temperature over night prior to delivery for research.
All eggs were laid the day before they were taken to the
laboratory where they were held at 4-5°C prior to use
within twelve hours. Eggs were sorted and only grade A
Large eggs were used. All eggs were dry cleaned by sand-

ing before packaging.

Preparation of Egg Yolk

Following removal from refrigeration, the eggs

were broken and separated by hand. Each yolk was carefully

40

rinsed in distilled water, which was deionized by means
of an Illco-Way Research Model deionizer (product
impurities < 0.5 ppm.). The yolks were rolled on four
layers of cheesecloth to remove adhering white, and the
chalazae were removed by hand. The vitelline membrane
was punctured and the yolk contents allowed to drain
through two layers of cheesecloth into a tared 2000 ml
beaker. The yolks from four dozen eggs were mixed by
stirring three minutes with a rubber spatula, afterwhich
135 i 0.01 g of the native yolk was weighed on a Torbal
torsion balance (Model PL-800, 800 g-capacity) into a
labeled one-half pint polyethylene freezer container.
The native yolk was stored covered at 4-5°C until divided

and analyzed.

Separation of Egg Yolk Fractions

A modification of the technique employed by Evans
and Bandemer (1957) was used to separate three crude egg
yolk protein and lipoprotein fractions which were desig-
nated as lipovitellin, lipovitellenin, and livetin. Walker
(1967), who used a similar method of separation, listed the
components of the three fractions. Thus, according to
Walker's classification, the crude lipovitellin used in
this experiment actually consisted of d-lipovitellin,
B-lipovitellin, phosvitin, and low—density lipoprotein.

The crude lipovitellenin was fairly pure and the crude

41

livetin consisted of a-, B-, and y-livetins in addition to

small amounts of unrecovered lipovitellenin.

Lipovitellin

 

A Toledo Balance (Model 4030, 5 kg-capacity) was
used to weigh the mixed egg yolks to the nearest 1.0 g.
Deionized water in an amount equal to twice the weight of
the egg yolk was added, and the diluted egg yolk was mixed
three minutes with a rubber spatula, before being placed
into eight 50 ml Teflon centrifuge tubes and centrifuged
in a Servall angle centrifuge, Type SS-l, equipped with a
refrigeration unit which maintained a temperature of
0 i 2°C in the interior chamber during centrifugation.
The centrifuge timer was set for thirty minutes and the
speed was brought to 10,000 rpm as quickly as possible
(approximately three minutes) without exceeding a 5 amp
reading on the ammeter. The 10,000 rpm speed was main-
tained until the centrifuge automatically stopped at the
end of the preset time. Since four centrifuge runs were
necessary for each replication, the Saran-covered diluted
egg yolk mixture was held at 4-5°C between runs, and
stirred for thirty seconds with a rubber spatula before
additional tubes were filled.

The supernatant solutions from all eight tubes
were combined in a 1000 ml beaker before any further sepa-

ration steps were begun. The creamy, white precipitate in

42

each centrifuge tube was rinsed with approximately 5.m1

of deionized water and the rinsings were discarded before
the precipitate was removed from the tubes with a metal
spatula. All precipitates were mixed in a tared one-half
pint polyethylene freezer container before weighing to the
nearest 0.01 g. The container was tightly covered,
labeled, and stored at 4-5°C until division, analyses,

and objective testing. This creamy, white precipitate

was designated as lipovitellin.

Lipovitellenin

 

The supernatant solution obtained from the lipo-
vitellin preparation was used to prepare the crude lipo-
vitellenin. The combined supernatant solution from each
set of eight centrifuge tubes was placed in 36 inches of
seamless, cellulose dialyzer tubing (1 1/8 inches diameter,
inflated). Dialysis was carried out against deionized
water at 4-5°C until a negative test for chloride was
obtained. Qualitative determination of chloride was made
according to the method outlined by Caldwell and King
(1961). Three drOps of concentrated HNO3 were added to
5 m1 of the dialysis water. One drop of approximately
0.1 N AgNO3 was added. The presence of a white precipi-
tate was indiCative of chloride presence and dialysis was

continued until a clear solution was obtained on AgNO3

addition.

43

The contents of all dialysis tubes were mixed in
a 2000 ml beaker and placed on a Cenco Magnetic Stirrer.
Using a 1 3/4 inch Teflon-coated stirring bar, the mixture
was stirred at medium speed throughout the pH adjustment.
A Beckman Zeromatic pH Meter equipped with a thermocompen-
sator and standardized with a pH 7 buffer solution was
used to measure the pH of the solution while 0.169 N NaOH
(pH 12.0) was added gradually from a 25 m1 buret supported
over the center of the solution. Sufficient NaOH
(approximately 10 ml) was added until the solution reached
pH 6.2.

The solution was placed in 50 ml Teflon centrifuge
tubes which were balanced and placed in a Servall angle
centrifuge at 0 i 2°C. The timer was set for ten minutes
and the speed increased to 10,000 rpm as rapidly as
possible without exceeding a 5 amp reading on the ammeter
(approximately three minutes). Following centrifugation,
a sticky, yellow, low-density fraction was suspended in
the tubes above a clear solution. The contents of the
tubes were placed in funnels (4 inch diameter) fitted with
Whatman No. 4 filter paper (24.0 cm diameter) which were
set into 500 m1 Erlenmeyer flasks. The top of each funnel
Was covered with Saran and filtration was allowed to con-
tinue overnight at 4-5°C. The sticky yellow low-density
fraction which collected on the filter paper was designated

as lipovitellenin. It was carefully removed from the filter

44

paper, placed in a labeled one pint polyethylene freezer
container and weighed to the nearest 0.01 g. The lipo-
vitellenin was stored at 4-5°C until division, analyses,

and objective testing were conducted.

Livetin

The filtrate remaining in the Erlenmeyer flasks
after lipovitellenin removal was designated as livetin.
It was mixed in a tared and labeled one quart polyethylene
freezer container and weighed to the nearest 0.01 g on a
Torbal torsion balance. Until division, analyses, and
objective testing were conducted, the livetin was stored

at 4—5°C.

Freezing of Egg Yolk and Egg Yolk Fractions

The experimental material for each replication was
divided into two parts.~ One-half of the native yolk and
each of the three fractions were weighed to the nearest
0.01 g on a Torbal torsion balance into labeled poly-
ethylene freezer containers, using one-half pint containers
for the native yolk, lipovitellin, and lipovitellenin and
a one pint container for the livetin. The half-portions of
the native yolk and the three fractions were frozen and
held at -23°C for one week, after which they were removed

from the freezer and thawed six hours at room temperature.

45

Chemical analyses and objective testing were
conducted on the frozen samples following the same proce-
dures used on fresh samples with one exception. No deter-
mination of total protein was made on the frozen samples,
since total protein has been shown to remain constant when
egg yolk is frozen (Evans and Davidson, 1953). Changes in
moisture content, however, made necessary recalculation of

the percentage protein contained in the frozen samples.

Chemical Evaluation

Fresh egg yolk and yolk fractions were analyzed
for total protein and total moisture content to determine
the proportions to be used in emulsion preparation. Frozen
egg yolk and yolk fractions were analyzed for total mois-
ture content. Percentage protein in the frozen egg yolk
and yolk fractions was recalculated using revised sample

sizes due to changes in moisture content.

Moisture

Total moisture was determined by the AOAC vacuum
oven method 16.3 (a) (1955). Sample weights of native
yolk, lipovitellin, lipovitellenin, and livetin were
approximately 2 g and drying was carried out at 90°C with
25-30 in. vacuum for six hours in a Hotpack vacuum oven,
Model 633. An average of two determinations for each

Sample was recorded.

46

Protein

Total reduced nitrogen was determined by the
Kjeldahl method as modified by Gunning and Arnold (Triebold
and Aurand, 1963). The oxidation catalyst used consisted
of an 8:1 mixture of K2804 and CuSO4. Sample sizes were
determined so that the titration step would require
20-40 ml of 0.1 N HCl. The approximate weights used were
as follows: lipovitellin, l g, lipovitellenin, 3 g,
livetin, 30 g, and native yolk, l 9. Wet oxidation of
the samples was accomplished by a three hour digestion
period. Ammonia was distilled into 150 m1 2% boric acid
and the solution in the receiving flasks was titrated with
standard 0.1 N HCl using a mixed indicator consisting of
0.083 g methylene blue and 0.125 9 methyl red per 100 m1
ethanol. The average percentage of protein in each sample
was calculated using the general conversion factor, 6.25.
An average of two determinations for each sample was

recorded.

Objective Measurements

Objective tests were performed on fresh and frozen
native yolk and yolk fractions to determine differences in
relative viscosity, pH, emulsion stabilizing power, and

electrophoretic behavior of egg yolk proteins.

47

Emulsion stability

 

Emulsions stabilized by native yolk and yolk
fractions were studied to determine the stability each
fraction and all combinations of yolk fractions imparted
to an oil-in-water emulsion system and to determine the
effects of freezing on the emulsion stabilizing properties

of the fractions.

Emulsion formula.--The emulSion formula selected

 

to test the emulsion stability imparted by all possible
combinations of the egg yolk fractions was based on the
formula used by Jordan gt El° (1962), which consisted of
15 9 egg yolk, 15 g corn oil, and 85 g deionized water.
It was necessary to modify the formula for each emulsion
mixture to maintain desired protein ratios and a constant
moisture content.

An emulsion containing 15 g egg yolk contains
2.45 g protein, using 16.4 as the average percentage
protein in egg yolk (Sweetman and MacKellar, 1959). The
average amount of moisture contained in 15 9 egg yolk is
7 g, based on 49.4 per cent moisture in egg yolk (Sweetman
and MacKellar, 1959). Adding this amount of water to the
85 9 recommended by the basic formula resulted in a 92 g
meisture content for the emulsion mixture. The amount of
mOisture contained in the yolk, yolk fraction or combina-

tion of fractions used in each emulsion mixture was

48

subtracted from 92 g to determine the amount of water to
be used in each preparation.

‘Two emulsions were prepared for each replication
using fresh and frozen native yolk as the emulsion stabi-
lizing agent. Protein and moisture contents of the native
yolk were used to determine the amount used to control the
protein content of each emulsion to 2.45 9. These emul-
sions were prepared for comparison to emulsions containing
proportional amounts of the three yolk fractions. Table 5
lists the weights of ingredients used in the native yolk

emulsions.

TABLE 5. Formulae used in preparation of native yolk

 

 

 

emulsionsa
Storage Ingredients
Treatment Native Yolk Deionized Water COrn Oil
9 m1 9
Fresh 14.90 - 15.25 85 15
Frozen 14.96 - 15.04 85 15

 

aThe amount of native yolk was corrected for
variance in moisture and protein content between replica-
tions.

Lipovitellin, lipovitellenin, and livetin each.
Contribute to the total protein of the egg yolk. For each

replication, the proportional amounts of protein contrib-

Uted by each fraction were calculated. .Using the results

49

of the analyses for total protein contained in each
fraction, the amount of each fraction to be used in the
emulsion mixture was determined.

Preparation of sixteen yolk fraction emulsions
was required to complete each replication of the 24 fac-
torial design. Table 6 lists the weights of the ingre-
dients used in the yolk fraction emulsions.

1 of

A sufficient quantity of a commercial brand
corn oil for all replications was procured at the begin-
ning of the investigation and stored at 4-5°C until before
a set of emulsions was prepared, when it was removed from
refrigeration and thoroughly mixed. A Torbal torsion
balance was used to weigh 15 i 0.01 g oil into each of

nine 25 ml pyrex test tubes. The tubes were tightly

covered with Parafilm until the oil was used.

Preparation of emulsions.--The nine emulsions
composing the fresh or frozen portion of each replication
were prepared as one set. The same order of emulsion
preparation was followed throughout the investigation.
Also maintained throughout the experiment was the time
elapsing from the day the eggs were laid until the day the

emulsions were prepared. This time was seven days for the

lMiesel Brand, Stabilized Pure Corn Oil distributed
by Geo. Miesel & Son, Detroit, Michigan.

50

 

 

 

 

TABLE 6. Formulae used in preparation of yolk fraction emulsionsa
Emulsion Ingredients
Typeb Yolk Fractionsb Deionized Corn
A B C Water Oil
9 9 m1 9
O - - 92 15
A 4.27 - 4.96 - 90 - 89 15
B - 11.02 - 12.80 86 - 84 15
C - - 29.63 34.79 63 - 58 15
Fresh
. AB 4.27 - 4.96 11.02 - 12.80 83 - 82 15
BC - 11.02 - 12.80 29.63 34.79 56 - 51 15
AC 4.27 - 4.96 - 29.63 34.79 60 - 55 15
ABC 4.27 - 4.96 11.02 - 12.80 29.63 34.79 54 - 48 15
0 - - 92 15
A 4.25 - 4.95 - 90 - 89 15
B - 11.08 - 12.82 86 - 84 15
C - - 28.40 34.78 64 - 59 15
Frozen
AB 4.25 - 4.95 11.08 - 12.82 83 - 82 15
BC - 11.08 - 12.82 28.40 34.78 57 - 50 15
AC 4.25 - 4.95 - 28.40 34.78 61 - 55 15
ABC 4.25 - 4.95 11.08 - 12.82 28.40 34.78 55 15

- 48

 

3The amount of each yolk fraction and deionized water was corrected
for variance in moisture and protein content between replications.

bFractions are labeled as follows: 0 = no fractions present,
A ‘ lipovitellin, B = lipovitellenin, and C = livetin. Emulsion type
refers to both storage treatment and stabilizer composition.

51

fresh yolk and fresh yolk fraction emulsions and fourteen
days for the frozen yolk and frozen yolk fraction emul-
sions.

The predetermined amounts of the yolk and yolk
fractions for each set of emulsions were weighed to the
nearest 0.01 g into tared one-half pint glass jars which
were then covered until mixing was started. Weighed corn
oil at room temperature was added to the yolk or yolk
fractions and the oil tube was allowed to drain in a
vertical position for thirty seconds, before addition of
the required amount of deionized water, at room tempera-
ture, from a 100 ml graduate cylinder.

The blade of an Osterizer blender (Model 452) was
attached and the jar containing the emulsion ingredients
was inverted on the blender assembly. The blender was
connected to a Fisher Scientific Powerstat (Type 3PN116)
to control blender speed, and to a GraLab timer (Model 171)
to control blending time. Blending was begun and con-
tinued for one minute with the powerstat set at 55. The
powerstat was then adjusted to 110 and the emulsion
homogenized for five minutes. If after one minute at the
higher speed, particles still adhered to the container,
timing was stOpped and the particles were scraped from the
Sides and bottom of the container with a metal spatula.
Mixing was then resumed for the remainder of the five

minutes.

52

Measurement of emulsion stability.--Immediately

 

after the preparation of each emulsion, two 15 ml gradu—
ated centrifuge tubes were filled with the emulsion
mixture, covered with Parafilm to prevent surface drying,
and placed in a vertical position in a test tube rack.
Emulsion stability was determined by measuring the amount
of separation of the oil and water phases at four time
intervals: thirty, sixty, ninety, and one hundred and
twenty minutes after the emulsions were poured. The
amount of drainage to the nearest 0.05 ml was recorded.
The average drainage of the two tubes for each of the
four times was calculated and used in statistical analysis.

pH of emulsion mixtures, native yolk,
and yolk fractions

 

 

The pH of the emulsion mixture remaining in each
container after the tubes had been filled was determined
using a Beckman Zeromatic pH meter. The pH value was’
recorded to the nearest 0.1 pH unit when the meter needle
maintained its position on the scale. The pH values of
the yolk and the three yolk fractions, before and after

frozen storage, were determined in the same manner.

' Viscosity of native yolk and yolk fractions

 

Measurements of relative viscosity were made on
samples of native yolk and the three yolk fractions, before

and after frozen storage using a Brookfield Syncho-lectric

53

Viscometer, Model RVT. Lipovitellin, lipovitellenin, and
native yolk samples were placed in 50 ml tapered pyrex
centrifuge tubes, to a depth one-half inch from the top.
The tubes were supported in a vertical position by a
150 ml beaker and spindle 7 was lowered to its indentation
mark into the center of the sample. Care was taken to
avoid the entrapment of air bubbles. It was necessary to
adjust the rotating speed of the viscometer from sample to
sample depending upon the composition of the sample and its
storage treatment. A table of rotation speeds for each
variable and for each replication is included in the Appen-
dix. Three readings were recorded for each sample; the
first reading was taken after one minute of rotation, and
additional readings were taken at thirty second intervals.
Livetin samples were placed in 600 m1 beakers to a depth
of 8.0 cm. Spindle l and a constant speed of 100 rpm were
used for both fresh and frozen samples. After lowering the
spindle carefully into the liquid until its indentation
mark was reached, three readings were recorded following
the procedure previously described.

Since the speed and spindle sizes varied between
samples, values were converted to poises to facilitate
comparison. The three values for each sample were

averaged.

54

Paper electrOphoresis

 

Samples of lipovitellin, lipovitellenin, and native
yolk were prepared for electrOphoresis by dispersing 1 g of
the yolk or yolk fraction in l g deionized water. The yolk
or fraction and water were weighed into a 50 m1 beaker and
hand stirred with a wooden applicator stick until the mix-
ture was homogeneous. Livetin was sufficiently dilute to
be used as obtained. Samples were placed in 10 x 75 mm
tubes, covered with Parafilm, and allowed to reach room
temperature before application to filter paper (Schleicher
and Schuell, 2043—A mgl, 3.0 x 30.6 cm) strips.

Filter paper electrophoresis was conducted using a
ridgepole electrOphoresis cell, Spinco Model R. Veronal
buffer (15.40 g sodium barbital and 2.76 g barbital per
liter), pH 8.6, ionic strength 0.075 was used. The filter
paper strips were wet with buffer solution and the cell
was allowed to equilibrate thirty minutes before a micro-
pipette and sample applicator were used to apply 0.006 ml
of sample to each strip. For each sample duplicate strips
were run. A constant current of five milliamperes was
applied for eighteen hours at room temperature after which
the strips were dried for fifteen minutes at 120°C.

To facilitate analysis of protein mobility, the
denatured protein on the strips was stained. Following a
six minute pre-rinse in methanol, the strips were immersed

in alcoholic bromphenol blue solution (1 g dye per liter

55

of methanol) for thirty minutes. The strips were rinsed
three times for fifteen minutes each in five per cent
acetic acid before being blotted on filter paper and dried
fifteen minutes at 120°C. Color was fixed by holding the
strips in NH4OH vapors for fifteen minutes.

In order to determine the electrophoretic patterns,
the stained strips were analyzed using a Spinco Model RB
Analatrol equipped with two 500 mu filters and a B-5 cam,
using a 1.0 mm slit width. Percentages of mobile and non-
mobile fractions were calculated following visual examina-
tion of the electrophoretic patterns. Migration distance
was measured in millimeters from the point of application

to the most distant portion of the curve.

Analyses of Data

Two computer programs designed to perform statis-
tical analysis using the CDC-3600 Computer at Michigan
State University were used to calculate the desired
statistical tests on the objective data. The Rand Routine
and AOV Routine were used to calculate analysis of variance
and standarddeviations of the means. Significant
differences detected_by analyses of variance were further
examined by graphing of interaction means and use of

Studentized range tests (Duncan, 1957).

RESULTS AND DISCUSSION

The present investigation was primarily concerned
with identifying the emulsifying properties of egg yolk
and three crude egg yolk fractions: lipovitellin, lipo-
vitellenin, and livetin. A second objective was to study
the effects of freezing and thawing upon the emulsifying
prOperties of egg yolk and all combinations of the three
fractions.

Test emulsions were prepared by emulsifying oil
in water using combinations of the three fractions in
amounts prOportional to those normally found in the egg
yolk as well as using native yolk. Emulsion stability
was determined for all emulsions by recording drainage
at thirty-minute intervals for two hours. Similar emul-
sions, prepared with frozen egg yolk and egg yolk
fractions, were evaluated in the same way. The viscosity
and the electrophoretic mobility of egg yolk and the three
fractions were measured by standard procedures before and
after the samples had been frozen and thawed to determine

the effects of gelation upon these parameters.

56

57

Chemical Analyses

Chemical determination of moisture and protein
content of the native yolk and the three fractions--
lipovitellin, lipovitellenin, and livetin-—were conducted
for three reasons. First, the determinations were neces-
sary in order to correctly balance the emulsion formulae.
Second, since a different lot of eggs from the same flock
of hens was used in each replication, these determinations
were conducted to detect differences in egg yolk composi-
tion between replications, and finally, these determina-
tions were conducted on both fresh and frozen materials to
determine whether changes in moisture or protein content
accompanied the gelation reaction. Replicate averages for
moisture and protein content and pH determinations of both
fresh and frozen native yolk and fractions appear in the

Appendix.

Moisture;

The results of the analysis of variance for mois-
ture is included in Table 7. As expected, lipovitellin,
lipovitellenin, livetin, and native yolk were different
from one another in respect to moisture content at the
0.1% level of probability (Table 8). There was no
significant difference in moisture attributed to
replications. Lipovitellin, lipovitellenin, and livetin

all exhibted higher moisture contents than native yolk.

58

.sunannmnoum to Hm>mH mH.o may um ucmonmflcmnm
an

{.1

 

 

Hm.o No.m mm Houum
mo.o mm.o m m x Em
«axom.mama «gaaa.>omm m mcoflpomum
Ho.o mo.o H usefinmmna mmmnoum
vH.H H5.H m mcowumoflammm
5v Hmuoa

smouomm musumwoz Eoommum mommanm>

 

noncommIcmwz

mo mwmnmmo

mo mousom

 

cmuonm tom ammuniumusmfiummnu mmmnoum

ozu spas cwpm>fla tom .cwcmaamufl>omfla .cflaamuw>omfia .xaom m>wumc mcoss
ucmucoo musumflofi one sflmuonm ca moosmHmMMMo How moccanm> mo mmmMHmsm .h mqm<e

59

.nmaa .cmocson

.maugu an am>am

 

 

 

 

 

 

 

 

 

 

 

 

 

mcofluma>muonm .COADMH>mo tumocmum A Down new we tmmmmnmxw memos unmaummuem
mo.o A ma.o mm.o om.H
OAmAMA< H H H H cmuoum
om.ma Hm.o hm.b mn.hm
. cflmuoum
mH.o «H.o mm.o om.H
OAmAMAm H H H A smmnm
_Hm.wH Hm.o mm.h Hm.hm
mm.o om.o vo.m mm.H
MAmAmAU H H H H cmnonm
ma.mv mm.hm mv.mm mm.mm
. mnoumfloz
mm.o mh.o mm.m mm.o
wAmAmAO H H In A ammum
so.ms Hm.sm m~.mm 1 sv.mm
nam>ma wH.o an s o m a
moosmummmwo ucmfipmmue uouomm
unmowmacmwm mmcoHDMH>mo onmosmum\msmmfi psmsummse .momnoum
mucosummuu mmmnoum 03p suds va xaom m>Humc cam .AOV swum>wa
.Amv casmaamufl>omfla .A¢V QHHHmDH>0mHH mo ucmusoo,sflmuoum can muspmfloa
Hon moosmHmMMMo.usmowmwsmflm cam .msOHpmw>mt custsmpm .msmmE usmfipmmua .m mqmde

60

This was due to the dilution involved in the separation
procedure. The moisture of these fractions is certainly
higher under these experimental conditions than it is
when the fractions are found in their combined state as
native yolk.

There were no significant differences in moisture
content between the fresh and frozen fractions and between
fresh and frozen samples of native yolk. This indicates
that freezing and thawing did not change the binding of
water in such a way that more or less of the water was

removed by the moisture determination employed.

Protein

The analysis of variance for protein (Table 7)
indicated a very highly significant difference in protein
content among the three fractions and native yolk. Mean
protein content and standard deviations are included in
Table 8.

The lipovitellin fraction contained the highest
percentage of protein on a wet basis and accounted for
52.36 per cent of the egg yolk's total protein. Lipo-
vitellenin contributed 35.70 per cent of the yolk's total
protein and the livetin fraction being extremely dilute,
represented 11.09 per cent of the yolk's total protein.

The protein contents of the fractions and native

yolk did not differ significantly among replications and

61

were not significantly greater after freezing and thawing.
These findings are in agreement with those of Evans and
Davidson (1953) who found no difference in protein content
between fresh and stored shell eggs which had been layed

at the same time by the same hens.

PE

The mean pH and standard deviations for the three
fractions and native yolk for both storage treatments are
shown in Table 9. Freezing did not appear to change the
TABLE 9. Means and standard deviations for pH determina-

tions of fresh and frozen samples of native
yolk, lipovitellin, lipovitellenin, and

 

 

livetin
1 u 1
Storage Fraction Mean/standard deviation
Treatment
Native yolk 5.9 i 0.08
Lipovitellin 5.9 i 0.16
Fresh
Lipovitellenin 6.3 i 0.22
Livetin 6.0 t 0.23
Native yolk 5.8 i 0.10
Lipovitellin 5.8 i 0.08
Frozen
Lipovitellenin 6.4 i 0.22
Livetin 6.1 i 0.25

 

62

pH of the native yolk, lipovitellin, lipovitellenin, or
livetin. This is in agreement with the work of LOpez
gt gt. (1954) who found that freezing did not alter the
pH of egg yolk. The pH of lipovitellin was the same as
that of the native yolk, whereas the pH values of lipo-
vitellenin and livetin were more neutral due to the

adjustment of pH necessary to effect their separation.

Emulsion Stability

The drainage data from the emulsion studies were
analyzed to determine which factors influenced the sta-
bility of emulsions prepared with native yolk, or all
combinations of lipovitellin, lipovitellenin, and livetin.
The effect of storage treatment was determined by com-
paring drainage data from emulsions prepared with fresh

and frozen egg yolk or fractions.

Recombined fractions vs. nativeyolk

 

The results of the analyses of variance for
determining differences in emulsion stabilizing power.
between fresh and frozen native yolk and recombined fresh
and frozen lipovitellin, lipovitellenin, and livetin
appear in Table 10.

No significant differences in drainage between
native yolk and recombined fraction emulsions or between

the two storage treatments occurred at the thirty minute

63

 

 

 

 

 

TABLE 10. Analyses of variance for differences in emulsion
stabilizing power between fresh and frozen
native yolk and fresh and frozen recombined
lipovitellin, lipovitellenin, and livetin

Drainage Source of Degrees of Mean
Time Variance Freedom Squares
Total 23
Replications 5 0.01
30 minutes Storage Treatment 1 0.02
Fractions 1 0.01
ST x F l 0.02
Error 15 0.01
Total 23
Replications 5 0.03
60 minutes Storage Treatment 1 0.12*
Fractions 1 0.17***
ST x F 1 0.07
Error 15 0.02
Total 23
Replications 5 0.03
90 minutes Storage Treatment 1 0.12
Fractions 1 0.45***
ST x F l 0.04
Error 15 0.04
Total 23
Replications 5 0.04
120 minutes Storage Treatment 1 0.13
Fractions 1 0.83***
ST x F 1 0.02
Error 15 0.06

 

*
Significant at the 5% level of probability.

**
Significant at the 1% level of probability.

**

*
Significant at the 0.1% level of probability.

64

reading. However, after sixty, ninety, and one hundred
and twenty minutes, emulsions prepared with native yolk
had significantly less drainage than those prepared with
the recombined lipovitellin, lipovitellenin, and livetin.
The emulsions prepared with fresh yolk and recombined
fractions drained less than those prepared with frozen
yolk and frozen recombined fractions, but this difference
waS-significant only for the sixty minute reading.

The mean drainage for these two emulsion formulae
is plotted against time in Figure 2. It should be noted
that although the two emulsion formulae differed signifi-
cantly in stability, total drainage was small in both
cases (Table 11). These findings indicate that the sepa-
ration techniques may have changed the fractions in such
manner that the emulsions prepared with recombined frac-
tions decreased in stability to a greater extent as time
increased than did those emulsions prepared with native
yolk, with both emulsion formulae imparting equal stability
at thirty minutes.‘ Freezing influenced both emulsion
formulae to the same extent, significantly increasing
drainage at sixty minutes in both cases.

The differing emulsion stability of the two
formulae may also be explained by the differences in pH
of the emulsion mixtures. Replicate averages, means, and
standard deviations for pH determinations of the emulsion

formulae appear in the Appendix. The mean pH of emulsions

65

mcofluomum cmcflnfioomn

cmuonm can nmmnm can xaow m>wpms cmuonm tam smoum mo omuflawnmum
mGOHmHsz How mafia mmmcfimno pmsflmmm omuuon mmmsflmnt mo accosd

Ammuosflfiv mEHB.mvmcfimua

OJH om JG

 

 

Macs m>numz

xaow m>Hpmz cmnonm

msofluomum omsHoEoomm.

msowpomum \\

Umsfiaeoowm cmnoum \\

 

 

.N musmfim

(sxeqttttttm) ebsutsrq

66

 

 

 

mH.o H mm.o mo.o H m~.o mo.o H mH.o oo.o H oH.o s
cmnoum
mm.o H mm.o mm.o H mm.o sm.o H ms.o mH.o H mH.o oma
40.0 H Hm.o no.0 H mm.o Ho.o H HH.o Ho.o H HH.o w
ammum
G~.o H mm.o mH.o H HH.o Go.o H H~.o mo.o H mo.o oma
omH om _om . . on “an emHHHHanm HamsnmmHa
AGHEV mHm>Hmpcfl mafia “50m um AHEV mmmcflmua macamasfim mmmuoum

bl

Aomdv sflum>fla com .cfiswaamufl>omfla

.cHHHmuH>omHH omcHnEoown cmuonm cam ammum new Awe Mao»

m>Humc cmuoum com swanm suHB venomous mcomeSEm scum mam>nmucfl
wasp noon um mmmcflmuo How mc0fluma>mo cumocmpm one memos usefiummna .HH momma

67

prepared with native yolk was 5.7 in comparison to a pH
of 5.9 for emulsions prepared with the recombined frac-
tions. More research is necessary to determine the effect
of pH on the stability of emulsions prepared with egg yolk
and egg yolk fractions.

Replicate averages for drainage from emulsions
prepared with fresh and frozen native yolk and the recom-
bined fractions appear in the Appendix.

Combinations of lipovitellin,
lipovitellenin, and livetin

 

 

The results of the analysis of variance for
determination of the effects of the presence of lipo-
vitellin, lipovitellenin, and livetin as well as the
effects of storage treatment, replication, and drainage
time on the stability of oil-in-water emulsions appear
in Table 12. A table of the means used in the interpreta-

tion of the analysis appears in the Appendix.

Effects of lipovitellin, lipovitellenin, and

 

livetin on emulsion drainage.--The presence of lipovitellin,

 

lipovitellenin, or livetin in the emulsion formula reduced
emulsion drainage significantly, at the 0.1% level of
probability. The effects of the protein fractions pgt-gg
are depicted in Figure 3. Although all three fractions
were effective emulsion stabilizers, lipovitellenin reduced

drainage by 7.1 m1 and thus, provided the best overall

68

TABLE 12. Analysis of variance for determination of the effects of
fractions, storage treatment, replications, and drainage
time on the stability of emulsions prepared with all
possible combinations of lipovitellin, lipovitellenin,
and livetin

 

 

Source of Variance D.F. Mean Squares
Total 383
Replications 5 0.85
Storage Treatment 1 2.39
Drainage Time 3 24.24***
Aa 1 328.39***
1 4815.67***
1 136.83***
x B 1 69.91***
x C l 27.14***
x C l 24.24***
x B x C 1 0.56
x ST 1 2.54
x ST 1 1.22
x ST 1 0.98
x B_x ST 1 4.31
x C x ST 1 2.78
x C x-ST 1 0.01
x B x C x ST 1 0.21
T x DT 3 0.01
x DT 3 0.03
x OT. 3 0.30
x DT 3 0.02
x B x DT 3 3.94***
x C x DT 3 2.02***
x C x DT 3 0.07*
x B x C x DT 3 0.22***
x ST x DT 3 0.03
.x ST x OT 3 0.04
x ST x UT 3 0.04
x_B x ST x DT 3 0.03
x C x ST x DT 3 0.01
x C x ST x DT 3 0.00
x B x C x ST x DT 3 0.06
emaining Error 240 0.03

21>?»m>om>>vw>nw>m>ww>om>w>w>oon

*Significant at the 5% level of probability.
***Significant at the 0.1% level of probability.
aA denotes lipovitellin.

B denotes lipovitellenin.
C denotes livetin.

ST denotes storage treatment.

DT denotes drainage time.

69

 

 

 

 

 

 

10.0__
9.0__
m 8.0t__
H
3
H 7.0___
H
.H
:1 6.0_
.,.g '—
2
c 5.0
"-1 fi—
8 4.0
m 'fi-
I:
'3 3 o
H ‘ ———
G
2.0__
1.0——

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Absent Present Absent Present Absent Present
Lipovitellin Lipovitellenin Livetin

Figure 3. Bar-graph of emulsion drainage depicting the
main effects of lipovitellin, lipovitellenin,
and livetin in emulsion stabilization

stabilizing effect of the three. Lipovitellin reduced

drainage by 1.3 m1, and livetin reduced drainage by 1.1 ml

in effects which were similar.

Very highly significant first-order interactions
occurred when any two of the three fractions were combined.

These interactions showed that when two fractions were

combined, their emulsion stabilizing powers were not

independent.

70

Table 13 indicates the responses of lipovitellenin
and lipovitellin to one another. The presence of both
lipovitellin and lipovitellenin in the emulsion formula
reduced emulsion.stability by 0.3 ml, in comparison to
the effects of both fractions alone.

TABLE 13. Interaction responses of lipovitellin and
lipovitellenin to one another

 

 

 

Lipovitellin Lipovitellenin Response to Presence
Absent Present of Lipovitellenin

m1 ml m1

Absent 8.1 1.5 -6.6

Present ~ 6.8 0.5 -6.3

Response to Interaction

Presence of -1.3 -1.0 -0.3

Lipovitellin

 

 

 

 

Table 14 indicates the responses of lipovitellenin
and livetin in respect to the presence of one another.-
Lipovitellenin and livetin were also antagonistic toward
one another in emulsion stabilization and the magnitude of
this antagonism was -l.0 ml.

The responses of lipovitellin and livetin to one
another in emulsion formulae appear in Table 15. In this

case, the antagonistic interaction effect was also -1.0 m1.

71

TABLE 14. Interaction responses of lipovitellenin and
livetin to one another

 

 

 

Livetin Lipovitellenin ~ Response to Presence
Absent Present of Lipovitellenin

ml ml ml

Absent 9.0 1.4 -7.6

Present. 7.3 0.7 -6.6

Response to Interaction

Presence of -l.7 —0.7 -1.0

Livetin.

 

 

 

 

TABLE 15. Interaction responses of lipovitellin and
livetin to one another

 

 

 

Livetin Lipovitellin . Response to Presence
Absent Present of Lipovitellin
m1 ml m1
Absent 6.3 4.0 -2.3
Present 4.6 3.3 -1.3
Response to Interaction
Presence of -1.7 -0.7 -l.0
Livetin

 

 

 

 

These antagonistic interactions which occurred
when two fractions were present in the emulsion mixture
suggested that a major decrease in stability might be
observed when all three fractions were present in the

emulsion system. This was not the case. The lack of any

72

significant interaction when all fractions were present

in the emulsions indicated that all of the fractions in
this case reduced drainage in the manner to be expected
from the consideration of fractions pgt gg. This may be
partially explained by a consideration of the mean drain-
age from the eight emulsion formulae (Table 16). Combina-
tions of any two fractions promoted greater stability than
either of the fractions alone, although the very highly
significant first-order interactions indicated that the
stability of these emulsions was not as great as would be
expected from independent action of the fractions pgt gg.
When all fractions were recombined, stability was-the
best, being equal to the combined independent action of
the fractions.

Becher (1965) stated that in oil-in-water
emulsions, inversion to water-in-oil will occur if the
lecithin/cholesterol ratio falls below 8:1. Lipo-u
vitellenin contains approximately thirty-eight per.cent
lecithin and six percent cholesterol for a lecithin/
cholesterol ratio of 6.33:1, and yet this fraction was
the most effective of the three fractions in promoting
emulsion stability. Since most-of the cholesterol of
the egg yolk is obtained from the lipovitellenin fraction,
further imbalance of the lecithin/cholesterol ratio when

another fraction is added is unlikely.

73

TABLE 16. Mean drainage and standard deviations for drainage from
emulsions prepared from fresh and frozen lipovitellin (A),
lipovitellenin (B), and livetin (C) and read at four time

 

 

 

inteévals
Storage Egg Yolka Mean/Standard Deviation for Drainage at:

Treatment Fractions 30' 60' 90' 120r

ml m1 ml ml
0 10.8 i 0.44 10.9 i 0.44 11.0 i 0.44 11.0 i 0.46
A 6.6 t 0.72 7.2 t 0.59 7.5 t 0.54 7.7 i 0.52
B 0.7 t 0.44 1.7 i 0.98 2.3 i 0.87 2.7 i 0.91
C 7.7 i 0.36 8.3 t 0.34 8.5 i 0.32 8.7 i 0.29
Pres“ AB 0.1 t 0.04 0.3 x 0.16 0.7 1 0.26 1.0 z 0.28
BC 0.2 i 0.10 0.7 i 0.26 1.1 i 0.41 1.5 i 0.51
AC 4.7 i 1.25 5.9 i 1.05 6.3 t 1.00 6.6 i 0.85
ABC 0.1 i 0.05 0.2 r 0.06 0.4 i 0.18 0.6 t 0.26
0 10.1 t 0.54 10.3 i 0.53 10.3 t 0.50 10.4 t 0.52
A 6.7 i 0.40 7.3 i 0.21 7.7 i 0.36 8.1 1 0.53
B 0.9 i 0.33 2.1 i 0.55 2.8 t 0.59 3.3 i 0.55
C 7.6 i 0.78 8.3 t 0.58 8.6 i 0.51 8.7 r 0.42
Fr°zen AB 0.3 t 0.19 0.6 i 0.33 1.0 x 0.51 1.2 1 0.59
BC 0.4 t 0.09 0.9 i 0.18 1.3 t 0.27 1.7 t 0.35
AC 5.6 i 0.84 6.5 t 0.68 6.9 t 0.63 7.2 i 0.57
ABC 0.2 t 0.13 0.5 t 0.27 0.6 i 0.32 0.8 t 0.38

 

aLetters denote the fractions used in the oil-in-water emulsions.

0 a no fractions.

74

Harkins and Zollman (1926) stated that oil-in-water
emulsions form readily if interfacial‘tension is below ten
dynes. The research of Vincent gt gt. (1966) indicated
that lipovitellin has a high interfacial tension and
livetin a low interfacial tension. Yet the stability of
emulsions prepared with lipovitellin and livetin were
similar, with lipovitellin being slightly more effective
in promoting emulsion stability. Solutes which lower.
interfacial tension are efficient emulsifiers, but low
interfacial tension apparently does not influence emulsion
stability.~

The findings of-this study, indicate that the
mechanism of emulsion stabilization by egg yolk is complex,
and do not-contradict the statement by Yeadon EEJEL' (1958)
that "those phosphatide preparations possessing good
emulsifying characteristics are presumably mixtures or

complexes of the phosphatides with other substances."

 

Drainage between storage treatments.--No signifi-
cant difference-in drainage was detected between emulsions
prepared with fresh yolk fractions and emulsions prepared
with fractions which had been frozen. In this experiment,
freezing was not detrimental to the emulsion stabilizing
power-of any fraction or fraction combination, however,
when the mean drainage from the fresh emulsions was

compared to that of the frozen emulsions, (Table 16) there

75

was a trend toward greater drainage when frozen fractions-
were used. This trend may become a significant decrease
in stability in emulsions where only a small proportion

of egg yolk is employed as the stabilizing agent. Miller
and Winter (1951) stated that the amount of frozen egg
yolk in mayonnaise could be reduced from 13.5 per cent to
8.6 per cent of the formula with no effect on stability.
The trend for frozen yolk emulsions to be less stable than
fresh yolk emulsions, in the present study, does not lend
supportho their suggestion. It appears that as Kilgore
(1935) suggested, more frozen than fresh yolk would pro—
duce mayonnaises of equal stability, although equal
quantities of fresh and frozen yolk could produce products
with acceptable stability. The viscosity of the emulsions
in this study were not examined, and while no great differ-
ences in viscosity were noted by this investigator, frozen
yolk may significantly effect the viscosity of the emulsion

system of mayonnaise.

Drainage variance among time intervals.--The time

 

at which drainage from the emulsions was read produced a
very highly_significant main effect on the magnitude of
the_drainage (Table 12). The mean drainage curve for all
emulsions (Figure_4) showed that drainage from the emul-
sions was greatest in the first thirty minutes, after

which there was a leveling off with a significant and

76

Drainage in Milliliters

 

A l l

 

. . +
30 60 90 12
Time of Reading in Minutes

0

Figure 4. Mean drainage from all emulsions plotted against
the times at which the readings were taken

nearly linear increase in drainage from thirty to one
hundred and twenty minutes.

The use of frozen fractions in the emulsions had
no effect on the rate of drainage from the emulsions, as
indicated by the low mean square for the interaction of
storage treatment with drainage time (Table 12). Since
storage treatment had no significant effect, drainage data
from both fresh and frozen fraction emulsions were averaged
in the explanation of significant interactions of drainage

and fraction combinations.

Interaction of drainage time and fraction

combinations.—-The lack of significant interactions of

 

drainage time with each of the three fractions indicated

77

that the magnitude of drainage for these fractions was
the same between thirty and one hundred and twenty
minutes, although as previously stated, the presence of
any fraction significantly reduced drainage.

The magnitude of significance in the second-order-
interactions of drainage time with combinations of any two
of the three fractions and an examination of the first-
order interaction mean squares for drainage time and
fractions revealed that the presence of lipovitellenin
was an important, though not exclusive, factor in the
second-order interactions. The mean square for the
interaction of drainage time and lipovitellenin, while
not significant at the 5% level of probability, was
greater than the mean squares of corresponding inter-
actions of drainage time with lipovitellin or-livetin.

The second-order interaction of drainage time with
livetin and lipovitellin was significant at the 5% level
of probability, whereas corresponding second-order
interactions which included lipovitellenin were signifi-
cant at the 0.1% level of probability.

The interaction of drainage time with lipovitellin
and lipovitellenin is depicted in Figure 5. It is apparent
that the initial drainage--from zero to thirty minutes--
was greatest when the lipovitellenin fraction was not

present.

78

cflcmaampfl>omwa one cwaamuw>omfla Spas mEHu wmmcwmuo mo newuomnmucH

mousse: as mcflommm mo mafia

ONH om om cm

i _ H 1
ucmmmnm cwcmaamuw>omflq tom cflaamufl>omflq .VIIIIIIIIIIIIIII

D

‘II

ucmmonm cflcwaamufl>omflq

ucmmmum CHHHmuH>oqu

 

 

1‘

Hammna cHamHHwHH>oaHa can cHHHmHH>omHH

 

.m mudmfih

ttttw ut abeurelq

sxenII

79

Figure 6 depicts the interaction of drainage time
with lipovitellenin and livetin. Again, the presence of
lipovitellenin greatly reduced the initial drainage, while
increasing the rate of drainage from thirty to one hundred
and twenty minutes.

The interaction of drainage time, lipovitellin,
and livetin, significant at the 5% level of probability,
is depicted in Figure 7. Lipovitellin and livetin both
reduced initial drainage, although not to the extent of
the lipovitellenin reduction (Figures 5 and 6). The
rate of drainage from thirty to one hundred and twenty
minutes was essentially the same for these emulsion
formulae, indicating that neither lipovitellin, livetin,
nor their combination was very effective in reducing the
rate of drainage from thirty to one hundred and twenty
minutes.

A comparison of Figures 5, 6, and 7 indicates
that the presence of lipovitellenin reduced the drainage
rate from zero to thirty minutes, but increased the rate
of drainage from thirty to one hundred and twenty minutes.
Lipovitellin interacted with lipovitellenin (Figure 5)
to reduce the rate of drainage from thirty to one hundred
and twenty minutes, while it did not greatly reduce this
rate when present alone. Livetin interacted with lipo-
vitellenin in a similar manner (Figure 7), but did not

reduce drainage in the presence of lipovitellenin

80

cflp0>fla tom cflcwHHmuH>omflH nufl3 08w» momswmuo mo cofluomnmuoH

mounts: ca guacamm mo MEHB
omH om om om

l? n n
1 u u 8

ucmmoum cwum>fiq can cacmHHmUH>omHa

ucmmmnm cflcmaawpfl>omfiq

 

 

ucmmmum cfium>flq

“nomad sfiuo>flq one GHGmHHmDH>OQHA

 

 

.m gunman
IoH
firm m
P
l t.
o m m
5
I. e
or. r.
U
Io.m m.
T.
I n
o.w I
It
.4.
.I . R
,o A s
105
lea
r'
83

81

cflum>fla cam :HHHmuH>OQHH nus? m8wu.mmmcwmuo mo cowuomnmch

mousse: as unwcmmm mo mafia
ONH om om on
,7 m .i _

usmmmum cfluw>flq one GHHHmuH>omfiq

 

dcmmmum cflaaoufiwomflq

ucmmmum cflum>flq

.‘ll

ucmmhm sflum>wq one GHHHmuH>OQHA

ill.

 

.h mnomflm

Sieqttttttw u: absutexq

82

as much as the lipovitellin-lipovitellenin combination.
Although both livetin and lipovitellin reduced initial
drainage somewhat, neither of them nor their combination
effected a reduction in drainage from thirty to one hundred
and twenty minutes.

The third-order interaction of drainage time with
lipovitellin, lipovitellenin, and livetin is depicted in
Figure 8. Drainage from each fraction combination emulsion
from zero to thirty minutes and the increase in drainage
from thirty to one hundred and twenty minutes is shown in
Table 17. Examination of this interaction gives the best
overall picture of the manner in which the three fractions
function to promote emulsion stability.

The highest initial drainage occurred when none of
the fractions were present, although in this case subse-
quent drainage was low. Thus, the maximum drainage which
could occur in this emulsion system was apparently achieved
in the early stages of drainage. Lipovitellenin was more
effective in reducing initial drainage than lipovitellin
or livetin. When more than one fraction was-present, the
comparison of initial drainage to subsequent drainage is
found to be more than a simple effect of different rates
of drainage toward a maximum. ,Both lipovitellin and
1ivetin,when present with lipovitellenin, further reduced
initial drainage, while counteracting lipovitellenin's

effect to increase subsequent drainage. While the

83

qumHHmHH>oaHH .Aav

ONH

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84

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mmmcflmno ca mmmmnocfl may one manages human“ on ones Eoum mmmcflmnn .ha mamfla

85

lipovitellenin and livetin fractions together did reduce
drainage from_thirty to one hundred and twenty minutes,

the reduction was only slightly less than that of livetin
alone. In the presence of both lipovitellin and livetin,
initial drainage was slightly reduced, while subsequent
drainage was greater than that observed when either lipo-
vitellin or livetin was present alone. Only when all

three fractions were present was the best emulsion sta-
bility obtained. All three fractions functioned to reduce
initial drainage to a minimum. The high drainage from
thirty to one hundred and,twenty minutes which was apparent
when only lipovitellenin or the combination of lipovitellin
and livetin were present was, in this case, compensated by
the ability of lipovitellin-lipovitellenin and lipo-
vitelleninelivetin combinations to reduce subsequent
drainage. When all fractions were present, these complex
interactions, which can be resolved only when drainage

time is considered, produced effects upon total drainage

which suggested independent action of the three fractions.

Relative Viscosity

Replicate averages for the relative viscosity of
fresh and frozen lipovitellin, lipovitellenin, livetin
and native yolk appear in the Appendix. Means and
standard deviations are listed in Table 18. An analysis

of variance test was not conducted since the prerequisite

86

TABLE 18. Means and standard deviations for the relative
viscosity of both fresh and frozen lipo-
vitellin, lipovitellenin, livetin, and
native yolk

 

 

Storage
Fraction Treatment Mean Standard Deviation-
poises poises
Fresh 1.64 x 106 0.84 x 106
Lipovitellin 6 6
Frozen 14.26 x 10 27.00 x 10
5 5
Fresh 8.14 x 10 6.38 x 10
Lipovitellenin. 5 5
Frozen 44.71 x'lO 45.71 x 10
Fresh 0.16 0.03
Livetin
Frozen 0.18 0.05
2 2
Fresh 1.37 x 10 0.11 x 10-
Native Yolk 6 6
Frozen 1.62 x 10 0.25 x 10

 

of equal variance was obviously not fulfilled. The
extremely large standard deviations for relative viscosity
determinations of fresh and frozen lipovitellin and lipo-
vitellenin indicated that these fraCtions differed greatly
in viscosity from one replication to another. This could
have been due to several factors. The spindle of the
Brookfield viscometer tended to cut a path within these
highly viscous samples, rather than rotate through them.
Also, the small differences in moisture content between

replications for these samples may have resulted in larger

87

changes in relative viscosity. Finally, the consistency
of the lipovitellin fraction samples particularly, tended
to remain somewhat lumpy, despite efforts of homogeniza—
tion. An examination of the means and replicate averages
for relative viscosity does, however, establish definite

patterns of viscosity change.

Native yolk

 

Freezing and thawing increased the relative

2 to 1.62 x 106

viscosity of native yolk from 1.37 x 10
poises. This increase, indicating that gelation had
occurred, is a sign of protein denaturation as expressed
by Gortner (1949). Forsythe gt gt. (1953) found that the
thick gel-like consistency of frozen whole egg fortified
with yolk had no adverse effects on its performance in
sponge cakes, however, Pearce and Lavers (1949) pointed
out that viscous frozen yolk is difficult to combine with
other ingredients and Jordan gt gt. (1952) were unable to

prepare satisfactory custards or plain cakes with untreated

frozen egg yolk.

Lipovitellin

 

The relative viscosity of lipovitellin was influ-
enced in an unpredictable manner by freezing and thawing.
The relative viscosity of frozen lipovitellin for two

replications was actually lower than that of the unfrozen

88

sample, although in all other replications, relative
viscosity increased after freezing and thawing with the
average increase of 1.26 x 107 poises being the greatest
change associated with any fraction. Powrie gt gt. (1963)
examined the relative viscosity of a lipovitellin solution

containing CaCl They found that the relative viscosity

2.
of the solution increased after freezing and thawing,
indicating that structural changes of the lipovitellin's
proteins had occurred. Lea and Hawke (1952), in their
study on the effects of freezing on the solubility of

lipovitellin also had a great deal of variability in their

results.

Lipovitellenin.

 

Frozen and thawed lipovitellenin had a higher
relative viscosity than fresh lipovitellenin for all
replications. The average increase was 3.66 x 106 poises--
approximately a four-fold increase in relative viscosity.
The lipovitellenin fraction contributed to the high
relative viscosity of frozen native yolk as indicated by
Fevold and Lausten (1946) and Feeney and Hill (1960).

Saari gt gt. (1964b), studying the effects of freezing on
a low-density lipoprotein solution (pH 4.0), indicated
that freezing and thawing low-density egg yolk lipoprotein

resulted in gel formation. Such a gel was noted by this

89

investigator, in conjunction with a definite loss of
glossiness after the lipovitellenin fraction had been

frozen and thawed.

Livetin

The relative viscosity of livetin remained
essentially the same after freezing and thawing. Gelation
does not seem to involve livetin, perhaps because it is
not a lipoprotein.

It is apparent that the increase in relative
viscosity which occurs when native yolk is frozen and
thawed is due to changes in both the lipovitellin and
lipovitellenin fractions of egg yolk. Further studies
of the effects of the gelation reaction are necessary,
using highly purified and further-sub-divided samples

of lipovitellenin and lipovitellin.

Paper Electrophoresis

Filter paper electrophoresis was conducted to
determine the effect of freezing and thawing on the
proteins of lipovitellin, lipovitellenin, livetin, and
native yolk. This procedure also aided in identifying
the components of the three crude protein fractions.
Typical electrophoretic patterns are depicted in Figure 9.
Migration distances and percentages of mobile and non-

mobile protein for each replication are listed in the

90

   

Frozen Fresh Frozen Fresh Frozen Fresh Frozen Fresh
Livetin Lipovitellenin Lipovitellin Native Yolk

Figure 9. Typical dyed paper strips for samples of both
fresh and frozen livetin, lipovitellenin,
lipovitellin, and native yolk

91

Appendix. Migration to the left of the origin was
calculated, but since it was considered to be mainly due
to spreading of the sample, it was included as part of

the non-mobile protein. The migration to the left could
also have been due to a very slight contamination of the
samples with egg white lysozyme, which becomes colored
with dye in this location, but does not contribute greatly
to the area under the curve of the electrophoretic pattern.
An analysis of the proportional composition of the native
yolk was not attempted, as difficulty in the application

of some samples led to an indeterminate sample volume.

Native yolk

 

Typical electrOphoretic patterns of fresh and
frozen native yolk (Figure 10) indicated that the proteins
of egg yolk were changed by the gelation phenomenon. Fresh
native yolk contained an average of 35.0 per cent non-
mobile protein and 65 per cent mobile protein, while in
frozen samples, the average amount of non-mobile protein
increased to an average of 51.3 per cent and mobile protein
decreased to 48.7 per cent. These findings are in agree-
ment with those of Meyer and Woodburn (1965) who indicated
that freezing increased the proportion of non-mobile
protein in native yolk. The large ranges in migration
distance are consistant with the findings of Evans gt gt.

(1958) that the yolk proteins of eggs from individual hens

92

 

 

 

 

 

 

 

__J . 4

Fresh Frozen.
Mean Migration - 48.9 mm Mean Migration - 48.3 mm
Mobile Protein - 65.0% Mobile Protein - 48.7%
Non-Mobile Protein - 35.0% Non-Mobile Protein - 51.3%

Native Yolk

Figure 10. Typical paper electrophoretic patterns of fresh and frozen
native yolk

93

vary greatly in speed of migration. A comparison of the
typical stained paper strips of lipovitellin, lipo-
vitellenin, livetin, and native yolk (Figure 9) indicated
that overlap occurs when all fractions are combined as
was indicated by Sugano (1958).

The fraction responsible for the change in protein
mobility of native yolk after freezing may be found by an
examination of the electrophoretic data from the fresh

and frozen fractions.

 

Lipovitellin

The mean migration distance of lipovitellin
remained essentially unchanged after freezing, and little
change in the mobility was noted (Figures 9 and 11).

These findings indicated that lipovitellin was not mobile
either before or after freezing, under the conditions of
this research.. The findings of Bernardi and Cook (1960a)
indicated that the slight mobility observed was due to

the presence of y-livetin, phosvitin, and the low-density
lipoprotein in the crude lipovitellin fraction. These
results substantiate the non-mobile nature of lipovitellin

found by Powrie gt gt. (1963).

Lipovitellenin

 

Freezing caused a dramatic change in the electro-

phoretic patterns of lipovitellenin (Figure 12). The

94

 

 

 

 

J

Fresh Frozen
Mean Migration - 44.3 mm' Mean Migration - 46.8 mm
Mobile Protein - 28.7% Mobile Protein - 26.7%
Non-Mobile Protein - 71.3% Non-Mobile Protein - 73.3%
Lipovitellin

Figure 11. Typical paper electrophoretic patterns of fresh and frozen
lipovitellin

95

 

 

 

__.J hf

Fresh Frozen
Mean Migration - 37.6 mm . Mean Migration - 24.3 mm
Mobile Protein - 89.3% Mobile Protein - 32.6%
Non-Mobile Protein - 10.7% Non-Mobile Protein - 67.4%
Lipovitellenin

Figure 12. Typical paper electrophoretic patterns of fresh and frozen
lipovitellenin

96

majority of protein in this fraction was mobile when
fresh samples were analyzed, but after freezing, the
majority of the protein was non-mobile. Examination of
typical stained paper strips for fresh and frozen lipo-
vitellenin samples (Figure 9) revealed the presence of a
slow-migrating protein after freezing, in results similar
to those of Powrie gt gt. (1963). These researchers
showed that this slow-migrating protein was not lipo-
vitellenin, which became non-mobile after freezing. They
indicated that in fresh samples the slow-migrating frac-
tion, which is unidentified at present, was carried a

greater distance by the more mobile lipovitellenin.

Livetin

Typical electrophoretic patterns of livetin
(Figure 13) indicated the presence of a- and B-livetins
in this fraction. The increase in non—mobile protein
after freezing was probably due to contamination with
lipovitellenin in the separation technique. Migration
distances remained essentially the same for fresh and

frozen samples.

97

____,A _, /\ J\ fi/\_

 

 

Fresh Frozen
Mean Migration - 54.8 mm Mean Migration - 55.8 mm
Mobile Protein - 95.9% Mobile Protein - 87.6%
Non-Mobile Protein - 4.1% Non-Mobile Protein - 12.4%
Livetin

Figure 13. Typical paper electrophoretic patterns of fresh and frozen
livetin

SUMMARY AND CONCLUS IONS

This investigation was primarily concerned with
identifying the emulsifying properties of three crude
egg yolk fractions: lipovitellin, lipovitellenin, and
livetin. A second objective was to study the effects of
freezing and thawing upon the emulsifying properties of
egg yolk and all combinations of the three fractions.

Test emulsions were prepared by emulsifying oil
in water using combinations of the three fractions in
amounts proportional to those normally found in the egg
yolk, as well as using native yolk. Emulsion stability
was determined for all emulsions by recording drainage
at thirty-minute intervals for two hours. Similar emul-
sions, prepared with frozen egg yolk and egg yolk fractions
were evaluated in the same manner. The viscosity and elec-
trophoretic mobility of egg yolk and the three fractions
were measured by standard procedures before and after the
samples had been frozen and thawed, to determine the
effects of gelation upon these parameters. All data
reported are the average of six replications.

Lipovitellin, lipovitellenin, livetin and native

yolk differed from one another in respect to moisture and

98

99

protein content (0.1% level of probability). Freezing
and thawing did not significantly influence these deter-
minations.

A comparison of the emulsion stabilizing powers
of native yolk and recombined fractions revealed no sig-
nificant difference in emulsion drainage between the two
formulae or the fresh and frozen storage treatments after
thirty minutes of drainage. However, after sixty, ninety,
and one hundred and twenty minutes, emulsions prepared
with native yolk were more stable than those prepared
with the recombined fractions (0.1% level of probability).
Emulsions prepared with fresh yolk and recombined fresh
fractions drained less than those prepared with frozen
yolk and frozen recombined fractions, but this difference
was only significant (5% level of probability) for the
sixty minute reading. Although the two emulsion formulae
differed in stability, total drainage was small in both
cases. The difference may be explained by changes in the
fractions during their separation or by differences in the
pH of the emulsion mixtures.

The presence of lipovitellin, lipovitellenin, or
livetin in the emulsion formula reduced emulsion drainage
significantly at the 0.1% level of probability. Although
all of the fractions were effective emulsion stabilizers,
lipovitellenin provided the best overall stabilizing effect

of the three.

100

Very highly significant interactions occurred when
any two of the three fractions were combined, indicating
that emulsion stabilizing powers were not independent.
Close examination of these interactions revealed that,
while combinations of any two fractions promoted greater
stability than either of the fractions alone, the stability
of the emulsions was less than would be expected from inde-
pendent action of the fractions EE£;§E°

Freezing and thawing was not detrimental to the
emulsion stabilizing power of any of the fractions or
fraction combinations, although there was a trend toward
higher drainage when frozen fractions were used, which
might become significant in an emulsion system of different
proportions.

Drainage increased throughout the two hour testing
time. Emulsion mixtures without any stabilizing agent
drained a large amount initially, apparently reaching the
drainage maximum early in the drainage period. The pres-
ence of lipovitellenin reduced initial drainage, but
increased the rate of drainage from thirty to one hundred
and twenty minutes. Livetin and lipovitellin reduced
initial drainage somewhat and interacted with lipovitellenin
to reduce subsequent drainage, although the latter effect
was not apparent when lipovitellin and livetin were present

alone or combined with one another. The best emulsion

101

stability was observed when all three fractions were
recombined.

Determinations of relative viscosity, before and
after lipovitellin, lipovitellenin, livetin, and native
yolk were frozen, revealed that freezing and thawing
greatly increased the viscosity of native yolk. The
increased relative viscosity of lipovitellenin after
freezing contributed to the thickness of the frozen yolk.
The lipovitellin fraction had the greatest mean relative
viscosity increase after freezing, but it was quite
variable and in two replications the relative viscosity
of lipovitellin was decreased by freezing and thawing.

Paper electrophoresis was unable to detect any
change in the mobility of the proteins of the lipovitellin
fraction after freezing which would account for its change
in relative viscosity. Lipovitellenin, however, produced
electrophoretic patterns which indicated that a majority
of its proteins were changed in such a manner that they
became non-mobile after freezing and thawing. It was
concluded that livetin did not contribute to gelation, as
its relative viscosity and electrophoretic patterns re-
mained the same after freezing and thawing.

The results of this investigation, while elucidat-
ing the emulsifying properties of three crude egg yolk
fractions, indicated that research is needed in the

following areas: (1) an analysis to determine the effect

102

of the lipid portion of lipovitellin and lipovitellenin
on their emulsion stabilizing powers; (2) a study to
determine the effects of various concentrations of egg
yolk proteins and lipids on emulsion stability; (3) an
investigation to determine the emulsifying properties of
further separated and more highly purified egg yolk pro-
teins; (4) an investigation of the emulsion stability
imparted to mayonnaise by the egg yolk proteins and lipo—
proteins; (5) an investigation to determine the effects
of varying pH on the stability of egg yolk emulsions;

(6) development of a more accurate tool for determining
the relative viscosity of frozen native yolk and the yolk
fractions which are thickened by gelation; (7) further
studies to determine the cause of the increase in relative
viscosity which lipovitellin and lipovitellenin undergo
after freezing and thawing; and (8) a study to examine
other functional properties of crude egg yolk protein

fractions, such as coagulation and foaming.

LITERATURE CITED

AOAC, 1955. Official Methods of Analysis. 8th ed.
Assoc. Offic. Agr. Chemists, Washington, D.C.

Bancroft, W. D., 1913. The theory of emulsification, V.
J. Phys. Chem. 17: 501-520.

Bancroft, W. D., 1915. The theory of emulsification, VI.
J. Phys. Chem. 19: 275-309.

Bancroft, W. D. and C. W. Tucker, 1927. Gibbs on
emulsification. J. Phys. Chem. 31: 1681-1692.

Barmore, M. A., 1934. The influence of chemical and
physical factors on egg white foams. Colo. Agr.
Expt. Sta. Bull. No. 9.

Bateman, G. M. and P. F. Sharp, 1928. Some observations
on the consistency of cream and ice cream mix-
tures. J. Dairy Sci. 11: 380-396.

Becher, P., 1965. Emulsions: Theory and Practice.
2nd ed. Reinhold Publishing Corporation, New
York.

Bernardi, G. and W. H. Cook, 1960a. An electrophoretic
and ultracentrifugal study of the proteins of the
high density fraction of egg yolk. Biochim.
et Biophys. Acta, 44: 86-96.

Bernardi, G. and W. H. Cook, 1960b. Separation and
characterization of the two high density lipo-
proteins of egg yolk, a- and B-lipovitellin.
Biochim. et Biophys. Acta, 44: 96-105.

Bernardi, G. and W. H. Cook, 1960c. Molecular weight
and behavior of lipovitellin in urea solution.
Biochim. et Biophys. Acta,-44: 105-109.

Blackwood, J. H. and G. M.-Wishart, 1934. The hydrolysis

of lecitho-vitellin by pepsin and trypsin-kinase.
Biochem. J. 28: 550-558.

103

104

Block, R. J., E. L. Durrum, and G. Zweig, 1955. A Manual
of Paper Chromatography and Paper Electrophoresis.
Academic Press, Inc., New York.

Burley, R. W., 1962. Interactions of chloroform with
a- and B-lipovitellin and other egg yolk constitu-
ents. Can. J. of Biochem. Physiol. 41: 389-395.

Burley, R. W. and W. H. Cook, 1961. Isolation and
composition of avian egg yolk granules and their
constituent a- and B-lipovitellin. Can. J.
Biochem. Physiol. 39: 1295-1307.

Burley, R. W. and W. H. Cook, 1962a. The dissociation of
a-.and B-lipovitellin in aqueous solution. Part I.
The effect of pH, temperature, and other factors.
Can. J. Biochem. Physiol. 40: 363-372.

Burley, R. W. and W. H. Cook, 1962b. The dissociation of
a- and B-lipovitellin in aqueous solution. Part II.
Influence of protein phbsphate groups, sulfhydryl
groups and related factors. Can. J. Biochem.
Physiol. 40: 373-379.

Caldwell, W. E. and G. B. King, 1961. A Brief Course in
Semimicro Qualitative Analysis. 2nd ed. American
Book Company, New York.

Calvery, H. O., 1929. Some chemical investigations of
embryonic metabolism. III. A study of the nitro-
gen distribution in the developing hen's egg by
the modified Van Slyke procedure. J. Biol. Chem.
83: 231-241.

Calvery, H. 0., 1930. Some chemical investigations of
embryonic metabolism. V. The tyrosine, tryptophane,
cystine, cysteine, and uric acid content of the
developing hen's egg. J. Biol. Chem. 87:

691-700.

Calvery, H. O. and A. White, 1931-32. Vitellin of hen's
egg. J. Biol. Chem. 94: 635-639.

Chargaff, E., 1942a. A study of lipOproteins. J. Biol.
Chem. 142: 491-504.

Chargaff, E., 1942b. The formation of the phosphorus
compounds in egg yolk. J. Biol. Chem. 142:
505-512.

105

Clayton, W., 1928. The Theory of Emulsions and Their
Technical Treatment. P. Blakiston's Son and
Company, Inc., Philadelphia.

Clayton, W., 1932. Colloid Aspects of Food Chemistry
and Technology. P. Blakiston's Son and Company,
Inc., Philadelphia.

Clayton, W. and J. F. Morse, 1939. Direct observation of
emulsification. Chem. & Ind. 17: 304-306.

 

Colmer, A. R., 1948. The action of Bacillus cerus and :3
related species on the lecithifi’complex of egg *
yolk. J. Bact. 55: 777-781. 3

Cook, W. H. and W. G. Martin, 1962. Composition and
properties of soluble lipoproteins in relation to
structure. Can. J. Biochem. Physiol. 40:
1273-1285.

w; A sun—bun re
' 0‘

Cook, W. H. and R. A. Wallace, 1965. Dissociation and
sub-unit size of B-lipovitellin. Can. J. Biochem.
43: 661-669.

Dawson, L. E., 1968. Information on egg procurement.
(Private Communication). Michigan State Univer-
sity, East Lansing, Michigan.

Dumas, and Cahours, 1842. Sur 1es matiéres azotées
neutres de 1'organisation. Ann. Chim. et Phys.
6(3): 385-448.

Duncan, D. B., 1957. Multiple range test for correlation
and heteroscadastic means. Biometrics, 13:
164-176.

Evans, R. J. and S. L. Bandemer, 1957. Separation of
egg yolk proteins by paper electrophoresis.
Ag. and Food Chem. 5(11): 868-872.

Evans, R. J. and J. A. Davidson, 1953. The protein
content of fresh and stored shell eggs. Poultry
Sci. 32: 1088-1089.

Evans, R. J., J. A. Davidson, D. H. Bauer, and S. L.
Bandemer, 1958. Distribution of proteins in
fresh and stored shell eggs. Poultry Sci. 37:
81-89.

Feeney, R. and R. M. Hill, 1960. Protein chemistry and
food research. Advances in Food Research, 10:
23-73.

 

106

Fennema, O. and W. D. Powrie, 1964. Fundamentals of low-
temperature food preservation. Advances in Food
Research, 13: 219-347.

Fevold, H. L. and A. Lausten, 1946. Isolation of a new
lipoprotein, lipovitellenin, from egg yolk.
Arch. Biochem. 11: 1-7.

Fisher, W. R. and R. M. Hill, 1964. Structure of lipo-
proteins: covalently bound fatty acids. Sci.
143: 362-363.

Forsythe, R. H., R. W. Kline, F. Pohley, and F. Wheeler,
1953. The relationship between "body“ and
functional performance of frozen egg products.
Poultry Sci. 32: 899.

Gortner, R. A., 1949. Outlines of Biochemistry. John
Wiley and Sons, Inc., New York.

Griswold, R. M., 1962. The Experimental Study of Foods.
Houghton Mifflin Company, Boston.

Hanahan, D. J., 1954. A convenient route to (disteroy1)-
L-a- and B-monostearoyllecithin. J. Biol. Chem.
”211: 321-325.

Harkins, W. D. and H. Zollman, 1926. Interfacial tension
and emulsification. I. The effects of bases, salts,
and acids upon interfacial tension between aqueous
sodium oleate solutions and benzene. J. Am. Chem.
Soc. 48: 69-80.

Hui, S. F. and R. H. Common, 1966. Studies on the
livetins of egg yolk. III. Heterogeneity and
immunoelectrophoretic behavior of beta-livetin.
Can. J. Biochem. 44: 1357-1364.

Jordan, R., B. N. Luginbill, L. E. Dawson, and C. J.
Echterling, 1952. The effect of selected pre-
treatments upon the culinary qualities of eggs
frozen and stored in a home type freezer. I.
Plain cakes and baked custards. Food Research,
17: 1-7.

Jordan, R., G. E. Vail, R. C. Rogler, and W. J. Stadelman,
1960. Functional properties and flavor of eggs
laid by hens on diets containing different fats.
Food Technol. 14: 418-422.

107

Jordan, R., G. E. Vail, J. C. Rogler, and W. J. Stadelman,
1962. Further studies on eggs from hens on diets
differing in fat content. Food Technol. 16:
118-120.

Jordan, R. and E. S. Whitlock, 1955. A note on the effect
of salt (NaCl) upon the apparent viscosity of egg
yolk, egg white, and whole egg magma. Poultry
Sci. 34: 566-571.

Joubert, F. J. and W. H. Cook, 1958a. Separation and
characterization of lipovitellin from hen egg
yolk. Can. J. Biochem. Physiol. 36: 389-398.

Joubert, F. J. and W. H. Cook, 1958b. Preparation and
characterization of phosvitin from hen egg yolk.
Can. J. Biochem. Physiol. 36: 399-408.

Jukes, T. H., 1933. The fractionation of the amino acids
of livetin. J. Biol. Chem. 103: 425-437.

Jukes, T. H. and H. 0. Kay, 1932. The basic amino acids
of livetin. J. Biol. Chem. 98: 783-788.

Kay, H. D. and P. D. Marshall, 1928. The second protein
(livetin) Of egg yolk. Biochem. J. 22: ~
1264-1269.

Kilgore, L. B., 1935. Egg yolk "makes" mayonnaise.
Food Inds. 7: 229.

King, A., 1941. Some factors governing the stability of
oil-in-water emulsions. Trans. Faraday Soc. 37:
168-180.

Lea, C. H. and J. C. Hawke, 1952. Lipovitellin 2. The
influence of water on the stability of lipovitellin
and the effects of freezing and of drying.

Biochem. J. 52: 105-114.

LeClerc, J. A. and L. H. Bailey, 1940. Fresh, frozen,
and dried eggs and egg products (their uses in
baking and for other purposes). Cereal Chem. 17:
279-311.

Levene, P. A. and T. Mori, 1929. The carbohydrate group
of ovomucoid. J. Biol. Chem. 84: 49-63.

Levene, P. A. and A. Rothen, 1929. On the molecular
size of the carbohydrate obtained from egg
proteins. J. Biol. Chem. 83: 63-68.

108

Lopez, A. C., R. Fellers, and W. D. Powrie, 1954. Some
factors affecting gelation of frozen egg yolk.
J. Milk and Food Technol. 17: 334-339.

Lopez, A. C., C. R. Fellers, and W. D. Powrie, 1955.
Enzymic inhibition of gelation of frozen egg,
yolk. J. Milk and Food Technol. 18: 77-80.

Lovelock, J. E., 1957. The denaturation of lipid-protein
complexes as a cause of damage by freezing. Proc.
Roy. Soc. (London), 147: 427-433.

Lowe, B., 1955. Experimental Cookery. 4th ed. John
Wiley and Sons, Inc., New York.

Marion, W. M., 1958. An investigation of the gelation
of frozen egg yolk after thawing. Ph.D. Thesis.
Purdue University.

If. - *-¢—.A—~_.- 2...»; sq‘ ._—_-..__._._.'_q
a q ‘ < I.

McCully, K. A., W. A. Maw, and R. H. Common, 1959. Zone
electrophoresis of the proteins of fowl's serum
and egg yolk. Can. J. Biochem. Physiol. 37:
1457-1468.

McIndoe, W. M., 1959a. A lipophosphoprotein complex in
hen plasma associated with yolk production.
Biochem. J. 72: 153-159.

McIndoe, W. M., 1959b. Lipoprotein of high lipid content
from egg yolk. Biochem. J. 73: 45P.

McNally, E. H., 1959. Observations on the dispersion and
precipitation of egg yolk. Poultry Sci. 38:
1227-1228.

Martin, W. J. and W. H. Cook, 1958. Preparation and
molecular weight of y-livetin from egg yolk.
Can. J. Biochem. Physiol. 36: 153-160.

Martin, W. J., K. J. Turner, and W. H. Cook, 1959.
Macromolecular properties of vitellenin from egg
yolk and its parent complex with lipids. Can. J.
Biochem. Physiol. 37: 1194-1207.

Martin, W. G., J. E. Vandegaer, and W. H. Cook, 1957.
Fractionation of livetin and the molecular weights
of the alpha and beta components. Can. J. Biochem.
Physiol. 35: 241-250.

109

Mattil, K. F., F. A. Norris, A. J. Stirton, and D. Swern,
1964. Bailey's Industrial Oil and Fat Products.
3rd ed. Interscience Publishers, a division of
John Wiley and Sons, Inc., New York.

Mecham, D. K. and H. S. Olcott, 1948. An egg protein
containing 10% phosphorus. Fed. Proc. 7: 173.

Mecham, D. K. and H. S. Olcott, 1949. Phosvitin, the
principal phosphoprotein of egg yolk. J. Am. Chem.
Soc. 71: 3670.

Meyer, D. D. and M. Woodburn, 1965. Gelation of frozen
defrosted egg yolk as affected by selected
additives--viscosity and electrophoretic findings.
Poultry Sci. 44: 437-446.

Miller, C. and A. R. Winter, 1950. The functional
properties and bacterial content of pasturized
and frozen whole eggs. Poultry Sci. 29: 88-97.

Miller, C. and A. R. Winter, 1951. Pasturized frozen
whole egg and yolk for mayonnaise production.
Food Research, 16(1): 43-49.

Mok, C. C. and R. H. Common, 1964. Studies on the
livetins of hen's egg yolk. I. Identification of
paper-electrophoretic and immunoelectrophoretic
livetin fractions with serum protein antigens by
immunoelectrOphoretic analysis. Can. J. Biochem.
42: 871-881.

Moran, T., 1925. Effect of low temperature on hen's
eggs. Proc. Roy. Soc. (London), B98: 436.

Osborne, T. B. and D. B. Jones, 1909. Hydrolysis of
vitellin from the hen's egg. Am. J. Physiol. 24:
153-160.

Osborne, T. B. and . . Campbell, 1900. The proteoids of
egg yolk. J. Am. Chem. Soc. 22: 413.

Ostrander, J. G., R. Jordan, W. J. Stadelman, and J. C.
Rogler, 1960. The ether extract of yolks of
eggs from hens on feed containing different fats.
Poultry Sci. 39: 746-750.

Payawal, S. R., B. Lowe, and G. F. Stewert, 1946.
Pasturization of liquid-egg products. II. Effect
of heat treatment on appearance and viscosity.
Food Research, 11: 246-260.

110

Pearce, J. A. and C. G. Lavers, 1949. Liquid and frozen
egg. V. Viscosity, baking quality, and other
measurements of frozen egg products. Can. J.
Res. 27F: 231-240.

Plimmer, R. H. A., 1908. The proteins of egg yolk.
J. Chem. Soc. 93: 1500-1505.

Powrie, W. D., H. Little, and A. C. Lopez, 1963.
Gelation of egg yolk. J. Food Sci. 28(1):
38-46.

Privett, O. S., M. L. Blank, and J. A. Schmit, 1962.
Studies on the composition of egg lipid.
J. Food Sci. 27: 463-468.

Radomski, M. W. and W. H. Cook, 1964. Fractionation and
dissociation of the avian lipovitellins and their
interactions with phosvitin. Can. J. Biochem.
42: 395-406.

Rhodes, D. N. and C. H. Lea, 1956. Composition of egg
phospholipids. Nature, Lond. 177: 1129-1130.

Rhodes, D. N. and C. H. Lea, 1957. Phospholipids 4.
On the composition of hen's egg phospholipids.
Biochem. J. 65: 526-533.

Rhodes, D. N. and C. H. Lea, 1958. On the composition
of the phospholipids of cows' milk. J. Dairy
Research, 25: 60-69.

Rolfes, T., P. Clements, and A. R. Winter, 1955. The
physical and functional properties of 1ypholized
whole egg, yolk, and white. Food Technol. 9:
569-572.

Romanoff, A. L. and A. J. Romanoff, 1949. The Avian Egg.
John Wiley and Sons, Inc., New York.

Saari, A., W. D. Powrie, and O. Fennema, 1964a.
Isolation and characterization of low-density
lipoproteins in native egg yolk plasma. J. Food
Sci. 29: 307-315.

Saari, A., W. D. Powrie, and O. Fennema, 1964b.
Influence of freezing egg yolk plasma on the
properties of low-density lipoprotein. J. Food
Sci. 29: 762-768.

111

Saito, Z., W. G. Martin, and W. H. Cook, 1965. Changes
in the major macromolecular fractions of egg yolk
during embryogenesis. Can. J. Biochem. 43:
1755-1770.

Scheraga, H. A., 1961. Protein Structure. Academic
Press, New York.

Schjeide, O. A. and M. R. Uhrist, 1960. Proteins
induced in plasma by estrogens. Nature, 188:
291-293.

Schmidt, G., M. J. Bessman, M. D. Hickey, and S. J.
Thannhauser, 1956. The concentration of some
constituents of egg yolk in its soluble phase.
J. Biol. Chem. 223: 1027-1029.

Shepard, C. C. and G. A. Hottle, 1949. Studies of the
composition of the livetin fraction of the yolk
of hen's eggs with the use of electrophoretic
analysis. J. Biol. Chem. 179: 349-357.

Smith, D. B. and K. J. Turner, 1958. N-terminal amino
acids of the proteins of the floating fraction
of egg yolk. Can. J. Biochem. Physiol. 36:
951-952.

Snell, H. M., A. G. Olsen, and R. E. Kremers, 1935.
Lecithoprotein--the emulsifying ingredient in
egg yolk. Ind. Eng. Chem. 27: 1222-1223.

Sugano, H., 1957. Studies on egg yolk proteins. J.
Biochem. 44: 205-215.

Sugano, H., 1958. Studies on egg yolk proteins. III.
Preparation of two major lipoproteins of the
egg yolk, a- and B-lipovitellin. J. Biochem. 45:
393-401.

Sugano, H., 1959. Studies on egg yolk proteins. IV.
Electrophoretic and ultracentrifugal investiga-
tions on the homogeneities and some properties
of a- and B-lipovitellin. J. Biochem. 46:
417-424.

Sugano, H. and I. Watanabe, 1961. Isolation and some
properties of native lipoproteins in egg yolk.
J. Biochem. 50: 473-479.

112

Sutheim, G. M., 1947. Introduction to Emulsions.
Chemical Publishing Company, Inc., Brooklyn,
New York.

Sweetman, M. D. and I. MacKellar, 1959. Food Selection
and Preparation. 4th ed. John Wiley and Sons,
New York.

Thomas, A. W. and M. I. Bailey, 1933. Gelation of egg
magma. Ind. Eng. Chem. 25: 669-674.

Tongur, V. and E. Ragosin, 1944. Avoidance of gelation
in egg mixtures. Chem. Abstracts, 38: 174.

Triebold, H. O. and L. W. Aurand, 1963. Food Composi-
tion and Analysis. D. VanNostrand Company, Inc.,
Princeton, New Jersey.

Turner, K. J. and W. H. Cook, 1958. Molecular weight
and physical properties of a lipoprotein from the
floating fraction of egg yolk. Can. J. Biochem.
Physiol. 36: 937-949.

Urbain, O. M. and J. N. Miller, 1930. Relative merits
of sucrose, dextrose, and levulose as used in the
preservation of eggs by freezing. Ind. Eng. Chem.
22: 355-356.

Vandegaer, J. E., M. E. Reichman, and W. H. Cook, 1956.
Preparation and molecular weight of lipovitellin
from egg yolk. Arch. Biochem. and Biophys. 62:
328-337.

Vincent, R., W. D. Powrie, and O. Fennema, 1966. Surface
activity of yolk, plasma, and dispersions of yolk
fractions. J. Food Sci. 31(5): 643-648.

Walker, N. E., 1967. Growth and development of chick
embryos nourished by fractions of yolk. J.
Nutrition, 92: 111-117.

Wallace, R. A., D. W. Jared, and A. Z. Eisen, 1966. A
general method and purification of phosvitin from
vertebrate eggs. Can. J. Biochem. 44: 1647-1655.

Watt, B. K. and A. L. Merrill, 1963. Composition of
Foods--Agricu1ture Handbook No. 8. Consumer and
Food Economics Research Division, Agricultural
Research Service, U.S. Department of Agriculture,
Washington, D.C.

113

Weinman, E. O., 1956. Lipide-protein complex in egg
yolk. Fed. Proc. 15: 381.

White, A., P. Handler, and E. L. Smith, 1959. Principles
of Biochemistry. 3rd ed. McGraw-Hill Book
Company, New York.

Wittcoff, H., 1951. The Phosphatides. Book Division,
Reinhold Publishing Corporation, New York.

Yeadon, D. A., L. A. Goldblatt, and A. M. Altschul, 1958.
Lecithin in oil-in-water emulsions. J. Am. Oil
Chem. Soc. 35: 435-438.

Ziemba, J. V., 1955. Five egg-quality properties
important to food processers. Food Eng. 27:
85, 191-192.

APPENDIX

114

115

Diet of Hens Producing Eggs for Research

Tables 19 and 20 give the composition and

analysis of the diet fed to the hens producing the eggs

for this research.

TABLE 19. Composition of ration identified as LB-63

(Dawson, 1968)

 

Ingredient

Lbs. per ton

 

Ground yellow corn

Soybean meal, dehulled, 50% protein
Alfalfa meal, dehyd., 17% protein
Meat and bone scraps, 50% protein

Fish meal, Vitaproil (with solubles
added, 55% protein)

Dried whey

Ground limestone

Dicalcium phosphate (24% Ca, 18.5% P)
Salt, iodized

Vitamin trace mineral premixa
Choline chloride 25%

Zinc oxide (73-80% zinc)

Animal fat

Oyster shells

1320.00
310.00
60.00

50.00

60.00
40.00
100.00
20.00
6.00

5.00 to 10.00

 

aDawes Vitefac No. 1 (10 lbs. per ton) or Nopco

M—4 (5 lbs. per ton) or equivalent product.

116

 

 

 

 

 

 

TABLE 20. Chemical analyses of ration identified as
LB-63 (Dawson, 1968)
Component %
Phosphorus 0.76
Calcium 2.73
Crude fiber 3.28
Ether extract 4.82
Protein 19.00
Water 7.72
TABLE 21. Percentage of total protein contributed by
each of the three fractions
Lipovitellin Lipovitellenin Livetin
Rep. Fresh Frozen Fresh Frozen Fresh Frozen
% % % % % %
1 55.43 55.52 34.94 34.93 9.63 9.55
2 52.62 53.14 32.76 32.36 14.62 14.50
3 54.65 53.58 33.14 34.14 12.21 12.28
4 51.13 51.46 36.26 35.87 12.61 12.67
5 49.74 50.74 39.90 39.31 10.37 9.95
6 49.89 50.46 37.73 37.19 12.38 12.35
Mean 52.36 35.70 11.07

 

117

 

 

 

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TABLE 28. Replicate averages for emulsion drainage at four time intervals from
emulsions prepared with no stabilizer (O) and with fresh lipovitellin
(A), lipovitellenin (B), and livetin (C) combinations as stabilizers

 

Average drainage in ml at:

 

 

Rep. Fraction
30 min. 60 min. 90 min. 120 min.
0 10.55 10.55 10.60 10.60
A 6.85 7.35 7.70 7 90
B 0.65 2.40 2.50 2 95
1 C 7.55 8.10 8.35 8 60
AB 0.05 0.18 0 40 0 65
BC 0.20 0.85 l 25 1.50
AC 5.20 6.50 7.00 7.20
ABC 0.10 0.30 0.60 0.85
O 10.65 10.85 10 95 10.95
A 7.55 8.00 8 30 8.50
B 0.35 1.15 1 40 1.75
2 C 6.90 7.90 8 20 8 45
AB 0.10 0.25 0.45 1 00
BC 0.10 0.30 0.70 0 90
AC 2.35 3.90 4.45 5 05
ABC 0.10 0.15 0.20 0 30
O 11.10 11.15 11.20 11 20
A 6.95 7.30 7.65 7 95
B 1.50 3.30 3.90 4 30
3 C 7.60 8.25 8 40 8 70
AB 0.15 0.60 l 00 1 40
BC 0.40 1.00 1 65 2 10
AC 4.45 5.55 6.05 6 40
ABC 0.15 0.25 0.65 1 00
O 11.50 11.60 11.65 11 70
A 6.05 6.80 7.30 7 50
B 0.75 1.45 2.05 2 4O
4 C 7.90 8.40 8.60 8.85
AB 0.15 0.40 0.80 1.00
BC 0.20 0.50 0.85 1.05
AC 5.00 6.20 6.60 6.90
ABC 0.10 0.20 0.40 0 60
O 10.95 11.00 11.05 11.15
A 5.50 6.25 6.65 6.95
B 0.30 0.55 1.75 1.95
5 C 7.80 8.20 8.40 8.50
AB 0.10 0.20 0.50 0.85
BC 0.15 0.75 1.55 2.05
AC 5.10 6.30 6.60 6.90
ABC 0.00 0.13 0.25 0.50
O 10.10 10.35 10.40 10 40
A 6.60 7.25 7.45 7.65
B 0.45 1.40 2.20 2 75
6 C 8.30 8.90 9.10 9 25
AB 0.10 ' 0.40 0.90 1 30
BC 0.15 0.55 0 80 l 20
AC 6.00 6.80 7.20 7 40
ABC 0.05 0.20 0.35 0 45

 

124

TABLE 29. Replicate averages for emulsion drainage at four time intervals from
emulsions prepared with no stabilizer (O) and with frozen lipovitellin
(A), lipovitellenin (B), and livetin (C) combinations as stabilizers

 

Average drainage in ml at:

 

 

Rep. Fraction
30 min. 60 min. 90 min. 120 min.
0 9.75 9.95 10.25 10.25
A 6.60 7.25 7.65 7.95
B 0.60 1.70 2.60 3.00
l C 8.10 8.50 8.70 8.85
AB 0.25 0.60 1.05 1.25
BC 0.30 0.75 0.95 1.30
AC 4.70 5.70 6.25 6.65
ABC 0.15 0.35 0.60 0.85
O 9.80 9.95 10.05 10.05
A 6.45 7.15 7.45 7.80
B 1.50 2.90 3.70 4.00
2 C 7.20 8.00 8.35 8.55
AB 0.65 1.25 1.90 2.25
BC 0.40 0.90 1.30 1.80
ABC 0.45 1.00 1.25 1.55
O 10.80 10.95 10.95 11.00
A 7.35 7.60 8.35 9.10
B 0.80 1.90 2.60 3.00
3 C 7.35 8.15 8.55 8.65
AB 0.20 0.50 0.75 0.95
BC 0.40 1.05 1.45 2.05
AC ‘5.60 6.40 6.85 7.15
ABC 0.15 0.40 0.55 0.65
O 10.60 10.65 10.65 10.65
A 6.25 7.05 7.30 7.60
B 1.10 2.55 3.45 3.95
4 C 6.40 7.30 7.70 ‘ 8.00
AB 0.35 0.70 1.15 1.40
BC 0.50 1.05 1.70 2.15
AC 5.50 6.90 6.90 7.10
ABC 0.10 0.30 0.40 0.60
O 10.40 10.50 10.60 10.70
A 7.00 7.50 7.70 8.00
B 0.70 1.70 2.25 2.85
5 C 8.45 8.80 9.00 9.00
AB 0.15 0.50 0.65 0.90
BC 0.40 0.85 1.35 1.80
AC 6.95 7.45 7.85 8.05
ABC 0.13 0.30 0.40 0.50
O 9.45 9.55 9.55 9.55
A 6.70 7.35 7.65 7.90
B 0.90 1.55 2.40 2.85
6 C 8.25 8.85 9.10 9.20
AB 0.15 0.30 0.45 0.55
BC 0.25 0.60 1.05 1.35
AC 5.90 6.85 7.15 7.50
ABC 0.15 0.35 0.60 0.80

 

125

TABLE 30. Main effect and significant interaction means from the analysis of
variance for determination of the effects of fractions, replication,
storage treatment, and drainage time on the stability of oil-in—water
emulsions

 

Factor and Categorya

 

 

Rep. Lipovitellin Lipovitellenin Livetin Storage Time Mean Drainage (m1)
1 0 0 0 0 0 4.52
2 0 0 0 0 0 4.42
3 0 0 0 0 0 4.77
4 0 0 0 0 0 4.59
5 O 0 0 0 O 4.57
6 0 0 0 0 0 4.55
0 0 0 0 Fresh 0 4.49
0 0 0 0 Frozen 0 4.65
0 0 0 0 0 30' 3.91
0 0 0 0 0 60' 4.47
0 0 0 0 0 90' 4.81
0 0 0 0 0 120' 5.08
0 - 0 0 O 0 5.48
0 + 0 O 0 0 3.66
0 0 - 0 0 0 8.11
0 0 + 0 0 0 1.03
0 0 0 - 0 0 5.17
0 0 0 0 0 3.97
0 - - 0 0 0 9.45
0 - + 0 0 0 1.51
0 + - 0 0 0 6.77
0 + + 0 0 0 0.54
0 0 - - 0 O 8.97
0 0 - + 0 0 7.25
0 0 + - 0 0 1.36
0 0 + + 0 0 0.70
0 - 0 - O O 6.33
O - 0 + O 0 4.63
0 + 0 - 0 O 4.00
0 + 0 + 0 0 3.31
0 - - 0 0 30' 9.06
0 - - 0 0 60' 9.43
0 - - 0 0 90' 9.60
0 - - 0 0 120' 9.70
0 - + 0 O 30' 0.54
0 - + 0 O 60' 1.32
0 - + 0 0 90' 1.89
0 - + 0 0 120' 2.29
0 + - 0 0 30' 5.89
0 + - 0 0 60' 6.72
0 + - 0 0 90' 7.09
0 + - 0 O 120' 7.40
0 + + o 0 30' 0.17
0 + + 0 0 60' 0.41
0 + + 0 O 90' 0.68
O + + 0 0 120' 0.92
O 0 - - 0 30' 8.56
0 0 - - 0 60' 8.91
0 0 - - O 90' 9.13
0 0 - - 0 120' 9.29
0 0 - + 0 30' 6.39
0 0 - + 0 60' 7.24
0 0 - + 0 90' 7.56
0 0 - + 0 120' 7.81
0 0 + - 0 30' 0.50
0 0 + ‘ 0 60' 1018

 

126

TABLE 30. Continued.

 

Factor and Categorya

 

 

Rep. Lipovitellin Lipovitellenin Livetin Storage Time Mean Drainage (ml)
0 0 + - 0 90' 1.70
0 0 + - 0 120' 2.05
0 0 + + 0 30' 0.21
0 0 + + O 60' 0.55
0 0 + + 0 90' 0.87
0 0 + + 0 120' 1.16
0 0 - 0 30' 5.64
0 - 0 - 0 60' 6.23
0 0 - 0 90' 6.61
0 - 0 - 0 120' 6.83
0 0 + 0 30' 3.97
0 0 + 0 60' 4.52
0 - 0 + 0 90' 4.88
0 - 0 + O 120' 5.16
0 + 0 - 0 30' 3.43
0 + 0 - 0 60' 3.86
0 + 0 - 0 90' 4.21
0 + 0 - 0 120' 4.51
0 + 0 + 0 30' 2.63
O + 0 + 0 60' 3.26
0 + 0 + 0 90' 3.55
0 + 0 + 0 120' 3.81
0 - - 0 30' 10.47
0 - - - 0 60' 10.59
0 - 0 90' 10.66
0 - - - 0 120’ 10.68
0 - + 0 30' 7.65
0 - - + 0 60' 8.28
0 - - + 0 90' 8.54
0 - - + 0 120' 8.72
0 - + - 0 30' 0.80
0 - + - 0 60' 1.88
0 - + - 0 90' 2.57
0 - + - 0 120' 2.98
0 + + 0 30' 0.29
0 - + + 0 60' 0.76
0 - + + 0 90' -1.22
0 - + + 0 120' 1.60
0 + - - 0 30' 6.65
0 + - - 0 60' 7.24
0 + - - 0 90' 7.60
0 + - - 0 120' 7.90
0 + - + 0 30' 5.12
0 + - + 0 60' 6.20
0 + - + 0 90' 6.58
0 + - + 0 120' 6.90
0 + + - 0 30' 0.20
0 + + - 0 60' 0.49
0 + + - O 90' 0.83
0 + + - 0 120' 1.13
0 + + + o 30' 0.14
0 + + + 0 60' 0.33
0 + + + 0 90' 0.52
0 + + + 0 120' 0.72

 

8The following are the factors and an identification of the categories of

each of the factors:

Rep.--replications one thru six.
Lipovitellin--absence (-) or presence (+).
Lipovitellenin--absence (-) or presence (+).
Livetin--absence (-) or presence (+).
Storage-~fresh or frozen.

Time--Reaaing at 30, 60, 90, or 120 minutes.

0 denotes that these factors were not separated when the mean was determined.

127

 

 

 

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I128

TABLE 32. Calculated average migration distances, mobile and non-mobile
protein fractions of fresh and frozen native yolk as
separated by paper electrophoresis

 

 

 

 

 

 

 

 

 

 

Storage a Migrationb MobilityC

Treatment Rep. Left Right Non-Mobile Mobile

mm mm 8 %

1 a 10 46 35.2 64.8

b 9 43 23.9 76.1

2 a 10 47 31.3 68.7

b 10 51 25.1 74.9

3 a 4 47 33.2 66.8

b 7 56 37.4 62.6

Fresh

4 a 9 44 44.7 55.3

b 11 46 23.8 76.2

5 a 15 49 45.6 54.4

b 13 53 34.8 65.2

6 a 13 55 39.9 60.1

b 14 50 44.7 55.3

Mean 10.0 48.9 35.0 65.0
Range 4-15 43-56 23.8-45.6 54.4-76.2

1 a 10 44 38.8 61.2

b 11 46 53.9 46.1

2 a 6' 54 55.8 44.2

b 6 49 55.3 44.7

3 a 8 52 55.1 44.9

b 9 46 57.8 42.2

Frozen

4 a 8 54 58.3 41.7

b 8 52 52.8 47.2

5 a ll 54 58.4 41.6

b 10 52 51.8 48.2

6 a 8 38 53.1 46.9

b 7 39 24.0 76.0

Mean 8.5 48.3 51.3 48.7
Range 6-11 38-54 24.0-58.4 41.6-76.0

a

Two strips (a and b) run for each replication.
bMigration from origin.
cNon-Mobile includes any mobility to the left.

.129

TABLE 33. Calculated average migration distances, mobile and non-mobile
protein fractions of fresh and frozen lipovitellin as
separated by paper electrophoresis

 

 

 

 

 

 

 

 

 

 

Storage a Migrationb ‘ .1 Mobilityc
Treatment Rep. Left Right Non-Mobile Mobile
mm mm .8 8
1 a 9 27 67.1 32.9
b 10 30 80.5 19.5
2 a 6 51 ' 77.3 22.7
b 5 44 69.7 30.3
3 a 7 47 57.3 42.7
b 8 58 62.4. 37.6
Fresh
4 a 9 41 72.7 27.3
b 10 45 73.1 26.9
5 a 7 40 79.0 21.0
b 10 40 79.6 20.4
6 a 12 55 71.2 28.8
b 9 53 65.7 34.3
Mean 8.5 44.3 71.3 28.7
Range 5-12 27-58 62.4-80.5 19.5-37.6
1 a 8 51 69.2 30.8
b 11 29 83.3 16.7
2 a 5' 51 80.3 19.7
b 6 53 79.3 20.7
3 a 5 50 54.6 45.4
b 4 62 51.2 48.8
Frozen
4 a 6 33 ' 90.1 9.9
b 9 37 88.0 12.0
5 a 6 51 ' 50.8 49.2
b 4 53 52.9 47.1
6 a 10 45 \ 90.8 9.2
b 32 46 88.9 11.1
Mean 8.8 46.8 ' 73.3 26.7
Range 4-32 29-62 50.8-90.8 9.2-49.2
a

Two strips (a and b) run for each replication.
bMigration from origin.
cNon-Mobile includes any mobility to the left.

.130

 

 

 

 

 

 

 

 

 

TABLE 34. Calculated average migration distances, mobile and non-mobile
protein fractions of fresh and frozen lipovitellenin as
separated by paper electrophoresis

Storage a Migrationb Mobilityc

Treatment Rep. Left Right Non-Mobile Mobile

mm mm 8 8
1 a 5 32 14.9 85.1
b 4 33 15.5 84.5
2 a 5 35 6.0 94.0
b 7 44 8.1 91.9
3 a 5 39 6.7 93.3
b 4 42 2.5 97.5
Fresh
4 a 5 49 6.5 93.5
b 4 52 8.3 91.7
5 a 13 33 20.3 79.7
b 8 43 13.5 86.5
6 a 9 23 15.4 84.6
b 11 26 10.5 89.5
Mean 6.7 37.6 10.7 89.3
Range 4-13 23-52 2.5-20.3 79.7-97.5
1 a 8 20 80.0 20.0
b 7 19 70.8 29.2
2 a 9 24 79.2 20.8
b 5 23 70.4 29.6
3 a 27 70.8 29.2
b 21 71.4 28.6
Frozen
4 a 6 30 69.2 30.8
b 7 26 58.8 41.2
5 a 7 27 59.4 40.6
b 7 26 54.8 45.2
6 a 28 24 75.9 24.1
b 6 25 48.5 51.5
Mean 8.5 24.3 67.4 32.6
Range 5-28 19-30 48.5-80.0 20.0—51.5

 

aTwo strips (a and b) run for each replication.
bMigration from origin.
cNon-Mobile includes any mobility to the left.

131

 

 

 

 

 

 

 

 

 

TABLE 35. Calculated average migration distances, mobile and non-mobile
protein fractions of fresh and frozen livetin as separated by
paper electrophoresis

. . b . . c
Storage a Migration Mobility
Treatment Rep. Left Right Non-Mobile Mobile
mm mm % %
1 a 4 50 5.0 95.0
b 4 50 4.3 95.7
2 a 3 56 14.3 85.7
b 2 54 0.0 100.0
3 a 2 58 0.0 100.0
b 5 60 0.0 100.0
Fresh
4 a 5 47 0.0 100.0
b 5 51 0. 100.0
5 a 4 56 0.0 100.0
b 7 58 8.3 91.7
6 a 8 61 8.7 91.3
b 7 57 9.1 90.9
Mean 4.7 54.8 4.1 95.9
Range 2-8 47-61 0.0-14.3 85.7-100.0
1 a 3 51 33.3 66.7
b 6 54 13.6 86.4
2 a 4 52 16.7 83.3
b 4 56 15.4 84.6
3 a 3 59 13.3 86.7
b 4 59 7.4 92.6
Frozen
4 a 3 62 0.0 100.0
b 2 64 0.0 100.0
5 a 2 53 16.7 83.3
b 4 67 12.5 87.5
6 a 4 48 12.1 87.9
b 3 45 7.5 92.5
Mean 3.5 55.8 12.4 87.6
Range 2-6 45-67 0.0-33.3 66.7-100.0

 

aTwo stripe (a and b) run for each replication.
bMigration from origin.
cNon-Mobile includes any mobility to the left.