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THESIE’

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

\‘ LIBRAIQ y

Michigan State
”musty

 

This is to certify that the
thesis entitled
FACTORS AFFECTING THE FUNCTIONAL
PROPERTIES OF FISH MUSCLE PROTEINS
presented by

Ab de lb ary Ahmed Dawood

has been accepted towards fulfillment
of the requirements for

 

Ph . D . degree in Wee and
Human Nutrition

zfi.

Major professor

    
 

Date January 23, 1.98

0-7639

 

 

 

 

OVERDUE FINES:
25¢ per day per item

RETURNING LIBRARY MATERIALS:

Place in book return to remove
charge from circulation records

 

FACTORS AFFECTING THE FUNCTIONAL PROPERTIES

OF FISH MUSCLE PROTEINS

BY

Abdelbary Ahmed Dawood

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1980

ABSTRACT

FACTORS AFFECTING THE FUNCTIONAL PROPERTIES
OF FISH MUSCLE PROTEINS

BY

Abdelbary Ahmed Dawood

Freshwater suckers (Catostomidae family), an
underutilized kind of fish, were obtained from Lake Huron
(Saginaw Bay, Michigan) to study the possibility of
developing new products utilizing its flesh. Suckers
were mechanically deboned, then blast frozen and stored
at -29°C. Minced sucker was analyzed for fat, protein,
and moisture content. The effects of storage at 3°C for
7 days and -29°C for 90 days on the solubility of myo—
fibrillar and sarcoplasmic proteins as well as nonprotein
nitrogen of sucker flesh were also studied.

Experiments were designed to add sodium chloride,
sodium tripolyphosphate and soy protein isolate in vary—
ing concentrations and combinations to study the influence
on protein solubility, pH, percent swelling and gel
formation of frozen mechanically deboned sucker flesh.
The functional properties of fish sausages and canned

minced fish were evaluated in terms of water holding

Abdelbary Ahmed Dawood

capacity using a centrifuge technique, texture using an
Instron Universal Testing Machine and cooking loss.

Results showed that suckers have a lower caloric
content than do red meats or poultry and, therefore, are
an ideal source of animal protein for use in low calorie
diets. Solubility of myofibrillar proteins decreased
due to either refrigeration (3°C) or freezer (-29°C)
storage, while sarcoplasmic proteins and nonprotein
nitrogen were essentially unchanged by both storage
methods as compared to that of prerigor muscle.

Results also indicated that both sodium chloride
and sodium tripolyphosphate influence the solubility of
fish muscle proteins. Total extracted protein and salt
soluble protein increased with the addition of either
sodium chloride or sodium tripolyphosphate, while the
percentage of water soluble protein and nonprotein nitro—
gen decreased. Myosin heavy chain was solubilized in
the presence of 3.0% and 3.6% sodium chloride and also
with the addition of 0.45% sodium tripolyphosphate. The
highest solubility of myosin heavy chain, that obtained
from samples containing 0.45% sodium tripolyphosphate
alone, was directly related to the high pH (7.8) of
extraction solution. Solubility did not seem to be
largely related to the ionic strength, since the ionic

strength of 0.45% sodium tripolyphosphate is insufficient

Abdelbary Ahmed Dawood

to solubilize myosin. Apparently the 2% and 4% soy pro-
tein isolate added did not completely solubilize, since
none of the protein fractions increased by 2% or 4% over
a control containing no soy protein isolate.

Results revealed that changes in pH, swelling,
and gel formation of sucker flesh were largely due to the
addition of sodium tripolyphosphate and sodium chloride.
Adding sodium tripolyphosphate increased pH and swelling
and also improved gel formation, while those values
decreased by the addition of sodium chloride. Adding soy
protein isolate did not change either pH or gel formation,
but did cause increased swelling. The functionality tests
indicated samples that have the highest pH also have the
highest values of solubility of myosin, greatest swelling,
and the best gel forming ability.

In comparing the influences of sodium chloride
and sodium tripolyphosphate, sodium chloride increased
protein solubility, while sodium tripolyphosphate
appeared to be more beneficial. For example, it increases
myosin solubility, pH, swelling, and gel forming ability.

Comparing the water holding capacity, texture, and
cooking loss of either sausage or canned product treat-
ments, both product types showed low binding characteris-
tics. However, adding corn meal and soy protein isolate
in combination with sodium chloride and sodium tripoly-

phosphate improved their water holding capacity, texture

Abdelbary Ahmed Dawood

and cook yield. It was concluded that sodium tripolyphos-
phate, corn meal, and fat should be used in manufacturing

minced sucker products.

DEDICATION

To Ahmed Aly Dawood, my father.

His memory helped me complete this

study and will be with me forever.

ii

ACKNOWLEDGMENTS

The author expresses sincere appreciation and
gratitude to Dr. James Price for his advice, ideas, and
suggestions throughout this study. His assistance in
preparing this dissertation was particularly valuable.

In addition, because of his guidance and advice
in the initial stages of this study, I would like to
acknowledge Dr. Estes Reynolds.

I am proud to have had as a guidance committee
Dr. Lawrence Dawson, Dr. Albert Pearson, and Dr. Niles
Kevern, a fine group of scientists and professionals
of national repute, who freely gave me their time and
interest.

Appreciation is also expressed to my excellent
friends Cherine LeBlanc, Dietrich Schaaf, Don Mulvaney
and my wife Fatma for their personal help. Fatma espe-
cially gave me a tremendous amount of love and support.

The greatest appreciation, which cannot be
expressed in words alone, goes to my parents and to my
uncle, Dr. Ibrahim Abdalla. I am most appreciative to
the College of Agriculture, AlMinya University, Egypt, for
providing the opportunity to do this study and their con-
tinuing financial support throughout this undertaking.

iii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . .

LIST OF APPENDICES . . . . . . . .

INTRODUCTION . . . . . . . . . . . .
LITERATURE REVIEW . . . . .

The Structure and Composition of Fish

Muscle . . .
Functional Classification of Muscle Pro—
teins . .

The Extractabiltiy of Muscle Proteins
Effect of Low Temperature Storage on

Protein Extractability . .
Effect of Sodium Chloride and Phosphates
on Protein Extractability . .
Effect of Heat on the Extractability of
Muscle Proteins . . . .
Functional Properties of Muscle Proteins
Swelling . . . . . .
Binding Properties and Gel- -Forming
Ability . . . . .

Water Holding Capacity . . . .
Texture . . . . . .
Functional Properties of Soy Protein Iso-

late in Meat Systems . . . . . . .
MATERIALS AND METHODS‘ . . . . . .
Materials . . . . . . . . . .
Fish . . . . . . . . .
Additives and Spices . . .
Preparation of Mechanically Deboned
Fish Flesh . . . . . . .

iv

Page

vi

xiii

I2
20
23
26
26
28
35
44
49
57
57
57
57

58

Page

Methods of Analysis . . . . . . . . 59
Protein Fractionation . . . . 59
Extraction of Soluble Components from

Mechanically Deboned Frozen Fish . . 6O
Fractionation of Soluble Components from
Mechanically Deboned Frozen Fish . . 61
Sodium Dodecylsulphate, Polyacrylamide
Gel Electrophoresis . . . . . . 65
Total Nitrogen . . . . . . . . . 68
Kjeldahl Method . . . . . . . . 68
Moisture Determination . . . . . . 68
Ether Extraction . . . . . . . . 69
Gel—Forming Measurement . . . . . . 69
Swelling Measurement . . . . . . . 70
pH Measurement . . . . . . . . . 71
Chemicals . . . . . . . . 71
Smokehouse Shrinkage . . . . . . . 71
Baking Loss . . . . . . . 71
Measurement of Water Holding Capacity . 71
Internal Texture Measurement . . . . 72
Processing Procedures . . . . . . 73
Statistical Analysis . . . . . . . 77
RESULTS AND DISCUSSION . . . . . . . . . 78

Yield of Mechanically Deboned Fish and Its
Chemical Composition . . . . . . . 78
Effects of Refrigeration and Freezing
Storage on the Extractability of Fish
Muscle Proteins . . . . . . . . 79
Effects of Additives on the Protein
Extractability of Frozen Mechanically

Deboned Fish . . . . . . . . . 85
Total Extracted Protein . . . . . . 85
Salt Soluble Protein . . . . . . . 94
Water Soluble Protein . . . . . . 97
Nonprotein Nitrogen . . . . . . . 104
Myosin Heavy Chain . . . . 106
Effects of Additives on pH of Frozen
Mechanically Deboned Fish . . . . 123
Effects of Additives on Percent Swelling
of Frozen Mechanically Deboned Fish . . 128

Effects of Additives on Gel Forming
Ability of Frozen Mechanically Deboned
Fish . . . . . . . . . . . . 133

Page

Further Processing of Frozen Mechanically

Deboned Fish . . . . . . . . . 140
Fish Sausages . . . . . . . . . 140
Canned Minced Fish . . . . . . . 152
SUMMARY AND CONCLUSION . . . . . . 159
APPENDICES . . . . . . . . . . . 163
REFERENCES . . . . . . . . I82

vi

Table

LIST OF TABLES

Treatment Design and Formulations for the
Measurement of Various Protein Fractions,
pH, Swelling, and Gel-Forming Ability .

Design and Formulation of Smoked Fish
Sausage Treatments . . . . . . .

Smokehouse Cooking Schedule . . . . .

Design and Formulation of Canned Minced
Fish Treatments . . . . . . . .

Average Percentage Yields Per Total
Weight of Fish for Dressing and Deboning
Operations, and Average Percentage of
Chemical Composition and Standard Devia-
tion of Mechanically Deboned Freshwater
Sucker Flesh . . . . . . . . . .

Mean and Standard Deviation of Protein
Fractions Extracted from Prerigor, Refrig—
erated and Frozen Sucker Flesh by the
Helander Method . . . . . . . .

Analysis of Variance of the Effect of Low
Temperature Storage on Extracted Salt
Soluble Protein . . . . . . . .

EffectsanAdding Sodium Chloride and
Sodium Tripolyphosphate on the Amount of
Total Extracted Protein of Frozen Mechani-
cally Deboned Fish . . . . . . .

Effect of Adding Soy Protein Isolate on

the Amount of Total Extracted Protein of
Frozen Mechanically Deboned Fish . . .

vii

Page

62

74

75

76

79

8O

83

86

92

Table

10.

ll.

12.

l3.

14.

15.

l6.

17.

Analysis of Variance of the Effects of
Sodium Chloride, Sodium Tripolyphosphate
and Soy Protein Isolate in Varying Con-
centrations and Combinations on Total
Extracted Protein of Frozen Mechanically
Deboned Fish . . . . . . . .

Effects of Adding Sodium Chloride and
Sodium Tripolyphosphate on the Amount of
Salt Soluble Protein Extracted from
Frozen Mechanically Deboned Fish . . .

Analysis of Variance of the Effects of
Sodium Chloride, Sodium Tripolyphosphate
and Soy Protein Isolate in Varying Concen-
trations and Combinations on Salt Soluble
Protein Extracted from Frozen Mechani-
cally Deboned Fish . . . . . .

Effect of Adding Soy Protein Isolate on
the Amount of Salt Soluble Protein
Extracted from Frozen Mechanically
Deboned Fish . . . . .

Effects of Adding Sodium Chloride and
Sodium Tripolyphosphate on the Amount of
Water Soluble Protein Extracted from
Frozen Mechanically Deboned Fish . .

Effect of Adding Soy Protein Isolate on
the Amount of Water Soluble Protein
Extracted from Frozen Mechanically
Deboned Fish . . . . . . . . .

Analysis of Variance of the Effects of
Sodium Chloride, Sodium Tripolyphosphate
and Soy Protein Isolate in Varying Con-
centrations and Combinations on Water
Soluble Protein Extracted from Frozen
Mechanically Deboned Fish

Effects of Adding Sodium Chloride and
Sodium Tripolyphosphate on the Soluble
Nonprotein Nitrogen of Frozen Mechani-
cally Deboned Fish . . . . .

viii

Page

93

94

98

99

100

103

105

106

Table

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

Effect of Adding Soy Protein Isolate on
the Nonrpotein Nitrogen of Frozen Mechani-
cally Deboned Fish . . . . . .

Analysis of Variance of the Effects of
Sodium Chloride, Sodium Tripolyphosphate
and Soy Protein Isolate in Varying Concen-
trations and Combinations on Nonprotein
Nitrogen Extracted from Frozen Mechani-
cally Deboned Fish . . . . . .

Effects of Adding Sodium Chloride and
Sodium Tripolyphosphate on Solubility of
Myosin Heavy Chain of Frozen Mechanically
Deboned Fish . . . . . . . .

Effect of Adding Soy Protein Isolate on
the Solubility of Myosin Heavy Chains of
Frozen Mechanically Deboned Fish

Analysis of Variance of the Effects of
Sodium Chloride, Sodium Tripolyphosphate
and Soy Protein Isolate in Varying Concen-
trations and Combinations on Mysoin Heavy
Chain Total Area Extracted from Frozen
Mechanically Deboned Fish

Effects of Adding Sodium Chloride and
Sodium Tripolyphosphate on pH of Frozen
Mechanically Deboned Fish . . . .

Effect of Adding Soy Protein Isolate on pH
of Frozen Mechanically Deboned Fish .

Analysis of Variance of the Effects of
Sodium Chloride, Sodium Tripolyphosphate
and Soy Protein Isolate, in Varying Con-
centrations and Combinations, on pH of
Frozen Mechanically Deboned Fish

Effects of Adding Sodium Chloride and
Sodium Tripolyphosphate on Percent Swel-
ling of Frozen Mechanically Deboned Fish

Effect of Adding Soy Protein Isolate on

Percent Swelling of Frozen Mechanically
Deboned Fish . . .

ix

Page

107

108

116

119

122

123

126

127

128

131

Table

27.

28.

29.

30.

31.

32.

33.

Effect of Adding Soy Protein Isolate on
Percent Swelling of Frozen Mechanically
Deboned Fish . . . . . . . . . .

Analysis of Variance of the Effects of
Sodium Chloride, Sodium Tripolyphosphate,
and Soy Protein Isolate, in Varying Con-
centrations and Combinations, on Swelling
of Frozen Mechanically Deboned Fish . .

Effects of Adding Sodium Chloride and
Sodium Tripolyphosphate on the Least Con—
centration Endpoint of Frozen Mechani-
cally Deboned Fish Extracted Protein

Effect of Adding Soy Protein Isolate on
Least Concentration Endpoint of Frozen
Mechanically Deboned Fish Extracted Pro-
tein . . . . . . . . . . .

Analysis of Variance of the Effects of
Sodium Chloride, Sodium Tripolyphosphate
and Soy Protein Isolate, in Varying Con—
centrations and Combinations on Least
Concentration Endpoint of Total Extracted
Protein of Frozen Mechanically Deboned
Fish . . . . . . . . . . . .

Average Percentages and Standard Devia-
tions of Cooking Loss, Water Holding
Capacity and Shear Force of Smoked Fish
Sausages . . . . . . . . . . .

Average PercentageszmuiStandard Devia-
tions<ofBaking Loss, Water Holding
Capacity and Shear Force of Canned
Minced Fish . . . . . . . . .

Page

131

134

135

138

141

145

155

Figure

LIST OF FIGURES

Effect of Low Temperature Storage on Salt
Soluble Protein, Water Soluble Protein and
Nonprotein Nitrogen Extracted from Sucker
Flesh by the Helander Method . . . .

Effect of Sodium Chloride in Varying Con-
centrations on the Protein Fractions of
Frozen Minced Fish . . . . . . . .

Effects of Sodium Chloride and Sodium Tri-
polyphosphate, in Varying Concentrations
and Combinations, on the Total Extractable
Protein of Frozen Mechanically Deboned
Fish . . . . . . . . . . . .

Effects of Sodium Chloride and Sodium Tri—
polyphosphate, in Varying Concentrations
and Combinations, on the Salt-Soluble Pro-
tein Fraction Extracted from Frozen
Mechanically Deboned Fish . . . . . .

Effect of Sodium Chloride in Varying Con-
centrations on the Ratio Between the
Amounts of Salt Soluble Protein and Water
Soluble Protein Extracted from Frozen
Minced Fish . . . . . . . . . .

Effect of Sodium Tripolyphosphate in Vary-
ing Concentrations on the Ratio Between
the Amounts of Salt Soluble Protein and
Water Soluble Protein Extracted from
Frozen Minced Fish . . . . . . .

SDS Polyacrylamide Gel Electrophoresis of

Total Protein Extracted from Frozen
Mechanically Deboned Fish . . . . .

xi

Page

81

87

91

96

101

102

109

Figure Page

8. SDS Polyacrylamide Gel Electrophoreses of
Total Protein Extracted from Frozem Mechani—
cally Deboned Fish with 3.0% Sodium
Chloride . . . . . . . . . . . . 112

9. SDS Polyacrylamide Gel Electrophoresis of
Total Protein Extracted from Frozen Mechani-
cally Deboned Fish with 3.6% Sodium
Chloride . . . . . . . . . . . . 114

10. Typical Absorbance Peak for Heavy Chain
Total Area of Myosin Extracted from Frozen
Mechanically Deboned Fish . . . . . . 117

11. Effects of Sodium Chloride, Sodium Tripoly—
phosphate and Soy Protein Isolate, in Vary-
ing Concentrations, and Combinations on
Myosin Heavy Chain Total Area per 20 pg
Protein Sample Extracted from Frozen
Mechanically Deboned Fish . . . . . . 120

12. Effects of Sodium Chloride and Sodium Tri-
polyphosphate, in Varying Concentrations
and Combinations on pH of Frozen Mechani—
cally Deboned Fish . . . . . . . . . 125

13. Effects of Sodium Chloride and Sodium Tri—
polyphosphate in Varying Concentrations
and Combinations on the Swelling of Frozen
Mechanically Deboned Fish . . . . . . 130

14. Effects of Sodium Chloride and Soy Protein
Isolate in Varying Concentrations and Com-
binations on the Swelling of Frozen
Mechanically Deboned Fish . . . . . . 132

15. Effects of Sodium Chloride and Sodium Tri-
polyphosphate, in Varying Concentrations
and Combinations, on Least Concentration
Endpoint of Total Extracted Protein Frac-
tion Extracted from Frozen Mechanically
Deboned Fish . . . . . . . . . . . 137

16. Whole Fish Sausages and Cross-Sections . . 143

xii

 

Figure Page

17. Typical Force/Distance Curve for Central
Mass of Fish Products . . . . . . 151

18. Cross-sections of Canned Minced Fish . 157

xiii

 

LIST OF APPENDICES

Appendix Page
1. Standard Curve for Myosin Heavy Chain
Total Peak Area . . . . . . . . 164
2. Average Percentage of Total Protein
Extracted from Mechanically Deboned
Frozen Fish . . . . . . . . . 166

3. Average Percentage of Salt—Soluble Pro-
tein Extracted from Mechanically

Deboned Frozen Fish . . . . . . . 168
4. Average Percentage of Water—Soluble

Protein Extracted from Mechanically

Deboned Frozen Fish . . . . . . . 170

5. Average Percentage of Nonprotein Nitro-
gen Extracted from Mechanically Deboned

Frozen Fish . . . . . . . . . 172
6. Mean Values of Myosin Heavy Chain Total

Area Extracted From Mechanically

Deboned Frozen Fish . . . . . . . 174
7. Average of pH of Mechanically Deboned

Frozen Fish . . . . . . . . . 176

8. Average Percent of Swelling of Mechani-
cally Deboned Frozen Fish . . . . . 178

9. Average of Least Concentration Endpoing
of Mechanically Deboned Frozen Fish . 180

xiv

INTRODUCTION

In today's world there is major emphasis on effi-
ciency and maximum utilization of energy resources. From
a nutritional standpoint, new sources of protein are
needed, and one source that is often overlooked is fish.
Underutilized fish species and those species having low
acceptance due to flavor defects, poor appearance, or
problems of separating lean from bony tissue can be used
successfully as raw materials for new fish products. In
Japan during the last 25 years machines have been devel-
oped which separate edible flesh from skin and bones of
eviscerated and deheaded fish and skeletal frames from
filleting operations to produce minced fish muscle
(Tanikawa, 1963). An increased amount of meat available
for human food, increased market value of bony fish and
savings in labor are some of the major benefits attributed
to mechanical deboning. Mechanically separated minced
muscle has been used in food products such as Kamaboko
fish cake and fish sausages. In 1970, over one million
metric tons of Kamaboko and fish sausages were produced
in Japan (Okada et a1., 1973). Also, other countries have
recently begun to show interest in developing new food
products from mechanically separated fish flesh.

1

 

Baker (1978) states that about 80-90% of the fish
resources from the Great Lakes are wasted. So-called
"trash" fish considered pollutants, are routinely thrown

back by fishermen. The silver redhorse sucker (Moxostoma

 

anisurum) and white sucker (Catostomus commersoni) taken

 

from the Great Lakes were selected for this study on
underutilized freshwater fish. These species are not
used now commercially because of their bony nature, which
complicates the separation of lean from skeletal tissue,
lower consumer acceptability and the inferior binding
properties of their flesh.

Binding properties of fish flesh are important
not only from the standpoint of production practices
but also because they influence quality characteristics
such as texture, tenderness, juiciness, appearance, and
palatability in finished products.

Freezing is generally used to preserve fish flesh,
but freezing can cause physio-chemical changes in the
colloidal structure of fish proteins. These changes lead
to protein denaturation resulting in flesh that is infer-
ior in functional performance as indicated by solubility
of the myofibrillar proteins (mainly myosin) and water
binding and holding characteristics. Technological prob-
lems induced by freezing include drip loss, and with pro-

longed storage, toughening of the muscle tissue causing

reduced acceptability and economic loss (Warrier et al.,
1975).

Basic studies performed on frozen sucker muscle
proteins have helped to define processing techniques that
maintain quality and improve the economics of production.
The use of phosphate salts and sodium chloride to improve
water holding capacity and firmness of canned meat, and
to obtain better structure and consistency in manufactured
sausages, is common practice in the meat industry. Addi—
tion of soy protein isolate reduces cooking losses by
absorbing some of the free water not bound by the func-
tional proteins.

The aim of this study was to investigate the
effect of added sodium chloride, sodium tripolyphosphate
and soy protein isolate (Cenpro—P), in varying combina-
tions and levels, on the functional prOperties of mechani-
cally deboned frozen sucker flesh. These properties
were evaluated in terms of the amount of different pro-
tein fractions extracted and the degree of swelling and
gel formation of fish muscle proteins. Finally, canned
and sausage—type products were developed to promote the
utilization of suckers. Both products were analyzed for
water holding capacity, cooking loss and texture. It was
not within the scope of this study to adjust appearance

and flavor of the products. However, these

characteristics could be altered to meet consumer demand

by adding various flavor ingredients to the minced flesh.

 

LITERATURE REVIEW

The Structure and Composition of
Fish Muscle

 

 

Muscle is composed of fibers which are several
centimeters in length and 0.01 to 0.1 millimeters in
diameter. Individual muscle fibers are surrounded by
membranes, called sarcolemma, and are arranged in bundles
which enclose connective and fatty tissue. Structurally,
these fibers include myofibrils, multiple nuclei, mito-
chondria, the fluid sarcoplasm, the sarcoplasmic reti-
culum and other bodies such as ribosomes, lysosomes and
glycogen granules (Cassens, 1972). The proximate com-
position of fish muscle varies widely from species to
species. Even within the same species the fat content of
one individual may be more than ten times greater than
that of another. The main constituents of the edible
portion (skin and bone-free fillet) are as follows:
moisture 28-90%, protein 6-28%; fat 0.2-64%, ash 0.4-l.5%.
These percentages representing all species of fish illus-
trate individual variations due to season and geographical
area of harvest, as well as age, sex and size of fish
(Stansby and Olcott, 1963). The total protein of fresh-

water fish as classified by Moorjani et al.,(1962),

consists of myofibrillar (59-73%), sarcoplasmic (about
35%) and connective tissue (4-6%) proteins. According to
Goll et_al.,(l974) proteins identified thus far in the
myofibrillar fraction are myosin (SO-55%), actin (15-20%),
tropomyosin (5-8%), troponin (5-8%), actinin (2-3%),
B-actinin (0.5—1%), component C (2—3%), and M-Line pro-
teins (3—5%). According to Connell and Howgate (1959)

the ratio of myosin to actin in cod actomyosin is in the
range of 2 to 4. Cod muscle myosin contributes 40% of
the total protein (Connell, 1964), while actin contributes
15-20% of cod actomyosin (Connell and Howgate, 1959).

The myosin molecule is an elongated structure
about 1600Ao in length and 15-40Ao in diameter (Rice,
1961; Zobel and Carlson, 1963). Its two major parts are
the rod—liked tail and globular head. The tail contains
2 or 3 stranded helical coiled coil, 12—1300Ao in length
and 15A° in diameter, while the globular head is 250—
350X° in length and 40X° in diameter (Rice, 1961 and 1964).
The head when split from the tail of the molecule is
referred to as heavy meromyosin (HMM), and the rod—like
tail is called light meromyosin (LMM). The molecular
weight of myosin is about 500,000 dalton, and the weight
of the head is about twice that of the tail (Gergely,

1966).

Functional Classification of Muscle Proteins

 

The role of muscle proteins in functional per-
formance can be specified more precisely by dividing
these into three major classes on the basis of their
solubility (Goll, et al.,l974). The first class consists
of the myofibrillar proteins which form the myofibril or
contractible part of muscle. Ionic strengths greater
than 0.30 are required to solubilize myofibrils. Because
some myofibrillar proteins are soluble in water, they
cannot be accurately defined on the basis of their solu—
bility alone. The second class contains sarcoplasmic
proteins, which are soluble at ionic strengths of 0.05 or
less. They generally comprise the proteins of the muscle
cell cytOplasm. Third are the stroma proteins which are
those proteins insoluble in neutral solutions. Collagen
and elastin constitute a large part of the stroma protein
fraction.

Myofibrillar proteins are responsible for the
quality attribute of texture and also have been identified
with the properties essential for water binding, gel for—
mation and emulsification characteristics in comminuted
sausages (Briskey and Fukazawa, 1971; Goll et al.,l974).
Bai and Radola (1977) reported that sarcoplasmic proteins
of fish muscle have received great attention because they

produce compounds responsible for flavor and color and

 

also because they contain enzymes causing post-mortem
biochemical changes. [The water soluble proteins have a
greater effect on functional properties of muscle pro-
teins than has been previously thought (Schut and Brouwer,
1974).] The effect of these proteins on water binding,
gel formation and emulsifying capacity is relatively minor
compared to myofibrillar proteins (Hamm, 1960; Swift and
Sulzbacher, 1963; and Schut and Brouwer, 1974).

Stroma proteins have poor water and fat binding
ability. They shrink in the temperature range of 60-65°C,
and form gelatin at higher temperatures causing problems
such as instability and formation of jelly pockets in
finished products. However, they are useful in reduction
of the cost of finished products (Kramlich, 1972; Saffle
et_al.,l964).

The role of fish proteins has been described as
being similar to that of red meat proteins (Learson
gt_§1.,l97l). Also, the regulatory proteins such as
trOpomyosin, troponin and d-actinin affect the physical
and chemical properties of actin and the combined actin-
myosin complex on their capacity for heat-gelling or
emulsification (Briskey and Fukazawa, 1971). Information
reported on myofibrillar proteins, specifically for myosin
and actin has aided in our understanding of how these

proteins function in food systems.

 

The Extractability of Muscle Proteins

Protein extractability and solubility are useful
parameters for determining the quantity of proteins avail-
able for emulsification (Saffle, 1968). A relationship
between protein solubility, muscle color and hydration
capacity was demonstrated by Borchert and Briskey (1965).
Determination of soluble protein was used in another
study as an index of frozen fish quality (Love, 1958).
There is some confusion in the literature as to whether
functionality depends upon the existence of these pro-
teins in the soluble state. It has been shown that
soluble proteins are required for good water binding
(Sayre and Briskey, 1963; Cook, 1967). Sherman (1961)
found a relationship between protein solubility and water
retention as measured by water release during centrifuga-
tion. Trautman (1964) found that salt soluble proteins
were the major emulsifying components in ham muscle.
Saffle (1968) and Kramlich (1972) concluded that the
presence of extensive amounts of protein in solution is
required for good emulsification. Trautman (1966a) also
concluded that protein solubility may be extremely impor-
tant in finely comminuted sausages.

The extractability of protein from different
muscles as a function of time post-mortem and physiologi-

cal condition has been studied by a number of

10

investigators (Ironside and Love, 1958; Sayre and
Briskey, 1963; Sc0pes, 1964; Saffle and Galbreath, 1964;
BorchertamxiBriskey, 1965; Cook, 1967; Dixon and Webb,
1966; Meinke e£;al,,1972). Factors which influence the
extractability of meat proteins are cited as post-mortem
age, pH, ionic strength, degree and type of masceration,
extractant to muscle ratio, extraction time and tempera—
ture and freezing (Helander, 1957; Bard, 1965; Saffle,
1968). The extractability of muscle proteins is generally
reduced during the early post—mortem period when pH is
low and muscle temperature is still high (Borchert and
Briskey, 1965). In 1964, Trautman developed a system of
studying the effect of pH on muscle protein extracts. He
found that a decreasing pH decreased the solubility of
both water and salt soluble proteins in a linear manner.
In this study the amount of salt soluble proteins
extracted from pre-rigor muscle was42% compared to 39%
extracted from post-rigor muscle. Conversely, Cook (1967)
found no difference in protein solubility of bovine
muscle between pre- and post-rigor stretched tissue and
noted that the contractile state of myofibrillar proteins
greatly influences their solubility. Holding muscle at
high temperature and low pH during the onset of rigor
mortis reduces sarcoplasmic and myofibrillar protein

solubility by about 55% and 75%,respective1y compared to

 

 

11

amounts found immediately post-mortem (Sayre and Briskey,
1963). No change was reported for myofibrillar protein
solubility however, and only a 17% reduction in sarco-
plasmic protein solubility was found when pH remained
high at the onset of rigor mortis. According to Meinke
et al.,(1972) solubility of fish muscle protein was mini-
mal in the pH range 5.5-6.0 but increased on both the
acidic and basic sides of this range. They found that
only 20% of fish protein was soluble at pH 5.5, while 70%
was soluble at pH 3 or pH 10. That the amount of
extracted protein increases as muscle pH increases has
been demonstrated by Saffle and Galbreath (1964).
Trautman (1964) reported that the amount of
extractable salt soluble meat proteins is influenced by
time post—mortem, while the amount of extractable water
soluble and salt insoluble proteins is not influenced by
this factor. Biochemical changes in fish muscle during
rigor mortis have been reviewed by Nazir and Magar (1963).
These authors found decreasing levels of glycogen, adeno—
sine triphosphate and creatine phosphate, but a rise in
inorganic phosphate in fish stored in crushed ice at
about 2°C. The effects of fish size, season of harvest,
reproductive cycle and starvation, on the amounts of
extracted soluble proteins, were studied by Ironside and

Love (1958). Saffle and Galbreath (1964) recorded the

 

 

12

influence of muscle collagen and elastin content on the
amount of salt soluble meat protein extracted. The amount
of extractable protein is known to increase with more
muscle destruction because the structural protein of
muscle becomes more available for solubilization as cell
disruption and breakage of muscle becomes more extensive
(Acton, 1972a).

Effect of Low Temperature

Storage on Protein
Extractability

 

 

 

Although frozen meat is used to a large extent in
today's processed meat formulations (Kramlich, 1972), it
has been shown to be less stable than fresh meat in
sausage-type emulsions (Morrison et al.,l971). Jarenback
and Liljemark (1975a) noted the disorder of myofibrils
and a reduction in interfilament spacing after frozen
storage of meat. This reduction and the observed dis-
orders within and between myofibrils may reflect an
impaired ability to reabsorb water following thawing.
Freezing temperature has been shown to be an important
factor in this problem. Jarenback and Liljemark (1975b)
studied the effect of three temperatures on actomyosin
filaments extracted from frozen cod. Decreases in number
of actomyosin filaments as well as in length and amount
of attached myosin to actomyosin were most extensive at

—10°C, less at -20°C and hardly noticeable at -30°C.

13

These authors suggested that such reductions in extract-
able protein might be due to the effect of freezing and
thawing rather than to storage above (-30°C), since
freezing and thawing without storage and storage only

at -30°C were not different in terms of reducing the
amount of extractable proteins.

Changes in ice—stored fish are subtle and unlike
those observed during frozen storage. Baliga et_al.
(1962) studied changes in soluble protein nitrogen of
freshwater fish during iced storage. While total protein
solubility decreased 20% after 15 days, sarcoplasmic pro-
tein decreased only slightly and nonprotein nitrogen
increased. Moorjani et_al.,(l962) also recorded decreases
in protein solubility for fish held in ice. They noted
that non-protein nitrogen remained almost unchanged for
16 days of storage. Later, Baliga et_al.,(l969) reported
a 10% decrease in protein solubility by the 5th day in
ice followed by increases with further storage up to 13
days. The fall in solubility on the 5th day occurred
at the same time as the highest level of precipitated
actomyosin.

With increasing storage time, there is a progres-
sive fall in the amount of protein extractable by neutral
salt from frozen flesh. Several studies have been pub-

lished confirming the decrease of protein extractability

14

during frozen storage. Solubility of protein decreased

at a steady rate when fish fillets were stored at ~14°C
for 14 weeks, then decreased no further even in fillets
stored for over 4 years (Love and Ironside, 1958).

Poulter and Lawrie (1977) found that protein solubility
decreased more rapidly during storage at —8°C than dur-
ing storage at -30°C. The rate of decline in solubility
varied from species to species, being much more rapid

in some, while pH increased in all the species studied.
Iwata and Okada (1971) noted that cold storage at ~10°C
decreased the amount of extractable fish protein. Pro-
tein solubility decreased from 72% to 45% in cod fillets
stored for up to 82 months at -29°C (Cowie and Little,
1966). In 1968, Cowie and Mackie studied the effect of
three temperatures (-7°, —14°, and ~29°C) for various
periods of times on the amount of extractable protein
using two different extraction methods. After 20 weeks of
storage at -7°C they reported that extractable protein con-
tent had fallen to 25-40% while after 34 weeks of storage
at —l4°C it dropped only to 40-60%. Protein extractability
remained at 80% up to 49 months of storage at -29°C,

then dropped gradually to about 70% after 82 months. On
the other hand, contrasting results have been reported
for both red meat and fish flesh. Acton and Saffle (1969)

found no difference in the protein extractability of

15

frozen and corresponding unfrozen meat. As reported by
Kato et al.,(l974) there was also no change in extract-
ability of myofibrillar protein in partially frozen fish
muscle at -3°C. Sarc0plasmic proteins of fish remain
mostly unchanged during prolonged frozen storage (Connell,
1960a; Babbitt et al.,l972; and Kato et al.,l974).

Several researchers have cited the decreased
extractability of myofibrillar proteins during frozen
storage as indicative of the denaturation of these pro—
teins (Connell, 1960a; Cowie and Little, 1966; and Kato
et al.,l974). This was questioned by Connell (1962),
however, who found that the amount of actomyosin extracted
from fish muscle falls during the development of rigor
mortis and then increases again. Since this would imply
a reversal of denaturation, Connell proposed that a
change in the type of association between essentially
native protein molecules was responsible. Hence, the use
of tissue protein extraction for monitoring denaturation
is complicated by the interaction of different proteins.
The denaturation mechanism of myosin B (Actomyosin) was
explained by Okitani et;al.,(l967) in two ways: Spon-
taneous aggregation and irreversible dissociation to
myosin A and actin and denaturation caused by storage con-

ditions such as temperature, pH and ionic strength.

l6

Connell (1960a) found that the decrease in the
amount of extractable protein from frozen fish is due
mainly to a decrease in the amount of actomyosin dis-
solved. Dyer and Morton (1956) reported that extractable
actomyosin appears to be a useful quality index for frozen
fish. Myosin B extracted from pre—rigor muscle gave
relatively high values for ATPase activity, ATP-sensitivity
and reduced viscosity when compared to that extracted from
post—rigor muscle (Fujimaki et_a1., 1965b). Kato e£_31.,
(1974) recorded a decrease in ATPase activity during
storage of fish at -3°C and in ice. On the other hand,
Connell (1960b) found no marked change in adenosine
triphosphate (Myosin-ATPase) activity during frozen stor-
age of cod flesh for up to 170 weeks at -29°C although a
definite loss of activity was detected at -22° and -14°C.
Seagran (1956) studied the changing properties of actomyo-
sin from fish muscle that had been frozen and reported
that solubility reached a maximum after one month of
storage and declined continuously thereafter. Dyer and
Morton (1956) and Oguni et_al., (1975) confirmed the
decreasing solubility of actomyosin with lengthening
frozen storage time.

Myosin is the least stable component of frozen
stored fish protein with denaturation intensified by

repeated freezing and thawing of the muscle (Nikkila and

17

Linko, 1953). Connell (1960a) reported that cod myosin
is a very unstable protein because it undergoes rapid
denaturation and aggregation both in unfrozen solution at
low temperature and, especially in frozen solution. Later
Connell (1962) found that 70-80% of myosin from cod flesh
became non-extractable at a rate similar to the extracta-
bility of myofibrillar protein during storage of this
species at —14°C. Early in 1950, Snow reported that
freezing and storing of myosin in the frozen state pro-
duced complex aggregation and reactions. According to
Connell (1960a) the aggregation reaction involved two
steps: a first—order step which does not affect the
sedimentation coefficient, followed by a second or higher
order step. Buttkus (1970) studied the aggregation of
rabbit and trout myosin in frozen solutions and suggested
a mechanism which involves disulphide-sulfhydryl exchange
reactions between the activated myosin molecule and aggre-
ates. This was concluded from observed behavior of
sulfhydryl groups in the monomeric myosin molecule and
from the influence of reducing conditions on high molecule
weight aggregations.

Chu and Sterling (1970) characterized degree of
myosin denaturation in fish. on the basis of protein
solubility, intrinsic ‘viscosity, isoelectric point,

ATPase activity, ultra-Violet absorption spectrum and

l8

optical rotatory dispersion of the purified myosin extract.
These measurements showed that denaturation increased in
the order: Fresh < Frozen < Frozen stored.

Actin, on the other hand, has a more stable
nature. The amount extracted from cod flesh remained
unchanged for up to 30 weeks of storage at -l4°C (Connell,
1960C). It might be the globular structure of actin
that make it seem to have characteristics as of sarco-
plasmic proteins, which remain largely undenatured and
soluble throughout storage.

Fatty acids reduce myofibrillar protein extract-
ability. Jarenback and Liljemark (1975c) demonstrated
that small quantities of linolic acid hydroperoxides
caused precipitation of isolated myofibrillar proteins.
Babbitt et_al.,(1972) explained the large decrease in
total extractable protein of minced muscle after 60 days
of frozen storage on the basis of higher concentrations
of fatty acids in these samples. Anderson and Ravesi
(1970) also found that extractability of protein decreased
as free fatty acid content increased. These authors sug-
gested that large amounts of free fatty acids could be
formed during iced storage before freezing. In this study,
protein extractability decreased most rapidly in the first

eight to ten weeks of storage (-12°C and -18°C).

 

 

19

Noguchi and Matsumoto (1975a, b) and Noguchi
et al.,(l975) studied the preventive effect of amino
acids, peptides, sulfur compounds, carboxylic acids,
sorbitol and glucose on the freezing denaturation of pro—
teins by determining solubility and superprecipitation of
carp actomyosin. Acetylation, peptide formation and
position and number of active groups were involved in the
preventive effect of these compounds. Frozen samples
containing some of these additives produced good quality
kamaboko jelly after frozen storage.

Myofibrillar proteins of frozen cod were extracted
at an ionic strenth as low as 0.1 in the presence of
sodium pyruvate (Tran, 1975). In frozen samples kept at
-20°C for up to 14 days, added sodium pyruvate allowed
greater protein extraction than potassium chloride.
During subsequent storage, however, the reverse was true.

Protein-Protein interaction of tobacco mosaic
virus has been studied by Lauffer (1964). Protein mole-
cules interact with each other, therefore individual
molecules influence and are influenced by other neighbor-
ing molecules. Associations between various proteins may
be anything from specific covalent chemical bonds to
weak intermolecular forces. Protein—Protein interactions
have been found to be responsible for the formation of

liquid crystalline phases (Lauffer, 1938). Other types

 

20

of protein-protein interaction result in the precipitation
of proteins from solution (Oncley et al.,1952). According
to Lauffer (1964) protein-protein interaction occurs
between the molecules of a single protein in both amor-
phous and crystalline precipitations. Certain proteins

in solution can exist in different states of polymeriza—
tion depending upon composition, temperature, pressure and
other characteristics of the solution (Oncley et al.,1952).
Effect of Sodium Chloride and

Phosphates on Protein
Extractability

 

 

 

Rigor mortis involves a type of aggregation of
muscle protein in situ, characterized by the disorganiza—
tion of tissue and loss of ATP from myofibrils. These
changes may prevent the distribution of proteins into
solution when salt solutions are used (Dixon and Webb,
1961). According to Asghar and Yeates (1974), the effect
of rigor mortis on protein solubility apparently disap-
peared with the addition of phosphate and appropriate
conditions of ionic strength, pH, and temperature during
muscle protein extraction. HermanssonznuiAkesson (1975b)
explained the influences of added salt on the properties
of proteins in three ways: (1) by specific ion binding,
(2) by influencing the ionic strength, and (3) by chang-

ing the properties of the solvent. In 1973, however,

21

Brotsky and Everson found that while a 2.5% sodium
chloride concentration has considerable ionic strength,
its ability to spread apart filaments is limited because
of cross-linkages. Helander (1957) reported that with a
4.5% salt concentration a higher extractability of myo—
fibrillar protein might be expected. Trautman (1964)
extracted only 42 and 39% of salt soluble proteins from
pre- and post-rigor pork muscle,respectively,using a

0.67 M sodium chloride solution, while Dyer et_al.,(l950)
extracted up to 95% of fish muscle protein with 0.85 M
sodium chloride. A pH of 7-9 and salt in the range of

3 to 5% represented optimum conditions for protein extrac-
tion in the latter study, with myosin the largest fraction
extracted. It is recognized that the addition of sodium
chloride to meat decreases its pH. Hamm (1957) found that
adding sodium chloride to post-rigor ham muscle consis-
tently lowers pH. Meinke e£_al.,(1972) studied the rela—
tionship between protein solubility, salt concentration
and pH. Their data indicated that protein solubility
increased with increasing salt concentration at approxi-
mately pH 6, while the amount of extracted protein gradu-
ally decreased at 3.5, 4.2, and 11.1 pH values with
increasing salt concentration up to 1.0 N. When salt

was added to muscle tissue it dissolved in the water

associated with the protein. Duerr and Dyer (1952) found

22

that myosin swelling was greatest in 2 to 5% salt solu-
tions and that water becomes more firmly bound to myosin
micelles in the tissues at these concentrations. These
authors recorded the denaturation of myosin when salt
concentrations reached 8 to 10% in muscle tissue.

The role that sodium pyrophosphate and sodium
tripolyphosphate have in increasing protein solubility
has been recognized by many investigators. Lee and Toledo
(1976) found the solubility of myosin to increase in the
presence of polyphosphates. Bendall (1954) explained the
effect of these phosphates by the splitting of actomyosin
into actin and myosin, and predicted that effective poly-
phosphates should have chemical structures similar to that
of ATP. Salt-induced denaturation of Baltic Herring myo-
sin was inhibited by the addition of various alkaline
phosphates in a study by Linko and Nikkila (1961). When
the concentration of salt in meat systems increases,
higher concentrations of phosphates are required to pre-
vent the denaturation of myosin. Mechanisms by which
phosphates are able to inhibit the reduction of myosin
solubility by sodium chloride and at the same time
increase the hydration of this protein can be explained
as follows: phosphates may be taken up directly by pro-
tein molecules, thereby increasing the number of polar

groups on the protein and phosphates may be bound to

23

actomyosin, thereby increasing the myosin negative charge
which causes its dissociation. Yasui et al.,(1964b)
reported that tripolyphosphate can dissociate actomyosin
into its components after it has been converted to pyro—
phosphate by enzymatic hydrolysis. Yasui et al.,(1964a)
studied the effect of three inorganic polyphosphates on
the solubility ofnnmmflxlB (natural actomyosin) and on the
extractability of structural protein from myofibrils in
various conditions. Pyrophosphate or tripolyphosphate,
which are low molecular weight inorganic polyphosphates,
react with salt—free myosin B as a salt. The affinity
of these inorganic salts for myosin B is greatly improved
in the presence of high salt concentrations and divalent
cations. Hexametaphosphate,on the other hand, is a
highly polymerized polyphosphate compound whose binding
with salt-free myosin is direct but somewhat inhibited
by the presence of high salt concentration and divalent
cations. According to Brotsky and Everson (1973) a
polyphosphate concentration of 0.5% can break bonds, but
cannot raise ionic strength sufficiently to spread

apart filaments.

Effect of Heat on the Extract-
ability of Muscle Proteins

 

 

Heating of meat causes the denaturation of sarco—

plasmic and myofibrillar proteins with the degree of

24

denaturation dependent upon heating temperature (Hamm and
Deatherage, 1960; Hamm, 1966). Solubility of actomyosin
is reduced by heating and the greatest decrease occurs at
temperatures between 40°C (104°F) and 60°C (140°F). The
myofibrillar proteins became almost insoluble above 60°C
(140°F) (Hamm, 1966). According to Hamm and Deatherage
(1960) the effect of heat on extractability of muscle

proteins expressed as percentage of fresh tissue was as

 

follows:
Heating Sarcoplasmic Myofibrillar
Temperature Proteins Proteins
(°C) (%) (%)
20 100 100
40 86 69
60 23 2
80 1 12*

 

*Increase may be due to some breakdown of connective
tissue.

In the study by Hamm and Deatherage (1960), most sarco-
plasmic proteins coagulated between 40°C (104°F) and 60°C
(140°F). At 50°C (122°F) a large part of these proteins
are still soluble while at 66°C (150.8°F) only a few are
not yet coagulated (Bendall, 1964). Cheng and Parrish

(1979) studied the effect of heat on the solubility of

 

25

myofibrillar proteins in pre-rigor and post—mortem bovine
muscle by using SDS-polyacrylamide gel electrophoresis.
They indicated that d—actinin and myosin are heat labile,
becoming insoluble at 50°C and 55°C,respectively. Actin,
tropomyosin and troponin were more heat resistant, how—
ever. Actin became insoluble between 70°C and 80°C
while trOpomyosin and troponin became insoluble above
80°C. These data are in agreement with Locker (1956) who
reported.thatmyosin was completely coagulated at 55°C.
Acton (1972b) recorded an 84 to 89% reduction in protein
solubility when internal temperatures of meat reached
75-94°C. During cooking the quantity of protein extracted
decreased slightly between 4°C—35°C, then decreased
sharply between 35°C and 75°C. Further heating of the
meat caused only slight changes in protein extractability.
In 1966 Hamm outlined several changes that occur
when muscle protein is heated. He recorded no changes
in the colloidal—chemical properties of tissue or in the
solubility of ion—binding of muscle proteins at 20°C to
30°C, although the ATPase activity of myosin decreases
at 30°C. Some changes in the myofibrillar proteins occur
between 30°C-50°C which affect solubility, pH, ionic
strength, water holding capacity, Ca++ and Mg++ binding
capacity and the number of available sulfhydryl groups.

The ATPase is completely inactivated between 30°C and 50°C.

26

A small portion of sarc0plasmic proteins are denatured
between 30 and 40°C. At 50°C to 55°C there is a rearrange-
ment of the myofibrillar proteins which delays changes in
water-holding capacity and pH. At those temperatures new
cross linkages begin to form which are quite stable and
the denaturation of sarc0plasmic protein continues. At
55°C to 80°C most of the changes occurring at 50°C to
55°C continue to a lesser extent. At 65°C most myofibril-
lar and sarCOplasmic proteins are coagulated. Around 63°C
collagen shrinks and at higher temperatures it is partially
transformed to gelatin. Above 80°C the formation of
disulfide bonds by oxidation of actomyosin sulfhydryl
groups takes place and this continues with increasing
temperature.

Smoke is another factor changing protein solu-
bility and the electrophoretic behavior of meat proteins

(Randall and Bratzler, 1970).

Functional Properties of Muscle Proteins

 

Swelling

Swelling is defined as the spontaneous uptake of
water from any surrounding fluid (Hamm, 1960). Swelling
and gelation measurements were negatively correlated with
water loss (Hermansson and Akesson, 1975a). According to
Hermansson (1972) swelling is strongly dependent on pH

and ionic strength.

27

Several investigators found that phosphates have
an effect on meat swelling. Schults et_al.,(l972) showed
that swelling of meat was increased by adding phosphates,
which in turn raised pH. Shults and Wierbicki (1973)
also found that meat swelling was increased by the addi-
tion of different phosphate components. Shults et_al.,
(1972) mentioned that meat swelling increased when meat
was blended with low levels of sodium chloride (1-2%),
but increasing the level of salt up to 3-5% caused
decreases in swelling followed in turn by a rapid increase
in swelling when salt levels increased from 5% to 10%.
Even greater increases in meat swelling were caused by
adding 0.5% tripolyphosphate to the various sodium
chloride levels.

Bendall (1954) noted that the swelling effect of
pyrophosphate and tripolyphosphate salts on comminuted
whale meat was due to their ability to split actomyosin
into its component proteins, actin and myosin,resulting
in the uptake of water. Bendall noted that the effective
polyphosphates have chemical structures similar to that
of ATP. Shults et_al,,(l972) also reported that muscle
ATP-ase converted some tripolyphosphate to pyrophosphate
which had a specific swelling effect on meat in addition
to its effect on pH elevation and its ability to split

myosin B. Shults et al.,(l972) reported that according

28

to Wierbicki et_al.,(1963) the effect of sodium chloride
and polyphosphates at different levels and in varying
combinations could be explained by the replacement of
Ca++ by Na+ on meat proteins causing an increase in meat
swelling, while the exchange of Mg+ and K+ by Na+ caused
a decrease in meat swelling. The addition of high levels
of sodium chloride (5-10%) caused a rapid increase in
meat swelling due strictly to the ionic effect of sodium
chloride on meat proteins.

Binding Properties and Gel-
Forming Ability

 

 

The relationships between different protein frac-
tions and binding properties were studied by Hegarty
(1963) who attempted to discover which protein fractions
had the greatest binding ability. Hegarty's results indi-
cated that about 90% of actomyosin and myosin fractions
were involved in binding, while about 50% and 60% of
actin and sarcoplasmic protein respectively were used to
bind oil added to the meat system. Fujimaki and Nakajima
(1958) noted that the binding quality of sausage decreased
as the quantity of sarcoplasmic protein decreased.
Hegarty (1963) and Swift §E_al,,(l96l) found that oil bind-
ing increased as protein concentration decreased. McCready
and Gunningham (1971) indicated that the emulsifying

capacity of broiler dark meat which contains lower amounts

 

29

of total and salt—soluble proteins was higher than that

of broiler light meat, which was higher in total and salt-
soluble protein content. This higher emulsifying capacity
of dark meat was due to its greater pH (6.5) which
intensified certain salt-extractable protein fractions
most instrumental in emulsifying capacity. Fukazawa gt
a1,,(1961a, b, c) compared the binding capacity of
experimental beef sausage and its protein composition
before and after cooking. They used tensile strength
measurements as an index of binding capacity and found
that myosin was the key to desirable binding quality.

The binding quality of meats was reported in a
series of publications by Sato and Nakayama (1970) and by
Nakayama and Sato (1971a, b, c). Sato and Nakayama (1970)
stated that myofibrillar proteins and hydration were
related to binding quality, and that myosin was the essen-
tial protein in the binding quality of sausage. Nakayama
and Sato (1971a) found that native tropomyosin and actin
influenced the binding capacity of meats when pyrophos-
phates were added to meat in processing sausages. The
interaction between myosin A and F—actin was promoted by
the presence of native tropomyosin. This interaction
played an important role in protein fraction viscosity.

In a second study Nakayama and Sato (1971b) found that

heat set myosin and actomyosin gels had similar binding

30

properties but different physical prOperties. The bind-
ing quality of heated actomyosin gel increased with
increasing myosin concentration indicating that the bind-
ing quality of sausage was influenced by the actomyosin
content of meat. Nakayama and Sato (1971c) reported that
myosin-A, F—actin, and tropomyosin also were important
and should be controlled in sausage manufacture. The
binding qualtiy of heat-set gel increased when F-actin
was present in a particular ratio to myosin, and a
greater increase was found when tropomyosin was added.
Pepper and Schmidt (1975) found that the binding strengths
of beef rolls was increased as mixing time increased.

The heat gelling test is an excellent method to
determine the changes which occur in muscle proteins dur-
ing the transformation of muscle into meat. Trautman
(1966b) found a definite relationship between the minimum
concentration of protein able to form a gel by heating the
protein sol at 80°C and post-rigor muscle pH. Fujimaki
et al.,(1965b) studied the effect of post-mortem aging on
gel formation and emulsifying capacity. Myosin became
more easily dissociated with post-mortem aging. Thus, the
post-mortem age of muscle had a major influence on the
role of extracted protein in gel formation and emulsifi-
cation. Suzuki and Kanna (1963) found that myosin frac-

tion extracted with salt solution from either full rigor

 

31

or post rigor fish muscle turned into gel. According to
Nakayama and Sato (1971b); Samejima et_al., (1969);
Fujimaki et_al., (1965b)and Fukazawa et_al., (1961b) myo—
sin fractions have a major effect on gel formation. Tong
et_al., (1975) found that alkaline extractions (pH 11) of
squid protein concentrations were more effective than
protein extracted by 4% salt in forming gels. These
authors also indicated that the inability to form gels was
probably related to the high salt content of their samples.
In addition the poor gelation properties of salt extracts
probably were related to residual salt content. The
inability to form a gel at pH 6 was due primarily to the
low solubility of protein at this pH. Ueda et_al,, (1968)
reported that gel strength was determined during formation
of the setting (a Japanese technical term, "suwari"). It
is a well known phenomenon for ground muscles that homogen—
eous sols change into more viscous elastic structures when
placed at about 30°C for a short period of time. They
explain this phenomenon as follows: during heating of
ground muscle sols to about 30°C, the closely folded pep-
tide chains of myosin unfolded so that free radicals are
exposed. Unfolded coils then combine with each other and
form three-dimensional net structure. The strength and
weakness of a gel is determined by formation and deforma-

tion of the setting, and the setting temperature required

32

for protein gel strength was found to be different from
species to species. Myosin denaturation mechanisms,
explained earlier by Buttkus (1974), were responsible for
firming or gelling of protein structures in Kamaboko and
meats from fish and mammals during cooking. According to
Ferry (1948) formation of gels in denatured proteins
involved first an unfolding of protein into more asym-
metric shapes and exposure of reactive groups as well as
nonpolar groups making them less hydrophilic. Associa-
tion of the resulting chains by cross-links spaced some
distance apart and by localized and nonlocalized attrac-
tive forces (including hydrogen bonding) produces a
three-dimensional network throughout the sample in which
the "gel fraction" approaches unity and in which rigidity
is inhanced by further cross-linking taking place during
heating.

Salt is necessary to extract salt-soluble pro-
tein from muscle tissues. These proteins play a major
role in the binding properties of batter which in turn
effects the texture, flavor, and other quality character—
istics of finished sausage. Acton (1972a) found that
binding strength increased when grinding was utilized
for tissue breakdown. The increase of meat surface area
and closer contact between surfaces in addition to the

presence of salt and surface—soluble protein reduced

33

cooking loss and increased binding strength. Nakayama

and Sato (1971c) found that the presence of 0.6 M sodium
chloride in meat sol was necessary to produce high bind-
ing quality in heated actomyosin gel. Further study by
Nakayama and Sato (1972c) showed that binding quality was
greatly increased when gels prepared from "set" sols of
minced hen meat were stored with 3.5% sodium chloride.

The addition of sodium chloride and phosphates aided in
extracting protein and setting up the protein for good
binding properties (Brotsky and Everson, 1973; Swift and
Ellis, 1957). Moore et_al.,(l976) found that binding
strength of beef roll increased as salt level increased
from 1% to 3%. An additional increase in binding strength
was reported when 0.25% tripolyphosphate was added in
combination with various salt levels. Suzuki and Kanna
(1963) reported that fish myosin extracted with salt
solution from flesh in full rigor and post-rigor had

the ability to form gel. However, the presence of phos—
phate seemed to be essential for gel formation. Since
polyphosphates.havebeen shown to improve binding proper-
ties of red meats (Swift and Ellis, 1957), it is con-
ceivable that fish treated with polyphosphates may produce
fish products with improved binding prOperties. Fukazawa
et_al,,(l96lb) reported that the addition of polyphosphate

resulted in the greatest amount of extracted protein both

34

before and after storage increasing the binding quality
of sausage. Polyphosphates have been widely used in sea-
foods for many years. The phosphates commonly used today
are trisodium orthophosphate, tetrasodium pyrophosphate,
sodium tripolyphosphate and sodium metaphosphate. In

the processing of seafood products, sodium tripolyphos—
phate is the most commonly used component of polyphosphate
blends since it gives a desirable combination of proper—
ties such as improving texture and increasing water hold-
ing capacity (Brotsky and Everson, 1973). Improving the
binding properties of meat by adding phosphates was
studied by a number of investigators (Froning, 1965 a, b;
Fukazawa et_al.,l96la, b; Sato and Nakayama, 1971b).
Fukazawa et_al.,(l96lb) found that polyphosphates
increased the binding capacity of structural proteins

and thus provided a more stable emulsion. Polyphosphates
may do this as a result of the dissociation of actomyosin
into its components to increase the amount of available
myosin (Fukazawa et_al.,196lb, Yasui et_al.,1964).
Fukazawa et_al.,(l96lb) mentioned that in denatured myo-
fibrillar protein especially that of myosin A the effect
of phosphate on binding quality was due to increased myo-
sin A content rather than the quantity of extracted pro-
tein. According to Nakayama and Sato (1971b) improvements

in the binding quality of meat via addition of phosphate

35

resulted in increases in the amount of force to produce
the gel breaking point. This is due to decreases in gel
elasticity and actomyosin viscosity thereby preventing
formation of air pockets in stuffed sausage and producing

a stronger gel matrix.

Water Holding Capacity

 

The ability of meat, particularly processed meat,
to hold intrinsic or added moisture is an important fac-
tor in determining quality and product acceptability.
According to Hamm (1960) the tightly bound water of hydra-
tion in meat is not readily released except under condi-
tions resulting in extreme hydration such as protein
denaturation, rigor mortis, changes in muscle pH, masking
of charges of hydrophylic side chains, formation of salt
bonds between hydrophilic side groups and the presence of
salt. These factors also affect the water—holding capacity
of meat. Further study by Hamm (1975) showed that the
rheological behavior<mfbeef muscle homogenates was influ-
enced by time post-mortem, pH, addition of sodium chloride
and water. Hamm (1975) further indicated that water hold—
ing capacity and swelling, myosin and actin interaction
changes, and solubilization of myofibrillar proteins are
the main factors affecting rheological properties of minced
meat. Sherman (1961) reported that pH, time, temperature,

additive concentration and the ability of alkaline

36

phosphates to split the bond between myosin and actin are
factors influencing water binding capacity. Swift and
Ellis (1956) also reported that factors influencing
moisture retention of meat treated with phosphate salts
are those which also influence solubilization of muscle
proteins, such as temperature, time, ionic strength, and
pH of treatments.

Several investigators have found that pH affects
water retention and swelling of meat products. Minimum
hydration is near the isoelectric point of muscle (pH 5.5)
(Morse, 1955; Hamm and Deatherage, 1960; Miller §t_§l,y
1968), and hydration increases with increasing or decreas-
ing pH. Similarly, Mahon (1961) found that the volume of
cured meat was at a minimum at its isoelectric point
(pH 5—5.5). Heating raw meat at its isoelectric point
resulted in the lowest water holding capacity, while
heating meat with pH values higher or lower than its iso—
electric point resulted in higher water holding capacity
(Hamm and Deatherage, 1960). Hamm (1960) also found that
increasing the alkalinity of meat caused an increase in
water retention properties. Sato and Nakayama (1970)
reported that water holding capacity of batters was due
mainly to hydration of myofibrillar protin, and that it
decreased in proportion to decreases in breaking energy

and myofibrillar protein content after heating. These

.-J..|\.¢IL

 

,,.
A
d.

37

factors thus directly affect binding quality in finished
products.

Frozen storage of meat decreased its water hold-
ing capacity (Miller et_§l.,l968; Kato gt_al.,l974;
Bremner, 1977). Differences in the ability of pre-rigor,
post—rigor and frozen meat to hold intrinsic or added
moisture during cooking were studied by Miller et_al.,
(1968). These workers found that pre-rigor meat retained
more moisture than either post-rigor or frozen meat during
cooking. The water holding capacity of meat proteins is
also directly related to drip in freezing and thawing and
shrinkage on cooking. There is possibly a direct relation
between drip on freezing and thawing and shrinkage on
cooking (Wierbicki §t_al.,l957).

Coagulation of myofibrillar proteins during cook-
ing causes the release of juice because heat affects the
ability of meat to hold intrinsic or added moisture (Hamm,
1966). Most of the free water and that immobilized within
the network of protein filaments found to become able to
move freely and it is released from tissue during coagula—
tion of myofibrillar proteins. The mono— and multi-
molecular layers of water of hydration bound to polar
groups in muscle proteins are not influenced by heat

coagulation.

 

 

 

38

The pH of muscle tissue increases during heating
(Wierbicki et al., 1957; Bendall, 1964). This change may
be caused by charge changes or hydrogen bonding, or both,
within myofibrillar proteins, since pH changes occur in
both the tissue and myofibrils (Bendall and Pederson,
1962). Hamm (1960) studied the relationship between water
binding capacity and pH. Heating at 40°C to 45°C decreases
the water holding capacity of muscle tissue in its iso—
electric range only. The fact that muscle tissue does
not lose its water binding capacity at pH values 4.5 and
7.5 indicates that in heating up to 40°C the dehydration
and shrinkage cannot be caused by the formation of stable
cross linkages. t is probably that the release of juice
and shrinkage of tissue in this range of temperatures are
produced by unfolding of peptide chains. These changes
may cause the formation of new hydrogen bonds around iso-
electric point of actomyosin according to Hamm (1966).
According to Acton (1972b) binding strength increased as
internal temperature increased in the range of 35-82°C.
Acton found that there was either no cooking loss, or
less than 1% fluid loss at temperature up to 55°C, and
then greater loss occurred when internal temperature
ranged from 55-94°C. Cooking temperature has an impor-
tant effect on binding capacity in poultry meat (Vadehra
§E_gl.,l970). Stronger bonds were developed when meat

was heated for long periods at lower temperature.

39

The addition of phosphate and sodium chloride was
found to have an effect on the water holding capacity of
cooked meat (Mahon, 1961; Shults et_al.,l972; Brotsky and
Everson, 1973; Shults and Wierbicki, 1973; Schwartz and
Mandigo, 1976). Mahon (1961) concluded that salt concen-
tration and net pH adjustment was the key to obtaining
maximum water retention in meats. Wierbicki et_al.,(l957)
studied the influence of adding chlorides of sodium,
potassium, calcium, and magnesium to meat before heating
on its water holding capacity and found that these salts
increased water holding capacity, but shrinkage was less
with divalent cations than with monovalent cations. How—
ever, the combination of sodium and magnesium chloride
promoted the water holding capacity of cooked meat the
most. Swift and Ellis (1956) also showed that an increase
in water retention could be obtained by adding magnesium
chloride to meat. Nakayama and Sato (1971c) reported
that the presence of 0.6 M sodium chloride in solution
was necessary to produce high binding quality and high
water holding capacity in heated actomyosin gels. Sherman
(1961) studied the effect of sodium chloride, pyrophos-
phate and polyphosphate on the water retention of ground
pork at 0°C and 100°C. All three additives improved its
water holding capacity. There were positive correlations

between water holding capacity and anion absorption and

 

40

between pH with added phosphate and water holding capacity
at 0°C. Phosphates affected the amount of actomyosin

that entered solution at 0°C during aging. The greater
the concentration of actomyosin, the stronger the gel
formed at 100°C. This gel extends throughout the meat
mass and retains moisture. Swift and Ellis (1956)
reported that moisture retention of meat treated with
sodium chloride was linearly related to the ionic strength
of the solution applied. Water holding capacity of meat
increased more when sodium chloride was added to meat in
combination with pyrophosphate. Shults and Wierbicki
(1973) found shrinkage could be reduced by 9% when 0.5%
perphosphate was added, while the addition of 1.0% sodium
chloride combined with 0.5% pyrophosphate resulted in a
total shrinkage reduction of 18% in cooked meat. The
addition of 1.0% sodium chloride also decreased shrinkage
of meat. Shrinkage was reduced more, however, when salt
concentration was increased to the range of 3 to 5%.
Sodium chloride concentrations of more than 5% had an
adverse effect on meat shrinkage. A slight reduction in
meat shrinkage occurred when the concentration of sodium
tripolyphosphate was increased from 0.5 to 1.0%, while
Kena FP—28 (a mixture of sodium tripolyphosphate, tetra-
sodium pyrophosphate and sodium acid pyrophosphate) and

tetrasodium pyrophosphate showed reduction of meat

41

shrinkage in concentrations up to 1.0%. Further study
was done by Shults et_al.,(l976) on pork rolls and chops,
and these workers found cooking loss also was reduced by
the addition of sodium chloride and tripolyphosphate.
Also, Schwartz and Mandigo (1976) found decreases in
moisture loss during cooking when salt was added to meat.
Cooking loss decreased and juiciness increased as sodium
tripolyphosphate levels increased.

Seafood treated with polyphosphate is generally
more juicy and flavorsome on thawing and cooking, and
natural moisture is still firmly bound to the muscle pro-
tein with less aggregation of myofilaments (Buttkus,
1970). Sutton (1973) mentioned that using polyphosphates
in meat and fish products reduced cooking loss. It may
be concluded that (Bendall, 1954; Hellendoorm,1962; and
Shults and Wierbicki, 1973) pyrophosphate and tripoly-
phosphate in combination with sodium chloride showed the
highest increase in the water holding capacity of meat.
The greatest effect of perphosphate and sodium chloride
on moisture retention was due to the alkalinity of pyro-
phosphate solutions (Swift and Ellis, 1956).

Sutton (1973) studied the hydrolysis of triphos-
phate and pyrophosphate in muscle using P32 labelled tri-
phosphate and found that rapid hydrolysis occurred in
cod and beef muscle with the rate of hydrolysis approxi-

mately proportional to that protein concentration.

 

42

Several theories have been reported to explain
how hydration is affected by the addition of sodium
chloride or phosphates separately or in combination.
According to Mahon (1961), the addition of 3% sodium
chloride caused a detectable drop in pH. This may be
explained as follows. On the acidic side of the iso—
electric point proteins were positively charged and Cl-
ions were absorbed. While on the basic side of the
isoelectric point there was a negative charge and Na+ ions
were absorbed with the libration of some H+ ions causing
a drop in pH. Fish meat is normally on the alkaline side
of its isoelectric point (pH 5-5.5) and consequently
carries a net negative charge. Adding sodium chloride to
negatively charged protein reduces the net negative charge
to zero (Isoelectric point of meat), at which stage there
is minimum hydration of muscle. Adding more sodium chlor-
ide causes the adsorption of more Na+ ions and produce a
net positive charge on protein. This positive charge
reaches a maximum when 4 to 5% sodium chloride has been
added, and at this stage the hydration of meat is also
at a maximum. If yet more sodium chloride is added, C1-
ions will be absorbed and the net positive protein charge
is again reduced to zero. In the region of 10% salt the
hydration of meat will be at a minimum. Experimentally,

the maximum water holding capacity of meat has been

 

 

43

reached in the presence of 4% sodium chloride and 0.5%
tripolyphosphate (Mahon, 1961).

Hamm (1971) reported the different ways that
polyphosphate components affect meat hydration: (1) Rais-
ing pH of meat increases the negative charge on myofibril-
lar proteins. Negative charges on adjacent myofilaments
repel each other allowing more water to enter the gel
structure, (2) Increasing ionic strength up to certain
limits causes a repulsion of adjacent molecules with
resultant increases in hydration, (3) polyphosphates can
break certain of the cross-links between myofibrillar
proteins. Such as the calcium bonds between adjacent
myofilaments also, pyrophosphate can directly effect the
resolution.ofactomyosin into actin and myosin.

Several methods for measuring water holding
capacity of muscle foods have been developed. Some
methods measure the area occupied by moisture on filter
paper when meat samples are subjected to standardized
pressure for a specified time (Wiebicki and Deatherage,
1958; Karmas and Turk, 1975). Other workers have meas-
ured the amount of juice extracted from centrifuged meat
(Sherman, 1961; Bremner, 1977). None of these methods
give any physical-chemical quantity which identifies

immobilized part of the water.

 

44

Labuza and Lewicki (1978) developed a new method
of measuring water holding capacity of moist foods and
gels which can be easily deformed under pressure. These
authors placed the food to be measured in a special cup
layered with filter paper of known water content and
stored it at 6°C for 72 hr. A standard soil test cell
was used to measure the water lost from the filter paper

as a function of applied pressure.

Texture

According to Jowitt (1974), texture is an attri—
bute of a substance caused by a combination of physical
prOperties (including size and shape, number, nature, and
conformation of constituent structural elements) and per-
ceived by the senses of touch (including kinaesthesis and
mouth feel), sight and hearing. The texture profile of a
product can be described by studying its initial hardness,
cohesiveness, elasticity, hardness of cross-section, ease
of swallowing, moisture release, coarseness, and skin
texture (Webb et al.,l975). These researchers also men—
tioned that no instrumental method is able clearly to
assess all texture characteristics of a food product
accurately indicating the individual characteristics
contributing to the total textural properties, as per—

ceived by human senses. DeMan (1975) reported that

 

45

texture is not only a property of a food, but that it
depends on the person examining or consuming the food.
In this respect, texture is as difficult to evaluate
objectively as are the characteristics color and flavor.

Several types of instruments have been used to
evaluate the texture of foods, however. The Instron
Universal Testing Machine was applied to meat texture by
several workers, e.g., Lee and Toledo (1976), Webb et al.,
(1976). This type of equipment is becoming increasingly
popular with investigators since it has served a number
of different purposes. By simply interchanging different
test cells one can measure tensile strength, compress-
ability, shearing strength, elasticity, etc. The Kramer
Shear Press also has been used for meat texture measure-
ments.

Several investigators have studied factors asso—
ciated with meat texture, starting from the muscle com-
position of the intact animal and continuing through
slaughter, rigor mortis and aging to cooking. Lee and
Toledo (1976) indicated that amount of time spent in
comminution, addition of sodium chloride or sodium chlor-
ide and tripolyphosphates, effect of mechanical deboning,
temperature of cooking, and type of heating medium used
are the factors affecting textural characteristics of

fish sausage. Simon et a1” (1965) found that the firmness

 

46

of frankfurter products increased as meat protein content
increased. In terms of developing the desired texture of
comminuted products utilizing muscle proteins, fractiona-
tion of the various types of protein has been classified
as an important functional property (Webb, 1974). The
texture of cooked fish muscle depends to a large extent
on its pH (Cowie and Little, 1966; Love et_al.,l974).

It has been reported that storage temperature and the
process of freezing affect meat texture (Heen and Karsti,
1965). Frozen fish is utilized to a large extent in
canned fish today. Heen and Karsti (1965) indicate that
undesirable changes, such as lipid oxidation and texture
changes which may occur in canned salmon prepared from
frozen fish, may be minimized or eliminated by careful
handling of the frozen fish and limiting the storage
period. Dyer and Morton (1956) showed an increase in
toughness of fish flesh after frozen storage. This tough-
ness may be due to a decrease in extractable actomyosin
content. Ravesi and Anderson (1969), for example, found
a correlation between the content of soluble protein and
the texture of frozen fish muscle. Tokiwa and Matsumiya
(1969) also found a relationship between fragmentation of
myofibrils and changes in the texture of fish meat. Frag-
mentation was related directly to the quality of fish

flesh and affected greatly by storage conditions.

47

Freezing and storage at —15°C or -28°C caused myofibrillar
breakdown into small fragments more rapidly than storage
at 0°C. According to Tran (1975) toughening of fish
flesh during frozen storage could be prevented by treat-
ing the fish with sodium pyruvate.

Grabowska and Sikorski (1973) studied the differ-
ence between the texture of sausage made from fresh
mechanically deboned Baltic cod and that made from frozen
mechanically deboned Baltic cod. Sausages made from
fresh cod yielded an acceptable finished product texture,
while that made from frozen flesh had unappealing grainy
texture. Moreover, separation of water and fat occurred
in the finished product. Such poor texture character-
istics were not improved even with the addition of carbo-
hydrates, sodium chloride, and phosphates, separately or
in combination. This lack of improvement may have been
due to a decrease in protein solubility. Texture and
binding of comminuted fish sausages were found to follow
a pattern similar to solubility changes in myofibrillar
proteins in minced frozen fish. Hashimoto et_al.,(l959)
found that the'elasticity of products decreased mainly
in direct proportion to and in correspondence with the
denaturation of myosin B.

Webb et_al.,(l975) reported that temperature and

length of time spent chopping affected frankfurter texture

48

and solubility during cooking. Lee and Toledo (1976) also
studied the effect of chopping on the texture character-
istics of fish sausage. The texture of cooked comminuted
products was improved by up to 12 minutes of chopping;
however, longer periods resulted in a slight decrease in
textural quality. The addition of sodium chloride and
polyphosphates separately or in combination also improved
texture characteristics. Two percent salt in the product
increased shearing strength, compressibility and puncture
strengths. Addition of polyphosphates increased solu-
bilization of muscle protein and improved water binding
and texture. In general, the mechanical strength of
products was greatest with no additions, and declined
progressively with the addition of sodium chloride and a
sodium chloride—pyrophosphate mixture (Lee and Toledo,
1976).

Soy protein isolate improved the texture of meat-
balls as determined by instrumental and sensory evalua-
tion (Hermansson, 1975). Lee and Toledo (1976) reported
the same results indicating that soy protein isolate
improved the texture of fish products. Textured soy
flour also has been used in the formulation of acceptable
mullet sausages (Daley and Deng, 1978; Daley §£_al.,l978).

It is known that certain changes occur in muscle

when it is subjected to thermal energy, as mentioned

 

49

earlier. These changes affect meat texture during cook-
ing. Texture is also influenced by the type of cooking.
Lee and Toledo (1976) noted that cooking for long periods
at temperatures below 80°C caused weakening of texture

in smokehouse cooked products. Firmness of cooked com—
minuted fish muscle was generally greater in steamed
products, less in smokehouse cooked products. Simon et
al.,(l965) found that firmness was increased by reducing
the relative humidity in the smokehouse during processing.

Functional Properties of Soy Protein Isolate
in Meat Systems

 

 

In the American meat industry, isolated soy pro-
tein is a widely used additive in sausage batters. Soy
protein isolate has properties similar to those of meat
proteins (Brown, 1972), and the use of soybean products
in various foods is fashionable from the standpoint of the
world food economy and dietetics. For example, large
amounts of food grade soybean products are used in fish
product industries in Japan (Yasumatsu et_al.,l972b).

Soy protein isolates are the most refined forms of
soybean proteins. They contain 90% or more protein on a
dry basis and have a 2-4% ash content (Meyer, 1966).

Many workers have investigated the functional properties
of soy protein isolates utilized in different foods.

Functional properties such as water and fat absorption,

50

oil emulsification, viscosity and gel formation are the
most important characteristics of soy protein isolates
as they are used in ground and canned meat formulations.

Wolf (1970) found that soybean proteins are a
mixture of components with molecular weights ranging
from 8000 to 600,000 and that the major soybean proteins
are globulins which are insoluble at their isoelectric
point (near pH 4.5). However, they are soluble in water
or dilute salt solution at pH values above or below the
isoelectric point (Wolf, 1969). The addition of soy
protein isolate to a variety of foods supplies desirable
functional properties, such as emulsifiability, ability
to absorb fat, moisture holding capacity, and thickening
and foaming ability (Wolf, 1970).

Some workers have claimed that extenders such as
soy protein isolate increase emulsifying characteristics
in batters and improve binding properties (Inklaar and
Fortuin, 1969). This is questionable, but they may absorb
water not bound by the protein matrix, and reduce shrink-
age or water loss (Pearson, et_al.,l965; Van Eerd, 1971).

Soy protein isolates are prepared by extraction
from undenatured flakes or flour with water near neutral
pH, and precipitation of the proteins by adjusting the
extract to the protein isoelectric pH. After isoelectric

precipitation the isolates are no longer completely

 

 

51

soluble in solutions with an ionic strength of 0.5 at

pH 7.6. Wolf (1970) found that insolubilization may be
due to a portion of the proteins being held together by
disulfide bonds as aggregates, and further aggregate
formation during isoelectric precipitation. He reported
that this disulfide crosslinks must be broken to resolu—
bilize these proteins.

Solubility is an important physical pr0perty of
soy proteins, and is required if their desired functional
properties are to be achieved. Oil seed proteins in
general are a less soluble form of protein than animal
proteins (Hagenmaier, 1972). Solubility of native soy—
bean globulins strongly depends on pH (Wolf, 1970;
Hagenmaier, 1972), temperature (Wolf, 1969) and salt
content (Anderson e£;ald,l973, Van Megen, 1974). Even at
the isoelectric point, soy protein can be dissolved
easily up to very high concentrations with ionic strengths
exceeding 0.7 with sodium chloride (Van Megen, 1974).
However, Hermansson and Akesson (1975a) found a marked
decrease in solubility in meat systems containing the soy
protein isolate Promide-D, when sodium chloride was added.
Solubility in this case was positively correlated to
moisture loss. Pearson et_al., (1965) found that a soy
sodium proteinate was less soluble and was a poor emulsi—

fier at the pH of meat.

 

52

Gel—forming ability is one of the important func-
tional properties of isolated soy protein (Yasumatsu
et al.,l972a), and the most important property of added
proteins (Hermansson, 1975). Soy proteins form a gel
which act as a matrix to hold moisture and fat (Wolf,
1970). The gel forming ability of soy proteins is
dependent on temperature, length of time of heating,
ionic strength, pH and protein concentration. Hermansson
(1972) reported that when Promide-D solution (10%) was
heated gelation occurred and strong gels wereformed. Also
water could not be pressed out of these gels even under
very high pressure. The gel strength of Promide-D
increased to a maximum with increasing temperature up to
80°C thendecreased as the temperature was raised above
this point. Gel strength also decreased rapidly with
increasing ionic strength. At ionic strength of 0.5 gels
were no longer formed.

The important properties of edible soybean pro—
teins with respect to their use in food systems were
summarized by Circle et_31.,(1964). Rate of gelling and
gel firmness of a commercial edible isolated soy pro—
teinate are dependent primarily on temperature, length of
time of heating and protein concentration. In concen-
trations of 8-14%, gels are formed within 10 to 30 minutes

at 70-100°C, but are disrupted if overheated to 125°C.

 

 

53

At concentrations at 16 to 17% the gels are firm, resil-
ient and self-supporting and are less succeptible to
disruption by overheating.

Several sodium salt additives may affect the heat-
gelation of soy proteinate (Circle et al.,l964). At low
concentrations (0.05 to 0.1%) these salts have a rela-
tively minor effect on the viscosity of unheated and
heated dispersions, but at higher concentrations (0.5 to
1%) the effect of the salts is to lower the viscosity of
unheated dispersions and to raise that of the heated
ones. The situation is different for the two reducing
agents, sodium sulfite and cysteine, which markedly
reduce the apparent viscosities of both unheated dis-
persions and heated gels. According to Yasumatsu, et al.,
(1972b), the additional effects of soybean proteins in
fish products may be influenced kn! intrinsic amounts of
protein and not by the amounts of fiber and fat in the
soybean protein isolates. Saio et al.,(l974) found that
the expansion ratio of gels was increased as temperature
increased and that their maximum expansion characteris-
tics were obtained around 2.5 to 8.5 pH.

Hermanssonand.Akesson (1975b) showed that NaCl
had a marked influence on both meat systems and the func-
tional properties of added protein. In the absence of

sodium chloride solubility was well correlated with

 

54

higher swelling, viscosity and gel strength. A reduction
in swelling and gel strength was correlated with a
decrease in protein solubility caused by protein associa-
tion in the presence of salt. Hermanson and Akesson
(1975b) found that Promine-D gels were smooth, and that
it was impossible to press out water even under very high
centrifugation forces.

The problems involved in the gelation of soybean
proteins were discussed by Catsimpoolas and Meyer (1970)
who found that the highest viscosity values were obtained
at a neutral or mildly alkaline pH. An increase or a
decrease in pH produced weaker gels. During heating at
65°, 70°, 75° and 80°C, the progel viscosity first
increased rapidly with time and then continued to increase
at a lower rate. Catsimpoolas and Meyer (1970) also
studied the effect of ionic strength on gelation. At
temperatures above 70°C the viscosities of progels and
gels decreased with increasing concentrations of sodium
chloride.

Swelling of soy proteins is strongly dependent
on pH and ionic strength. Hermansson (1972) found a
good positive correlation between gel strength and swell-
ing ability, especially for soybean protein isolate. The
addition of salt caused about a 60% decrease in solubility

of Promide-D. This is understandable, as the amount of

 

55

water taken up decreased with ionic strength. Swelling
increased as pH increased in the range of 4-10. Further
study by Hermansson (1975) indicated that Bromine—D had
a high swelling capacity. Hermansson and Akesson (1975a)
obtained positive correlations between solubility, swell-
ing, viscosity, and moisture loss of model meat systems.
Meat systems in which part of the protein is
replaced by soy protein isolates may show complex behav-
ior with respect to water—binding properties. Fleming
et al.,(l974) reported that the water absorption and vis-
costiy characteristics of untreated slurries of soy flour
and concentrate were lower than those of the soy protein
isolate because water absorption and viscosities tended to
increase with the concentration of protein in a product.
Judge et al.,(l974) indicated that the addition of soy
proteins to beef patties reduced shrinkage on cooking.
Brown (1972) stated that Cook et al.,(l969) pointed out
that a meat—soy system will hold approximately 100% more
free water than a pure meat system to which excess water
was added. Yasumatsa et al.,(l972b) found that the
desirable effect of soy protein products in Kamaboko is
their ability to remain as particles which can hold water.
Cooked patties containing textured soy protein retained
more moisture and less fat than all beef patties (Seideman

et al., 1977). Karmas and Turk (1976) studied the water

 

56

binding properties of cooked fish containing various pro-
tein additives. The addition of soy protein to cooked
fish increased its water binding capacity, and this capac-
ity was not affected by sodium, calcium, and potassium
associated with the soy protein. Karmas and Turk (1976)
also indicated that fresh samples of soy protein had
significantly better water binding capacity than old
samples. Beef rolls containing 3% soy protein isolate
retained more moisture which resulted in a higher cooked
yield. Cooked yield of meat is closely associated with

water binding capacity (Moore et al.,l976).

 

MATERIALS AND METHODS

Materials

 

Fish

 

Approximately 500 lbs. of fresh water sucker from
Lake Huron (Saginaw Bay) were obtained for study in July
of 1978. Roughly 50 percent of the fish were of the Silver

Redhorse variety (Moxostoma anisurum), while the other half

 

were White Suckers (Catostomus commersoni).

 

Fish were removed from nets and either kept alive
in oxygenated tank for protein fractionation experiment
or stored in wooden boxes in an ice house for one day
before collection. They were then transported in ice to
the meat laboratory at Michigan State University where
they were held over night at 3°C and processed the follow-

ing morning.

Additives and Spices

 

All additives used in this investigation were food
grade materials. These consisted of "Cenpro-P-4540" soy
protein isolate (Central Soya Co., Ft. Wayne, Ind. 46802),
degermenated and enriched white cornmeal (Quaker Oats Co.,
Chicago, Ill. 60654), iodized sodium chloride (Hardy Salt
Co., St. Louis, Mo. 63166), granular sodium tripolyphos-
phate (FMC Corp., Carteret, N.J. USA), 99% pure

57

 

58

monosodium glutamate (Accent International, Inc., West-
wood, Mass. 02090) and sodium ascorbate of 91.7% purity
(Permacurate-Roche Chemical Division, Hoffman-La Roche

Inc., Nutley, N.J. 07110.

The spices employed were powdered onion, garlic,
ground white pepper, allspice and ginger (Asmus Spice Co.,
Detroit, Mi. 48213) and ground paprika (McCormick & Co.,
Inc., Baltimore, Md. 21202). A smoke flavor was imparted
to products by adding condensed liquid snwke (E. H. Wright
Co., Kansas City, MO. 64108).

Preparation of Mechanically
Deboned Fish Flesh

 

 

Fish were headed-gutted, rinsed under running
cold water, Split length wise, and passed through a
Bibun mechanical deboning machine (Type SDX 13, Bibun
Co., Fukuyama Hiroshima, Japan) equipped with a 5mm hole
size drum. The minced flesh was then passed through the
meat separator a second time to remove as many bone and
scale fragments as possible. Yields of both the dressing
and mechanical deboning operations were calculated.

Samples of minced flesh were collected after the
second pass and analyzed for moisture, fat, protein, and
nonprotein nitrogen. The sucker flesh was then vacuum
packaged in 5 and 20 1b. lots in Cryovac (polyvinylidene
chloride) bags which were blast frozen and stored at -29°C.

The frozen fish was thawed in a 3°C cooler as needed.

 

59

Methods of Analysis

 

Protein Fractionation

 

Protein fractionation was performed according to
a modification of the method of Helander (1957). All
samples were extracted twice in duplicate. Extractions
were carried out at 2 to 3°C with cold extracting solu-

tions.

Sarcoplasmic protein.--Five grams of ground pre—

 

rigor, chilled or frozen sucker muscle were weighed into
a 125 ml beaker. Fifty m1 of 0.015 M potassium phosphate
buffer (pH 7.4) were added to the sample which was then
extracted on a magnetic stirrer for 3 hrs. This was
followed by centrifugation at 1400 xg for 20 min. The
supernatant was filtered through eight layers of cheese
cloth into 100 ml graduated cylinder and the residue
resuspended in 50 ml of potassium phosphate buffer for

a repeat of the procedure described above. The volume
of the combined supernatants was recorded. Sarcoplasmic
protein nitrogen was determined in duplicate 15 m1

samples by the nicrokjeldahl method (A.O.A.C., 1975).

Nonprotein Nitrogen.--Fifteen m1 duplicate samples

 

were pipetted from the sarcoplasmic protein supernatant
into 50 m1 polyethylene centrifuged tubes, into which 5 m1

of 10% (W/V) trichloroacetic acid were added. The

 

60

mixture was allowed to stand for 2 to 4 hrs. at 3°C and
was then centrifuged at 12,000 X9 for 20 mins. The
supernatant was carefully decanted into kjeldahl flasks
for nonprotein nitrogen determinatiOn by the microkjeldahl

method.

Myofibrillar protein.--The residue from the sarco—

 

plasmic protein extraction was suspended in 50 m1 of 1.1 M
potassium iodide (KI) phosphate buffer (pH 7.4) and
extracted on a magnetic stirrer for 3 hrs, followed by
centrifugation at 1400}{g;for 20 mins. The supernatant
was filtered through 8 layers of cheese cloth into a 100
ml graduated cylinder. The residue was resuspended in

50 m1 of 1.1 M KI phosphate buffer and the procedure as
described above repeated. The volume of the combined
supernatants was recorded. Duplicate fifteen m1 samples
of the supernatant were used to determine the amount of
myofibrillar protein nitrogen by the microkjeldahl method.
Extraction of Soluble Compon-

ents from Mechanically

Deboned Frozen Fish
(MDFF)

 

 

 

Total soluble, salt soluble, and water soluble
proteins as well as nonprotein nitrogen, were extracted
by a modification of the method described by Trautman
(1964). Extractions were carried out two times at 3°C

using cold extracting solutions.

61

One hundred grams of fish flesh, with or without
soy protein isolate were blended with either six hundred
grams of plain water or 600 grams of water mixed with
sodium chloride, sodium tripolyphosphate or a combination
of these in varying levels (Table 1). This was done with
a Haring blender set at low speed (approximately 2300 rpm)
for 90 seconds. The homogenate was centrifuged at 9500
X gfor 10 minutes in an automatic refrigerator centrifuge
(Sorvall Type RC2B, Rotor GSA--5008, Ivan Sorvall Inc.,
Norwalk, Conn.). The supernatant (soluble components)
was then decanted and filtered through 8 layers of cheese
cloth.

Fractionation of Soluble Com-

ponents from Mechanically
Deboned Frozen Fish

 

 

 

Duplicate 15 m1 samples of the filtered super-
natant were used for the determination of nonprotein nitro—
gen as previously described. The remaining filtered
extract was divided into two parts to obtain the follow-

ing components.

Soluble protein extract.--One part of the fil—

 

tered extract was dialyzed against the same extracted
buffer so that water and salt soluble proteins were
retained in solution while dialyzable nonprotein nitrogen

was removed. The volume of the dialyzed extracts was

 

62

TABLE 1.--Treatment Design and Formulations for the
Measurement of Various Protein Fractions,
pH, Swelling, and Gel-Forming Ability

 

 

 

Code No.

A-l No sodium chloride (Nacl), No sodium Tripoly-

phosphate (STPP), and No soy protein Isolate
ISPI)

A-2 No NaCl, No STPP, 2% SPI

A-3 No NaCl, No STPP, 4% SPI

A—4 No NaCl, 0.225% STPP, No SPI
A-5 NO NaCl, 0.225% STPP, 2% SPI
A-6 No NaCl, 0.225% STPP, 4% SPI
A-7 No NaCl, 0.45% STPP, No SPI
A-8 No NaCl, 0.45% STPP, 2% SPI
A-9 No NaCl, 0.45% STPP, 4% SP1
B—l 3.0% NaCl, No STPP, No SPI

B-2 3.0% NaCl, No STPP, 2% SPI

B-3 3.0% NaCl, NO STPP, 4% SPI

B—4 3.0% NaCl, 0.255% STPP, No SPI
B-S 3.0% NaCl, 0.225% STPP, 2% SPI
B—6 3.0% NaCl, 0.255% STPP, 4% SPI
B—7 3.0% NaCl, 0.45% STPP, No SPI
B-8 3.0% NaCl, 0.45% STPP, 2% SPI
B-9 3.0% NaCl, 0.45% STPP, 4% SPI

63

TABLE l.--Continued

 

 

Code No. Treatment and Formulation
C-l 3.6% NaCl, No STPP, No SPI

C-2 3.6% NaCl, NO STPP, 2% SPI

C-3 3.6% NaCl, NO STPP, 4% SP1

C-4 3.6% NaCl, 0.225% STPP, No SPI

C-S 3.6% NaCl, 0.225% STPP, 2% SPI

C-6 3.6% NaCl, 0.225% STPP, 4% SPI

C-7 3.6% NaCl, 0.45% STPP, NO SPI

C-8 3.6% NaCl, 0.45% STPP, 2% SPI

C-9 3.6% NaCl, 0.45% STPP, 4% SPI

 

 

64

recorded. The total soluble protein present in duplicate

5 m1 samples was determined by the microkjeldahl method.
Results for soluble protein nitrogen were

expressed as a percentage of total protein nitrogen by

using the formula:

0\0

Extractable Protein Nitrogen =

Extractable N — Nonprotein N
X 100

 

Total N — Nonprotein N

Water-soluble protein.--The second part of the

 

filtered extract was dialyzed over night against 0.05 M
NaCl to remove nonprotein nitrogen and to insolubilize
the salt soluble protein. This was followed by centri-
fugation for 10 mins at 9500 Xg. The supernatant, con-
taining the water soluble proteins, was then dialyzed
against the same extraction buffer fl1r24 hours at 3°C.
The volume of the dialyzed extracts was recorded. The
total soluble protein present in duplicate 10 ml samples

was determined by the microkjeldahl method.

Salt—soluble protein.--The residue from the cen—

 

trifuged 0.05 M NaCl dialyzate containing the salt soluble
protein was resuspended in 0.05 M sodium chloride and
centrifuged again at 9500}{g for 10 mins. to remove any
contaminating water-soluble protein. The resuspention

in 0.05 M sodium chloride was repeated yet another time,

 

65

followed by centrifugation, after which the residue was
solubilized in 1.2 M sodium chloride and dialyzed against
the same extracted buffer for 24 hrs. at 3°C.

Sodium Dodecylsulphate (SDS)

Polyacrylamide Gel Electro-
phoresis

 

 

Protein fractions of fresh fish and all total
soluble protein fractions extracted from mechanically
deboned frozen fish were compared using the technique
described by Weber and Osborne (1969). In principle, the
use of sodium dodecylsulfate (SDS) in combination with
disulfide reducing agents (e.g., B—mercaptoethanol)
causes the disruption of forces maintaining the secondary
and tertiary structure of proteins resolving them into
single polypeptide chains. Because of this and the fact
that SDS combines with all proteins in a constant ratio,
imparting a uniform charge, electrophoretic mobility is
solely a function of protein size or molecular weight
(Reynolds and Tanford, 1970).

The total extracted protein was combined with
glycerol in a 50:50 ratio and stored at -80°C until
used. Glycerol is able to attach a considerable amount
of water to itself, rendering the latter unavailable for
ice formation (Lovelock, 1954). Bound water can still
act as a solvent for cell salts, however, so that the

concentration of cell salts in the frozen material is

 

66

kept low. Protein samples taken from the glycerol mixture
were prepared for electrophoresis by heating solutions
containing 20 ug of total protein ranging from 40 ml for
higher protein content samples to 80 pl for lower protein
content sample with 80 n1 oftracking dye, composed of 1.0%
SDS, 0.05 M Tris HCl at pH 7.1, 0.5% mercaptoetanol, 2%
glycerol, and 0.01% Pyronin Y, in an 80°C water bath for
30 mins.

Ten percent polyacrylamide gels were prepared for
a stock solution composed of 62.5g of electrophoresis
grade acrylamide and 0.625g of N, N methylenebisacrylamide
(0.25%) in 250 m1 of distilled water. Ten m1 of this
stock solution were combined with 5 m1 of tris—glycine
stock solution composed of 0.5 M Trisma and 1.5 M glycine.
Added to this were 1.25 ml of glycerol, 1 ml of 2.5% sodium
dodecylsulfate, 10 pl of N, N, N1, N1, tetramethylenedi-
amine (TEMED) and 6.75 ml of H20. Finally, 1 m1 of freshly
made 1% ammonium persulfate was added to the gel solution
to initiate polymerization, and the solution was deaerated
using a water suction pump. The gel solution was immedi-
ately poured into glass running tubes (5mm I.D x 10 cm)
which were held precisely vertical and then left for
polymerization.

The tubes were placed in an electrophoesis appara-

tus (BIO-RAD, Model 150A, Gel Electrophoresis cell).

 

67

Samples containing 20 mg of total soluble protein were
layered on the exposed upper surface of a gel. Chamber
buffer, composed of 100 m1 of 20 M Tris-Glycine, 40 ml

of 2.5% SDS and 860 ml water per liter, was carefully
layered over the sample. Lower and upper chambers were
filled with the same chamber buffer and a constant current
applied at 3 mA per gel. The SDS treated proteins migrated
toward the positive electrode in the lower chamber.

Approximately 18 hrs. were required for the com-
pletion of a run which was terminated when the tracking
dye (red color) reached 1/2 cm away from the end of a gel.
Gels were removed from running tubes by squirting water
from a syringe between the gel and the tube wall. They
were thenimmersed in staining solution (0.05 g comassie
brilliant blue, 75 m1 methanol, 65 ml water and 10.5 ml
glacial acetic acid) for 16 hrs., followed by destaining
( % methanol, 7.5% glacial acetic acid) for 4 days.

The relative amounts of myosin heavy chains were
estimated by measuring areas under corresponding curves
produced by scanning the gels. Gels were scanned at a
wavelength of 550 nm using scan speed of 1/2 cm per min.
on a Beckman Spectrophotometer (Model 2400) equipped with
a Gilford gel scanner (Model 2520). A horizontal
cuvette holder was attached to the scanner. A Hewlett

Packard Intigrator (Model 3380) was programmed to correct

 

 

68

for baseline drift by re-establishing baseline before and
after each peak. Chart speed was adjusted at 2cm/min.
Startckfljuzand slope sensitivity were adjusted at 0.25
min. and 3.00 mv/min. respectively.

A standard curve (Appendix 1) was prepared in
which areas of myosin heavy chain bands were plotted
against the total protein extracted from mechanically
deboned frozen fish samples. While this standard curve
loses linearity at areas above about 500,000 total area,
linearity of area could be achieved using mechanically
deboned frozen fish protein samples containing up to 30

pg total soluble protein.

Total Nitrogen

 

Total nitrogen was determined on approximately
0.5 grams of muscle by the microkjeldahl method (A.O.A.C.,

1975).

Kjeldahl Method

 

The American Instrument Co. microkjeldahl method
(1961) was used to determine both the concentration of
protein in the different protein fractions and the amount

of nonprotein nitrogen in the different treatments.

Moisture Determination
Approximately 5 grams of muscle sample were

weighed into previously dried and tared aluminum dishes

 

 

69

and dried in a 100°C air convection oven for 24 hrs.
Weight loss was recorded after cooling the samples in a
desicator and moisture was calculated as percentage of
fresh tissue (A.O.A.C., 1975). Dried samples were saved

for either extract determination.

Ether Extraction

 

The fat content of the fish muscle was determined
by extracting dried samples with anhydrous ether for 4 hrs.
on Goldfisch apparatus, as described by A.O.A.C (1975).

Percentage fat was calculated on a fresh tissue basis.

Gel-Forming Measurement

 

Gel formation was determined using the least
concentration endpoint (LCE) method reported by Trautman
(1966b). Ten m1 of total extracted protein containing
different protein concentrations were heated in an 80°C
water bath for 10 mins. and then transferred to an ice
water bath for 1 hr. The strength of the coagulation was
evaluated by inverting each tube, the least concentration
endpoint being the lowest protein concentration forming a
stable gel that remained in the inverted tube. Duplicate
samples for each concentration of total protein were used
to determine LCE, and all treatment measurements were

repeated an additional time in duplicate.

 

 

7O

Swelling Measurement

 

Muscle swelling was determined by blending 25
grams of muscle, with or without soy protein isolate,with
100 m1 of plain deionized water or with 100 m1 of water
containing sodium chloride, sodium tripolyphosphate or a
combination of these in varying levels (Table 1). This
was done with a waring blender set at low speed for
90 secs. Two homogenized 35 grams samples were weighed
into two 50 ml centrifuge tubes and centrifuged at 480
x g for 15 mins. Supernatant volume was recorded.

Percent swelling was determined according to the

following formula reported by Wierbicki et a1. (1963).

% Swelling = 400 - (14.3 X S)
where:
S = Supernatant in ml. (1 ml = 1 gram)
7 grams = Weight of meat in the slurry
(35-7) grams = amount of added weight in the slurry

[(35-7) - S]/7 X 100

[35-7
7

% Swelling

S
7] 100

S
[4 - 7] 100

400 - 14.298

 

71

pH Measurement

 

The pH of total extracted proteins was measured
with a pH meter (Beckman, Model 35600 Digital pH meter),

using a glass electrode.

Chemicals

 

All chemicals used in this investigation were of

reagent grade unless otherwise specified.

Smokehouse Shrinkage

 

The difference in weight of the stuffed product
before and after smokehouse cooking was calculated as
smokehouse shrinkage. Results are presented as percent

cooking loss.

Baking Loss

 

Baking loss was calculated by the weight differ-
ence of filled cans before and after baking. Results are

presented as percent baking loss.

Measurement of Water Holding

Capacity
Water holding capacity of the cooked fish flesh

 

was determined using the centrifuge technique of Bremner

(1974). Both the smoked sausages and canned fish product
were allowed to warm to approximately 25°C before testing.
Cylindrical shapes were prepared by coring the center of

products, providing a uniform cross section 13mm in

 

 

72

diameter. Ten grams of the core were weighed into 15 ml
glass CorexR (No. 844l)centrifuge tubes and centrifuged

at 39100 Xg for 60 minutes. The supernatant was drained
and the tubes inverted on paper toweling for 15 mins. to
allow complete drainage of the remaining fluid (Zapata,
1978). The weight of the meat residue was recorded and
water-holding capacity calculated according to the follow-

ing formula.

Water-holding Capacity = 100 x weight of meat

residue/10 g

Eight replicated measurements were made on each treatment

for both products (four replicate measurements per trial).

Internal Texture Measurement

 

The texture of both products, measured as shear
force, was determined on the Instron Universal Testing
Machine (Model TTBB, Instron Corp., Canton, Mass.). Sample
preparation, Fish Sausages: The edible casing and firm
surface layer under the casing were removed in order that
only resistance (i.e., internal texture) to the central
mass of sausage was measured as the cell travelled through.
The diameter of the skinless sausage was recorded. The
sausage was then cut into a 10cm long cylinder and sheared
using a compression load cell of the 1 to 50 kg range. A

shear cell was attached to the upper moving fixture.

73

Drive speed and chart speed were adjusted at 20cm/minute
and 5 cm/minute, reSpectively. The resistance force met
by the cell was expressed as Kg-f per cm of sausage.
TWelwereplicated measurements were done on products from
each treatment (smg< replicate measurements per trial).
Sample preparation, Canned Minced Fish: Canned
products were cored in the center to achieve a uniform,
cylindrical shaped sample, 18mm in diameter. Cylinders
10cm long were sheared across the width using the Instron

as described earlier.

Processing Procedures

I. Smoked Fish Sausages.--The following ingre-

 

dients were mixed at medium speed for 8 mins. in a Kitchen
Aid Food Preparer (Model A-200, Hobart Mfg. Co., Troy,
Ohio) with a paddle attachment (Table 2): Mechanically
Deboned Frozen Fish, sodium chloride, sodium tripolyphos-
phate, monosodium glutamate, sodium ascorbate, condensed
smoke and spices, either with or without soy protein iso-
late and white corn meal or the combination of these two
included. Spices consisted of 0.10% garlic powder,

0.30% white pepper powder, 0.30% paprika pepper powder and
0.05% allspice. Hydrogenated vegetable oil was added and
the mixture blended at low speed for 7 mins. more. The

fish paste was stuffed into collagen casings (Brechteen,

74

 

 

 

ing additives and spices are present:
0.1% monosodium glutamate,
and the spices.

tripolyphosphate,
ascorbate,

In all formulations

0.5%

condensed smoke,

TABLE 2.--Design and Formulation of Smoked Fish
Sausage Treatments
ggde Treatment and Formulationsl
A 97% Mechanically Deboned Frozen Fish (MDFF),
2% sodium chloride (Nacl), No hydrogenated
vegetable oil (HVO).
B 88% MDFF, 3.0% Nacl and HVO
C 84% MDFF, 3.0% Nacl, 4% soy protein isolate
(SPI) and 8% HVO
D 84% MDFF, 3.0% Nacl, 4% white corn meal and
8% HVO
E 80% MDFF, 3.0% Nacl, 4% SPI, 4% white corn meal
and 8% HVO
l

(A, B, C. D, E) the follow-

0.45% sodium
0.05% sodium

 

75

Box 411, Mt. Clemens, Mich.) 35mm in diameter which were
then linked.

Sausages were cooked in an Elek—Trol laboratory
Smokehouse (Drying Systems Inc., Chicago, Ill.) according
to the schedule shown in Table 3. The cooking process

was followed by a 10 min. cold water shower.

TABLE 3.--Smokehouse Cooking Schedule

 

Temperature (°F)

 

 

 

11?: ) Relative Humidity %
. Dry Bulb Wet Bulb
60 120 84 22
120 140 100 25
90 160 120 30
60 175 133 30
60 190 154 40
II. Canned Frozen Minced Fish.--The following

 

ingredients were mixed in the same manner described
earlier for sausages (Table 4): Mechanically Deboned
Frozen Fish, ground beef tripe, sodium chloride, sodium
tripolyphosphate and spices, either with or without

soy proteinisolate and white corn meal or the combination
of these two included. Hydrogenated vegetable oil was
added and the mixture blended at low speed for 7 mins.

more. The fish paste was then placed into size 211 x 304,

 

 

 

76

TABLE 4.—-Design and Formulation of Canned Minced Fish

 

 

Treatments
Code m . l
N ler ireatment and Formulations
A 86.5% Mechanically Deboned Frozen Fish (MDFF)
B 82.5% MDFF, 4% soy protein isolate (SPI)
C 82.5% MDFF, % white corn meal
D 78.5% MDFF, 4% SPI, 4% white corn meal

 

1In all formulations (A, B, C. D) the following

additives and spices are present: 4% beef tripe, 8%
hydrogenated vegetable oil, 1% sodium chloride, 0.45%
sodium tripolyphosphate, 0.30% white pepper powder, 0.15%
onion powder, 0.15% garlic powder, 0.30% paprika pepper
powder and 0.05% ground ginger.

c-enamel coated cans containing about 15% zinc oxide
(American Can Company, Greenwich, Conn.). The filled
cans were baked in a dry air oven (Thelco, Model 18, GCA/
Precision Scientific, Chicago, Ill.) for 30 mins at 167°F
(75°C). The product was then pressed and drained to
remove any free liquid. Immediately after baking cans
were closed using an automatic closing machine (Canning
Devices Inc., Manitowoc, Wis.).

The sealed cans were processed in a still retort
(Food Machinery Corp., Sprague-Sells Division, Hoopeston,
Ill.) at 250°F (121°C) for 75 mins. The processed cans
were immediately cooled in water until the temperature
at the center of the cans was reduced to approximately

100°F.

77

Statistical Analysis

 

A factorial experiment was designed with three
levels of sodium chloride, three levels of sodium tri-
polyphosphate and three levels of soy protein isolate.
Analysis of variance (Steel and Torrie, 1960) was per-
formed to assess the effect of main variables and their
interactions. When significant differences were observed
between more than two means, either Duncan's Multiple
Range Test (Duncan, 1955), the student "t" test or com-
paring all means with a control (Dunnett's procedure)
were performed to determine which means were significantly
different. Analysis of variance was conducted at the
Michigan State University Computer Center using SPSS
(Statistical Package for the Social Science) version 7.0

(MSU, March 18, 1978).

 

 

RESULTS AND DISCUSSION

Yield of Mechanically Deboned Fish and Its
Chemical Composition

 

 

Table 5 shows some of the physical and chemical
characteristics of mechanically deboned sucker flesh after
it was processed by the deboning machine twice, for a
yield of 47.60% of the round weight of the fish. This
amount is similar to that reported by Morris (1977) and
Zapata (1978), who obtained a 50% yield of mechanically
deboned flesh for the same fish species using the same
technique. However, in this study eviscerated and deboned
suckers yielded approximately 80% of their weight as
mechanically deboned flesh. Yield is an important consid—
eration in determining the final costs of a given product.
In general, yields vary considerably depending on the
species, amount of waste from heading and gutting and the
type of machinery used.

Moisture, fat and protein content of minced sucker
flesh (Table 5) is similar to that reported by Zapata
(1978). According to Stansby and Olcott (1963), fresh—
water suckers are among those fish with high protein low
oil content (15-20% and less than 5%, respectively).

Suckers have a lower caloric content than do red meats or

78

 

 

79

TABLE 5.--Average Percentage Yields Per Total Weight of
Fish for Dressing and Deboning Operations, and
Average Percentage of Chemical Composition and
Standard Deviation of Mechanically Deboned
Freshwater Sucker Flesh (Catostomus and
Moxostoma Spp.)

 

 

 

 

Characteristics Percentage
Eviscerated and deheaded fish 67.60

(% of round weight)

Mechanically deboned meat 47.60

(% of round weight)

Moisture 80.70 i 0.52
Nitrogen x 6.25 16.65 i 0.02
Fat 2.03 i 0.15
Nonprotein Nitrogen 1.70 i 0.05

 

poultry, and thus are an ideal source of animal protein
for use in low caloric diets. Generally, up to one—third
of the oil in fish is comprised of polyunsaturated fatty
acids with four to six double bonds per fatty acid (Oster-
haug et_al, 1963). Due to the relatively high moisture
content of suckers (80.70%), the ratio of moisture to pro-
tein content (16.65%) is approximately 5 to 1 (Table 5).

Effects of Refrigeration and Freezing Storage
on the Extractability of Fish Muscle Proteins

 

 

Seasonal fluctuations in supplies of fish make it
necessary to store quantities of minced fish for future

use. Unless the raw fish is utilized immediately, freezing

80

is the first choice of storage methods to preserve proper
gelling of the fish proteins for production of texturized
products. According to the generally accepted concepts of
freezing technology, slow freezing usually causes a serious
deterioration of fish flesh due to the rupture of cells by
the formation of large ice crystals in the muscle tissue.
However, fast freezing is thought not to have this effect
on fish flesh (Slavin, 1963). Since refrigeration is so

important in the fish industry, Table 6 and Figure 1

TABLE 6.——Mean* and Standard Deviation of Protein Fractions
Extracted from Prerigor, Refrigerated and Frozen
Sucker flesh (Catstomus and Moxostoma spp.) by
the Helander Method (N = 4).

 

 

 

 

Salt Soluble Water Soluble Nonprotein
Protein Protein Nitrogen
Prerigor 50.15a : 1.38 29.04a i 0.43 12.28a'i0.53

H-
H-

Refrigerated 43.11b 0.47 27.57a 1.34 11.66a :0.45

0.32 29.53a 3.09 11.99a :0.52

H-
H-

Frozen 37.20C

 

*Meansin.the same column not followed by the same
superscript are significantly different (p < 0.01).

compare refrigeration and fast freezing treatments to
prerigor muscle with respect to the degree of muscle pro-
tein denaturation. The extractability of myofibrillar
protein (salt soluble protein) from frozen and refriger-

ated muscle was less than that from prerigor muscle

81

a
e m <
-_—-

.Uole

pm :wNon u U tam Oom um pmumummflummm u m .uomflumud u 4 uponumz
pmpcmfimm mnu an A.Qmm mEuoumoxOE tam mDEODMODMUV nmmam meosm Eoum
pmuomuuxm Azmzv cmmouuez Camuoumcoc tam Amway :HmDOMm mansaom pmumz

 

 

.Ammmv cflmuoum mandaom pamm so mmmwoum musumummfime 304 m0 pomMMMII.H musmfim

z 2 am; mmm

 

 

 

 

 

 

 

 

 

 

 

 

O

O
<-

o
(\J
Apuqmoenxg warned

O
to

82

indicating that some denaturation occurred in the myo-
fibrillar proteins. In refrigerated muscle stored at 3°C
for seven days the rate and extent of the protein denatura-
tion were much smaller. There was approximately a 7%
reduction in extracted myofibrillar protein compared to
13% in the frozen muscle. Moreover, neither refrigeration
nor freezing significantly affected the amount of sarco—
plasmic protein (water soluble protein) or nonprotein
nitrogen extracted from muscle (Table 6). An analysis of
variance (Table 7) indicated that freezing and refrigera-
tion significantly decreased the amount of myofibrillar
protein extracted from fish muscle (p < 0.01). Results
of the studentized range test showed that the amount of
myofibrillar protein extracted from either prerigor or
refrigerated muscle was significantly higher than that
extracted from frozen muscle (p < 0.01). Freezing had
greater an effect in decreasing the extractability of
myofibrillar protein (Table 6).

These results are in agreement with the findings
of Dyer (1951), Connell (1964), and Babbitt et_§1. (1972).
According to Goll et_al. (1969), prerigor conditions are
optimal for myofibrillar protein extraction due to the
presence of ATP which maintains disassociation between
actin and myosin. Rigor and post rigor extractability,

according to these workers, would be reduced by

83

 

mmmm.mvm HH HmpOE

 

. . mmsouw

came 0 Namm m m cflspflz

Hoooo.o Amem.emm Hmma.eee Heem.mmm N md2090
cmwspmm

m mo mSHm>Im mHMSWm and: mmumsqm Sowmmum mosseum>
mOCMOHMHcmHm mo 83m mo mmmummo mo mOHSOm

 

so mmmuoum

newness mensaom beam empomnpxm
mHSMMMmmE®B Boa mo pommmm map mo moanesm> mo mflmhamc<ll.h mamme

 

84

interactions between myofibrillar proteins, particularly
those between myosin and the relatively inextractable
actin, which result in the rate of myosin extraction
becoming dependent upon the rate of actin extraction. In
a review of fish muscle proteins Connell (1964) indicated
that the myofibrillar protein (actomyosin) fraction of
fish flesh is subject to the greatest protein insolubili—
zation by freezing, whereas the solubility of sarco-
plasmic proteins is not influenced by freezing. Protein
denaturation is defined as a change in protein such that
it is no longer soluble or extractable by salt solutions
under conditions in which native protein would be soluble
or extractable (Dyer and Dingle, 1961). The amount of
denaturation depends primarily on temperature and pH.
Fish protein is sensitive to denaturation, being more
labile than most mammalian muscle, and will become
denatured at lower temperature and higher pH than
required for red meat protein denaturation (Forrest et al.,
1975). Denaturation of fish muscle proteins at low
temperatures is caused by the formation of a strong

salt solution in the muscle at the eutectic point (Dyer,
1951; Love, 1958). This occurs because inorganic salts
of cells, such as magnesium chloride, can remain in solu—
tion even at very low temperature. It, therefore, seems

likely that the dehydration of muscle cells during frozen

85

storage is the main phenomenon favoring conditions for
denaturation (Dyer, 1951).
Effects of Additives on the Protein

Extractability of Frozen
Mechanically Deboned.Fish

 

 

 

The value of measurements of protein solubility
in various salt solutions lies in the functional role of
proteins in processing high quality products. Since rigor
mortis is thought to decrease extractability of some pro—
teins (Dixon and Webb, 1961) and because phosphates or
increasing ionic strength enhance extractability, the
effects of adding sodium chloride, sodium tripolyphosphate

and soy protein isolate were investigated.

Total Extracted Protein

 

Sodium chloride is the most common nonmeat ingred-
ient added to fish products. This salt serves many func-
tions in meat systems, one of which is to solubilize
myofibrillar proteins. The effects of adding several con-
centrations of sodium chloride (0.0, 3.0, and 3.6%) on
total protein extracted from minced frozen fish are pre-
sented in Table 8andHAppendix2. Total protein fraction
was isolated by dialyzing the filtered extract to retain
both salt soluble and water soluble proteins, while dialy-
zable nonprotein nitrogen was removed. Total extracted

protein increased significantly (p < 0.05) when sodium

86

TABLE 8.—-Effectscf Adding Sodium Chloride and Sodium
Tripolyphosphate on the Amount of Total
Extracted Protein of Frozen Mechanically
Deboned Fish (Total Extracted Protein = mg
Extracted Protein/100 mg Total Protein)

 

o\°

Sodium Chloride

 

0.0 3.0 3.6 Meanb

 

 

 

0.000 13.67a 67.40 62.17 47.63

 

% Sodium 0.225 15.20 54.70 54.81 41.57
Tripolyphosphate 0.450 47.84 58.80 61.11 55.91
Meanb 25.48 60.30 59.36
aStandard error for each cell mean (N = 12) is
0.07

bStandard error for each row or column mean
(N = 36) is 0.04.

chloride was added. The greatest increase occurred when
3.0% sodium chloride was used in the extraction.

Comparison between the highest extractability
values obtained from frozen fish samples with addition of
sodium chloride (Table 8), without taking into account
optimum pH and ionic strengths, and those values obtained
earlier by Helander's (1957) method (Table 6) indicated
that both methods yielded almost the same amount of pro-
tein.

Total extracted protein curves (Figure 2) indi—

cate a minimum solubility of minced frozen fish at zero

 

 

87

Percent Extractability

 

 

Percent Sodium Chloride

Figure 2.--Effect of Sodium Chloride in Varying Concen-
trations on the Protein Fractions of Frozen
Minced Fish. 0-0 (Total Extracted Protein),
0- -0 (Salt Soluble Protein), X---X (Water
Soluble Protein) and. --- - (Nonprotein
Nitrogen).

88

percent sodium chloride (data from this study). Using an
extraction solution without sodium chloride, only 14—48%
of the fish protein was solubilized, the amount depend—
ing on the phosphate level. Much of this solubility value
was due to the presence of sodium tripolyphosphate, since
only 13.67% total protein was extracted with plain water.
Table 8 shows a rapid increase in protein extractability
from 0.0% to 3.0% sodium chloride, a phenomenon noted also
by Dyer et_al. (1950). The effect of increasing sodium
chloride from 3.0 to 3.6% on protein solubility was small,
and may be associated with slight decreases in solubility
in the absence of tripolyphosphate. Adding sodium chlor-
ide to the extraction solution increased ionic strength
and high ionic strengths are required to solubilize myo-
fibrillar proteins (Dyer et_al., 1950). According to
Bendall (1954), solutions of 3.0% and 3.6% sodium chloride
have ionic strengths of 0.51 and 0.61 respectively. Most
myofibrillar proteins require ionic strengths greater
than 0.3 to be solubilized (Goll et_al., 1974); or as
noted by Helander (1957) and G011 gt_al., (1969), myo-
fibrillar proteins are soluble only in solutions with
ionic strengths ranging from 0.4 to 1.5. In addition to
providing the necessary ionic strength, sodium chloride
has the ability to spread protein filaments apart in the

extraction solution (Brotsky and Everson, 1973). Thus,

 

 

89

the increase in total extracted protein is a result of
adding sodium chloride.

Sodium tripolyphosphate is the most widely used
component of phosphate blends in the manufacture of
processed meats since it has a desirable combination of
properties. In samples containing no sodium chloride,
approximately 50% of the fish proteins were extracted
when 0.45% sodium tripolyphosphate was added, but only 13%
and 15% respectively were obtained when 0.0% and 0.225%
sodium tripolyphosphate were added (Table 8). However,
with sodium chloride concentrations of 3.0% and 3.6%,
total extracted protein was lower when sodium tripoly-
phosphate was used. The low extractability (Table 8)
obtained with sodium tripolyphosphate alone might be due
to low ionic strength (Dyer et_§l., 1950), and low pH.
Therefore, the very low extractability values of 15%
(Table 8) obtained might be due to both low ionic strength
and lower pH. Meinke et_al. (1972) noted that the high-
est solubilization of fish myofibrillar proteins required
extreme pH conditions (< 4.0 but > 8.0). The lower
extractability of protein at lower ionic strengths was
attributed to the presence of strong associations between
myofibrillar proteins (Goll et_al., 1969). Sodium tri-
polyphosphate concentrations of 0.225% and 0.45% were

not strong enough to spread protein filaments apart at

90

low ionic strength. However, 0.45% sodium tripolyphos-
phate raised the pH to 7.8, which was more favorable for
extraction of fish muscle proteins (Meinke §E_al., 1972),
as indicated by the sharp increase in the percent extracta-
bility of fish protein (Figure 3). There were no

increases in the amounts of extracted protein by adding
sodium tripolyphosphate in the presence of sodium chloride
(Figure 3).

There was no effect on the total extracted protein
when 2% and 4% soy protein isolate was added to samples
containing no sodium chloride (Table 9). However, an
analysis of variance revealed highly significant differ-
ences between soy protein isolate treatments (Table 10).
With further analysis using Dunnet's Test, it was estab-
lished that significant differences existed between
individual treatments. The effects of 2% and 4% soy pro—
tein isolate treatments were significantly greater
(p < 0.05) than that of the control (0.0% soy protein
isolate). Adding 2% and 4% soy protein isolate did not
increase the extracted protein greatly since soy protein
isolate contains approximately 90% protein. According
to Wolf (1970), insolubilization of soy protein isolates
may be caused by that portion of those proteins held
together by disulfide bonds as aggregates, and more aggre-

gates are formed during isoelectric precipitation.

91

 

PERCENT EXTRACTABILITY

 

0.226 O45

PERCENT TRIPOLYPHOSPHATES

Figure 3.--Effects of Sodium
polyphosphate, in
and Combinations,
Protein of Frozen
0-0 0.0% Nacl,

Chloride and Sodium Tri-
Varying Concentrations

on the Total Extractable
Mechanically Deboned Fish.
-0 3.0% Nacl, X--—X 3.6%

 

92

TABLE 9.—-Effect of Adding Soy Protein Isolate on the
Amount of Total Extracted Protein of Frozen
Mechanically Deboned Fish (Total Extracted
Protein = mg Extracted Protein/100 mg Total

 

 

 

 

 

Protein)
% Sodium Chloride
0.0 3.0 3.6 Meanb
% Soy 0.0 25.35a 58.87 56.74 46.99
Protein Isolate 2.0 25.20 60.53 60.28 48.67

4.0 25.83 61.36 61.08 49.42

 

aStandard error for each cell mean (N = 12) is
0.07.

bStandard error for each column mean (N = 36)
is 0.04.

Disulfide crosslinks must be broken to resolubilize these
proteins. As described earlier, protein molecules inter-
act with each other, and one type of interaction results
in the precipitation of protein from solution (Oncley,

et al., 1952). Precipitation of proteins may have occur—

 

red when soy protein isolate was added to frozen minced
fish since large quantities of pellets were found in
centrifuge bottles in samples containing soy protein
isolate. Pearson et_al. (1965) found that a soy sodium
proteinate was less soluble at the pH of meat. There were
significant interactions (p < 0.01) between either sodium

chloride and sodium tripolyphosphate or sodium chloride

93

 

 

mae.ee eee.eaoe see Hence

oeo.o mem.e Hm Hmseemmm

Hoe.o Nee.eae Hme.ee oom.emoe eN emcemHexm
Heo.e eso.e eem.e eem.e m m> x N> x as
Heo.o eom.a oam.o oem.N e m> x N>
Hoo.o mmm.e mam.o mem.e e m> x H>
Hee.e mem.oee eem.ee mem.mme e N> x H>
Am>v mumHOmH

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Am>v mumnmmonm

Hoo.o Hem.emme emm.em ame.mee N r>HOEAee aseeom
AH>V

Heo.o moe.NeeN eee.mam mmm.eme N meepoHeo aseeom
m mo 00cmoeeecmem 05Hm>rm mummwm mwmmmww mo Mwmwmwm MMHWMMMMW

 

 

chem Umconmo SHHMOHcmnomz
ammoum mo :Hmuoum pmuomuuxm Hmuoe so mcoHMMCAQEOU paw mcoflu
Immucwosou mafimum> CH mpmHomH :Hmpoum mom paw mpmnmmonmmaomflue
Edepom .mpHHOH30 Esflpom mo mpommmm may mo messanm> mo mHmSHMCMII.oa mamme

94

and soy protein isolate or sodium tripolyphosphate and
soy protein isolate on the extractability of protein from

frozen minced fish (Table 10).

Salt Soluble Protein

 

In samples without sodium chloride, no salt solu-
ble protein was extracted unless sodium tripolyphosphate
was present;(Tablell and Appendix 3% Salt soluble protein
comprised approximately 51% and 54% of the total extracted
protein in samples containing 3.0 and 3.6% sodium chloride,
respectively, while about 8-50% of the total extracted pro-
teins were salt soluble in the presence of sodium tripoly-

phosphate alone (Table 11).

TABLE ll.——Effects of Adding Sodium Chloride and Sodium
Tripolyphosphate on the Amount of Salt Solu-
ble Protein Extracted from Frozen Mechanically
Deboned Fish (Salt Soluble Protein = mg salt
Soluble Protein/100 mg Total Extracted Pro-

 

 

 

 

 

tein)
% Sodium Chloride
0.0 3.0 3.6 Meanb
0.000 0.00 47.65 51.47 33.04
% Sodium 0 225 8.17a 57.02 54.84 40.01
Tripolyphosphate 0.450 52.17 47.75 55.59 51.83
Meanb 20.11 50.80 53.96

 

aStandard error for each cell mean (N = 6) is 0.08.

b
Standard error for each row or column mean

(N 13) is 0.05.

95

As noted earlier, salt soluble protein extracted
from frozen minced fish increased as sodium chloride con-
centration increased, with the largest increase obtained
between 0.0% and 3.0% sodium chloride (Figure 2). The data
indicated that salt soluble protein increased as sodium
tripolyphosphate concentration increased. The data also
showed that salt soluble protein comprised 8.2% to 57.0%
of the total extracted protein in samples containing
sodium tripolyphosphate, while lower percentages of the
total extracted protein were salt soluble protein in the
samples without sodium tripolyphosphate (Table 11). In
addition, the data suggest that adding sodium tripoly-
phosphate sharply increased the extractability of salt
soluble protein in the absence of sodium chloride (Fig-
ure 4). However, the addition of sodium tripolyphosphate
in the presence of 3.0% sodium chloride had slight
adverse effect on the extractability of salt soluble pro-
tein. These results may be explained by the fact that
pyrophosphate can directly effect the resolution of acto-
myosin into its components actin and myosin and increase
the solubility of those dissociated proteins (Fukazawa
e£_al., 1961b). Sodium tripolyphosphate can cause this
after it has reverted to pyrophosphate by enzymatic
hydrolysis (Yasui et_al., 1964b). An analysis of vari-

ance revealed significant effects on salt soluble protein

96

PERCENT EXTRACTABI LlTY

   

O l.
O 0.226 0.46
PERCENT TRI POLYPH OSPHATES

Figure 4.--Effects of Sodium Chloride and Sodium Tripoly-
phosphate, in Varying Concentrations and
Combinations, on the Salt-Soluble Protein
Fraction Extracted from Frozen Mechanically
Deboned Fish. 0-0 0.0% Nacl, 0- -I 3.0%

Nacl and X—-—X 3.6% Nacl.

 

 

97

caused by adding either sodium chloride or sodium tri-
polyphosphate (p < 0.01) (Table 12).

The data do not indicate a very large change in
the amounts of salt soluble protein from frozen minced
fish attributable to addition of soy protein isolate
(Table 13). However, analysis of variance (Table 12)
shows a significant effect caused by adding soy protein
isolate (p < 0.01). Here again, further analysis com—
paring the effects of 2% and 4% soy protein isolate to
the control using Dunnet's test indicated that both levels
significantly increased the extractability of salt soluble
protein (p < 0.05).

The interactions between sodium chloride and
sodium tripolyphosphate or between sodium tripolyphosphate
and soy protein isolate were significant (p < 0.01)

(Table 12). However, salt soluble protein was not sig-
nificantly affected by interaction between sodium chloride

and soy protein isolate.

Water Soluble Protein

 

Water soluble protein fractions were obtained from
the supernatant of the filtered protein extract, which
was dialyzed overnight against 0.05M sodium chloride to
remove nonprotein nitrogen and to insolubilize the salt
soluble proteins. Adding sodium chloride decreased the

percentage of extracted water soluble protein in the total

98

 

 

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Hoo.o mam.mfl mam.o maa.m m m> x N> x H>
Hoo.o mma.m mmm.o Noe.a a m> x N>
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Am>v mumHOmH

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Am>v mnmammond

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AH>V

Hoo.o omm.smm mam.em omw.am m moHHoHno asflwom
m mo mocwoflmacmam msHm>Im mumdwm mmumswm Eoommum mocwflum>
. . com: mo Esm mo mmmummo mo mousom

 

swam pmconmo mHHMOHcmnomz cmmoum
Eoum Umpomupxm camuoum mansaom Hamm co mcoHpMCHQEOU Ucm mcoflu
Imuycmocou mcfi>Hm> CH mpMHOmH cambonm mom can mumcmmonmwaomflue
asfloom .mofluoHno asflwom no muommum ms» mo mocmaum> mo mammamcau-.ma mamas

99

TABLE 13.—-Effect of Adding Soy Protein Isolate on the
Amount of Salt Soluble Protein Extracted
from Frozen Mechanically Deboned Fish (Salt
Soluble Protein = mg salt soluble protein/
100 mg total extracted protein)

 

% Sodium Chloride

 

 

 

 

 

0.00 3.00 3.60 Meanb
% Soy 0.0 18.82a 51.55 52.63 41.00
Protein Isolate 2.0 20.66 49.88 55.62 42.05
4.0 20.87 50.98 53.63 41.82

aStandard error for each cell mean (N = 6) is 0.08.

bStandard error for each column mean (N = 18) is

extracted protein. Water soluble protein decreased from
approximately 77% to 40% and 43% of the total extracted
protein by adding 3 and 3.6% sodium chloride, respectively
(Table 14). This effect was expected, since salt soluble
protein content increased as sodium chloride concentration
increased (Figure 5). The quantity of water soluble pro-
tein also increased as sodium chloride concentration
increased (Figure 2 and Appendix 4). The increase in water
soluble protein caused by adding sodium chloride may be

due to the lack of precipitation of myofibrillar protein

during dialysis. This interpretation is in agreement with

100

TABLE 14.--Effects of Adding Sodium Chloride and Sodium
Tripolyphosphate on the Amount of Water Solu-
ble Protein Extracted from Frozen Mechanically
Deboned Fish (Water Soluble Protein = mg Water
Soluble Protein/100 mg Total Extracted Protein)

 

Sodium Chloride

 

 

 

 

0.00 3.00 3.60 Meanb

0.000 99.21 36.68 35.13 57.00

% Sodium 0.225 88.91 46.11 50.51 61.84

Tripolyphosphate 0.450 44.26 37.41 43.78 41.82
Meanb 77.46 40.07 43.14

 

aStandard error for each cell mean (N = 6) is 0.08.

bStandard error for each row or column mean (N =
18) is 0.04.

the finding of 6011 et al., (1974), who reported that some

 

myofibrillar proteins, such as troponin, are soluble in

water. There is an increase in water soluble protein,

and a decline in the rate of increase in salt soluble

protein between 3.0 and 3.6% sodium chloride (Figure 5).

This may be caused by a mechanical effect during the

extraction process, or by development of cross-linked

structures inhibiting extraction of salt soluble protein.
As sodium tripolyphosphate increased, water soluble

protein decreased in samples without sodium chloride

(Table 14). The protein fraction extracted (99.21%) at

0.0% sodium chloride and 0.0% sodium tripolyphosphate was

101

 

D—I
CD
CD

40
20

Percent Extractability

 

O
O

1 2 3 4
Percent Sodium Chloride

Figure 5.--Effect of Sodium Chloride in Varying Con-
centrations on the Ratio between the Amounts
of Salt Soluble Protein and Water Soluble
Protein Extracted from Frozen Minced Fish.
0-0 Total Extracted protein, 0- -0 salt
soluble protein and X-—-X water soluble
protein.

 

 

102

 

100

00
C

CD
0

[\D
C)

Percent Extractability
.D>
O

 

O

O 0.225 0.45
Percent STP

Figure 6.——Effect of Sodium Tripolyphosphate (STP) in

Varying Concentrations on the Ratio Between
the Amounts of Salt Soluble Protein and Water
Soluble Protein Extracted from Frozen Minced
Fish. 0-0 Total Extracted Protein, 0- -0
Salt Soluble Protein and X-—-X water soluble
Protein.

103

water soluble protein. The mean values of water soluble

protein over all salt levels were 57.6%, 61.84% and 41.81%
of the total extracted protein in samples containing 0.0%,
0.225% and 0.45% sodium tripolyphosphate, respectively
(Table 14). Generally, water soluble protein was lowest
at the 0.45% level of sodium tripolyphosphate (Figure 6).
Once again, this effect was expected, since the amount of
salt soluble protein increased as sodium tripolyphosphate
increased (Figure 6).

Addition of soy protein isolate resulted in slight

increases in the water soluble protein extracted from

frozen minced fish (Table 15). Thiseffect was apparent

TABLE 15.--Effect of Adding Soy Protein Isolate on the
Amount of Water Soluble Protein Extracted from
Frozen Mechanically Deboned Fish (Water Soluble
Protein = mg Water Soluble Protein/100 mg Total
Extracted Protein)

 

% Sodium Chloride

 

 

 

 

0.0 3.0 3.6 Meanb

% Soy 0.0 74.78a 41.08 42.67 52.88
Protein Isolate 2.0 78.57 38.07 43.49 53.37
4.0 78.91 41.05 43.26 54.41

 

aStandard error for each cell mean (N = 6) is 0.08.

bStandard error for each column mean (N = 18) is

0.04.

 

104

only in samples without sodium chloride, since water
soluble protein increased from 74.89% to 78.57% and
78.91% by adding 2% and 4% soy protein isolate.

An analysis of variance established that adding
soy protein isolate significantly affected the extracta—
bility of water soluble protein (p < 0.01). A Dunnet
test showed that 2% and 4% soy protein isolate treatments
resulted in significantly (p < 0.05) higher amounts of
water soluble protein being extracted. There were also
significant interactions (p < 0.01) between sodium chlor-
ide and either sodium tripolyphosphate or soy protein
isolate (Table 16). The interaction between sodium tri-
polyphosphate and soy protein isolate was not signifi—

cant.

Nonprotein Nitrogen

 

Microkjeldahl nitrogen analysis was used to
determine the amount of nonprotein nitrogen in 15 m1 sam—
ples of filtered total extracted protein of the superna-
tant after precipitation of proteins with trichloracetic
acid (TCA). Average percentages of soluble nonprotein
nitrogen decreased significantly (p < 0.05) as either
sodium chloride or sodium tripolyphosphate concentrations
increased (Table 17). The largest amount of nonprotein
nitrogen was obtained from samples with plain water

(Table 17 and Appendix 5).

 

 

105

 

 

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106

TABLE l7.--Effects of Adding Sodium Chloride and Sodium
Tripolyphosphate on the Soluble Nonprotein
Nitrogen of Frozen Mechanically Deboned Fish
(Nonprotein Nitrogen = mg Nonprotein Nitrogen/
100 mg Total Protein "N x 6.25")

 

% Sodium Chloride

 

 

 

0.0 3.0 3.6 Meanb
% Sodium 0.000 7.77a 6.11 5.15 6.34
Tripolyphosphate 0.225 6.07 6.07 6.06 6.07
0.450 5.75 5.60 5.61 5.65

Meanb 6.53 5.93 5.61

 

aStandard error for each cell mean (N = 12) is

bStandard error for each row or column mean
(N = 36) is 0.005.

Adding soy protein isolate significantly (p < 0.05)
decreased the amount of soluble nonprotein nigrogen
(Table 18).

Analysis of variance (Table 19) indicated that
there were significant interaction between sodium chloride
and sodium tripolyphosphate, and also between sodium

chloride and soy protein isolate (p < 0.01).

Myosin Heavy Chain

 

Myosin heavy chain bands did not appear on SDS gel
electrophoresis patterns of samples of total extracted

protein without either sodium chloride or sodium

107

TABLE l8.-—Effect of Adding Soy Protein Isolate on the
Nonprotein Nitrogen of Frozen Mechanically
Deboned Fish (Nonprotein nitrogen = mg non-
protein nitrogen/lOO mg Total Protein

 

 

 

 

 

"N x 6.25")
% Sodium Chloride
0.0 3.0 3.6 Meanb
0.0 6.75a 6.21 5.89 6.28
% Soy 2.0 6.62 5.88 5.64 6.05
Protein Isolate 4.0 6.22 5.69 5.29 5.75
aStandard error for each cell mean (N = 12) is
0.01.
bStandard error for each column mean (N = 36) is
0.005.

tripolyphosphate and in samples containing 0.225% sodium
tripolyphosphate (gels 1-6, Figure 7). These data indi—
cated that myosin heavy chains insolubilize at low ionic
strength. However, 0.45% sodium tripolyphosphate solu-
bilized myosin (gels Nos. 7, 8 and 9, Figure 7). The
effect of the latter concentration of sodium tripolyphos-
phate may be due to the high pH (7.8) of the extraction
solution, since the ionic strength of 0.45% sodium tri-
polyphosphate is insufficient to solubilize myosin (Goll,
et_al., 1969). In combination or alone, 3 percent sodium
chloride with sodium tripolyphosphate extracted myosin,

but myosin heavy chain bands were light (gels Nos. 1, 2

108

 

 

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111

and 3) when 3.0% sodium chloride alone was used (Figure 8).
Adding 3.6% sodium chloride with or without sodium tri—
polyphosphate also extracted myosin from frozen minced
fish (Figure 9).

Changes in the total peak area of soluble myosin
heavy chains from frozen mechanically deboned fish are
shown in Table 20. The gel scanner was equipped with an
integrator to give total peak area of myosin heavy chain
bonds. Base line positioning (A and B, Figure 10) is
critical in obtaining accurate peak areas (Figure 10).

As sodium chloride increased from 3.0% to 3.6%, myosin
heavy chain total area increased from 0.74 x 105 to 1.00

x 105 in samples containing no sodium tripolyphosphate.

On the other hand, myosin heavy chain total peak area
decreased as sodium chloride increased in samples contain—
ing either 0.225% or 0.45% sodium tripolyphosphate. These
results are in agreement with those of Linko and Nikkila
(1961) who found that salt induced denaturation of Baltic
Herring myosin was inhibited by the addition of various
alkaline phosphates. When salt concentration in meat
systems increased, higher concentrations of phosphates

are required to prevent the denaturation of myosin. This
effect might be due to the lowering of pH, since pH
decreased as sodium chloride increased (Table 23). It

was considered that the findings of Meinke et al. (1972)

 

 

 

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117

 

 

 

 

Kt

 

 

 

Figure lO.--Typical Absorbance Peak for Heavy Chain Total
Area of Myosin Extracted from Frozen Mechani-
cally Deboned Fish.

A = Baseline before establishing myosin
heavy chain peak

ED
ll

Baseline after establishing myosin
heavy chain peak.

 

 

118

could offer an interpretation of the results. They noted
that the highest solubilization of fish myofibrillar pro-
teins required extreme pH conditions (< 4.0 or > 8.0),
and as mentioned earlier, that myosin comprised 40% of
Fish (Cod) muscle protein (Connell, 1964). In general,
adding either sodium chloride or sodium tripolyphosphate
increased the solubility of myosin heavy chains (Table 20).

Soy protein isolate had no effect on myosin heavy
chain solubility (Table 21).

Figure 11 and Appendix 6 show the effects of
sodium chloride, sodium tripolyphosphate and soy protein
isolate in varying concentrations and combinations on the
solubility of myosin heavy chain. The largest myosin
heavy chain peak area was obtained from samples containing
0.45% sodium tripolyphosphate alone (Nos. 7, 8, and 9,
Figure ll-A).

An analysis of variance (Table 22) indicated
significant differences in peak area caused by sodium
chloride, sodium tripolyphosphate and soy protein isolate.
There was also significant interaction between sodium
chloride and sodium tripolyphosphate (P <(LCHJ. However,
peak area was not significantly affected by interactions
between either sodium chloride and soy protein isolate
or sodium tripolyphosphate and soy protein isolate

(Table 22).

 

119

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120

Figure ll.-—Effects of Sodium Chloride, Sodium Tripoly-

N

U‘l

oo

.5

phosphate and Soy Protein Isolate, in Vary-
ing Concentrations, and Combinations on
Myosin Heavy Chain (MHC) Total Area per

20 Mg Protein Sample Extracted from Frozen
Mechanically Deboned Fish.

A = No sodium chloride, B = 3.0% sodium
chloride and C = 3.6% soidum chloride

and 3 of A, B and C were 0.0% Sodium Tripoly—
phosphates.

and 6 of A, B and C were 0.225% Sodium Tri-
polyphosphates.

and 9 of A, B and C were 0.45% Sodium Tri-
polyphosphates.

and 7 of A, B and C were 0.
Isolate

O

% Soy Protein

N
O
o\0

and 8 of A, B and C were
Isolate

Soy Protein

and 9 of A, B and C were 4.0% Soy Protein
Isolate

 

121

 

 

 

 

 

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123

Effects of Additives on pH of Frozen
Mechanically DebonedgFish

 

 

It is interesting to note changes in the pH of
frozen minced fish flesh caused by adding varying concen-
trations and combinations of sodium tripolyphosphate,
sodium chloride and soy protein isolate, since protein
solubility, swelling, gel formation and water holding
capacity in fish muscle are affected by pH (Hermanson,
1972; Trautman, 1964; Hamm, 1960). The minimum fish
protein solubility falls within a pH range of 5.5-6.0;
however, protein solubility increases on both the acidic
and basic sides of this'range (Meike, et al., 1972).

In general, as sodium chloride was increased,

the pH of frozen deboned fish decreased (Table 23). These

TABLE 23.--Effects of Adding Sodium Chloride and Sodium
Tripolyphosphate on pH of Frozen Mechanically
Deboned Fish

 

% Sodium Chloride
0.0 3.0 3.6 Meanb

 

 

0.000 6.86a 6.20 6.14 6.40

 

% Sodium 0.225 7.27 6.41 6.38 6.69
Tripolyphosphate 0.450 7.80 6.61 6.67 7.05
Meanb 7.31 6.43 6.40
aStandard error for each cell mean (N = 6) is
0.002.
b

Standard error for each row or column mean (N =
18) is 0.001.

 

124

results agree with those of Hamm (1957), who found that
adding sodium chloride to post-rigor ham muscle consis-
tently lowered its pH. This reduction in pH was

explained as follows by Mahon (1961). On the acidic side
of the isoelectric point protein is positively charged

and C1- ions are absorbed, while on the basic Siderthere are
negative charges and Na+ ions are absorbed with the libera-
tion of some H+ ions causing a drop in pH. Frozen mechani-
cally deboned fish has a pH of 6.68 which is on the
alkaline side of the isoelectric point of some proteins,
and consequently carries a net negative charge. Adding
sodium chloride to negatively charged protein causes Na+
ions to be absorbed with the liberation of H+ ions,

causing a drop in pH.

As sodium tripolyphosphate increased, pH also
increased.(Tab1e12 andAppendix 7). pH increased to 7.8
with the addition of 0.45% sodium tripolyphosphate in
samples without sodium chloride (Table 23). Brotsky and
Everson (1973) reported similar results. Adding sodium
tripolyphosphate to meat increased its pH, which in turn
increased the negative charge on myofibrillar proteins.

The data also showed that addition of sodium tripolyphos-
phate raised the pH of frozen minced fish even in the
presence of sodium chloride with the largest increase

when no sodium chloride was added (Figure 12). There

 

 

125

 

O 0.225 0.450
PERCENT TR IPOLYPHOSPHATES

Figure 12.--Effects of Sodium Chloride and Sodium Tri-
polyphosphate, in Varying Concentrations and
Combinations on pH of Frozen Mechanically
Deboned Fish. 0-0 0.0% Nacl, O- -O 3.0%
Nacl and X—--X 3.6% Nacl.

126

were no remarkable changes in pH caused by adding soy
protein isolate to frozen minced fish in samples with or

without sodium chloride (Table 24).

TABLE 24.--Effect of Adding Soy Protein Isolate on pH of
Frozen Mechanically Deboned Fish

 

% Sodium Chloride

 

 

 

0.0 3.0 3.6 Meanb
0.0 7.27a 6.46 6.37 6.70
% Soy 2.0 7.33 6.42 6.40 6.72
Protein Isolate 4.0 7.33 6.39 6.42 6.71
aStandard error for each cell mean (N = 6) is

0.002.

bStandard error for each column mean (N = 18) is
0.001.

From the results of an analysis of variance
(Table 25), it was evident that blending either sodium
chloride, sodium tripolyphosphate, or soy protein iso-
late significantly affected the pH of frozen minced
fish (P < 0.01). The analysis also indicated that there
were significant effects on the pH of fish muscle caused

by the interaction of those three additives.

127

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128
Effects of Additives on Percent Swelling of
Frozen Mechanically Deboned Fish
The effects of sodium chloride, sodium tripoly-
phosphate and soy protein isolate on frozen minced fish
swelling are shown in Appendix 8. As sodium chloride
increased, swelling decreased, with the lowest mean
values observed in the presence of 3.6% sodium chloride

(Table 26). Shults et al., (1972) reported similar

TABLE 26.—-Effects of Adding Sodium Chloride and Sodium
Tripolyphosphate on Percent Swelling of Frozen
Mechanically Deboned Fish

 

% Sodium Chloride

 

 

0.0 3.0 3.6 Meanb
0.000 55.68a 53.34 47.27 52.10
% Sodium 0.225 72.60 59.78 48.45 60.28

Tripolyphosphate 0.450 108.40 61.57 63.95 77.85

Meanb 78.77 58.23 53.22

 

aStandard error for each cell mean (N = 12) is
1.10.

bStandard error for each row or column mean
(N - 36) is 0.63.

results, stating that meat swelling decreased with the

addition of 3-5% sodium chloride. The decrease in swell-

ing was attributed to the exchange of Mg++ and K+ by
Na+ .

 

 

 

129

As amounts of sodium tripolyphosphate blended
with frozen minced fish increased, swelling also increased
(Table 26). Bendall (1954), Hermansson (1972), and Shults

et al., (1972) recorded similar findings. It is thought

 

that sodium tripolyphosphate causes frozen mechanically
deboned fish to swell because of its relative effect on
pH. Swelling increased as pH increased in the range of
4—10 (Hermansson, 1972). Moreover, Bendall (1954) stated
that swelling is due to the ability of inorganic pyro—
phosphate to split actomyosin into its component proteins,
actin and myosin, resulting in the uptake of water. This
phenomenon resembles the action of adenosine triphosphate
on disassociation of actomyosin. The data indicated that
swelling increased sharply in the presence of both levels
of sodium tripolyphosphate in samples without sodium
chloride (Figure 13). Swelling was also caused by the
combined effect of sodium tripolyphosphate and sodium
chloride (Table 26).

Swelling of frozen minced fish increased as soy
protein isolate levels increased, with the highest values
occurring at the 4.0% level of soy protein isolate
(Table 27, Figure 14). Hermansson (1975) found that soy
protein isolate has a high capacity to cause swelling.
Pearson gt_gl., (1965) also noted the tendency of this

substance to absorb water.

 

130

HO
I00
90
80
70
60
50
40

PERCENT SWELLING

 

0
0 0.225 0.45
PERCENT TRIPOLYPHOSPHATES

Figure l3.--Effects of Sodium Chloride and Sodium Tri-
polyphosphate in Varying Concentrations
and Combinations on the Swelling of Frozen
Mechanically Deboned Fish. 0-0 0.0% Nacl,
O- -O 3.0% Nacl and X---X 3.6% Nacl.

 

131

TABLE 27.--Effect of Adding Soy Protein Isolate on Per—
cent Swelling of Frozen Mechanically Deboned

 

 

 

 

Fish
% Sodium Chloride

0.0 3.0 3.6 Meanb
0.0 72.19a 49.65 42.50 54.78
% Soy 2.0 73.48 54.42 52.63 60.18
Protein Isolate 4.0 88.97 70.62 63.54 74.38
aStandard error for each cell mean (N = 12) is

1.10

bStandard error for each column mean (N = 36) is
0.63

Proteins have the ability to absorb large amounts
of water, and the ratio of water to protein can be very
important to the binding properties of protein. Brown
(1972), citing Cook §E_al., (1969), pointed out that a
meat-soy system will hold approximately 100% more free
water than a pure meat system to which excess water was
added.

Proteins vary widely in their ability to bind
water, and therefore in their degree of hydration. As
with electric charge, hydration is a source of stability

to protein macromolecules in an aqueous system. Water

132

 

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Figure l4.--Effects of Sodium Chloride and Soy Protein
Isolate in Varying Concentrations and Com-
binations, on the Swelling of Frozen
Mechanically Deboned Fish. 0—0 0.0%

Nacl, O- -I 3.0% Nacl and X---X 3.6% Nacl.

 

 

 

133

is held on proteins by hydrogen bonidng to backbone
structure, and by hydration of polar side chains.

Lauffer (1964) suggested that ions on the protein sur—
face may bind water by electrostriction or that water may
be organized in "iceberg" form around hydrophobic groups
or that both phenomena occur at the same time. Also,
insoluble proteins possibly absorb water, changing the
degree of hydration, and consequently alterating their
physical and chemical properties. This could have occurred
when soy protein isolate was blended with frozen minced
fish and water. Therefore, soy protein isolate, itself,
was responsible for the uptake of a considerable quantity
of added water.

An analysis of variance (Table 28) indicated sig-
nificant differences in swelling caused by sodium chloride,
sodium tripolyphosphate and soy protein isolate. There
were also significant interactions between those three

additives (p < 0.01).

Effects of Additives on Gel Forming Ability
of Frozen Mechanically Deboned Fish

 

 

One of the most important functional properties
of comminuted fish meat is its ability to form gels which

are stable after heating in the presence of salts. It is

 

 

 

134

 

 

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mmN.HH NNO.NHHH Hm Hmschmm

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Hoo.o amH.Nm Hmm.Nmm OHN.NONH m N> x N> x H>
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AN>V mumHOmH

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. HN>V mnmcamoaa

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HH>V

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mwmsqm mmHmsqm Eoowwum GOHuMHHm>

m Mo OUQMOHchmHm ODHM>Im cmoz mo Esm mo mwmummm mo OOHSOm

 

ESHUOm

4mawHOmH cflwpoum wow 6cm
HOUHHOHnu ESHUom mo muommmm mnu wo wocmHHm> mo mflmmHms<ll.mm mqmfie

gmflm bwconwo
%HHMOHcmLooz coNon mo mcHHHme co .mcoHHHCHQEOU paw mcoHu
IMHHCOUCOU mcflxum> CH

.wumnmmoamwaomHHB

135

well known that the rheological characteristics of such
gels depend primarily on the concentration of muscle pro—
tein and its physiochemical state, which is, in turn,
related to the ionic strength and pH of the muscle.

The effects of varying concentrations and combina-
tions of sodium chloride, sodium tripolyphosphate and soy
protein isolate on the gel forming ability of minced
frozen fish are given in Appendix 9. The least concen—
tration endpoint is the minimum protein concentration
required to form a gel. The least concentration endpoint
of total extracted protein required for gelation increased
as the concentration of Sodium chloride increased (Table

29). Poor gelation properties of total extracted

TABLE 29.——Effects of Adding Sodium Chloride and Sodium
Tripolyphosphate on the Least Concentration
Endpoint of Frozen Mechanically Deboned Fish
Extracted Protein

 

9 Sodium Chloride

O

 

 

 

0.00 3.00 3.60 Meanb
0.000 1.41a 4.36 5.62 3.40
% Sodium 0.225 1.40 5.53 6.87 4.60
Tripolyphosphate 0.450 1.22 7.75 7.31 5.43
Meanb 1.34 5.88 6.60
aStandard error for each cell mean (N = 12) is

bStandard error for each row or column mean
(N = 36) is 0.06.

 

136

protein in samples containing sodium chloride were proba—
bly related to high ionic strength and possibly lower pH
in these samples. These results agree with those of Tong
et_gl., (1975), who reported that poor gelation proper—
ties were related to residual salt content.

The least concentration endpoint was decreased by
adding 0.45% sodium tripolyphosphate only in samples con-
taining no sodium chloride (Table 29 and Figure 15).
Tong, et_al., (1975) also noted that alkaline protein
extracts were more effective than salt protein extracts
in gel forming ability. In the present study, those
samples with only 0.45% Sodium tripolyphosphate had pH
values as high as 7.8. Improved gelling ability with
0.45% sodium tripolyphosphate may also be related to
myosin content, since the largest myosin heavy chain
total peak areas were obtained only from protein samples
extracted from fish in the presence of 0.45% sodium
tripolyphosphate (Figure 11). These findings are similar
to those of Fukazawa et_al., (1916c) and Nakayama and
Sato (1971b), who noted that myosin had a major effect on
gel formation. However, in samples containing sodium
chloride the presence of sodium tripolyphosphate increased
least concentration endpoint values. The least concen—
tration endpoint significantly increased (p < 0.01) as

sodium chloride increased (Table 29).

 

 

PERCENT PROTEIN REQIRED FOR GELLING

”1
|'-‘
LO
{3
H
(D
H
U1

137

 

 

 

O 0.226 0.45
PERCENT TRIPOLYPHOSPHATES

.--Effects of Sodium Chloride and Sodium Tri-
polyphosphate, in Varying Concentrations
and Combinations, on Least Concentration
Endpoint of Total Extracted Protein Fraction
Extracted from Frozen Mechanically Deboned
Fish. 0-0 0.0% Nacl, 0- -O 3.0% Nacl and
x---x 3.6% Nacl.

 

 

138

It has been shown that soy protein isolate has
the ability to form a gel when heated (Hermansson, 1972).
Soy protein isolate slightly improved gel forming ability
in samples of frozen minced fish without sodium chloride.
The least concentration endpoint decreased slightly from
1.49% to 1.19% and 1.34% with the addition of 2% and 4%,

respectively, of soy protein isolate (Table 30).

TABLE 30.-—Effect of Adding Soy Protein Isolate on Least
Concentration Endpoint of Frozen Mechanically
Deboned Fish Extracted Protein

 

o\0

Sodium Chloride

 

0.0 3.0 3.6 Meanb

 

 

% Soy 2.0 1.19 6.31 6.74 4.75
Protein Isolate 4.0 1.34 6.09 6.74 4.72
aStandard error for each cell mean (N = 12) is
0.11
bStandard error for each column mean (N = 36) is
0.06

Gelation of soy protein isolate does not occur
at concentrations of 6% or lower, although there is a
small increase in viscosity (Circle et_al., 1964). The
lower levels of 2% and 4% soy protein isolate were

selected for blending with frozen minced fish to avoid a

 

139

strong soy flavor in finished products. Moreover, since
fish flesh contained approximately 16% total protein con-
tent, there was no need to add higher concentrations of
soy protein isolate for gelation. As noted above, in
samples containing sodium chloride the presence of soy
protein isolate did not improve gel formation (Table 30)
and the least concentration endpoint was increased by
adding soy protein isolate. Catsimpoolas and Meyer (1970)
reported similar results, noting that at temperatures
above 70°C the viscosities of soy protein gels decrease
as sodium chloride concentration increase from 1.18% to
11.76%. This reduction in gel strength in the presence
of sodium chloride was correlated with a decrease in
protein solubility caused by protein aggregations such as
the precipitation of soy proteins (Hermansson and Akesson,
1975b) and a decrease in pH (Catsimpoolas and Meyer,
1970). These workers found that the highest viscosity
values were obtained at a neutral or mildly alkaline pH.
The water soluble protein fraction extracted from
frozen minced fish with plain water formed gels at a low
protein concentration (1.60%) with the application of
heat at 80°C for 10 minutes (Appendix 9). This is an
indication that freezing had no major effect on the physio-
chemical properties of sarcoplasmic protein in mechani-

cally deboned fish.

140

Analysis of variance indicated that there were
significant effects on gel formation caused by sodium
chloride, sodium tripolyphosphate and soy protein isolate
blended with frozen minced fish (p < 0.01, Table 31).
There were also significant interactions between those
additives on the gel forming ability of fish muscle pro-
tein (p < 0.01).

Further Processing of Frozen Mechanically
Deboned Fish

 

 

Fish Sausages

 

The development of new products such as sausage
from frozen mechanically deboned fish requires a basic
knowledge of the functionality of muscle proteins, since
denaturation during frozen storage causes changes in the
quality of finished products. In addition, studies done
on protein solubility, swelling, gel formation and the pH

of frozen mechanically deboned fish indicated that 3%

\0

sodium chloride, 0.456 sodium tripolyphosphate and 4% soy
protein isolate were adequate for fish sausage formulation.
The study further indicated that those concentrations are
needed for quality maintenance and improvement in the
economics of sausage production. However, 4% soy protein
isolate did not improve the functional properties of
frozen mechanically deboned fish as was expected, and the

use of more than 4% soy protein isolate in product

 

141

 

 

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, Am>v mumnmmosm

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HH>V

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mumsqm mmumsqm Eocmmum mOCMHHw>

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mo uCHompCm COHHMHmeoCOU Hmmmq Co mCoHHMCHQEou pCm mCOHamup
ICwOCOO mCH>Hm> CH .mumHOmH CHmpoum mom UCm mumcmmonthomHHB
ECHpom .mpHHoHCO ECHpom mo muommmm may mo wOCMHHm> mo mHthMC¢11.Hm mqmfie

 

142

formulation imparted a soy flavor to the finished product.
Because of this flavor, 4.0% white corn meal was used in
product formulation.

In comparing treatments, fish sausages containing
only 2.0% sodium chloride and 0.45% sodium tripolyphos—
phate (Treatment-A, Table 2 and Figure 16) shrank more
when heated to 82°C internal temperature for 30 minutes
than any of the suasages made with 3.0% sodium chloride,
0.45% sodium tripolyphosphate and 8% hydrogenated vegetable
oil and any of the combinations of soy protein isolate and
white corn meal (i.e., treatments B, C, D and E, Table 2).
Among sausages made according to the five treatments and
heated to an internal temperature of 82°C, the one with
only salt and phosphate (treatment A) had poor physical
structure and shape, whereas the others appeared normal
(Figure 16).

The effect of overheating on the binding character-
istics of sausages is well known in the meat industry.
According to Swift and Ellis (1957), heating to internal
temperatures much in excess of 65.5°C should be avoided.
However, regulation No. 541.2 of the Michigan Department
of Agriculture, Food Inspection Division, requires that
all smoking should be accomplished so that the internal
temperature of the coldest part of a fiShpreparation

remains at 82°C for not less than 30 minutes. Also,

 

143

 

Figure l6.--Whole Fish Sausages and Cross-sections:1

A - 2%
and 8%
C — 3%
and 8%
8% HVO
and 8%
1

sodium chloride (NaCl), B - 3% NaCl
hydrogenated vegetable oil (HVO),
NaCl, 4% soy protein isolate (SPI)
HVO,D - 3.0% NaCl, 4% corn meal and
and E - 3% Nacl, 4% SPI, 4% corn meal
HVO .

In all sausages (A, B, C, D, and E) the

following additives and spices are present: 0.45%
sodium tripolyphosphate, 0.1% mono-sodium glutamate,
0.05% sodium ascorbate, 0.5% condensed smoke, and

mixed spices.

 

144

U. S. Government regulations require an F of 30

180
minutes to control Clostridium botulinum type B found

 

 

naturally in fish (FDA, 1970).

Adding 8% hydrogenated vegetable oil, 4% soy pro-
tein isolate and 4% corn meal separately or in combina-
tion reduced the cooking loss of fish sausages (Table 32),
but in comparing mean differences between treatments B,

C, D and E only treatment E, which contained 4% soy pro—
tein isolate and 4% corn meal, significantly decreased
cooking loss (p < 0.05).

The quality of the meat used is one of the most

important concerns facing the processor in manufacturing

sausage (Hasimoto et al., 1959). Sodium tripolyphos-

 

phate is thought to improve certain physical character-
istics of sausage products and, permits wider latitude
in utilizing raw materials that naturally lack the
ability to bind.

Sodium chloride and sodium tripolyphosphate also
can be used in fish products to reduce percentage of
cooking loss during processing. The greatest cooking loss
that occurred with frozen mechanically deboned fish
containing only 2% sodium chloride and 0.45% sodium
tripolyphosphate (Treatment-A) may be caused by the poor

binding capacity and higher moisture content of fish

 

145

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Homz NN n N

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meCCOHMHCmHm mum HmHHomHomsm mEmm man an UmBOHHom uOC 30H oEmm map CH wmmquoHom ommuw>¢8

 

ANHHZV

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Hmnzv

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xNuzv
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m D U m ¢_ muCoEpmmHB

 

mommmsmm CmHm waoam MD A& HMCoHpomm Eo o.m\m1mmv wouom Hmofim UCC
quommmo mCHUHom Hopmz .mmoq mCHxOOO mo mCoHHmH>wQ UHMUCMpm UCm NmommNHCoouom mmMHm>¢II.Nm mqm<e

 

146

muscle. Higher moisture content also causes shrinkage

to be greater during cooking. Therefore, addition of 2%
sodium chloride and 0.45% sodium tripolyphosphate did not
improve the water binding capacity of frozen mechanically
deboned fish, as was also reported by Grabowska and Sikor-
ski (1973), who found that the addition of 0.6%-5.0%
sodium chloride and 0.2%-0.5% polyphosphate, separately
and in combination, did not improve the binding quality of
mechanically deboned frozen fish. Nevertheless, high
shrinkage observed in this study (34.65%) does not mean
that sodium chloride and sodium tripolyphosphate did not
reduce cooking loss, sinCe there were no samples without
either sodium chloride or sodium tripolyphosphate for
comparison. One purpose of this study was to investigate
improvement in frozen mechanically deboned fish sausages
quality, but not to assess the effect of sodium chloride
and sodium tripolyphosphate on quality. According to
Hashimoto e£_al., (1959). Sausages made from poor quality
meat are usually not acceptable, mainly because the water
and fat content of the meat have been released from the
products. Grabowska and Sikorski (1973) investigated the
water and fat binding capacities of mechanically deboned

fish and found that its suitability for sausage emulsions

 

147

deteriorates during frozen storage more rapidly than that
of fillets or round fish. It may be that denaturation of
myofibrillar protein, mainly actomyosin, is closely
related to the quality of the sausage since actomyosin

is the largest constituent of muscle protein (Hashimoto
gt;§1., 1959).

The quality of fish sausages made during this
study was evaluated by estimating water holding capacity
(WHC) and texture of the finished products (Table 32).

It will be noted that all additives, including hydro—
genated vegetable oil, soy protein isolate, and corn meal,
improved WHC of sausages. Adding corn meal produced a
greater increase 1J1 the WHC of the sausage more than

the addition of soy protein isolate. The greatest water
holding capacity (99.53%) was obtained when frozen
mechanically deboned fish contained a combination of the
higher salt level and 0.45% sodium tripolyphosphate,
vegetable oil and both soy protein isolate and corn meal
(Treatment—E). It was established that adding either 4%
soy protein isolate or 4% corn meal separately or in com-
bination significantly increased WHC (p < 0.05). In
products based on comminuted meats, such as sausages,
sodium chloride is important not only for its antimicro-
bial action, but also because it aids in the improvement

of WHC. The ability of muscle proteins to hold fat as

 

148

well as water is also very important in the sausage
industry, and one of the functions of sodium chloride is
to enhance the power of myofibrillar proteins to bind
water and emulsify fat.

Regulation No. 541 of the Michigan Department of
Agriculture, Food Inspection Division, indicated that
all smoked fish should be processed so that the water
phase portion of the finished product has a sodium chlor-
ide content of not less than 5%. This means that the
sodium chloride content of the finished product, when
divided by the sum of the sodium chloride and moisture
contents and multiplied by 100, will yield a number of at
least 5. Adding 3% sodium chloride to minced fish brings
sodium chloride in the water phase portion of the finished
product to approximately 5%. Sodium chloride and sodium
tripolyphosphate increase WHC of sausages through the com-
bined effects of relatively high ionic strength (Hamm,
1960) and an increase in the solubility of muscle pro-
teins, caused by the ability of phosphate to dissociate
actomyosin into myosin and actin (Bendall, 1954).

When the sodium chloride concentration of minced
fish was increased to 3% and 8% hydrogenated vegetable
oil was added, WHC improved by 7% (Treatment—B). An
unexpected result of this study was that the WHC of
sausage increased with increasing fat content, for fat

should not contribute to water binding capacity

 

149

(Hamm, 1960). Swift et_al., (1954) noted this effect of
fat on WHC up to a fat to protein ratio of 2.8:1. At
higher ratios (>3.4:1) the WHC decreased. Hamm (1960)
noted that the amount of sodium chloride required in
sausage formulas is usually calculated in proportion to
the total amount of lean meat and fat. Fat takes up much
less sodium chloride than muscle tissue, and adding fat to
meat increases the amount of sodium chloride per unit
weight of lean meat. This, in turn, increases the WHC

of muscle. Sodium chloride may also improve the emulsify-
ing properties of muscle proteins because of high hydra-
tion, and perhaps begins a dissolution process on the
surfaces of protein particles, thereby promoting inter-
facial reaction between proteins and fat.

Soy protein isolate is thought to bind water and
fat when it forms a gel upon heating. Adding soy protein
isolate improved swelling of minced fish as mentioned
earlier (Table 27), and it significantly increased WHC
of fish sausage (p < 0.05, Table 32). Hamm (1960) found
a highly significant correlation between swelling and
WHC. Increased swelling is a desirable effect because
high absorption usually tends to increase the unit yield
of the finished product (Yasumatsu e£_al., 1972b). The
latter researchers also reported that an increase in
water absorption is directly proportional to the amount

of protein added. Corn meal, however, is not classified

 

.111 i L

150

as a binder, although it may bind water it is incapable of
emulsifying fat. Adding 4% corn meal to fish sausage
formulations significantly increased their WHC (p < 0.05).
According to Kramlich (1972) the ability of starch to bind
water may be affected adversely by meat amylases. These
enzymes are capable of degrading starch, but degradation
did not occur when starch was heated above 80°C, the
inactivation temperature of meat amylases.

The texture of the central mass of sausages was
determined as a shear value, obtained from the resultant
force/distance curve (Figure 17). The force measured at
the yield point (A, Figure 17) indicated firmness (Table
32), and the greater the shear force, the greater the
firmness of the product. Texture and binding in comminuted
fish sausage has been found to follow a pattern similar to
that of solubility changes in myofibrillar proteins in
minced frozen fish (Grabowska and Sikorski, 1973). The
relationship between texture, binding and the solubility
of myofibrillar proteins might explain the effect of
sodium chloride and sodium tripolyphosphate on texture,
since the solubility of myofibrillar proteins is enhanced
by adding sodium chloride and sodium tripolyphosphate to
frozen minced fish.

Adding 8% hydrogenated vegetable oil to fish

sausage decreased its firmness (Table 32). Swift et al.,

, 151

 

Force —>

 

 

Distance —>

Figure l7.—-Typical Force/Distance Curve for Central
Mass of Fish Products. A = Yield point
indicative of firmness.

 

 

152

(1954), reported similar results, noting that variations
in fat content produced changes in the tenderness of
bologna. They found that as fat content increased,
product tenderness also increased. On the other hand, in
this study the greatest firmness in fish sausage (1.52Kg-f)
was obtained with a blend of 4% soy protein isolate and

4% corn meal in addition to 3% sodium chloride and 0.45%
sodium tripolyphosphate. Texture measurements indicated
that adding either 4% soy protein isolate or 4% corn meal,
or a combination of both significantly increased the
firmness of sausages (p < 0.05). Increased firmness may
be caused by improved sWelling due to the presence of soy
protein isolate, as reported earlier (Table 27), and also

by the absorption of water by corn meal.

Canned Minced Fish

 

Canned fish is commercially sterile since it is
heated to temperatures at which most microorganisms and

their spores, particularly Clostridium botulinum, are

 

killed. Such high temperatures cause denaturation,
amino acid destruction, H28 liberation, metmyoglobin
formation, collagen solubilization, Maillard-type brown—
ing, and accompanying undesirable changes in flavor.
These effects are more marked in canned products than in
normal cooked fish products (Lawrie, 1968). It is gen-

erally recognized that canned foods produced from frozen

 

153

fish especially from fish held for long periods on ice
or in refrigerated brine, are inferior in quality to
those made from absolutely fresh fish. Denaturation of
protein which occurs in round fish and frozen minced
fish during such a holding period may affect the canned
product in many ways. Generally, holding frozen fish
results in decrease protein solubility, lower water and
fat binding capacities, and poor texture. Water holding
capacity and textural properties were investigated in
this study of canned minced fish. The earlier study
reported in this dissertation indicated that the addi-
tion of 0.45% phosphate'without sodium chloride extracted
approximately 50% of the total protein, solubilized the
greatest quantity of myosin heavy chains, produced the
highest pH and swelling, and gave the best gel forming
ability to frozen minced fish (Tables 8, 20, 23, 26, and
29). Therefore, it was decided to use the same blend of
frozen minced fish in a canned product, with the addition
of 1.0% sodium chloride to improve product taste and to
obtain the improved functional properties mentioned
earlier under practical conditions. In addition to the
amounts of sodium chloride and sodium tripolyphosphate
mentioned above, the canned minced fish paste blend con—
tained 4% beef tripe and 8% hydrogenated vegetable oil.

Some of the batter also contained 4% soy protein isolate

 

154

or 4% corn meal, or a combination of these two ingredients.
The product was processed at 121°C:fixr75 minutes. After
the cans were filled, the fish paste was baked in a dry
air oven at 75°C for 30 minutes to remove most of the
oxygen and portions of its moisture content in order to
obtain the proper texture.

The average percentages of baking loss for each
treatment were 0.75, 0.69, 0.41, and 0.33 (Table 33).
These values were calculated by averaging the cooking loss
percentage of two trials. There was a slight reduction
in mositure content (0.33% to 0.75%),which resulted from
evaporation during heating. The fast surface coagulation
occurring during the dry heating process prevented much
evaporation from fish paste. Fish paste that contained
4% soy protein isolate and 4% corn meal in addition to
the other ingredients (Treatment-D) had the lowest baking
loss (0.33%). This reduction in baking loss may be due
to an improvement in the water binding capacity of fish
paste through the addition of soy protein isolate and
corn meal. Coagulation of protein and gelatinization of
starch that occurred at the product surface also pre-
vented baking loss. Moreover, the moisture content of
fish paste containing soy protein isolate and corn meal
was less than that of the other treatments since some of
the fish flesh was replaced in them by these two ingredi-

ents, both of which contain little moisture. Baking loss

 

 

 

 

155

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156

of fish paste was significantly decreased (p < 0.05) by
adding corn meal or a combination of corn meal and soy
protein isolate, but not by soy protein isolate alone.

Canned minced fish containing 1.0% sodium chloride,
0.45% sodium tripolyphosphate, 4% beef tripe and 8% hydro—
genated vegetable oil (Treatment-A) shrank more than that
containing 4% soy protein isolate and/or 4% corn meal in
addition to the other ingredients. When the finished
products had been processed at 121°C for 75 minutes, all
but those subject to treatment A showed relatively firm
structure with few air pockets (Figure 18). Treatment-A
containing neither soy protein isolate nor corn meal pro-
duced more meat exudate. When fish paste was heated,
moisture was released due to the denaturation of muscle
proteins and the syneresis of gelation gel. Meat that
has a high water holding capacity should reabsorb some of
the released moisture. Canned minced fish that contained
either soy protein isolate or corn meal or a combination
of both reabsorbed the released moisture, whereas minced
fish alone failed to do so.

Treatment A showed approximately 80% water hold-
ing capacity. Further improvement in water holding
capacity was obtained by adding soy protein isolate and/
or corn meal (Treatment A vs. B, C and D). In this

study corn meal appeared to be a better water binder than

\

 

 

 

157

   

Figure 18.--Cross-sections of Canned Minced Fish.

A1 — 86.5% minced fish, Bl — 82.5% minced
fish and 4% soy protein isolate (SPI),
Cl — 82.5% minced fish and 4% corn meal,
Dl - 78.5% minced fish, 4% SPI and 4% corn
meal.

1In all formulations (A, B, C and D) the
following additives are present: 4% beef tripe,
8% hydrogenated vegetable oil, 1.0% sodium
chloride, 0.45% sodium tripolyphosphate and mixed
spices.

 

158

soy protein isolate in fish paste. This may be because
corn meal has a greater capacity to swell and form gels
than soy protein isolate. The greatest water holding
capacity (98.79%) was obtained by using 4% soy protein
isolate and 4% corn meal in combination with the other
ingredients.

The force required to shear a 1.8 cm sectional
diameter of canned fish product made according to treat-
ment A was 180 grams. This product was soft and mushy
and difficult to slice. Firmness increased with the addi-
tion of 4% soy protein isolate (Treatment-B), 4% corn
meal (Treatment-C) and a combination of the two (Treat-
ment—D). The force needed to shear 1.8 cm sectional
diameters of products made according to treatments B, C
and D was 290, 310, and 410 grams, respectively. Statis-
tical analysis indicated that each of the four treatment
means for firmness were significantly different from

each other (p < 0.05).

 

 

 

SUMMARY AND CONCLUSION

Some of the physical and chemical characteristics
of minced sucker flesh were examined. Prerigor sucker
muscle protein was fractionated into myofibrillar and
sarcoplasmic proteins as well as nonprotein nitrogen.

The effects of refrigeration and freezer storage on the
extractability of those protein fractions were examined.
It was found that the solubility of myofibrillar proteins
decreased during both refrigerator and freezer storage,
but that the solubility of sarcoplasmic proteins and non-
protein nitrogen tmas not influenced by either method of
storage.

Frozen minced sucker protein was fractionated into
salt and water soluble proteins and nonprotein nitrogen
fractions, as well as myosin heavy chain to study the
interrelated effects of adding sodium chloride, sodium
tripolyphosphate and soy protein isolate on protein solu-
bility, pH, swelling and gel forming properties. Total
extracted protein and salt soluble protein increased with
the addition of either sodium chloride or sodium tripoly-
phosphate. On the other hand, levels of water soluble

protein and nonprotein nitrogen decreased when either

159

 

 

160

sodium chloride or sodium tripolyphosphate were added to
extraction solution. Myosin heavy chain was solubilized
with the addition of 3% or 3.6% sodium chloride and also
in the presence of 0.45% sodium tripolyphosphate, but not
when a concentration of 0.225% of this chemical was used.
The values of total extracted protein, water soluble and
salt soluble proteins and nonprotein nitrogen extracted
from frozen minced fish containing 2% and 4% soy protein
isolate indicated that soy protein isolate did not com-
pletely solubilize, since none of the protein fractions
increased by 2% or 4% over a control containing no soy
protein isolate.

Swelling and pH of frozen minced fish increased
as sodium tripolyphosphate concentration increased, but
as sodium chloride concentration increased swelling and
pH decreased. Adding soy protein isolate did not change
the pH, but did cause increased swelling. Poor gelation
prOperties resulted from adding sodium chloride, but the
addition of sodium tripolyphosphate alone improved gel
forming ability. Measurement of gel formation did not
seem to be a suitable way to estimate the effect of adding
soy protein isolate on the quality of frozen minced fish.

The functionality tests indicated that frozen
minced fish has some desirable properties. Samples which

had the highest ionic strength did not exhibit the highest

 

161

overall ratings of functional properties. It is not cer—
tain that added sodium chloride is in itself responsible
for the resulting functional behavior of fish muscle pro-
tein. If this were the case, the presence of salt would
have both beneficial and detrimental aspects. _Taking
protein solublity as an example, the salt—rich samples
produced high values in solubility, but also showed

the lowest values for swelling, gel forming and pH. In
comparison, sodium tripolyphosphate appears to cause many
beneficial effects, such as increases in protein solubil—
ity, pH, swelling and gel forming ability.

Frozen minced sucker was blended with different
additives and sausage-type and canned products were
processed and their functional properties evaluated in
terms of water-holding capacity and texture. Fish sausages
and canned minced fish showed poor water holding capacity,
low binding characteristics and high cooking losses. How—
ever, 3.0% salt in fish sausages and 1.0% salt added to
canned products as well as 0.45% sodium tripolyphosphate
in combination with hydrogenated vegetable oil, soy pro-
tein isolate and corn meal in both products improved
their functional properties. Since soy protein isolate
has an objectionable flavor, and because the addition of
corn meal improved water holding capacity, texture and

cooking losses at least as much as soy protein isolate,

162

it was concluded that corn meal should be used in manu—
facturing processed fish products.

If canned minced fish were accepted throughout the
world, especially in the less—developed countries, it
would significantly contribute to the efficient use of
underutilized fish species for food, and extend world

supplies of quality protein.

 

APPENDICES

163

 

APPENDIX 1

STANDARD CURVE FOR MYOSIN HEAVY CHAIN

TOTAL PEAK AREA

164

 

X1O
7.0 -

6.0 -
5-O -
4.0 '-
3.0 -
2.0 -
1.0 -

0.0 I l 1 l 1 1 l n
O 10 20 3O 4O

 

MHC Total Area

 

Micrograms Total Protein

Appendix l.--Standard Curve for Myosin Heavy Chain Total
Peak Area.

165

 

 

APPENDIX 2

AVERAGE PERCENTAGE OF TOTAL PROTEIN EXTRACTED

FROM MECHANICALLY DEBONED FROZEN FISH

166

 

APPENDIX 2.--Average Percentage and Standard Deviation
of Total Protein Extracted from Mechanically
Deboned Frozen Fish (N = 4)

 

Percent Additives

 

 

 

Sodium Soy Sodium Chloride

Tripoly— Protein

phosphate Isolate 0.00 3.00 3.60
0.000 0.0 l3.65§0.25 67.81:l.92 58.54:0.68
0.000 2.0 13.45i0.20 66.14:2.22 64.26:l.28
0.000 4.0 l3.9li0.l4 68.24i1.53 63.72:0.74
0.225 0.0 14.17:0.08 49.13il.04 52.79il.l8
0.225 2.0 14.83i0.36 55.99il.64 55.79:0.51
0.225 4.0 16.6li0.06 58.9512.40 55.85i2.24
0.450 0.0 49.24t2.12 59.67il.24 58.90i2.41
0.450 2.0 47.31il.66 59.74:l.08 60.78il.66
0.450 4.0 46.96:2.27 56.88i0.97 63.66:l.64

 

*Total extracted protein = mg extracted protein/
100 mg total muscle protein.

167

 

APPENDIX 3

AVERAGE PERCENTAGE OF SALT-SOLUBLE PROTEIN

EXTRACTED FROM MECHANICALLY DEBONED

FROZEN FISH

APPENDIX 3.—-Average Percentage and Standard Deviation
of Salt—Soluble Protein Extracted From
Mechanically Deboned Frozen Fish (N = 2)

 

Percent Additives

 

 

 

 

Sodium Soy Sodium Chloride

Tripoly— Protein

phosphate Isolate 0.00 3.00 3.60
0.000 0 0 0.00 26.57*i0.98 29.97i0.51
0.000 2 0 0.00 32.70 i2.42 34.53i0.55
0.000 4.0 0.00 37.07 i0.40 31.53i0.25
0.225 0.0 0.78i0.00 32.07 i2.46 29.37i0.20
0.225 2.0 1.17i0.04 32.19 i0.85 30.33i1.87
0.225 4.0 1.85i0.08 28.47 i0.51 30.45il.02
0.450 0.0 25.10i4.l6 29.97 :l.95 30.09i0.68
0.450 2.0 25.59il.l9 25.52 i0.51 35.73i0.8l
0.450 4.0 24.17i0.55 28.62 i1.23 36.04i1.08

*Salt soluble protein = mg salt soluble protein/
100 mg total muscle protein.

169

 

APPENDIX 4
AVERAGE PERCENTAGE OF WATER-SOLUBLE
PROTEIN EXTRACTED FROM MECHANICALLY

DEBONED FROZEN FISH

170

 

APPENDIX 4.—-Average Percentage and Standard Deviation
of Water-Soluble Protein Extracted from

 

 

 

 

 

Mechanically Deboned Frozen Fish (N = 2)
Percent Additives

Sodium Soy Sodium Chloride

Tripoly— Protein

phosphate Isolate 0.00 3.00 3.60
0.000 0.0 13.39*i0.00 25.9li0.39 19.28:0.17
0.000 2.0 13.39 10.00 22.3l:0.38 22.04i0.30
0.000 4.0 13.96 :0.04 26.00:0.43 24.32i0.51
0.225 0.0 13.18 :0.04 22.94:2.12 27.81i2.37
0.225 2.0 13.06 13.18 24.83i0.55 29.67i0.l7
0.225 4.0 14.23 i0.34 27.89:0.03 25.52i0.55
0.450 0.0 16.54 :0.81 22.88i0.93 24.98i0.34
0.450 2.0 22.76 :0.25 21.59i6.06 26.13i0.04
0.450 4.0 23.99 i0.38 21.47:3.26 29.25:0.89

*Water soluble protein mg water soluble protein/
100 mg total muscle protein.

171

APPENDIX 5

AVERAGE PERCENTAGE OF NONPROTEIN NITROGEN

EXTRACTED FROM MECHANICALLY DEBONED

FROZEN FISH

172

 

 

APPENDIX 5.--Average Percentage and Standard Deviation
of Nonprotein Nitrogen Extracted from
Mechanically Deboned Frozen Fish (N = 4)

 

Percent Additives

 

 

 

Sodium Soy Sodium Chloride

Tripoly- Protein

phosphate Isolate 0.00 3.00 3.60
0.000 0.0 8.00*i0.45 6.59i0.l7 5.48:0.29
0.000 2.0 8.15 i0.11 5.88:0.07 5.10:0.21
0.000 4.0 7.15 :O.14 5.87:0.20 4.88:0.23
0.225 0.0 6.36 :0.00 .16:0.20 6.35i0.16
0.225 2.0 5.97 i0.03 .08i0.l6 6.13:0.10
0.225 4.0 5.88 i0.05 .96:0.13 5.69:0.16
0.450 0.0 5.88 10.05 .88:0.16 5.84:0.20
0.450 2.0 5.73 i0.18 .67:0.10 5.70i0.18
0.450 4.0 5.63 i0.18 5.23:0.08 5.30i0.06

 

*Nonprotein nitrogen = mg nonprotein nitrogen/100
mg total muscle protein (N)(6.25).

173

 

 

APPENDIX 6

MEAN VALUES OF MYOSIN HEAVY CHAIN
TOTAL AREA EXTRACTED FROM
MECHANICALLY DEBONED

FROZEN FISH

174

 

Appendix 6.-—Mean Values and Standard Deviation of Myosin
Heavy Chain Total Area Extracted from
Mechanically Deboned Frozen Fish (N = 3)

 

Percent Additives

 

 

 

Sodium Soy Sodium Chloride
Tripoly- Protein
phosphate Isolate 0.00 3.00 3.60
5 5
0.000 0.0 0.00 1.02 x 10 .14 x 10
i 0.01 i 0.23
0.000 2.0 0.00 0.66 x 105 .04 x 105
i 0.00 i 0.15
0.000 4.0 0.00 0.55 x 105 82 x 105
i 0.02 i 0.14
0.225 0.0 0.00 2.02 x 105 .02 x 105
i 0.08 i 0.07
0.225 2.0 0.00 2.55 x 105 25 x 105
i 0.14 i 0.16
0.225 4.0 0.00 1.85 x 105 .16 x 105
i 0.14 i 0.06
0.450 0.0 2 46 x 105 1.74 x 105 .14 x 105
i 0.30 i 0.16 i 0.03
0.450 2.0 2.66 x 105 1.82 x 105 46 x 105
+ 0.06 i 0.28 i 0.03
0.450 4.0 2.66 x 105 1.99 x 105 75 x 105
i 0.48 i 0.09 i 0.17

 

*Total peak area that measured by scanning myosin
heavy chain bond obtained from 20 micrograms total
extracted protein sample.

175

 

APPENDIX 7

AVERAGE OF PH OF MECHANICALLY

DEBONED FROZEN FISH

176

 

APPENDIX 7.--Average and Standard Deviation of pH of
Mechanically Deboned Frozen Fish (N = 2)

 

Percent Additives

 

 

 

Sodium Soy Sodium Chloride

Tripoly- Protein

phosphate Isolate 0.00 3.00 3.60
0.000 0.0 6.80i0.00 6.3010.00 6.09i0.03
0.000 2.0 6.90i0.00 6.20i0.00 6.15i0.00
0.000 4.0 6.90:0.00 6.10i0.00 6.19i0.01
0.225 0.0 7.20:0.00 6.40i0.02 6.35i0.01
0.225 2.0 7.30i0.00 6.39i0.01 6.39i0.02
0.225 4.0 7.30i0.00 6.43i0.01 6.40:0.01
0.450 0.0 7.80i0.00 6.68i0.03 6.67i0.02
0.450 2.0 7.80i0.00 6.68i0.01 6.67i0.02
0.450 4.0 7.80i0.00 6.65i0.00 6.68i0.00

 

177

APPENDIX 8

AVERAGE PERCENT OF SWELLING OF

MECHANICALLY DEBONED FROZEN

FISH

178

 

APPENDIX 8.-—Average Percent and Standard Deviation of
Swelling of Mechanically Deboned Frozen

Fish (N =

4)

 

Percent Additives

 

 

 

Sodium Soy Sodium Chloride

Tripoly- Protein

phosphate Isolate 0.00 3.00 3.60
0.000 0.0 65.7433.57 49.65:5.84 28.20i0.00
0.000 2.0 42.50:0.00 40.7li3.57 56.80:0.00
0.000 4.0 56.80t0.00 69.67i4.l3 56.80:0.00
0.225 0.0 57.7214.13 56.80t0.00 42.50i0.00
0.225 2.0 71.10i0.00 51.44:6.84 42.50:0.00
0.225 4.0 88.97i4.13 71.1030.00 60.37i4.l3
0.450 0.0 96.12i7.15 42.50i0.00 56.80i0.00
0.450 2.0 106 85:8.25 71.1030.00 56.58:3.57
0.450 4.0 121.15i5.84 71.10i0.00 76.46:6.84

 

179

 

APPENDIX 9

AVERAGE OF LEAST CONCENTRATION ENDPOINT

OF MECHANICALLY DEBONED FROZEN FISH

180

 

APPENDIX 9.--Average and Standard Deviation of Least Con-
centration Endpoint of Mechanically Deboned
Frozen Fish (N = 4)

 

Percent Additives

 

 

 

Sodium Soy Sodium Chloride

Tripoly- Protein

phosphate Isolate 0.00 3.00 3.60
0.000 0.0 1.60i0.56 4.45i0.40 5.19i0.39
0.000 2.0 1.43i0.29 4.65i0.00 6.04i0.65
0.000 4.0 1.20:0.28 3.99i0.14 5.63i0.24
0.225 0.0 1.81i0.10 4.45i0.47 7.12i0.22
0.225 2.0 0.82:0.11 7.07i0.93 6.82i0.21
0.225 4.0 1.57i0.23 5.06:0.47 6.67i0.49
0.450 0.0 1.07i0.24 6.82i0.66 6.66:0.36
0.450 2.0 1.33i0.52 7.21i0.11 7.35i0.36
0.450 4.0 1.25:0.70 9.2130.02 7.91:0.14

 

Note: Least concentration endpoint is the mini—
mun protein concentration required to form a gel.

181

bis v J

 

 

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182

 

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